Development of an active site titration reagent for α-amylases

α-Amylases are among the most widely used classes of enzymes in industry and considerable effort has gone into optimising their activities. Efforts to find better amylase mutants, such as through high-throughput screening, would be greatly aided by access to precise and robust active site titrating agents for quantitation of active mutants in crude cell lysates. While active site titration reagents designed for retaining β-glycosidases quantify these enzymes down to nanomolar levels, convenient titrants for α-glycosidases are not available. We designed such a reagent by incorporating a highly reactive fluorogenic leaving group onto unsaturated cyclitol ethers, which have been recently shown to act as slow substrates for retaining glycosidases that operate via a covalent ‘glycosyl’-enzyme intermediate. By appending this warhead onto the appropriate oligosaccharide, we developed efficient active site titration reagents for α-amylases that effect quantitation down to low nanomolar levels.

α-Amylases are among the most widely used classes of enzymes in industry and considerable effort has gone into optimising their activities.

Amylases are among the most common classes of enzymes employed in industrial settings, being used in detergents, bread, beer, biofuel, and many other sectors. Accordingly, α-amylases account for 25% of the world''s multi-billion dollar enzyme market.^1,2 α-Amylases are endo-acting enzymes that cleave starch into malto-oligosaccharides, which are further degraded by exo-acting α-glucosidases, glucoamylases, β-amylases and α-glucan phosphorylases and lyases. They are found in CAZy GH families 13, 57, 119 and 126, with the vast majority in the large GH13 family.³ GH13 enzymes adopt a (β/α)₈ fold with three highly conserved active site carboxylic acids.^4–6 They employ a classical double-displacement mechanism⁷ in which one of the glutamic acids provides acid catalytic assistance to the leaving group departure while an aspartate attacks the anomeric centre, forming a covalent glycosyl enzyme intermediate. In a second step, water attacks the anomeric centre with base assistance from the glutamate residue (Fig. 1A and B).Open in a separate windowFig. 1Koshland mechanism of retaining β- and α-glycosidases (A & B). The same mechanism has been observed for the hydrolysis of “β”-valienols (C), and for “α”-valienols (D).Given their industrial importance, a huge amount of attention has been given to the discovery and improvement of α-amylases to attain optimal performance for particular applications. These approaches typically require high-throughput analysis of large numbers of gene products or mutants thereof.^8–10 Identification of the best candidates then ideally requires high-throughput assay coupled with a method for determining the enzyme concentration in each sample. This can be a challenging task in the absence of purification, as would be the case for truly high-throughput approaches. The “gold standard” method to quantify active enzyme concentration is active site titration.¹¹ Active site titrants react stoichiometrically with their target enzymes and release one equivalent of a quantifiable agent, which is typically either a chromophore or fluorophore. For enzymes that operate via a covalent intermediate, such as retaining glycosidases, the active site titrants are usually chromogenic or fluorogenic substrates that form this intermediate with a rate constant (k_on) that is much greater than that for its hydrolysis (k_off) – ideally with k_off approaching zero.Our lab has previously developed active site titration reagents for several retaining β-glycosidases^12,13 and neuraminidases.^14,15 By replacing the substituent on the position adjacent to the anomeric centre of the sugar (the hydroxyl at C-2 for many monosaccharides) with a fluorine atom, both the formation and the hydrolysis of the glycosyl-enzyme intermediate are slowed, largely through inductive destabilisation of the transition state. Further incorporation of a reactive fluorogenic leaving group generates a reagent that, upon covalently inactivating the glycosidase, releases a stoichiometric and quantifiable amount of fluorophore. The fluorogenic response is then measured to determine the amount of active glycosidase that is present in solution.Unfortunately, this same strategy does not work for retaining α-glycosidases. In those cases, k_off remains greater than k_on, likely due to the inherently greater reactivity of the β-glycosyl-enzyme intermediate,^16,17 and the compounds are simply substrates with low turnover numbers. By use of 2,2-dihalosugars with yet more reactive leaving groups, this problem could be solved in some cases, but their synthesis is challenging, and inactivation rates were low, or non-existent in some cases.^18,19 Alternative approaches were called for.Recently, a new class of glycosidase substrates was reported in which the sugar moiety is replaced by an equivalently hydroxylated cyclohexene.^20–23 Hydrolysis of these enol ethers likely occurs via an allylic cation of almost identical reactivity to that of the equivalent oxocarbenium ion. Glycosidases cleave these substrates via the classical Koshland mechanism⁷ (Fig. 1C and D), but considerably more slowly than their natural substrates. However, incorporation of a good leaving group will accelerate, relatively, the first step such that, in some cases, they act as mechanism-based inactivators making them candidates for development of an active site titrant for α-amylases.Since α-amylases are endo-acting enzymes that do not usually cleave monosaccharide glycosides, an ‘extended” oligosaccharide version containing a total of 2 or 3 sugar/pseudosugar moieties would be needed. Substrates longer than this would be prone to internal glycoside cleavage. Since 2-chloro-4-nitrophenyl maltotrioside (CNP-G3) functions as a substrate for most amylases, we focused on addition of a maltosyl unit to a valienol moiety containing a 6,8-difluorocoumarin (F₂MU) leaving group at its “anomeric centre”. The low pK_a of this coumarin, 4.7,¹⁴ results in a greater reactivity of the reagent and also ensures the coumarin will be deprotonated and thus fluorescent, upon release at neutral pH.Synthesis of partially protected alcohol 2 from gluconolactone 1via literature methods²⁴ was followed by attachment of F₂MU via a Mitsunobu reaction and subsequent removal of the protecting groups under acidic conditions, generating known pseudo-glycoside 3.²³ To check this concept before we synthesized the longer version, we tested compound 3 as a titrant of a simple α-glucosidase and found that it did indeed titrate the enzyme (Fig. S5^†). Since elongation of this pseudosugar via classical organic synthesis would require substantial protecting group chemistry, we elected instead to employ an enzymatic coupling strategy using the GH13 cyclodextrin transglycosidase, CGTase. This enzyme can use glycosyl fluorides, such as α-maltosyl fluoride, to effect glycosyl transfer onto suitable acceptors. However, a significant competing reaction would involve self-condensation of glycosyl fluorides ultimately forming cyclodextrins. To avoid this problem, we employed a maltosyl fluoride donor (4), in which the 4′-hydroxyl had been capped with a methyl group.^25,26 Incorporation of 4′-methoxy groups does not alter the reaction with α-amylases, as this site in the normal substrate is occupied by additional sugar residues. Thus CGTase-catalysed glycosylation between known glycosyl fluoride 4 and pseudo-glycoside 3, gave the pseudo-trisaccharide 5 in 64% isolated yield (Scheme 1).Open in a separate windowScheme 1Synthesis of titration reagent 5.With this reagent in hand, we proceeded to screen its ability to inactivate a small panel of α-amylases. As shown in Fig. 2, time-dependent inactivation was observed for all enzymes tested, with the most industrially relevant enzymes, Effusibacillus pohliae amylase (EPA) and Aspergillus oryzae amylase (AOA), being inactivated the fastest.Open in a separate windowFig. 2Time-dependent inactivation of a small panel of amylases, showing remaining % activity versus time. Red box with X: AOA (91 nM); blue square: EPA (66.7 nM); purple cross: PPA (500 nM); green triangle: HPA (125 nM). AOA = A. oryzae amylase; EPA = E. pohliae amylase; HPA = human pancreatic amylase; PPA = porcine pancreatic amylase.Kinetic parameters for inactivation were then determined by directly monitoring the release of F₂MU by UV-Vis (Table 1). To determine k_on and k_off (Scheme 2), we monitored chromophore (F₂MU) release by absorbance at 370 or 380 nm (dependent on the concentration of 5 in the measurements of each enzyme). After mixing 5 with each enzyme individually, a burst phase followed by a steady-state phase was observed. For each enzyme, this was then repeated with varying concentrations of 5. Initial rates of F₂MU release versus concentration of 5 were fit to a Michaelis–Menten equation to provide k_on. The rate constant of cyclitol release, k_off, was determined by measuring rates of the steady-state region at a saturating concentration (5× K_i). We found that several amylases: Effusibacillus pohliae amylase (EPA), Aspergillus oryzae amylase (AOA), Rhizomucor pusillus amylase (RPA) and porcine pancreatic amylase (PPA), inactivated quickly (highest k_on, lowest k_off, and greatest k_on/K_i), and are therefore ideal candidates for titration with compound 5. Human pancreatic amylase (HPA), on the other hand, while inactivating rapidly, binds the reagent relatively poorly.Open in a separate windowScheme 2Kinetic parameters for the hydrolysis of 5 by several amylases (at 25 °C for EPA, AOA, and RPA and 30 °C for human pancreatic amylase [HPA] and porcine pancreatic amylase [PPA])

NMR solution structure of dsDNA containing a bicyclic D-arabino-configured nucleotide fixed in an O4'-endo sugar conformation

[3.2.0]bcANA is a D-arabino-configured bicyclic nucleotide with a 2'-O,3'-C-methylene bridge. We here present the high-resolution NMR structure of a [3.2.0]bcANA modified dsDNA nonamer with one modified nucleotide incorporated. NOE restraints were obtained by analysis of NOESY cross peak intensities using a full relaxation matrix approach, and subsequently these restraints were incorporated into a simulated annealing scheme for the structure determination. In addition, the furanose ring puckers of the deoxyribose moieties were determined by analysis of COSY cross peaks. The modified duplex adopts a B-like geometry with Watson-Crick base pairing in all base pairs and all glycosidic angles in the anti range. The stacking arrangement of the nucleobases appears to be unperturbed relative to the normal B-like arrangement. The 2'-O,3'-C-methylene bridge of the modified nucleotide is located at the brim of the major groove where it fits well into the B-type duplex framework. The sugar pucker of the [3.2.0]bcANA nucleotide is O4'-endo and this sugar conformation causes a change in the delta backbone angle relative to the C2'-endo deoxyribose sugar pucker. This change is absorbed locally by slight changes in the epsilon and zeta angles of the modified nucleotide. Overall, the [3.2.0]bcANA modifications fits very well into a B-like duplex framework and only small and local perturbations are observed relative to the unmodified dsDNA of identical base sequence.     

Interstrand communication between 2'-N-(pyren-1-yl)methyl-2'-amino-LNA monomers in nucleic acid duplexes: directional control and signalling of full complementarity

Very efficient interstrand communication systems in nucleic acid duplexes, based on pyrene excimer formation between 2'-N-(pyren-1-yl)methyl-2'-amino-LNA monomers, demonstrate the versatility of functionalized 2'-amino-LNA monomers for Angstrom-scale chemical engineering.     

Synthesis and molecular structure of piplartine (=piperlongumine)

The structure of piplartine (=piperlongumine) was established as ($\text{1}$)-$\text{3}$-3',4',5'-trimethoxycinnamoyl-5,6-dihydro-2(1H)-pyridone ($\text{13}$) by synthesis and by an X-ray crystallographic analysis. Model condensation of ($\text{E}$)-3,4,5-trimethoxycinnamoyl chloride and crotonamide gave not the expected cinnamoylcrotonamide, but ($\text{1}$)-$\text{N}$-3', 4', 5' -trimethoxycinnamoyl-3-chlorobutyramide ($\text{12}$).     

DNA-selective hybridization and dual strand invasion of short double-stranded DNA using pyren-1-ylcarbonyl-functionalized 4'-C-piperazinomethyl-DNA

Incorporation of a novel pyren-1-ylcarbonyl-functionalized 4'-C-piperazinomethyl-DNA monomer into oligodeoxynucleotides leads to increased thermal stability of duplexes with DNA complements but reduced thermal stability of duplexes with RNA complements. This DNA-selective hybridization is explored for recognition of double-stranded DNA by a novel dual strand invasion approach.     

alpha-L-LNA (alpha-L-ribo configured locked nucleic acid) recognition of DNA: an NMR spectroscopic study

We have used NMR and CD spectroscopy to study and characterise two alpha-L-LNA:DNA duplexes, a nonamer that incorporates three alpha-L-LNA nucleotides and a decamer that incorporates four alpha-L-LNA nucleotides, in which alpha-L-LNA is alpha-L-ribo-configured locked nucleic acid. Both duplexes adopt right-handed helical conformations and form normal Watson-Crick base pairing with all nucleobases in the anti conformation. Deoxyribose conformations were determined from measurements of scalar coupling constants in the sugar rings, and for the decamer duplex, distance information was derived from 1H-1H NOE measurements. In general, the deoxyriboses in both of the alpha-L-LNA:DNA duplexes adopt S-type (B-type structure) sugar puckers, that is the inclusion of the modified alpha-L-LNA nucleotides does not perturb the local native B-like double-stranded DNA (dsDNA) structure. The CD spectra of the duplexes confirm these findings, as these display B-type characteristic features that allow us to characterise the overall duplex type as B-like. The 1H-1H NOE distances which were determined for the decamer duplex were employed in a simulated annealing protocol to generate a model structure for this duplex, thus allowing a more detailed inspection of the impact of the alpha-L-ribo-configured nucleotides. In this structure, it is evident that the malleable DNA backbone rearranges in the vicinity of the modified nucleotides in order to accommodate them and present their nucleobases in a geometry suitable for Watson-Crick base pairing.     

Solvent effects on the singlet - triplet equilibrium and reactivity of a ground triplet state arylalkyl carbene

Results from intramolecular singlet and triplet specific reactivity in solvents of different Polarity suggest that the spin state equilibrium of 1,2-diphenyl-1-butylidene, a triplet ground state carbene. is largely susceptible to solvent polarity. The results are consistent with stabilization of the zwitterionic singlet state in solvents of high polarity.     

Synthesis strategies and properties of smart amphiphilic networks

This paper briefly surveys recent developments in the field of amphiphilic networks (APN) which are a new class of crosslinked polymer systems consisting of covalently bonded hydrophobic and hydrophilic chain segments. The covalent bonds between immiscible hydrophobic and hydrophilic polymer chains prevent demixing and yield polymer networks with unique structure and properties. Telechelic macromonomers provide the basis for the first generation of APNs obtained by copolymerization of the macromonomer with selected low molecular weight monomers. Synthesis of a variety of APNs using methacrylate-telechelic polyisobutylene (PIB) macromonomers prepared by living carbocationic polymerization (LCCP) and quantitative chain end derivatization is reviewed. The second generation of PIB-based amphiphilic networks is prepared by crosslinking of well-defined hydroxy-telechelic PIB and partially deprotected silylated poly(2-hydroxyethyl methacrylate) (PHEMA) precursor chains. Other opportunities providing better structural control of APNs by crosslinking of functional amphiphilic block copolymers (or precursors) obtained by combining living carbocationic and anionic polymerizations are outlined as well. Properties of APNs, such as control of swellability by composition, pH-response of swelling, fast surface structure reorganization by contacting with solvent, morphology, sustained release of drugs and bio- and blood compatibility, are also summarized.     

Synthetic approaches towards new polymer systems by the combination of living carbocationic and anionic polymerizations

This study summarizes recent efforts to obtain by combination of living carbocationic and anionic polymerizations block copolymers which are potential precursors for building new well-defined polymeric architectures with microphase separated morphology. Living carbocationic polymerization (LCCP) yields telechelic polyisobutylene (PIB) chains with a variety of useful endgroups, such as tert-chlorine, isopropenyl, primary hydroxyl, tolyl etc. When tolyl-ended PIB was used as precursor for macroinitiator of living anionic polymerization of 2-(tert-butyldimethylsilyloxy)ethyl methacrylate (tBuMe₂SiOEMA), mixtures of homopolymers and block copolymers were formed due to incomplete lithiation of this chain end. In another approach a new functionalization method was developed by end-quenching living PIB chains with 1,1-diphenylethylene (DPE). In the presence of BCl₃ a new telechelic PIB with 2,2-diphenylvinyl (DPV) endgroups was formed. A corresponding DPV model compound was synthesized from 2-chloro-2,4,4-trimethylpentane (TMPCl). Because of steric hindrance less than quantitative lithiation of this material occurred. Controlled deprotection of PtBuMe₂SiOEMA obtained by living anionic polymerization (LAP) was utilized to prepare a precursor network composed of partially deprotected PtBuM₂SiOEMA and hydroxyl-telechelic PIB by using a diisocyanate crosslinker. After network formation deprotection with HCl was completed and a new amphiphilic network (APN) containing PIB and poly(2-hydroxyethyl) methacrylate) (PHEMA) segments crosslinked by urethane linkages was obtained.     

Spin-orbit ab initio investigation of the photolysis of bromoiodomethane.

The photodissociation of bromoiodomethane has been investigated by spin-orbit ab initio calculations. The experimentally observed A- and B-bands and the corresponding photoproducts were assigned by multistate second-order multiconfigurational perturbation theory in conjunction with spin-orbit interaction through complete active space state interaction potential energy curves, vertical excitation energies, and oscillator strengths of low-lying excited states. The present conclusions with respect to the dissociation process in the B-band are different compared with those of previous studies. The reaction between the iso-CH(2)Br-I and iso-CH(2)I-Br species has also been studied. Finally, a set of stable excited states was identified for both isomers. These species might be of importance in the recombination process that follows the photodissociation in a solvent.