The fed-batch production of a thermophilic 2-deoxyribose-5-phosphate aldolase (DERA) in Escherichia coli by exponential feeding strategy control.

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes a sequential aldol reaction useful in synthetic chemistry. In this work, the effect of a feeding strategy on the production of a thermophilic DERA was investigated in fed-batch cultures of recombinant Escherichia coli BL21 (pET303-DERA008). The predetermined specific growth rate (micro(set)) was evaluated at 0.20, 0.15, and 0.10 h(-1), respectively. The DERA concentration and volumetric productivity were associated with micro(set). The cells synthesized the enzyme most efficiently at micro(set) = 0.15 h(-1). The maximum enzyme concentration (5.12 g/L) and total volumetric productivity (0.256 g L(-1) h(-1)) obtained were over 10 and five times higher than that from traditional batch cultures. Furthermore, the acetate concentration remained at a relatively low level, less than 0.4 g/L, under this condition which would not inhibit cell growth and target protein expression. Thus, a specific growth rate control strategy has been successfully applied to induce fed-batch cultures for the maximal production of the thermophilic 2-deoxyribose-5-phosphate aldolase.

Biocatalysis in development of green pharmaceutical processes.

Biocatalysis is one of the greenest technologies for the synthesis of chiral molecules due to exquisite regioselectivity and stereoselectivity in water under mild conditions. For example, protection and deprotection of functional groups are usually not required for biotransformations, high or low temperature reactions can often be circumvented and usage of organic solvents are drastically minimized. To truly empower the green chemistry feature of biocatalysis, it is essential to integrate enzymatic transformations strategically into chemical transformations at the retrosynthetic level. This review focuses on the development of novel biocatalytic pharmaceutical processes to replace chemical routes with poorer process efficiency and higher manufacturing costs.

Chemoenzymatic synthesis of small molecule human therapeutics.

Pharmaceuticals have historically been produced by either chemical synthesis or whole cell fermentation. The former is applied to synthetic small molecules while the latter to natural products. As a result of recent advances in rapid discovery of enzymes through genome mining and metagenomics, and their tunability in functions and stability through directed evolution, biocatalysis is emerging to be a transformational technology for drug discovery and production. Enzymes can catalyze reactions otherwise challenging by chemical approaches. Furthermore, enzymatic catalysis is a powerful tool for green chemistry development. This manuscript gives a brief overview of current status in integrating chemical and biological transformations for the synthesis of small molecular therapeutics.

Efficient chemoenzymatic synthesis of pelitrexol via enzymic differentiation of a remote stereocenter.

[structure: see text] An efficient chemoenzymatic process is described for the synthesis of pelitrexol, a novel GARFT inhibitor. The remoteness of this molecule's stereocenter in the tetrahydropterin moiety from the terminal carbonyl group provided a significant challenge in synthesis. The introduction of an oxalamic ester adjacent to the stereocenter dramatically enhanced an enzyme's enantioselectivity for hydrolysis at the terminal ester, producing the desired S-acid with high optical purity and yield. The recycling of the "wrong" enantiomer is achieved via a dehydrogenation/hydrogenation strategy.

CouO and NovO: C-methyltransferases for tailoring the aminocoumarin scaffold in coumermycin and novobiocin antibiotic biosynthesis.

During the biosynthesis of the streptomycete aminocoumarin antibiotics novobiocin and the dimeric coumermycin A(1), the bicyclic coumarin scaffold is C-methylated adjacent to the phenolic oxygen. The SAM-dependent C-methyltransferases NovO and CouO have been heterologously expressed and purified from Escherichia coli and shown to act after the aminocoumarin ring has been constructed by prior action of Nov/CouHIJK. Neither C-methyltransferase works on the tyrosyl-derived S-pantetheinyl intermediates tethered to NovH or on the subsequently released free aminocoumarin. NovL ligates the aminocoumarin to prenylhydroxybenzoate to yield novobiocic acid, which is the substrate for NovO before it is O-glycosylated by NovM. In coumermycin assembly, the corresponding ligase CouL makes the bis-amide by tandem ligation of two aminocoumarins to a dicarboxypyrrole. CouO works on both the mono- and bis-amides for mono- and di-C-methylation adjacent to the phenolic hydroxyl before it is glycosylated by CouM. Thus, the specific timing of C-methylation in the aminocoumarin antibiotic pathways is established.

An efficient and practical chemoenzymatic preparation of optically active secondary amines.

[reaction: see text] An efficient and practical chemoenzymatic method was developed for the preparation of a variety of chiral secondary amines. Here, oxalamic esters were identified as unique derivatives amenable to the enzyme-catalyzed kinetic resolution of racemic secondary amines. Both enantiomers of the amines were produced in high optical purity and yields after the cleavage of the oxalamic groups.

Tailoring of glycopeptide scaffolds by the acyltransferases from the teicoplanin and A-40,926 biosynthetic operons.

The teicoplanin acyltransferase (Atf) responsible for N-acylation of the glucosamine moiety to create the teicoplanin lipoglycopeptide scaffold has recently been identified. Here we use that enzyme (tAtf) and the cognate acyltransferase from the related A-40,926 biosynthetic cluster (aAtf) to evaluate specificity for glycopeptide scaffolds and for the acyl-CoA donor. In addition to acylation of 2-aminoglucosyl glycopeptide scaffolds with k(cat) values of 400-2000 min(-1), both Atfs transfer acyl groups to regioisomeric 6-aminoglucosyl scaffolds and to glucosyl scaffolds at rates of 0.2-0.5 min(-1) to create variant lipoglycopeptides. Using the teicoplanin glycosyltransferase tGtfA, tAtf, and GtfD, a glycosyltransferase from the vancomycin producer, it is possible to assemble a novel lipoglycopeptide with GlcNAc at beta-OH-Tyr(6) and an N(6)-acyl-glucosaminyl-vancosamine at Phegly(4). This study illustrates the utility of chemo- and regioselective acyltransferases and glycosyltransferases to create novel lipoglycopeptides.

Characterization of a regiospecific epivancosaminyl transferase GtfA and enzymatic reconstitution of the antibiotic chloroeremomycin.

Chloroeremomycin, a vancomycin family glycopeptide antibiotic has three sugars, one D-glucose and two L-4-epi-vancosamines, attached to the crosslinked heptapeptide backbone by three glycosyltransferases, GtfA, -B, and -C. Prior efforts have revealed that GtfB and -C in tandem build an epivancosaminyl-1,2-glucosyldisaccharide chain on residue 4 of the aglycone; however, the characterization of GtfA and glycosylation sequence of chloroeremomycin have been lacking. Here, we report the expression and purification of GtfA, as well as synthesis of its sugar donor, 2'-deoxy-thymidine 5'-diphosphate (dTDP)-beta-L-4-epi-vancosamine. GtfA transfers 4-epi-vancosamine from the chemically synthesized dTDP-4-epi-vancosamine to the beta-OH-Tyr6 residue of the aglycone, preferentially after GtfB action, to generate chloroorienticin B. With the preferred kinetic order of GtfB, then GtfA, then GtfC established, we have succeeded in reconstitution of chloroeremomycin from the heptapeptide aglycone by the sequential actions of the three enzymes.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 8. Pharmacological optimization of orally bioavailable 2-pyridone-containing peptidomimetics.

The optimization of the pharmacokinetic performance of various 2-pyridone-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors following oral administration to either beagle dogs or CM-monkeys is described. The molecules described in this work are composed of a 2-pyridone-containing peptidomimetic binding determinant and an alpha,beta-unsaturated ester Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. Modification of the ester contained within these compounds is detailed along with alteration of the P(2) substituent present in the peptidomimetic portion of the inhibitors. The pharmacokinetics of several inhibitors in both dogs and monkeys are described (7 h plasma concentrations after oral administration) along with their human plasma stabilities, stabilities in incubations with human, dog, and monkey microsomes and hepatocytes, Caco-2 permeabilities, and aqueous solubilities. Compounds containing an alpha,beta-unsaturated ethyl ester fragment and either an ethyl or propargyl P(2) moiety displayed the most promising combination of 3C enzyme inhibition (k(obs)/[I] 170 000-223 000 M(-1) s(-1)), antiviral activity (EC(50) = 0.047-0.058 microM, mean vs seven HRV serotypes), and pharmacokinetics following oral administration (7 h dog plasma levels = 0.248-0.682 microM; 7 h CM-monkey plasma levels = 0.057-0.896 microM).

Epimerization of an L-cysteinyl to a D-cysteinyl residue during thiazoline ring formation in siderophore chain elongation by pyochelin synthetase from Pseudomonas aeruginosa.

The thiazoline-containing siderophores pyochelin, yersiniabactin, and Micacocidin A all have D-thiazoline rings, participating in high-affinity chelation of ferric iron. However, studies with pyochelin (Pch) synthetase and yersiniabactin (Ybt) synthetase reconstituted from pure protein components have shown that only L-cysteine is activated and tethered as a covalent aminoacyl-S-enzyme intermediate. Nor are any of the canonical epimerase domains of nonribosomal peptide synthetase (NRPS) assembly lines found in the Ybt or Pch synthetase modules. Here, we report that the PchE subunit of the Pch synthetase exchanges solvent deuterium into the C(2) center of the thiazoline moieties during siderophore chain elongation. Both PchE and HMWP2, from Ybt synthetase, subunits have a 310-360-residue insert in their amino acid activation domains that look like defective methyltransferase (MT) domains. We suggest these inserts are noncanonical epimerase domains, reversibly deprotonating and reprotonating acyl-S-enzyme intermediates at the C(2) locus. The PchE subunit does not epimerize the Cys-S-enzyme intermediate, but once amide bond formation from a benzoyl-S-PchE donor is catalyzed by the cyclization (Cy) domain of PchE, the N-benzoyl-Cys-S-PchE intermediate is present as a D,L-mixture. The subsequent phenylthiazolinyl-S-PchE intermediate, arising from cyclodehydration of the N-benzoyl-Cys-S-PchE intermediate, is likewise a D,L-mixture on hydrolytic release and enantiomer analysis. These results suggest a default role for MT domains of NRPS assembly lines in generating alpha-carbanionic species from thioester intermediates during siderophore chain elongation.

Synthesis of proposed oxidation-cyclization-methylation intermediates of the coumarin antibiotic biosynthetic pathway.

[structure: see text] A chemoenzymatic synthesis was described to prepare proposed oxidation-cyclization-methylation intermediates of the coumarin antibiotic biosynthetic pathway. The successful synthesis of these fragile molecules relies heavily on mild enzymatic deprotection and efficient enzymatic kinetic resolution to minimize epimerization, decomposition, multiple orthogonal protections, and retro aldol reactions often encountered in their chemical synthesis.

Chemoenzymatic route to macrocyclic hybrid peptide/polyketide-like molecules.

Hybrid peptide-polyketides are a class of medically and biologically important natural products characterized by stereochemical and functional diversity. In their biosynthesis, hybrids are often macrocyclized to achieve rigid structures that populate bioactive conformations. We herein present a chemoenzymatic strategy to access the stereochemical and functional diversity found in macrocyclic hybrid natural products in a manner amenable to efficient library synthesis. Our method makes use of small building blocks in the form of Fmoc-protected epsilon-amino acids containing embedded polyketide functionality. The building block approach allows for combinatorial synthesis of linear molecules that can be activated as soluble thioesters or tethered to a solid-phase resin. We demonstrate that these linear molecules are substrates for macrocyclization by a tolerant catalyst, TycC TE, derived from a nonribosomal peptide synthetase. The method should allow for access to diverse structures with hybrid peptide-polyketide character that can be screened for improved or novel activities.

A Stereocontrolled Synthesis of Monofluoro Ketomethylene Dipeptide Isosteres.

A simple, stereocontrolled synthesis of monofluoro ketomethylene dipeptide isosteres has been developed. N-Tritylated ketomethylene dipeptide isosteres, prepared from N-tritylated amino acids, are converted to their Z-TMS enol ethers and fluorinated with Selectfluor. There is cooperative stereocontrol between the N-tritylamine group and the alkyl group at C-2. The method is short (six steps), diastereoselective (85 --> 95%), and enantioselective (>95%).