The Interaction of Isopenicillin N Synthase with Homologated Substrate Analogues δ‐(L‐α‐Aminoadipoyl)‐L‐homocysteinyl‐D‐Xaa Characterised by Protein Crystallography

Isopenicillin N synthase (IPNS) converts the linear tripeptide δ‐(L ‐α‐aminoadipoyl)‐L ‐cysteinyl‐D ‐valine (ACV) into bicyclic isopenicillin N (IPN) in the central step in the biosynthesis of penicillin and cephalosporin antibiotics. Solution‐phase incubation experiments have shown that IPNS turns over analogues with a diverse range of side chains in the third (valinyl) position of the substrate, but copes less well with changes in the second (cysteinyl) residue. IPNS thus converts the homologated tripeptides δ‐(L ‐α‐aminoadipoyl)‐L ‐homocysteinyl‐D ‐valine (AhCV) and δ‐(L ‐α‐aminoadipoyl)‐L ‐homocysteinyl‐D ‐allylglycine (AhCaG) into monocyclic hydroxy‐lactam products; this suggests that the additional methylene unit in these substrates induces conformational changes that preclude second ring closure after initial lactam formation. To investigate this and solution‐phase results with other tripeptides δ‐(L ‐α‐aminoadipoyl)‐L ‐homocysteinyl‐D ‐Xaa, we have crystallised AhCV and δ‐(L ‐α‐aminoadipoyl)‐L ‐homocysteinyl‐D ‐S‐methylcysteine (AhCmC) with IPNS and solved crystal structures for the resulting complexes. The IPNS:Fe^II:AhCV complex shows diffuse electron density for several regions of the substrate, revealing considerable conformational freedom within the active site. The substrate is more clearly resolved in the IPNS:Fe^II:AhCmC complex, by virtue of thioether coordination to iron. AhCmC occupies two distinct conformations, both distorted relative to the natural substrate ACV, in order to accommodate the extra methylene group in the second residue. Attempts to turn these substrates over within crystalline IPNS using hyperbaric oxygenation give rise to product mixtures.     

Isopenicillin N synthase binds δ-(L-α-aminoadipoyl)-L-cysteinyl-D-thia-allo-isoleucine through both sulfur atoms

Isopenicillin N synthase (IPNS) catalyses the synthesis of isopenicillin N (IPN), the biosynthetic precursor to penicillin and cephalosporin antibiotics. IPNS is a non‐heme iron(II) oxidase that mediates the oxidative cyclisation of the tripeptide δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐valine (ACV) to IPN with a concomitant reduction of molecular oxygen to water. Solution‐phase incubation experiments have shown that, although IPNS can turn over analogues with a diverse range of hydrocarbon side chains in the third (valinyl) position of its substrate, the enzyme is much less tolerant of polar residues in this position. Thus, although IPNS converts δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐isoleucine (ACI) and AC‐D ‐allo‐isoleucine (ACaI) to penam products, the isosteric sulfur‐containing peptides AC‐D ‐thiaisoleucine (ACtI) and AC‐D ‐thia‐allo‐isoleucine (ACtaI) are not turned over. To determine why these peptides are not substrates, we crystallized ACtaI with IPNS. We report the synthesis of ACtaI and the crystal structure of the IPNS:Fe^II:ACtaI complex to 1.79 Å resolution. This structure reveals direct ligation of the thioether side chain to iron: the sulfide sulfur sits 2.66 Å from the metal, squarely in the oxygen binding site. This result articulates a structural basis for the failure of IPNS to turn over these substrates.     

An Engineered Tryptophan Synthase Opens New Enzymatic Pathways to β‐Methyltryptophan and Derivatives

β‐Methyltryptophans (β‐mTrp) are precursors in the biosynthesis of bioactive natural products and are used in the synthesis of peptidomimetic‐based therapeutics. Currently β‐mTrp is produced by inefficient multistep synthetic methods. Here we demonstrate how an engineered variant of tryptophan synthase from Salmonella (StTrpS) can catalyse the efficient condensation of l ‐threonine and various indoles to generate β‐mTrp and derivatives in a single step. Although l ‐serine is the natural substrate for TrpS, targeted mutagenesis of the StTrpS active site provided a variant (βL166V) that can better accommodate l ‐Thr as a substrate. The condensation of l ‐Thr and indole proceeds with retention of configuration at both α‐ and β‐positions to give (2S,3S)‐β‐mTrp. The integration of StTrpS (βL166V) with l ‐amino acid oxidase, halogenase enzymes and palladium chemocatalysts provides access to further d ‐configured and regioselectively halogenated or arylated β‐mTrp derivatives.     

The Thioesterase Bhp is Involved in the Formation of β‐Hydroxytyrosine during Balhimycin Biosynthesis in Amycolatopsis balhimycina

The putative hydrolase gene bhp from the balhimycin biosynthetic gene cluster has been cloned and overexpressed in Escherichia coli. The corresponding enzyme Bhp was purified to homogeneity by nickel‐chelating chromatography and characterized. Although Bhp has sequence similarities to hydrolases with “haloperoxidase”/perhydrolase activity, it did not show any enzymatic activity with standard “haloperoxidase”/perhydrolase substrates (e.g., monochlorodimedone and phenol red), nonspecific esterase substrates (such as p‐nitrophenyl acetate, p‐nitrophenyl phosphate and S‐thiophenyl acetate) or the model lactonase substrate dihydrocoumarin. However, Bhp could be shown to catalyse the hydrolysis of S‐β‐hydroxytyrosyl‐N‐acetyl cysteamine thioester (β‐OH‐Tyr‐SNAC) with 15 times the efficiency of S‐L ‐tyrosyl‐N‐acetyl cysteamine thioester (L ‐Tyr‐SNAC). This is in agreement with the suggestion that Bhp is involved in balhimycin biosynthesis, during which it was supposed to catalyse the hydrolysis of β‐OH‐Tyr‐S‐PCP (PCP=peptidyl carrier protein) to free β‐hydroxytyrosine (β‐OH‐Tyr) and strongly suggests that Bhp is a thioesterase with high substrate specificity for PCP‐bound β‐OH‐Tyr and not a “haloperoxidase”/perhydrolase or nonspecific esterase.     

CGTase‐Catalysed cis‐Glucosylation of L‐Rhamnosides for the Preparation of Shigella flexneri 2a and 3a Haptens

We report the enzymatic synthesis of α‐D ‐glucopyranosyl‐(1→4)‐α‐L ‐rhamnopyranoside and α‐D ‐glucopyranosyl‐(1→3)‐α‐L ‐rhamnopyranoside by using a wild‐type transglucosidase in combination with glucoamylase and glucose oxidase. It was shown that Bacillus circulans 251 cyclodextrin glucanotransferase (CGTase, EC 2.1.4.19) can efficiently couple an α‐L ‐rhamnosyl acceptor to a maltodextrin molecule with an α‐(1→4) linkage, albeit in mixture with the α‐(1→3) regioisomer, thus giving two glucosylated acceptors in a single reaction. Optimisation of the CGTase coupling reaction with β‐cyclodextrin as the donor substrate and methyl or allyl α‐L ‐rhamnopyranoside as acceptors resulted in good conversion yields (42–70 %) with adjustable glycosylation regioselectivity. Moreover, the efficient chemical conversion of the products of CGTase‐mediated cis‐glucosylation into protected building blocks (previously used in the synthesis of O‐antigen fragments of several Shigella flexneri serotypes) was substantiated. These novel chemoenzymatic strategies towards useful, convenient intermediates in the synthesis of S. flexneri serotypes 2a and 3a oligosaccharides might find applications in developments towards synthetic carbohydrate‐based vaccine candidates against bacillary dysentery.     

Kinetic Study of the Scavenging Reaction of the Aroxyl Radical by Eight Kinds of Vegetable Oils in Solution

A detailed kinetic study was performed for the reaction of the aroxyl radical (ArO?) with eight vegetable oils 1–8, which contain different concentrations of α‐, β‐, γ‐, and δ‐tocopherols and ‐tocotrienols (‐Tocs and ‐Toc‐3s). The second‐order rate constants (k_s) and aroxyl radical absorption capacity (ARAC) values for the reaction of ArO? with vegetable oils 1–8 (rice bran 1, perilla 2, rapeseed 3, safflower 4, grape seed 5, sesame 6, extra virgin olive 7, and olive oils 8) were measured in ethanol/chloroform/D₂O (50:50:1, v/v/v) solution at 25 °C using stopped‐flow spectrophotometry. The k_s value (16.1 × 10^?3 L g^?1 s^?1) of rice bran oil 1 with the highest activity was 8.0 times larger than that (2.02 × 10^?3) of olive oil 8 with the lowest activity. The concentrations (in mg 100 g^?1) of α‐, β‐, γ‐, and δ‐Tocs and ‐Toc‐3s contained in the vegetable oils 1–8 were determined using high performance liquid chromatography‐mass spectrometry/mass spectrometry (HPLC‐MS/MS). From these results, it was clarified that the ArO?‐scavenging rates (k_s) (i.e., the relative ARAC value) obtained for the vegetable oils 1–8 may be well explained as the sum of the product of the rate constant () and the concentration ([AOH‐i]/10⁵) of AOH‐i (Tocs and Toc‐3s) included in vegetable oils. The results suggest that the ARAC assay method might be used in the evaluation of antioxidant activity of general food extracts.     

Activity of α‐Aminoadipate Reductase Depends on the N‐Terminally Extending Domain

L ‐α‐Aminoadipic acid reductases catalyze the ATP‐ and NADPH‐dependent reduction of L ‐α‐aminoadipic acid to the corresponding 6‐semialdehyde during fungal L ‐lysine biosynthesis. These reductases resemble peptide synthetases with regard to their multidomain composition but feature a unique domain of elusive function—now referred to as an adenylation activating (ADA) domain—that extends the reductase N‐terminally. Truncated enzymes based on NPS3, the L ‐α‐aminoadipic acid reductase of the basidiomycete Ceriporiopsis subvermispora, lacking the ADA domain either partially or entirely were tested for activity in vitro, together with an ADA‐adenylation didomain and the ADA domainless adenylation domain. We provide evidence that the ADA domain is required for substrate adenylation: that is, the initial step of the catalytic turnover. Our biochemical data are supported by in silico modeling that identified the ADA domain as a partial peptide synthetase condensation domain.     

Multi‐Enzymatic Synthesis of Optically Pure β‐Hydroxy α‐Amino Acids

A novel enzymatic production system of optically pure β‐hydroxy α‐amino acids was developed. Two enzymes were used for the system: an N‐succinyl L ‐amino acid β‐hydroxylase (SadA) belonging to the iron(II)/α‐ketoglutarate‐dependent dioxygenase superfamily and an N‐succinyl L ‐amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N‐succinyl aliphatic L ‐amino acids and produced N‐succinyl β‐hydroxy L ‐amino acids, such as N‐succinyl‐L ‐β‐hydroxyvaline, N‐succinyl‐L ‐threonine, (2S,3R)‐N‐succinyl‐L ‐β‐hydroxyisoleucine, and N‐succinyl‐L ‐threo‐β‐hydroxyleucine. LasA catalyzed the desuccinylation of various N‐succinyl‐L ‐amino acids. Surprisingly, LasA is the first amide bond‐forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L ‐leucine. By combining SadA and LasA in a preparative scale production using N‐succinyl‐L ‐leucine as substrate, 2.3 mmol of L ‐threo‐β‐hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β‐hydroxy α‐amino acids.

     

Enzymes in Organic Chemistry, 11:[1] Hydrolase‐Catalyzed Resolution of α‐ and β‐Hydroxyphosphonates and Synthesis of Chiral,Non‐Racemic β‐Aminophosphonic Acids

The enantioselective acylation of racemic diisopropyl α‐ and β‐hydroxyphosphonates by hydrolases in t‐butyl methyl ether with isopropenyl acetate as acyl donor is limited by the narrow substrate specificity of the enzymes. High enantiomeric excesses (up to 99%) were obtained for the acetates of (S)‐diisopropyl 1‐hydroxy‐(2‐thienyl)methyl‐, 1‐hydroxyethyl‐ and 1‐hydroxyhexylphosphonate and (R)‐diisopropyl 2‐hydroxypropylphosphonate. The hydrolysis of a variety of β‐chloroacetoxyphosphonates by the lipase from Candida cylindracea and protease subtilisin in a biphasic system gives (S)‐β‐hydroxyphosphonates (ee 51–92%) enantioselectively. (S)‐2‐Phenyl‐2‐hydroxyethyl‐ and (S)‐3‐methyl‐2‐hydroxybutylphosphonates (ee 96% and 99%, respectively) were transformed into (R)‐2‐aminophosphonic acids of the same ee.     

Surfactant Synthesis via Lipase Esterification of Methyl α-d-Glucopyranoside with Selected Aliphatic Carboxylic Acids

Lipase‐catalyzed esterification and properties of synthesized carbohydrate esters were investigated. Methyl α‐d ‐glucopyranoside was the acyl group acceptor and different carbon atom chain lengths of aliphatic carboxylic acids (C12, C14 and C16) as the acyl group donors were applied in the esterification. Physico‐chemical studies on the synthesized carbohydrate esters were carried out. It was found that melting point for the methyl 6‐O‐hexadecanoyl‐α‐d ‐glucopyranoside was the highest consecutively followed by methyl 6‐O‐tetradecanoyl‐α‐d ‐glucopyranoside and methyl 6‐O‐dodecanoyl‐α‐d ‐glucopyranoside. Liquid crystal properties of the synthesized carbohydrate ester synthesized were evaluated via optical polarized microscopy. It was found that the liquid crystal textures for mono‐substituted carbohydrate esters were of the smectic phase. In a quaternary system (carbohydrate ester/n‐butanol/n‐hexadecane/water), a maximum 34 % of water (by mass) was contained in the monophasic region of methyl 6‐O‐tetradecanoyl‐α‐d ‐glucopyranoside and a maximum of 52 % water (by mass) was contained in a monophasic methyl 6‐O‐dodecanoyl‐α‐d ‐glucopyranoside. For methyl‐6‐O‐dodecanoyl‐α‐d ‐glucopyranoside, its concentration at aggregation was 5.2 × 10^?4 mM, with minimum air/water surface tension of 26 mN m^?1. The Gibbs energy of micellization was calculated at ?50 kJ mol^?1. The maximum adsorption density of methyl 6‐O‐dodecanoyl‐α‐d ‐glucopyranoside was determined at 4 × 10^?6 mol m^?2 while its minimum area per surfactant molecule at the air/water surface was 47 Ų.     

Asymmetric synthesis and conformational analysis by NMR spectroscopy and MD of Aba- and α-MeAba-containing dermorphin analogues

Dermorphin analogues, containing a (S)‐ and (R)‐4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one scaffold (Aba) and the α‐methylated analogues as conformationally constrained phenylalanines, were prepared. Asymmetric phase‐transfer catalysis was unable to provide the (S)‐α‐Me‐o‐cyanophenylalanine precursor for (S)‐α‐MeAba in acceptable enantiomeric purity. However, by using a Schöllkopf chiral auxiliary, this intermediate was obtained in 88 % ee. [(S)‐Aba 3‐Gly 4]dermorphin retained μ‐opioid affinity but displayed an increased δ‐affinity. The corresponding R epimer was considerably less potent. In contrast, the [(R)‐α‐MeAba 3‐Gly 4]dermorphin isomer was more potent than its S epimer. Tar‐MD simulations of both non‐methylated [Aba 3‐Gly 4]dermorphin analogues showed a degree of folding at the C‐terminal residues toward the N terminus of the peptide, without however, adopting a stabilized β‐turn conformation. The α‐methylated analogues, on the other hand, exhibited a type I/I′ β‐turn conformation over the α‐MeAba 3 and Gly 4 residues, which was stabilized by a hydrogen bond involving Tyr 5‐H_N and D ‐Ala 2‐CO.     

Terpendole E and its Derivative Inhibit STLC‐ and GSK‐1‐Resistant Eg5

Terpendole E is first natural product found to inhibit mitotic kinesin Eg5, but its inhibitory mechanism remains to be revealed. Here, we report the effects of terpendole E and 11ketopaspaline (a new natural terpendole E analogue) on the Eg5–microtubule interaction and in several Eg5 mutants. 11‐Ketopaspaline is a shunt product from terpendole E, and it shows potent inhibitory activity against the microtubule‐stimulated ATPase activity of Eg5. Unlike other Eg5 inhibitors, such as S‐trityl‐L ‐cysteine (STLC) and GSK‐1, both terpendole E and 11‐ketopaspaline only partially inhibited Eg5–microtubule interaction. Furthermore, terpendole E and 11‐ketopaspaline inhibited several Eg5 mutants that are resistant to STLC (Eg5^D130A, Eg5^L214A) or GSK‐1 (Eg5^I299F, Eg5^A356T), but with the same extent of inhibition against wild‐type Eg5. Because Eg5^D130A and Eg5^L214A show cross‐resistance to most known Eg5 inhibitors, which bind the L5 loop, these results suggest that terpendole E and its analogues have a different binding site and/or inhibitory mechanism to those for L5 loop‐binding type Eg5 inhibitors.     

The inverse type II β-turn on D-Trp-Phe, a pharmacophoric motif for MOR agonists

Herein we propose the D ‐Trp‐Phe sequence within an inverse type II β‐turn as a new kind of pharmacophoric motif for μ‐opioid receptor (MOR) cyclopeptide agonists. Initially, we observed that c[Tyr‐D ‐Pro‐D ‐Trp‐Phe‐Gly] ( 4 ), an analogue of endomorphin‐1 (H‐Tyr‐Pro‐Trp‐Phe‐NH₂) lacking the crucial protonatable amino group of Tyr 1, is a MOR agonist with 10^?8 M affinity. Molecular docking analysis suggested that the relevant interactions with the receptor involve D ‐Trp‐Phe. The bioactive conformation of this region was investigated by selected derivatives of 4 designed to adopt an inverse type II β‐turn. These efforts led to c[Tyr‐Gly‐D ‐Trp‐Phe‐Gly] ( 14 ) and to the cyclotetrapeptide c[D ‐Asp‐1‐amide‐β‐Ala‐D ‐Trp‐Phe] ( 15 ), showing improved nanomolar affinity. Both 14 and 15 selectively bind MOR, as they have negligible affinity for the κ‐ and δ‐opioid receptors. Both 14 and 15 behave as partial MOR agonists in functional assays. Conformational and docking analyses confirm the role of the inverse β‐turn in binding. These results indicate that the D ‐Trp‐Phe inverse β‐turn structure can be used for designing non‐endomorphin‐like peptidomimetic opioid agonists in general, characterized by an atypical mechanism of interaction between ligand and receptor.     

A simple and efficient way to synthesize optically active polyamides by solution polycondensation of di‐O‐methyl‐L‐tartaryl chloride with diamines

A series of optically active polyamides containing di‐O‐methyl‐L ‐tartaryl moieties in the main chain were synthesized by polycondensation of di‐O‐methyl‐L ‐tartaryl chloride 5 with diamines and characterized by gel permeation chromatography, UV–vis, circular dichroism (CD), IR, and NMR spectroscopies. The polycondensation reaction could be carried out under mild conditions and the reaction time was short (2–3 h). The key monomer 5 prepared from L ‐tartaric acid via esterification, etherification, hydrolysis, and chlorination was easily purified by vacuum sublimation. These polyamides with number average molecular weights ranging from 14,000 to 35,000, displayed large optical activity in dimethyl sulfoxide solution, and their specific optical rotations oscillated between 87.2° and 210.7° depending on the structures of the diamines. The glass transition temperatures of these polyamides were in the range of 106–191°C, and the 10% mass loss occurred at temperature above 300°C. The polyamides derived from aromatic diamines exhibited higher T_g and thermal stability than those derived from aliphatic diamines. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

The β‐δ‐Phase Transition and Thermal Expansion of Octahydro‐1,3,5,7‐Tetranitro‐1,3,5,7‐Tetrazocine

Phase behavior of octahydro‐1,3,5,7‐tetranitro‐1,3,5,7‐tetrazocine (HMX) is investigated by X‐ray powder diffraction (XRD). The XRD patterns at elevated temperature show that there is a co‐existing temperature range of β‐ and δ‐phase during the phase transition process. Additionally, mechanical forces can catalyze the conversion from δ‐ back to β‐phase. Based on the diffraction patterns of β‐ and δ‐phase at different temperatures, we calculate the coefficients of thermal expansion by Rietveld refinement. For β‐HMX, the linear coefficients of thermal expansion of a‐axis and b‐axis are about 1.37×10⁻⁵ and 1.25×10⁻⁴ °C⁻¹. A slight decrease in c‐axis with temperature is also observed, and the value is about −0.63×10⁻⁵ °C⁻¹. The volume coefficient of thermal expansion is about 1.60×10⁻⁴ °C⁻¹, with a 2.2% change from 30 to 170 °C. For δ‐HMX, the linear coefficients of thermal expansion of a‐axis and c‐axis are found to be 5.39×10⁻⁵ and 2.38×10⁻⁵ °C⁻¹, respectively. The volume coefficient of thermal expansion is about 1.33×10⁻⁴ °C⁻¹, with a 2.6% change from 30 to 230 °C. The results indicate that β‐HMX has a similar volume coefficient of thermal expansion compared with δ‐HMX, and there is about 10.5% expansion from β‐HMX at 30 °C to δ‐HMX at 230 °C, of which about 7% may be attributed to the reconstructive transition.     

Oxygen utilisation by isopenicillin n synthase from penicillium chrysogenum

The enzyme isopenicillin N synthase (IPNS) converts δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N; an equimolar amount of oxygen is used in this oxidative ring closure reaction. Oxygen uptake rates of the reaction catalysed by partially purified IPNS from Penicillium chrysogenum SC 6140 and P2 were measured using an oxygen electrode. In contrast to published properties of Cephalosporium acremonium IPNS, the enzyme from P. chrysogenum was not stimulated by the addition of glutathione and showed reduced stimulation by Fe²⁺. The analysis of oxygen uptake rates showed the reaction to be first order with respect to oxygen concentration and the K_m for ACV to be 0·4 mmol dm^?3. The implications of these results for cell-free reactions using this enzyme and penicillin fermentations are discussed.     

Direct polycondensation of N‐trimellitylimido‐L‐isoleucine with aromatic diamines

N‐Trimellitylimido‐L ‐isoleucine (3) was prepared from the reaction of trimellitic anhydride with L ‐isoleucine [L ‐2‐amino‐3‐methylvalerianic acid or (2S,3S)‐2‐amino‐3‐methyl‐n‐valerinic acid] in an N,N‐dimethylformamide solution at the refluxing temperature. The direct polycondensation reaction of the monomer imide diacid (3) with 1,4‐phenylenediamine, 4,4′‐diaminodiphenylmethane, 4,4′‐diaminodiphenylsulfone, diaminodiphenylether, 1,5‐naphthalendiamine, 2,4‐diaminotoluene, and 1,3‐phenylenediamine was performed in a medium consisting of triphenyl phosphite, N‐methyl‐2‐pyrolidone (NMP), pyridine, and calcium chloride. The polycondensation was performed under two different conditions: in one method, the reaction mixture was heated in an NMP solution at 60, 90, and then 130°C for different periods of time, and in the other method, the reaction mixture was refluxed only for 1 min in the same solvent. The resulting poly(amide imide)s (PAIs), with inherent viscosities of 0.21–0.37 dL/g, were obtained in high yields. All of these compounds were fully characterized by IR spectra, elemental analyses, and specific rotation measurements. Some structural characterizations and physical properties of these new optically active PAIs were examined. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 116–122, 2003     

Structural investigations of (9‐ethyl‐carbazol‐6‐yl) methyl methacrylate/methyl acrylate copolymers using two‐dimensional NMR spectroscopy

(9‐Ethyl‐carbazol‐6‐yl) methyl methacrylate/methyl acrylate (E/A) copolymers of different compositions were prepared by solution polymerization by varying the molar infeed ratio, using AIBN as initiator at 60°C. The reactivity ratios calculated by Kelen–Tudos (KT) method were found to be r_E = 1.16 ± 0.02 and r_A = 0.69 ± 0.01 whereas those calculated from RREVM method were found to be r_E = 1.18 and r_A = 0.68. The molecular weights (M_w) and polydispersity index (PDI, M_w/M_n) were determined using gel permeation chromatography (GPC). Glass transition temperatures (T_g) for different compositions of E/A copolymers were determined using differential scanning calorimetry (DSC). Copolymer molar outfeed ratio (F_E) was calculated from ¹H NMR spectra. The α‐methyl, methine, backbone methylene, and quaternary carbon resonance signals of the copolymers were distinguished using ¹³C{¹H}, DEPT‐45, ‐90, and ‐135 NMR techniques. The α‐methyl and β‐methylene showed compositional and configurational sensitivity up to pentad and tetrad level, respectively, whereas methine showed only compositional sensitivity up to pentad level. Unambiguous assignments for ¹H and ¹³C{¹H} NMR spectra were done by correlating 1D (¹H, ¹³C{¹H}, DEPT) and 2D (HSQC, TOCSY) NMR data. The spectral assignments for carbonyl region were done by studying higher bond order couplings by heteronuclear multibond correlation (HMBC) spectra. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 5595–5606, 2006     

Walking a Fine Line with Sucrose Phosphorylase: Efficient Single‐Step Biocatalytic Production of l‐Ascorbic Acid 2‐Glucoside from Sucrose

The 2‐O‐α‐d ‐glucoside of l ‐ascorbic acid (AA‐2G) is a highly stabilized form of vitamin C, with important industrial applications in cosmetics, food, and pharmaceuticals. AA‐2G is currently produced through biocatalytic glucosylation of l ‐ascorbic acid from starch‐derived oligosaccharides. Sucrose would be an ideal substrate for AA‐2G synthesis, but it lacks a suitable transglycosidase. We show here that in a narrow pH window (pH 4.8–6.0, with sharp optimum at pH 5.2), sucrose phosphorylases catalyzed the 2‐O‐α‐glucosylation of l ‐ascorbic acid from sucrose with high efficiency and perfect site‐selectivity. Optimized synthesis with the enzyme from Bifidobacterium longum at 40 °C gave a concentrated product (155 g L^?1; 460 mm ), from which pure AA‐2G was readily recovered in ～50 % overall yield, thus providing the basis for advanced production. The peculiar pH dependence is suggested to arise from a “reverse‐protonation” mechanism in which the catalytic base Glu232 on the glucosyl–enzyme intermediate must be protonated for attack on the anomeric carbon from the 2‐hydroxyl of the ionized l ‐ascorbate substrate.     

Enzymatic preparation of L‐α‐glycerylphosphorylcholine in an aqueous medium

L ‐α‐Glycerylphosphorylcholine (L ‐α‐GPC) was successfully prepared from phosphatidylcholine (PC) of food‐grade soy lecithin powder using a novel enzymatic reaction in an aqueous medium. 94.5% yield of L ‐α‐GPC was obtained under the optimal conditions of 55°C, 6.67 mg/mL substrate, 2 mM CaCl₂, and 33.4 U/mL phospholipase A₁ (Lecitase Ultra). L ‐α‐GPC at 98% purity, 73.4% (wt%) recovery, and specific rotation ( ) of ?2.5° was achieved by silica gel column chromatography. Owing to its excellent catalytic efficiency, low cost, and ready availability, phospholipase A₁ (Lecitase Ultra) provides a very satisfactory option for converting PC to L ‐α‐GPC. Practical applications: L ‐α‐Glycerylphosphorylcholine (L ‐α‐GPC) has been studied recently for its potential use as a supplement that may support neurological functions, but it is only found in trace amounts in nature. The present results indicate that Lecitase Ultra can be used for producing L ‐α‐GPC from aqueous PC and suggest encouraging prospects for practical or industrial applications utilizing its notable catalytic performance, economy, and convenience.