Highly diastereoselective chemoenzymatic synthesis of (2′R)- and (2′S)-2′-deoxy[2′-H]guanosines

In an effort to develop an efficient synthetic method of highly diastereoselective (2′R)- and (2′S)-2′-deoxy[2′-²H]guanosines, chemoenzymatic conversion was investigated. The synthesis of (2′R > 98% de)-2′-deoxy[2′-²H]guanosine was achieved by biological transdeoxyribosylation using (2′R > 98% de)-2′-deoxy[2′-²H]uridine, 2,6-diaminopurine, and Enterobacter aerogenes AJ-11125, followed by treatment with adenosine deaminase. (2′S > 98% de)-2′-Deoxy[2′-²H]guanosine was synthesized from (2′S > 98% de)-2′-deoxy[2′-²H]uridine and 2,6-diaminopurine using thymidine phosphorylase and purine nucleoside phosphorylase instead of E. aerogenes AJ-11125.     

Chemo‐Enzymatic Synthesis of 3‐Deoxy‐β‐D‐ribofuranosyl Purines

9‐(3‐Deoxy‐β‐D ‐erythro‐pentofuranosyl)‐2,6‐diaminopurine ( 6 ) was synthesized by an enzymatic transglycosylation of 2,6‐diaminopurine ( 2 ) with 3′‐deoxycytidine ( 1 ) as a donor of 3‐deoxy‐D ‐erythro‐pentofuranose moiety. This transformation comprises i) deamination of 1 to 3′‐deoxyuridine ( 3 ) under the action of whole cell (E. coli BM‐11) cytidine deaminase (CDase), ii) the phosphorolytic cleavage of 3 by uridine phosphorylase (UPase) giving rise to the formation of uracil ( 4 ) and 3‐deoxy‐α‐D ‐erythro‐pentofuranose‐1‐O‐phosphate ( 5 ), and iii) coupling of the latter with 2 catalyzed by whole cell (E. coli BMT‐4D/1A) purine nucleoside phosphorylase (PNPase). Deamination of 6 by adenosine deaminase (ADase) gave 3′‐deoxyguanosine ( 7 ). Treatment of 6 with NaNO₂ afforded 9‐(3‐deoxy‐β‐D ‐erythro‐pentofuranosyl)‐2‐amino‐6‐oxopurine (3′‐deoxyisoguanosine; 8 ). Schiemann reaction of 6 (HF/HBF₄+NaNO₂) gave 9‐(3‐deoxy‐β‐D ‐erythro‐pentofuranosyl)‐2‐fluoroadenine ( 9 ).     

Expression of a <Emphasis Type="Italic">hemA</Emphasis> Gene from <Emphasis Type="Italic">Agrobacterium radiobacter</Emphasis> in a Rare Codon Optimizing <Emphasis Type="Italic">Escherichia coli</Emphasis> for Improving 5-aminolevulinate Production

The 5-aminolevulinate (ALA) synthase gene (hemA) from Agrobacterium radiobacter zju-0121, which was cloned previously in our laboratory, contains several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was picked out as the host to construct an efficient recombinant strain. Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the appropriate conditions. The results indicated that the activity of ALA synthase expressed in Rosetta(DE3)/pET-28a(+)-hemA was about 20% higher than that in E. coli BL21(DE3). Then the effects of precursors (glycine and succinate) and glucose, which is an inhibitor for ALA dehydratase as well as the carbon sources for cell growth, on the production of 5-aminolevulinate were investigated. Based on an optimal fed-batch culture system described in our previous work, up to 6.5 g/l (50 mM) ALA was produced in a 15-l fermenter.     

Electronic spectra of 2-aminopurine and 2,6-diaminopurine: phototautomerism and fluorescence reabsorption

Ultraviolet absorption, fluorescence and excitation spectra of 2-aminopurine (2AP) and 2,6-diaminopurine (2,6DAP) have been studied using aqueous solutions at different pH values. The molecules are found to fluoresce most efficiently near the neutral pH. It is also found that the fluorescence caused by the solutions of these compounds is partly reabsorbed. Molecular orbital calculations on 2AP, its tautomers, anion and cation, and excited state life-time measurements corresponding to the emissions of 2AP and 2,6DAP near 370 and 342 nm, respectively, have been carried out, and have been found to be useful in the interpretation of the experimental observations.     

Self-complementary purines by quadruple hydrogen bonding

The first discrete, self-complementary, quadruply hydrogen-bonded complexes based on the 2,6-diaminopurine (DAP) scaffold have been prepared; regioselective urea formation at the C2 amino group of the heterocycle allows intermolecular dimerization (K(dim) approximately 1-1.6 x 10(3) M(-1) in CDCl3) through a DADA hydrogen bonding motif.     

Molecular Cloning and Heterologous Expression of Laccase from Aeromonas hydrophila NIU01 in Escherichia coli with Parameters Optimization in Production

Prior studies disclosed that Aeromonas hydrophila NIU01 was a biodecolorization and bioelectricity bacterium which was isolated from a cross-strait of Taiwan. However, enzymatic function, laccase, involved in this strain had never been reported. This first attempt is to explore its laccase activity, the molecular cloning and heterologous recombinant expression in Escherichia coli. A full-length novel gene of 1,647 bp, LacA, encoding of 549 amino acids was successfully cloned by polymerase chain reaction. The recombinant pET-15b(+)-NIU-LacA expression was compared in different E. coli strains. By applying Taguchi’s L9 in culture optimization, the soluble laccase increased to 22.7 %, in which the conditions were obtained at 22 °C with initial shaking speed at 200 rpm, addition of lactose of 0.2 mM and CuSO₄ of 0.5 mM to the medium, and shaking off while cell mass reached to OD_600nm of 1.5. NIU-LacA was strongly inhibited by chloride ion. The optimal temperature was 60 °C and the optimum pH for ABTS (2,2′-azino-bis (3-ethylbenzthiazolinesulfonic acid) and 2,6-DMP (2,6-dimethoxyphenol) were pH 2.1 and pH 7.5 which enzymatic activity was 274.6 and 44.8 U/L, respectively. Further study in structural modeling of NIU-LacA showed the C terminal domain was the major variance in the three most closely A. hydrophila strains.     

Cloning and expression of L-asparaginase gene in Escherichia coli

The L-asparaginase (ASN) from Escherichia coli AS1.357 was cloned as a DNA fragment generated using polymerase chain reaction technology and primers derived from conserved regions of published ASN gene sequences. Recombinant plasmid pASN containing ASN gene and expression vector pBV220 was transformed in different E. coli host strains. The activity and expression level of ASN in the engineering strains could reach 228 IU/mL of culture fluid and about 50% of the total soluble cell protein respectively, more than 40-fold the enzyme activity of the wild strain. The recombinant plasmid in E. coli AS1.357 remained stable after 72h of cultivation and 5h of heat induction without selective pressure. The ASN gene of E. coli AS1.357 was sequenced and had high homology compared to the reported data.     

Substitution of Adenine by Purine‐2,6‐diamine Improves the Nonenzymatic Oligomerization of Ribonucleotides on Templates Containing Thymidine

A standard DNA sequencer was used as a novel and highly efficient tool to study the template‐controlled polymerization of RNA. When labeled with appropriate fluorescent dyes, primers and their extension products could be separated and quantified with excellent sensitivity, reproducibility, and speed. The new technique was applied to compare the template‐controlled incorporation of adenosine mononucleotide 2 and its purine‐2,6‐diamine analogue 3 , the latter being capable of forming three H‐bonds with thymidine or uridine residues. The rates and yields of incorporation are similar when only one thymidine unit is available for pairing in the template (see template 6 and Table 2). However, on template 7 with two consecutive thymidine residues, purine‐2,6‐diamine is clearly ahead of adenine (see Table 3). This advantage is most pronounced when the template contains stretches of three and four thymidine moieties (see templates 8 and 9 and Tables 4 and 5, resp.).     

On the syntheses of 8-Heteroaryl-substituted 9-(β-D-Ribofuranosyl)-2,6-diaminopurines through Pd-catalyzed coupling in the presence of cupric oxide

Convenient methods for the preparation of 9-(β-D-ribofuranosyl) derivatives of 8-(2- and 3-thienyl)-2,6-diaminopurine and of 8-(2- and 3-furyl)-2,6-diaminopurine, which are potential antiviral agents has been worked out. The key step was a Pd(0)-catalyzed Stille coupling between 2- and 3-tributylstannylthiophene and 2- and 3-tributylstannylfuran and trimethylsilyl protected 9-(β-D-ribofuranosyl)-2,6-diamino-8-bro-mopurine. The use of N,N-dimethylformamide as solvent at 110° and dichloro(diphenylphosphine-propane)palladium(II) [PdC1₂(dppp)] with cupric oxide as co-reagent was essential in order to obtain a fast reaction and high yields.     

Microcalorimetric research on recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> with high production of polyhydroxyalkanoates (PHAs)

The thermogenic curves of metabolism of the four strains of Escherichia coli pUC19cab/JM109, pUC19cab/XL-IBlue, JM109 and XL-IBlue were determined using the LKB-2277 BioActivity Monitor and the ampoule method at 37°C. The pUC19cab/JM109 and pUC19cab/XL-IBlue are recombinant E. coli strains bearing the same foreign plasmid pUC19cab, which confers the ability to produce polyhydroxyalkanoates (PHAs). The yield of PHAs of pUC19cab/XL-IBlue was higher than that of pUC19cab/JM109. XL-IBlue and JM109 were the host bacteria. The heat flow of these strains was XL-Blue≈JM109>pUC19cab/JM109>pUC19cab/XL-IBlue. These results indicate an obvious interrelation between the PHAs production and the heat flow rate of E. coli strains.     

Synthesis of pyrimidine acyclonucleosides

Nucleoside analogues of uridine, 5-bromo-, 5-iodo-, and 5-fluorouridines, thymidine and cytidine were prepared by condensing appropriately substituted 2,4-dimethoxypyrimidines with an acyclic side chain in the form of a benzoylated halo-ether, and subsequent removal of the protecting benzoyl group in base. The 2′-O-p-tosylates of these nucleoside analogues could then be modified to 2′-halo-, azido-, and amino derivatives. Many of these compounds are competitive inhibitors of uridine phosphorylase in vitro, the most active being 5-methyl-1-(2′-hydroxyethoxymethyl)uracil.     

Construction of recombinant Escherichia coli strains for polyhydroxybutyrate production using soy waste as nutrient

Construction and comparison of recombinant Escherichia coli strains harboring the polyhydroxybutyrate (PHB) operon from Ralstonia entropha using vectors possessing different promotors, as well as the production of PHB from soy waste by the recombinant strain, are reported. The lac promotor was the most efficient on expression of the phb operon among the three promotors studied: i.e., lac promotor, T7 promotor and the normal σ⁷⁰ promotor. The pKS/PHB was the most efficient plasmid for phboperon expression among the three plasmids used: i.e., pKS⁻, pAED4, and pJM9131. It was observed that isopropyl-β-d-thiogalactopyranoside was not required for the induction of the expression of phb operon. The cell dry wt and polyhydroxyalkan cote content by E. coli XL-1 Blue (pKS/PHB) were 3.025 g/L and 27.83%, respectively.     

In situ electrode modification for direct differential pulse polarography of 2,6-diaminopurine and its metabolite, 2,6-diamino-8-purinol

Preferential adsorption of allopurinol (1H-pyrazolo-[3,4-d]-pyrimidin-4-ol) at graphite electrodes from pH 7.0 phosphate buffer solutions of 2,6-diaminopurine and 2,6-diamino-8-purinol allowed in situ electrode modification. Modified electrodes were applied to the simultaneous determination of 2,6-diaminopurine and 2,6-diamino-8-purinol in phosphate buffer at pH 7.0. Differential pulse polarography allowed both compounds to be quantified in the concentration range 1 × 10^?6–5 × 10^?4 M. The relative standard deviation of the peak current is about 10%. Calibration curve characteristics are C (μM) = 0.0094 ± 0.0002 I (μA) + 0.22 ± 0.07 μA, with r = 0.9985 and C (μM) = 0.0065 ± 0.0001 I (μA) + 0.03 ± 0.04 μA with r = 0.9990 for 2,6-diamino-8-purinol and 2,6-diaminopurine, respectively.     

Synthesis and oligomerization of Fmoc/Boc-protected PNA monomers of 2,6-diaminopurine, 2-aminopurine and thymine

A Boc-protecting group strategy for Fmoc-based PNA (peptide nucleic acid) oligomerization has been developed for thymine, 2,6-diaminopurine (DAP) and 2-aminopurine (2AP). The monomers may be used interchangeably with standard Fmoc PNA monomers. The DAP monomer was incorporated into a PNA and was found to selectively bind to T (ΔT(m)≥ +6 °C) in a complementary DNA strand. The 2AP monomer showed excellent discrimination of T (ΔT(m)≥ +12 °C) over the other nucleobases. 2AP also acted as a fluorescent probe of the PNA:DNA duplexes and displayed fluorescence quenching dependent on the opposite base.     

Synthesis of a new class of uridine phosphorylase inhibitors

A new series of potent uridine phosphorylase inhibitors have been prepared from barbituric acid. Among them, 1-[(2-hydroxyethoxy)methyl]-5-)(m--benzyloxy)benzylbarbituric acid ( 37 , BBBA) is the most promising having a K_i value of 1.1 ± 0.2 nM with uridine phosphorylase from human liver. The new inhibitors are easily synthesized and are better inhibitors of human uridine phosphorylase than their uracil counterparts.     

Chain center‐functionalized amphiphilic block polymers: Complementary hydrogen bond self‐assembly in aqueous solution

Hydrogen bonding self‐assemblies were formed in an aqueous medium from a pair of an amphiphilic ABA triblock copolymer and a hydrophobic homopolymer, both with a triple hydrogen bonding site that was complementary to each other and precisely placed at the main‐chain center: (PEGMA)_m–(MMA)_n– ADA –(MMA)_n–(PEGMA)_m and (MMA)_p– DAD –(MMA)_p ( A = hydrogen acceptor; D = hydrogen donor; PEGMA: PEG methacrylate; MMA: methyl methacrylate). The polymers were synthesized by the ruthenium‐catalyzed living radial polymerization with bifunctional initiators (Br– ADA –Br and Cl– DAD –Cl) aiming at pinpoint chain center functionalization to give a symmetric segmental sequence; ADA and DAD initiators were derived from 2,6‐diaminopyridine and thymine, respectively. On mixed equimolar in tetrahydrofuran (THF), both polymers spontaneously associated, and the apparently 1:1 assembly further grew into higher aggregate particles on subsequent addition of water. The aggregates in water/THF were relatively stable and uniform in size, which most likely stems from the intermolecular complementary hydrogen bond interaction at polymer chain centers. In sharp contrast, an equimolar mixture of ADA ‐block polymer and DAD ‐free poly(MMA) in water/THF resulted in larger and irregular particles, and thus short‐lived to eventually collapse. These results indicate that, however structurally marginal, precise pinpoint functionalization of macromolecular chains allows stable self‐assemblies via complementary hydrogen bond interaction even in aqueous media. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013 , 51, 4498–4504     

Expression of human β-defensin-2 with multiple joined genes in Escherichia coli

Human β-defensin (HBD)-2 is a small cationic peptide with a broad range of antimicrobial activity. In this study, multiple copies of the hBD2 gene were linked in tandem, and a number of different Escherichia coli expression vectors were evaluated, including pQE-30, pBV220, pET-28a(+), and pGEX-4T-2. No expression of multiple joined genes was detectable in the pQE-30 expression system, whereas in pBV220 with one or two joined hBD2 genes and in pET-28a(+) with one, two, or four copies, target proteins were expressed at a low level. Only when pGEX-4T-2 was applied as expression plasmid with one or two joined hBD2 genes were target proteins expressed in high level, and the expressed fusion proteins account for 26 and 16% of the total insoluble proteins, respectively. In the pGEX-4T-2 and pET-28a(+) expression systems, the effects of multiple joined genes on the growth of host strains and plasmid stability were examined. Host cells containing plasmid carrying fewer copies of hBD2 genes were faster in cell growth. Plasmid stability decreased with an increase in multiple joined genes, which was especially noticeable in the pET-28a(+) system. Furthermore, the presence of glucose in culture medium brought about a positive effect on plasmid stability when using pET28-nhBD2 as expression vectors.     

Simulated Microgravity Affects Growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> and Recombinant β-<Emphasis Type="SmallCaps">d</Emphasis>-Glucuronidase Production

Effects of simulated microgravity (SMG) on bacteria have been studied in various aspects. However, few reports are available about production of recombinant protein expressed by bacteria in SMG. In this study growth of E. coli BL21 (DE3) cells transformed with pET-28a (+)-pgus in double-axis clinostat that could model low shear SMG environment and the recombinant β-d-glucuronidase (PGUS) expression have been investigated. Results showed that the cell dry weights in SMG were 16.47%, 38.06%, and 28.79% more than normal gravity (NG) control, and the efficiency of the recombinant PGUS expression in SMG were 18.33%, 19.36%, and 33.42% higher than that in NG at 19 °C, 28 °C, and 37 °C, respectively (P < 0.05).     

Construction of recombinant Escherichia coli strais for production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate)

Plastic wastes constitute a worldwide environmental problem, and the demand for biodegradable plastics has become high. One of the most important characteristics of microbial polyesters is that they are thermoplastic with environmentally degradable properties. In this study, pUC 19/PHA was cloned and transformed into three different Escherichia coli strains. Among the three strains that were successfully expressed in the production of polyhydroxyalkanoates (PHA), E. coli HMS174 had the highest yield in the production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-HV]). The cell dry weight and PHA content of recombinant HMS174 reached as high as 10.27 g/L and 43% (w/w), respectively, in fed-batch fermentor culture. The copolymer of PHA, P(HB-HV), was found in the cells, and the biopolymers accumulated were identified and analyzed by gas chromatography, proton nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. We demonstrated clearly that the E. coli host for PHA production has to be carefully selected to obtain a high yield. The results obtained indicated that a superior E. coli with high PHA production can be constructed with a desirable ratio of P(HB-HV), which has potential applications in industry and medicine.     

In Vitro Refolding of Triosephosphate Isomerase from <Emphasis Type="Italic">L. donovani</Emphasis>

The triosephosphate isomerase of Leishmania donovani (LdTIM) was expressed at high level in Escherichia coli. The TIM gene was cloned in expression vector pET-23(a) with C-terminal 6× His tag fused in frame, and expressed as a 27.6-kDa protein in E. coli as inclusion bodies. The recombinant LdTIM from E. coli lysate was solubilized in 6 M guanidine hydrochloride and purified by Ni-NTA chromatography. In the present study, the effect of bovine serum albumin on the reactivation of TIM was investigated. Furthermore, 8-anilino-1-naphthalene sulfonic acid was used to detect the structural changes induced by bovine serum albumin (BSA). Here, we conclude that BSA assists in the refolding and regain of LdTIM enzyme activity by providing framework for structure formation. This study indicates that numerous protein–protein contacts are constantly occurring inside the cell that leads to the formation of native protein.