Structure of the polyketide cyclase SnoaL reveals a novel mechanism for enzymatic aldol condensation

SnoaL belongs to a family of small polyketide cyclases, which catalyse ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. Several of these antibiotics are among the most used anti-cancer drugs currently in use. The crystal structure of SnoaL, involved in nogalamycin biosynthesis, with a bound product, has been determined to 1.35 A resolution. The fold of the subunit can be described as a distorted alpha+beta barrel, and the ligand is bound in the hydrophobic interior of the barrel. The 3D structure and site-directed mutagenesis experiments reveal that the mechanism of the intramolecular aldol condensation catalysed by SnoaL is different from that of the classical aldolases, which employ covalent Schiff base formation or a metal ion cofactor. The invariant residue Asp121 acts as an acid/base catalyst during the reaction. Stabilisation of the enol(ate) intermediate is mainly achieved by the delocalisation of the electron pair over the extended pi system of the substrate. These polyketide cyclases thus form of family of enzymes with a unique catalytic strategy for aldol condensation.     

Reversed micelle solvents as tools of enzyme purification and enzyme-catalyzed conversion

Reversed micelle solvents represent nanometer-sized aqueous droplets stabilized by surfactants inside the bulk organic solvents. The aqueous cores can host various hydrophilic solutes, including bioactive substances thus revealing a challenge to the biotechnology's needs of the safe media for bioseparations and bioconversions. This review discusses the structure and the properties of reversed micelle solvents in view of the parameters that can be easily operated in technology to achieve safe liquid-liquid extraction of proteins/enzymes or bioconversion of hydrophobic substrates. The paper highlights the importance of how the reversed micelle microenvironment should be arranged with respect to the preservation of the activity of the enzyme as target product or biocatalyst. The main aspects are demonstrated with own experimental results on alpha-amylase purification and lipase-catalyzed esterification using cationic reversed micelle solvents. The trials of performing continuous processes involving reversed micellar separation and reaction media are also reviewed and the current problems are addressed.     

Synthesis of proline-derived dipeptides and their catalytic enantioselective direct aldol reactions: catalyst, solvent, additive and temperature effects

A series of dipeptides of l-proline-l-amino acid and l-proline-d-amino acid were synthesized to evaluate the catalytic effect for asymmetric direct aldol reactions. In the direct aldol reaction, a catalyst of l-proline-l-amino acid achieves better enantioselectivity than the corresponding l-proline-d-amino acid catalyst. Solubility of the dipeptide catalysts in the solvents is a key point for achieving a better yield of the direct aldol reaction, while hydrogen bonding of solvent does not play an important role in attaining better enantioselectivity and yield. Yield and enantioselectivity of the direct aldol reaction in water were improved by NMM and SDS additives, but the results that were done in plain DMSO were even better.     

Synthesis of DL-standishinal and its related compounds for the studies on structure-activity relationship of inhibitory activity against aromatase

DL-Standishinal (1), an aromatase inhibitor isolated from Thuja standishii, was synthesized in 15 steps from p-formylanisole via aldol reaction of 12-hydroxy-6,7-secoabieta-8,11,13-trien-6,7-dial (2). In the present study, we found that the aldol condensation of 2 proceeded in excellent yield with the protonic catalyst such as d-camphorsulfonic acid in CH(2)Cl(2). Moreover, structure-activity relationship of 1 and its related compounds was studied and it was revealed that the isomers having cis-configuration on the A/B-ring generally exhibited more potent inhibitory activities against aromatase than those with trans-configuration.     

Asymmetric aldol reactions catalyzed by efficient and recyclable silica-supported proline-based peptides

A series of silica-supported proline-based peptides were synthesized and applied as catalysts for direct asymmetric intermolecular aldol reactions. Among these, a peptide with two L-proline units was found to be the most efficient one for the asymmetric aldol reactions between acetone and aromatic aldehydes. The reactions generated the corresponding products with satisfactory isolated yields (up to 97%) and enantiomeric excesses (up to 96%) in the presence of this catalyst (5 mol %). Furthermore, the silica-supported organocatalyst could be recovered and recycled by a simple filtration of the reaction solution and used for five consecutive trials without loss of its reactivity.     

The origin of enantioselectivity in aldolase antibodies: crystal structure, site-directed mutagenesis, and computational analysis

Catalytic aldolase antibodies, generated by reactive immunization, catalyze the aldol reaction with the efficiency of natural enzymes, but accept a much broader range of substrates. Two separate groups of aldolase antibodies that catalyze the same aldol reactions with antipodal selectivity were analyzed by comparing their amino acid sequences with their crystal structures, site-directed mutagenesis data, and computational docking of the transition states of the aldol reaction. The crystal structure of aldolase antibody 93F3 Fab' at 2.5A resolution revealed a combining site with two lysine residues, including LysL89 that reacts to form the covalent enamine intermediate. In contrast, antibody 33F12 has one active site lysine, LysH93. The reactive lysine residues in each group of antibodies are differentially located on the heavy and light chain variable regions in pseudo-symmetric opposite orientations, but both within highly hydrophobic environments. Thus, the defining feature for the observed enantioselectivities of these aldolase antibody catalysts is the respective location and relative disposition of the reactive lysine residues within the active sites of these catalysts.     

Preparation of Immobilized L‐Prolinamide Via Enzymatic Polymerization of Phenolic L‐Prolinamide and Evaluation of Its Catalytic Performance for Direct Asymmetric Aldol Reaction

Phenolic L‐prolinamide was allowed to participate in enzymatic polymerization with horseradish peroxidase as the catalyst, generating immobilized L‐prolinamide. The catalytic performance of the resultant polymer‐supported L‐prolinamide for direct asymmetric aldol reaction between aromatic aldehyde and cyclohexanone was studied. The results show that as prepared L‐prolinamide can catalyze the aldol reaction at room temperature in the presence of H₂O. Relevant aldol addition products are obtained with good yields (up to 91%), high diastereoselectivities (up to 6:94 dr), and medium enantioselectivities (up to 87% ee). Moreover, the title polymer‐supported catalyst can be recovered and reused for at least five cycles while the activity remains almost unchanged. Chirality 26:209–213, 2014. © 2014 Wiley Periodicals, Inc.     

Self-assembled micelles of biodegradable triblock copolymers based on poly(ethyl ethylene phosphate) and poly(-caprolactone) as drug carriers

A series of novel amphiphilic triblock copolymers of poly(ethyl ethylene phosphate) and poly(-caprolactone) (PEEP-PCL-PEEP) with various PEEP and PCL block lengths were synthesized and characterized. These triblock copolymers formed micelles composed of a hydrophobic core of poly(-caprolactone) (PCL) and a hydrophilic shell of poly(ethyl ethylene phosphate) (PEEP) in aqueous solution. The micelle morphology was spherical, determined by transmission electron microscopy. It was found that the size and critical micelle concentration values of the micelles depended on both hydrophobic PCL block length and PEEP hydrophilic block length. The in vitro degradation characteristics of the triblock copolymers were investigated in micellar form, showing that these copolymers were completely biodegradable under enzymatic catalysis of Pseudomonas lipase and phosphodiesterase I. These triblock copolymers were used for paclitaxel (PTX) encapsulation to demonstrate the potential in drug delivery. PTX was successfully loaded into the micelles, and the in vitro release profile was found to be correlative to the polymer composition. These biodegradable triblock copolymer micelles are potential as novel carriers for hydrophobic drug delivery.     

Mechanism of dihydroneopterin aldolase. NMR, equilibrium and transient kinetic studies of the Staphylococcus aureus and Escherichia coli enzymes

Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of 7,8-dihydro-D-neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8-dihydro-L-monapterin (DHMP). Whether the epimerization reaction uses the same reaction intermediate as the aldol reaction or the deprotonation and reprotonation of C2' of DHNP has been investigated by NMR analysis of the reaction products in a D2O solvent. No deuteration of C2' was observed for the newly formed DHMP. This result strongly suggests that the epimerization reaction uses the same reaction intermediate as the aldol reaction. In contrast with an earlier observation, the DHNA-catalyzed reaction is reversible, which also supports a nonstereospecific retroaldol/aldol mechanism for the epimerization reaction. The binding and catalytic properties of DHNAs from both Staphylococcus aureus (SaDHNA) and Escherichia coli (EcDHNA) were determined by equilibrium binding and transient kinetic studies. A complete set of kinetic constants for both the aldol and epimerization reactions according to a unified kinetic mechanism was determined for both SaDHNA and EcDHNA. The results show that the two enzymes have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them.     

Enhancement of RNA self-cleavage by micellar catalysis.

It has been reported recently that naturally occurring catalytic RNAs like hammerhead and hairpin ribozyme do not require metal ions for efficient catalysis. It seems that the folded tertiary structure of the RNA contributes more to the catalytic function than was initially recognized. We found that a highly specific self-cleavage reaction can occur within a small bulge loop of four nucleotides in a mini-substrate derived from Arabidopsis thaliana intron-containing pre-tRNA(Tyr) in the absence of metal ions. NH(4)(+) cations and non-ionic or zwitter-ionic detergents at or above their critical micelle concentration are sufficient to catalyze this reaction. The dependence on micelles for the reaction leads to the assumption that physical properties, i.e. the hydrophobic interior of a micelle, are essential for this self-cleavage reaction. We suggest that NH(4)(+)-ions play a crucial role for the entry of the negatively charged RNA into the hydrophobic interior of a detergent micelle. A change of the pattern of hydration or hydrogen bonds caused by the hydrophobic surrounding enhances the reaction by a factor of 100. These findings suggest that highly structured RNAs may shift pK(a) values towards neutrality via the local environment and thereby enhance their ability to perform general acid-base catalysis without the participation of metal ions.     

Synthesis of complex nucleoside antibiotics

Herbicidin B and fully prtected tunicaminyluracil, which were undecose nucleoside antibiotics, were synthesized using a samarium diiodide (SmI2) mediated aldol reaction with the use of alpha-phenylthioketone as an enolate. The characteristics of the SmI2-mediated aldol reaction are that the enolate can be regioselectively generated and the aldol reaction proceeds under near neutral condition. This reaction is proved to be a powerful reaction for the synthesis of complex nucleoside antibiotics. The synthesis of caprazol, the core structure of caprazamycins, was conducted by the strategy including beta-selective ribosylation without using a neighboring group participation and the construction of a diazepanone by a modified reductive amination. Our synthetic route would provide a range of key analogues with partial structures to define the pharmacophore, which can be a lead for the development of more effective anti-bacterial agents.     

SYNTHESIS OF COMPLEX NUCLEOSIDE ANTIBIOTICS

Herbicidin B and fully protected tunicaminyluracil, which were undecose nucleoside antibiotics, were synthesized using a samarium diiodide (SmI₂) mediated aldol reaction with the use of α-phenylthioketone as an enolate. The characteristics of the SmI₂-mediated aldol reaction are that the enolate can be regioselectively generated and the aldol reaction proceeds under near neutral condition. This reaction is proved to be a powerful reaction for the synthesis of complex nucleoside antibiotics. The synthesis of caprazol, the core structure of caprazamycins, was conducted by the strategy including β-selective ribosylation without using a neighboring group participation and the construction of a diazepanone by a modified reductive amination. Our synthetic route would provide a range of key analogues with partial structures to define the pharmacophore, which can be a lead for the development of more effective anti-bacterial agents.     

Improved polymerase ribozyme efficiency on hydrophobic assemblies

During an early step in the evolution of life, RNA served both as genome and as catalyst, according to the RNA world hypothesis. For self-replication, the RNA organisms must have contained an RNA that catalyzes RNA polymerization. As a first step toward recapitulating an RNA world in the laboratory, a polymerase ribozyme was generated previously by in vitro evolution and design. However, the efficiency of this ribozyme is about 100-fold too low for self-replication because of a low affinity of the ribozyme to its primer/template substrate. To improve the substrate interactions by colocalizing ribozyme and substrate on micelles, we attached hydrophobic anchors to both RNAs. We show here that the hydrophobic anchors led to aggregates with the expected size of the corresponding micelles. The micelle formation increased the polymerization yield of full-length products by 3- to 20-fold, depending on substrates and reaction conditions. With the best-characterized substrate, the improvement in polymerization efficiency was primarily due to reduced sequence-specific stalling on partially extended substrates. We discuss how, during the origin of life, micellar ribozyme aggregates could have acted as precursors to membrane-encapsulated life forms.     

嵌段共聚物聚乙二醇-聚苹果酸苄基酯载药胶束的制备及性质研究

目的：为构建聚合物胶束药物运载体系,制备嵌段共聚物聚乙二醇-聚苹果酸苄基酯载药胶束并测定其性质。方法：以L-天冬氨酸为原料,重氮化、环化后经开环聚合得到聚苹果酸苄基酯。氨基聚乙二醇通过酰胺键连接到β-聚苹果酸苄基酯上形成两亲性嵌段共聚物,喜树碱做药物模型制备载药胶束。动态光散射法测定胶束粒径、评价胶束稳定性,高效液相法测定喜树碱载药率和包封率,芘荧光法与动态光散射法测定临界胶束浓度。结果：喜树碱包封率72%,载药率6%,临界胶束浓度为40μg.mL-1。随着聚苹果酸苄基酯分子量减小,胶束稳定性增强。结论：聚乙二醇-聚苹果酸苄基酯在疏水链/亲水链分子量比值为2-4时在水中可自组装形成纳米胶束,可作为性能优良的聚合物药物载体。     

Structure-based mutagenesis approaches toward expanding the substrate specificity of D-2-deoxyribose-5-phosphate aldolase

2-Deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) catalyzes the reversible aldol reaction between acetaldehyde and D-glyceraldehyde-3-phosphate to generate D-2-deoxyribose-5-phosphate. It is unique among the aldolases as it catalyzes the reversible asymmetric aldol addition reaction of two aldehydes. In order to expand the substrate scope and stereoselectivity of DERA, structure-based substrate design as well as site-specific mutation has been investigated. Using the 1.05 A crystal structure of DERA in complex with its natural substrate as a guide, five site-directed mutants were designed in order to improve its activity with the unnatural nonphosphorylated substrate, D-2-deoxyribose. Of these, the S238D variant exhibited a 2.5-fold improvement over the wild-type enzyme in the retroaldol reaction of 2-deoxyribose. Interestingly, this S238D mutant enzyme was shown to accept 3-azidopropinaldehyde as a substrate in a sequential asymmetric aldol reaction to form a deoxy-azidoethyl pyranose, which is a precursor to the corresponding lactone and the cholesterol-lowering agent Lipitor. This azidoaldehyde is not a substrate for the wild-type enzyme. Another structure-based design of new nonphosphorylated substrates was focused on the aldol reaction with inversion in enantioselectivity using the wild type or the S238D variant as the catalyst and 2-methyl-substituted aldehydes as substrates. An example was demonstrated in the asymmetric synthesis of a deoxypyranose as a new effective synthon for the total synthesis of epothilones. In addition, to facilitate the discovery of new enzymatic reactions, the engineered E. coli strain SELECT (Deltaace, adhC, DE3) was developed to be used in the future for selection of DERA variants with novel nonphosphorylated acceptor specificity.     

Synthesis of well-defined amphiphilic block copolymers having phospholipid polymer sequences as a novel biocompatible polymer micelle reagent

To realize safer and effective drug administration, novel well-defined and biocompatible amphiphilic block copolymers containing phospholipid polymer sequences were synthesized. At first, the homopolymer of 2-methacryloyloxyethylphosphorylcholine (MPC) was synthesized in water by reversible addition-fragmentation chain transfer (RAFT) controlled radical polymerization. The "living" polymerization was confirmed by the fact that the number-average molecular weight increased linearly with monomer conversion while the molecular weight distribution remained narrow independent of the conversion. The poly(MPC) thus prepared is end-capped with a dithioester moiety. Using the dithioester-capped poly(MPC) as a macro chain transfer agent, AB diblock copolymers of MPC and n-butyl methacrylate (BMA) were synthesized. Associative properties of the amphiphilic block copolymer (pMPC(m)-BMA(n)) with varying poly(BMA) block lengths were investigated using NMR, fluorescence probe, static light scattering (SLS), and quasi-elastic light scattering (QELS) techniques. Proton NMR data in D2O indicated highly restricted motions of the n-butyl moieties, arising from hydrophobic associations of poly(BMA) blocks. Fluorescence spectra of N-phenyl-1-naphthylamine indicated that the probes were solubilized in the polymer micelles in water. The formation of polymer micelles comprising a core with poly(BMA) blocks and shell with hydrophilic poly(MPC) blocks was suggested by SLS and QELS data. The size and mass of the micelle increased with increasing poly(BMA) block length. With an expectation of a pharmaceutical application of pMPC(m)-BMA(n), solubilization of a poorly water-soluble anticancer agent, paclitaxel (PTX), was investigated. PTX dissolved well in aqueous solutions of pMPC(m)-BMA(n) as compared with pure water, implying that PTX is incorporated into the hydrophobic core of the polymer micelle. Since excellent biocompatible poly(MPC) sequences form an outer shell of the micelle, pMPC(m)-BMA(n) may find application as a promising reagent to make a good formulation with a hydrophobic drug.     

Synthesis of tunicaminyluracil derivatives

A tunicaminyluracil derivative, which is a key component of the tunicamycin nucleoside antibiotics, was synthesized using a samarium diiodide (SmI2) mediated aldol reaction and intramolecular Pummerer reaction as the key steps. The alpha-phenylthio ketone 11, the precursor of the samarium enolate, was prepared from D-galactose. Treatment of 11 with SmI2 at -40 degrees C resulted in complete conversion to the corresponding samarium enolate, and subsequent addition of uridine 5'-aldehyde 12 afforded the desired aldol products 13a,b. Compound 13a was converted to the sulfoxide 15 by a sequential diastereoselective reduction of the ketone and an oxidation with mCPBA. Activation of 15 with Tf2O provided the desired cyclized compound 17. In this reaction, the aldol product 13a was also obtained as a consequence of a competitive intramolecular version of DMSO-oxidation via a 7-membered ring intermediate. Compound 18 or 19 are ready for use as a glycosyl donor in glycosylations to provide a range of analogues as potential glycosyltransferase inhibitors as well as related natural products.     

Formation of core-shell type biodegradable polymeric micelles from amphiphilic poly(aspartic acid)-block-polylactide diblock copolymer

Poly(aspartic acid)-block-polylactide diblock copolymers (PAsp-b-PLAs) having both hydrophilic and hydrophobic segments of various lengths were synthesized. These PAsp-b-PLA diblock copolymers formed polymeric micelles consisting of a hydrophobic PLA core and a hydrophilic, pH-sensitive PAsp shell in aqueous solution. The effects of the segment length of both the PLA and the PAsp portions and the pH of the solution on the shapes and sizes of the PAsp-b-PLA polymeric micelles were investigated. The results indicated a balance between the effects of electrostatic repulsion, hydrogen bonding in the PAsp shell layer, and hydrophobic interactions in the PLA core determine the sizes of the PAsp-b-PLA polymeric micelles. Moreover, the PAsp-b-PLA polymeric micelles did not possess any cytotoxic activity against L929 fibroblast cells. The obtained polymeric micelle should be useful for biodegradable biomedical materials such as drug delivery vehicle.     

Fast bilayer-micelle fusion mediated by hydrophobic dipeptides

To understand the transition from inanimate matter to life, we studied a process that directly couples simple metabolism to evolution via natural selection, demonstrated experimentally by Adamala and Szostak. In this process, dipeptides synthesized inside precursors of cells promote absorption of fatty acid micelles to vesicles, inducing their preferential growth and division at the expense of other vesicles. The process is explained on the basis of coarse-grained molecular dynamics simulations, each extending for tens of microseconds, carried out to model fusion between a micelle and a membrane, both made of fatty acids in the absence and presence of hydrophobic dipeptides. In all systems with dipeptides, but not in their absence, fusion events were observed. They involve the formation of a stalk made by hydrophobic chains from the micelle and the membrane, similar to that postulated for vesicle-vesicle fusion. The emergence of a stalk is facilitated by transient clusters of dipeptides, side chains of which form hydrophobic patches at the membrane surface. Committor probability calculations indicate that the size of a patch is a suitable reaction coordinate and allows for identifying the transition state for fusion. Free-energy barrier to fusion is greatly reduced in the presence of dipeptides to only 4–5 kcal/mol, depending on the hydrophobicity of side chains. The mechanism of mediated fusion, which is expected to apply to other small peptides and hydrophobic molecules, provides a robust means by which a nascent metabolism can confer evolutionary advantage to precursors of cells.     

Synthesis and evaluation of a star amphiphilic block copolymer from poly(epsilon-caprolactone) and poly(ethylene glycol) as a potential drug delivery carrier

A star polymer composed of amphiphilic block copolymer arms has been synthesized and characterized. The core of the star polymer is polyamidoamine (PAMAM) dendrimer, the inner block in the arm is lipophilic poly(epsilon-caprolactone) (PCL), and the outer block in the arm is hydrophilic poly(ethylene glycol) (PEG). The star-PCL polymer was synthesized first by ring-opening polymerization of epsilon-caprolactone with a PAMAM-OH dendrimer as initiator. The PEG polymer was then attached to the PCL terminus by an ester-forming reaction. Characterization with SEC, (1)H NMR, FTIR, TGA, and DSC confirmed the star structure of the polymers. The micelle formation of the star copolymer (star-PCL-PEG) was studied by fluorescence spectroscopy. Hydrophobic dyes and drugs can be encapsulated in the micelles. A loading capacity of up to 22% (w/w) was achieved with etoposide, a hydrophobic anticancer drug. A cytotoxicity assay demonstrated that the star-PCL-PEG copolymer is nontoxic in cell culture. This type of block copolymer can be used as a drug delivery carrier.