Enzymatic Recognition Research Articles

Chondroitin sulfate-based anti-inflammatory macromolecular prodrugs

Macromolecular prodrugs of three non-steroidal anti-inflammatory drugs (NSAIDs), ibuprofen, ketoprofen, and naproxen, were prepared by the covalent attachment of the drugs onto chondroitin sulfate (ChS) using PEG 1000 as a spacer. Drug–PEG adducts were synthesized using 1,1′-carbonyl diimidazole as a coupling agent in dimethyl sulfoxide, followed by the reaction with ChS in highly dilute aqueous solution at pH 6.8 via N-(3-dimethylaminopropyl)- N′-ethylcarbodiimide hydrochloride (EDAC) as a conjugation agent. The drug–ChS conjugates were confirmed by FTIR, 1H NMR and 13C NMR and the molar percent of drug substitution onto ChS was characterized by 1H NMR using the peak areas of the three protons of –ΦCHC H 3 on the drugs to those of –NHCOC H 3 on ChS. All drug–ChS conjugates are water-soluble. The release amounts of the free drugs from their corresponding drug–ChS conjugates were evaluated in the presence or absence of either esterase or chondroitinase, and the both enzymes in pH 7.4 Tris-buffer solutions at 37 °C by high performance liquid chromatography (HPLC). Keto–ChS conjugates released ∼100% ketoprofen within 12 h in the presence of esterase, but the combination with chondroitinase did not accelerate the release rate. The degradation of Keto–ChS conjugates by chondroitinase was confirmed by gel permeation chromatography (GPC). The Keto–ChS conjugates still retained the enzymatic recognition even at the substitution of ketoprofen as high as 56 mol%. The inhibition percent of carrageenan-induced edema of Keto–ChS-56 was comparable to that of a simple blend of ChS and ketoprofen, suggesting that biologically active ChS and ketoprofen could be liberated from the conjugate.

Manometric transduction in enzyme biosensors

The combination of enzymatic recognition and manometric transduction is explored, using enzymes that consume or evolve a gas with low solubility in aqueous media. A design is discussed whereby change in partial pressure of a gas in the headspace is related to the turnover of analyte by the enzyme. Headspace and sample volume dimensions are considered, demonstrating the influence of flux at the air-water interface. The relative importance of diffusion and reaction for the enzyme solution is shown. When enzyme kinetics dominate, the concentration gradient is low and the overall kinetics are determined by the total amount of active enzyme, reducing either enzyme concentration or enzyme layer thickness will reduce the diffusion limitation. A Teflon-enzyme composite is presented to allow a reuseable immobilised enzyme preparation and a disc with stirring magnet identified as an efficient configuration. A glucose oxidase system was tested in the monitoring of glucose consumption during fermentation. Application to other enzyme systems is discussed.

Combined Application of Galactose Oxidase and β‐N‐Acetylhexosaminidase in the Synthesis of Complex Immunoactive N‐Acetyl‐D‐galactosaminides

AbstractA high‐yield preparatory procedure for the synthesis of p‐nitrophenyl 2‐acetamido‐2‐deoxy‐β‐D‐galacto‐hexodialdo‐1,5‐pyranoside (2) using the galactose oxidase from Dactylium dendroides in a batch reactor was developed. Enzymatic recognition of this aldehyde and the respective uronic acid 3 obtained by NaClO2 oxidation was studied using a set of 36 fungal β‐N‐acetylhexosaminidases from Acremonium, Aspergillus, Penicillium and Talaromyces genera. The aldehyde 2 was readily hydrolysed by all tested β‐N‐acetylhexosaminidases but neither the uronic acid 3 nor its methyl ester 4 were accepted. Molecular modelling with docking into the active centre of the β‐N‐acetylhexosaminidase from Aspergillus oryzae revealed that the aldehyde 2 is processed as a C‐6 geminal diol by the enzyme. The aldehyde 2 was tested for transglycosylation reactions using GlcNAc as an acceptor. The β‐N‐acetylhexosaminidase from Talaromyces flavus gave the best yields (37%) of the transglycosylation product 2‐acetamido‐2‐deoxy‐β‐D‐ galacto‐hexodialdo‐1,5‐pyranosyl‐(1→4)‐2‐acetamido‐ 2‐deoxy‐D‐glucopyranose, which was oxidised in situ to yield the final product 2‐acetamido‐2‐deoxy‐β‐D‐galactopyranosyluronic acid‐(1→4)‐2‐acetamido‐2‐deoxy‐D‐glucopyranose (6). Compounds 3 and 6 were shown to be high‐affinity ligands for two natural killer cell activation receptors, NKR‐P1A and CD69. For the latter receptor they turned out to be among the best ligands described so far. This increase was obviously due to the presence of a carboxy moiety.

Hybridization properties and enzymatic replication of oligonucleotides containing the photocleavable 7-nitroindole base analog

Universal DNA base analogs having photocleavable properties would be of great interest for development of new nucleic acid fragmentation tools. The photocleavable 7-nitroindole 2′-deoxyribonucleoside d(7-Ni) was previously shown to furnish a highly efficient approach to photochemically trigger DNA backbone cleavage at preselected position when inserted in a DNA fragment. In the present report, we examine its potential use as universal DNA nucleoside, by analogy with the 5-nitroindole analog that is generally considered as universal base. The d(7-Ni) phosphoramidite was incorporated into oligonucleotides. Hybridization properties of resulting 11mer duplexes indicated a behavior close to that of the 5-nitroindole analog. Enzymatic recognition by Klenow fragment exonuclease-free using 40mers containing the unnatural bases as templates indicated notably a decrease of the polymerase activity with preferential incorporation of dAMP opposite both the 7-Ni and 5-Ni bases. Incorporation of the d(7-Ni) triphosphate was also studied indicating absence of significant differences between the incorporation kinetics opposite each natural base in the template. All the hybridization and enzymatic data indicate that 7-nitroindole can be considered as a cleavable base analog, although not strictly fulfilling, like the 5-nitro isomer, all properties required for a universal base.

Sweetening Cyclic Peptide Libraries

Enzymatic macrolactamization of linear glycosidated peptides provides access to an important class of drug-like molecules. The work presented in this issue [1] shows that it may be possible to make complex libraries of glycosidated cyclic peptides by incorporating glycosidated amino acids into linear peptides via solid-phase peptide synthesis followed by thioesterase-mediated peptide cyclization.

The Role of Weak Specific and Nonspecific Interactions in Enzymatic Recognition and Conversion of Long DNAs

According to the currently accepted model, enzymes searching for specific recognition sequences or structural elements (modified nucleotides, breaks, single-stranded DNA fragments, etc.) slide at a high rate along DNA. Such sliding is possible only if the enzymes possess sufficiently high affinity for all DNA, sequence notwithstanding. Therefore, significant differences in their affinity for specific and nonspecific DNA sequences are unlikely, and the formation of a complex between an enzyme and its target DNA is not a basic factor of enzyme specificity. To elucidate such factors, we have analyzed many DNA replication, DNA repair, topoisomerization, integration, and recombination enzymes using a number of physicochemical methods, including the method of stepwise increase in ligand complexity developed in our laboratory. It has been shown that high affinity of all studied enzymes for long DNAs is provided by the formation of many weak contacts of the enzyme with all nucleotide units covered by the protein globule. The main role lies in the contact between positively charged amino acid residues and internucleoside phosphate groups; however, the contribution of each contact is very small, and the full contact interface usually resembles that characteristic of interactions between oppositely charged biopolymer surfaces. In some cases, a significant contribution to the affinity is made through hydrophobic and/or van der Waals interactions of the enzymes with nucleotide bases. On the whole, such nonspecific interactions provide for five to eight orders of enzyme affinity for DNA, depending on the enzyme. Specific interactions of enzymes with long DNAs, in contrast to their contacts with small ligands, are usually weak and comparable in efficiency with weak nonspecific contacts. The sum of specific interactions most often provides for approximately one or, rarely, two orders of affinity. According to structural data, DNA binding to any of the investigated enzymes is followed by a stage of DNA conformation adjustment, which includes partial or complete DNA melting, deformation of its backbone, stretching, compression, bending or kinking, eversion of nucleotides from the DNA helix, etc. The full set of such changes is specific for each individual enzyme. The fact that all enzyme-dependent changes in DNA are effected through weak specific (rather than strong) interactions is very important. Enzyme-specific changes in DNA conformation are required for effective adjustment of reacting orbitals to an accuracy of 10°–15°, which is possible only in the case of specific DNAs. A transition from nonspecific to specific DNA leads to an increase in the reaction rate (k cat) by four to eight orders of magnitude. Thus, the stages of DNA conformation adjustment and catalysis proper provide for the high specificity of enzyme action.

Study of a Bioreactor Based upon α‐Chymotrypsin

α‐Chymotrypsin (ACHT) was immobilized on an epoxide‐derivatized silica gel by an in situ immobilization process. Several racemates has been resolved according an enzymatic recognition mechanism on this ACHT chiral stationary phase. The loadability of the ACHT‐CSP, used as bioreactor, has been evaluated in both buffered and non‐buffered mobile phases under the enzymatic process.

A new linker for glucuronylated anticancer prodrugs

The synthesis, enzymatic hydrolysis and self decomposition of model glucuronylated prodrugs, incorporating a new linker with different aryl substituents, have been studied. Determination of kinetic parameters ( V max, K m and t 1/2) showed the important role of aromatic substitution in enzymatic recognition and linker decomposition.

Exploiting the enzymatic recognition of an unnatural base pair to develop a universal genetic analysis system.

With the invention of the DNA chip, genome-wide analysis is now a reality. Unfortunately, solid-phase detection systems such as the DNA chip suffer from a narrow range in quantification and sensitivity. Today the best methodology for sensitive, wide dynamic range quantification and genotyping of nucleic acids is real-time PCR. However, multiplexed real-time PCR technologies require complicated and costly design and manufacturing of separate detection probes for each new target. We developed a novel real-time PCR technology that uses universal energy transfer probes constructed from An Expanded Genetic Information System (AEGIS) for both quantification and genotyping analyses. RNA quantification by reverse transcription-PCR was linear over four orders of magnitude for the simultaneous analysis of beta-actin messenger RNA and 18S ribosomal RNA. A single trial validation study of 176 previously genotyped clinical specimens was performed by endpoint analysis for factor V Leiden and prothrombin 20210A mutation detection. There was concordance for 173 samples between the genotyping results from Invader tests and the AEGIS universal energy transfer probe system for both factor V Leiden and prothrombin G20210A. Two prothrombin and one factor V sample gave indeterminate results (no calls). The AEGIS universal probe system allows for rapid development of PCR assays for nucleic acid quantification and genotyping.

Efficient RNase H-directed cleavage of RNA promoted by antisense DNA or 2'F-ANA constructs containing acyclic nucleotide inserts.

The ability of modified antisense oligonucleotides (AONs) containing acyclic interresidue units to support RNase H-promoted cleavage of complementary RNA is described. Manipulation of the backbone and sugar geometries in these conformationally labile monomers shows great benefits in the enzymatic recognition of the nucleic acid hybrids, while highlighting the importance of local strand conformation on the hydrolytic efficiency of the enzyme more conclusively. Our results demonstrate that the duplexes support remarkably high levels of enzymatic degradation when treated with human RNase HII, making them efficient mimics of the native substrates. Furthermore, interesting linker-dependent modulation of enzymatic activity is observed during in vitro assays, suggesting a potential role for this AON class in an RNase H-dependent pathway of controlling RNA expression. Additionally, the butyl-modified 2'F-ANA AONs described in this work constitute the first examples of a nucleic acid species capable of eliciting high RNase H activity while possessing a highly flexible molecular architecture at predetermined sites along the AON.

Enzymatic asymmetric hydroxylation of unnatural substrates with soybean lipoxygenase

Surrogate substrates mimicking the natural substrate (linoleic acid) bearing a spacing prosthetic group with a non-ionic hydroxyl terminus undergo asymmetric hydroxylation with soybean lipoxygenase. The prosthetic modifier supplies the missing structural features needed for enzymatic recognition and controls the regiochemical outcome of the reaction by its high hydrophobic content. The effect of pH on the regiochemistry clearly shows that all the substrates can arrange themselves at the active site of soybean lipoxygenase in only one orientation leading to formation of hydroperoxides by oxygenation at the ω-6 carbon.

Structural and thermodynamic studies on mutant RNA motifs that impair the specificity between a viral replicase and its promoter

The 3′-end region of the genomic RNA of brome mosaic virus forms a tRNA-like structure that is critical for its replication. Previous studies have shown that in this region, a stem-loop structure, called SLC, is necessary and sufficient for the binding of the RNA replicase, and for RNA replication. Recently, we determined the high-resolution NMR structure of SLC, which demonstrated that a 5′-AUA-3′ triloop region is an important structural element for the enzymatic recognition. We proposed that the 5′-adenine of the triloop, which is rigidly fixed (“clamped”) to the stem, is a key recognition element for the replicase. To elucidate the role of this “clamped base motif” for the enzymatic recognition, we have now investigated the solution conformations of several stem-loop molecules with mutant triloops, 5′-UUA-3′, 5′-GUA-3′, 5′-CUA-3′ and 5′-UUU-3′, that destroy the enzymatic recognition. For the GUA and UUA mutants, we have obtained high-resolution solution structures using 2D NMR. All four mutants have very similar thermodynamic stabilities, and all have the same secondary structures, a triloop with a five base-paired stem helix. In addition, they have quite similar sugar puckering patterns in the triloop region. The NMR structures of the GUA and UUA show that the 5′ nucleotide of the triloop (G6 in GUA or U6 in UUA) lacks the strong interactions that hold its base in a fixed position. In particular, the U6 of UUA is found in two different conformations. Neither of these two mutants has the clamped base motif that was observed in the wild-type. While UUA also shows global change in the overall triloop conformation, GUA shows a very similar triloop conformation to the wild-type except for the lack of this motif. The absence of the clamped base motif is the only common structural difference between these two mutants and the wild-type. These results clearly indicate that the loss of function of the UUA and GUA mutants comes mainly from the destruction of a small key recognition motif rather than from global changes in their triloop conformations. Based on this study, we conclude that the key structural motif in the triloop recognized by the replicase is a solution-exposed, 5′-adenine base in the triloop that is clamped to the stem helix, which is called a clamped adenine motif.

Preparation and enzymatic recognition of guanine lesions induced by nitrogen oxides.

Xanthine (Xan) and oxanine (Oxa) are the major deamination products of guanine formed by the treatment with nitrogen oxides (e.g., NO and HNO2). In this study, 2'-deoxyribonucleoside 5'-triphosphates of Xan and Oxa were prepared by the NaNO2 treatment of dGTP. These modified nucleotides were incorporated into oligonucleotides by DNA polymerase reactions. The repair activities of various DNA N-glycosylases for Xan and Oxa were examined using these substrates.

Structure-specificity relationship of cardiac glycosides as a substrate for glucohydrolase II.

Cardenolide glucohydrolase II (CGH II) is a cardenolide-specific glucohydrolase obtained from Digitalis lanata leaves. We investigated the structure-specificity relationship of several cardenolide disaccharides as a substrate for CGH II. Conformation analysis of the substrates was performed using molecular mechanics calculations. The sugar chain conformation of two inert glycosides was significantly different from that of the other glycosides. The other two glycosides, which were weak substrates of CGH II, were suggested to have an intramolecular hydrogen bond between the sugar groups. It was deduced that this hydrogen bond restricts the conformational change of the sugar chain and prevents the glycosides from enzymatic recognition.

INTRACELLULAR SURFACTANT REMOVAL FROM PHAGOCYTIZED MINERALS: Development of a Fluorescent Method Using a BODIPY-Labeled Phospholipid

Lung surfactant serves as a protective coating when adsorbed on particle surfaces, so its removal or rate of removal in vivo may affect expression of mineral cytotoxicity. Removal of phospholipid surfactant components from the surface of mineral particles ingested by alveolar macrophages (AM) was measured using fluorescence microscopy. Dipalmitoylphosphatidylcholine with a fluorescent label (BODIPY) substituted for C1-C4 on the second acyl chain (DPPC*), was mixed with dioleoylphosphatidylcholine (DOPC) to coat respirable quartz and kaolin particles. Fluorescence from quartz or kaolin particles of 3-4, 5-6 and 8-9 µm size decreased in intensity with increasing ratios of DOPC/DPPC* for the same DOPC concentration of 0.4 mg/ml. There was a direct correlation between fluorescence and residual phospholipid surfactant remaining on particles using phospholipase A2 (PLA2) digestion in a cell-free system, indicating that the presence of the fluorophore on DPPC did not hinder enzymatic recognition. Lavaged primary AM obtained from male Fischer rats were challenged in vitro with DOPC/DPPC* (10:1 mol:mol) coated particles at 50 µg particles/106 cells. In contrast to the biexponential response seen in cell-free experiments, the rate of fluorescence decay from ingested coated quartz or kaolin particles over 7 days was monoexponential, with the same t1/2 (41 h) for each dust. This study suggests that the rate of phagolysosomal digestion and removal of the adsorbed surfactant is not a determinant of the different mineral-specific pathogenicities or toxicities of quartz and kaolin, although residual fluorescence remained on particles even after 7-8 days.

Probing Aplysia californica adenosine 5'-diphosphate ribosyl cyclase for substrate binding requirements: design of potent inhibitors.

Readily synthesized nicotinamide adenine dinucleotide (NAD(+)) analogues have been used to investigate aspects of the cyclization of NAD(+) to cyclic adenosine 5'-O-diphosphate ribose (cADPR) catalyzed by the enzyme adenosine 5'-O-diphosphate (ADP) ribosyl cyclase and to produce the first potent inhibitors of this enzyme. In all cases, inhibition of Aplysia californica cyclase by various substrate analogues was found to be competitive while inhibition by nicotinamide exhibited mixed-behavior characteristics. Nicotinamide hypoxanthine dinucleotide (NHD(+)), nicotinamide guanine dinucleotide (NGD(+)), C1'-m-benzamide adenine dinucleotide (Bp(2)A), and C1'-m-benzamide nicotinamide dinucleotide (Bp(2)N) were found to be nanomolar potency inhibitors with inhibition constants of 70, 143, 189, and 201 nM, respectively. However, NHD(+) and NGD(+) are also known substrates and are slowly converted to cyclic products, thus preventing their further use as inhibitors. The symmetrical bis-nucleotides, bis-adenine dinucleotide (Ap(2)A), bis-hypoxanthine dinucleotide (Hp(2)H), and bis-nicotinamide dinucleotide (Np(2)N), exhibited micromolar competitive inhibition, with Ap(2)A displaying the greatest affinity for the enzyme. 2',3'-Di-O-acetyl nicotinamide adenine dinucleotide (AcONAD(+)) was not a substrate for the A. californica cyclase but also displayed some inhibition at a micromolar level. Finally, inhibition of the cyclase by adenosine 5'-O-diphosphate ribose (ADPR) and inosine 5'-O-diphosphate ribose (IDPR) was observed at millimolar concentration. The nicotinamide aromatic ring appears to be the optimal motif required for enzymatic recognition, while modifications of the 2'- and 3'-hydroxyls of the nicotinamide ribose seem to hamper binding to the enzyme. Stabilizing enzyme/inhibitor interactions and the inability of the enzyme to release unprocessed material are both considered to explain nanomolar inhibition. Recognition of inhibitors by other ADP ribosyl cyclases has also been investigated, and this study now provides the first potent nonhydrolyzable sea urchin ADP ribosyl cyclase and cADPR hydrolase inhibitor Bp(2)A, with inhibition observed at the micromolar and nanomolar level, respectively. The benzamide derivatives did not inhibit CD38 cyclase or hydrolase activity when NGD(+) was used as substrate. These results emphasize the difference between CD38 and other enzymes in which the cADPR cyclase activity predominates.

Stereochemical resolution of racemates, in HPLC, using a chiral stationary phase based upon immobilized α-chymotrypsin. II. Enzyme chiral separations

Several racemates has been resolved according to an enzymatic recognition mechanism on an α-chymotrypsin chiral stationary phase. The loadability of the ACHT-CSP has been evaluated both in buffered and non-buffered mobile phases under the enzymatic process and compared with the loading capacity obtained by the structural recognition process.

Synthesis of Farnesol Analogues through Cu(I)-Mediated Displacements of Allylic THP Ethers by Grignard Reagents.

The synthesis of a family of farnesol analogues, incorporating aromatic rings, has been achieved in high yields through the development of a regioselective coupling of allylic tetrahydropyranyl ethers with organometallic reagents. The allylic THP group is displaced readily by Grignard reagents in the presence of Cu(I) halides but is stable in the absence of added copper. Thus, an allylic THP group can fulfill its traditional role as a protecting group or serve as a leaving group depending on reaction conditions. An improved synthesis of (2E,6E)-10,11-dihydrofarnesol also has been accomplished using this methodology, and some preliminary studies on the reactivity and regioselectivity of THP ether displacements were conducted. The farnesol analogues reported herein may be useful probes of the importance of nonbonding interactions in enzymatic recognition of the farnesyl chain and allow development of more potent competitive inhibitors of enzymes such as farnesyl protein transferase.

Induction of KDNase Sm, a Deaminoneuraminic Acid (KDN) Residue-Specific Sialidase fromSphingobacterium multivorum,Using Synthetic KDN-Glycosides

Various aryl and alkyl α-glycosides of KDN were synthesized and tested as substrates for their susceptibility to a deaminoneuraminic acid (KDN)-specific sialidase fromSphingobacterium multivorum,designated KDNase Sm. The synthetic KDN-glycosides were all hydrolyzed by the action of KDNase Sm. A hydroxyl group at C-5 position of KDN was required for the recognition by the enzyme, and was shown not to be replaced by an amino- or an acylamino group for the enzymatic recognition. These synthetic KDN-glycosides were also examined for their inducing activity of KDNase inS. multivorumand were shown to induce the KDNase activity effectively when the bacterium was cultured in minimum salt medium containing both 0.1% glucose and 0.1% various KDN-glycosides. No KDNase activity was induced by the KDN-glycosides without 0.1% glucose. This is the first case of using synthetic KDN-glycosides as inducers of KDNase Sm.

Environmental Control of the Deformability of the DNA Double Helix

The deformability of DNA is of crucial importance in a number of processes and interactions, such as its enzymatic recognition, its packaging into chromosomes, its interactions with drugs, and the formation of photodimers in it. Here we have studied this property by following the formation of excited-state molecular complexes (excimers) between adjacent bases of poly(dA-dT).poly(dA-dT) at 20 degrees C. The force that drives the helix distortion appears to stem mainly from charge-resonance interaction. The results indicate that the deformability of the helix on the nanosecond time scale is considerable at normal solvent viscosities, whereas it is greatly reduced by frictional forces at high viscosities (attained through sucrose addition) at which the excimer has a much less favorable geometry: the difference in the interaction energies between 1 cP and 58 cP is about 6 kcal/mol, a value that is similar to the base stacking energies for the undistorted helix. This behavior parallels the modulation by the solvent viscosity of the thermally driven motions of the bases of poly(dA).poly(dT), which we recently reported on the basis of time-resolved intrinsic fluorescence anisotropy measurements (S. Georghiou et al., Biophys, J., 70, 1909-1922, 1996). It is inferred that environmental impediments to molecular motion can modulate the conformation and dynamics of DNA. Such modulation might play a role in gene regulation: particular base configurations, which can be enzymatically recognized, may be attained as dictated by the prevailing viscosity conditions and/or geometric constraints. By contrast, up to 3 M NaCl or 0.1 M MgCl2 do not significantly reduce the deformability of the helix. The considerable plasticity of this polynucleotide is probably linked to the significant flexibility of the TA step that may account for the wide-spread use of the TATA sequence in transcription, site-specific recombination and the initiation of DNA replication.