Improved production and function of llama heavy chain antibody fragments by molecular evolution

The aim of this study was to improve production level of llama heavy chain antibody fragments (V_HH) in Saccharomyces cerevisiae while retaining functional characteristics. For this purpose, the DNA shuffling technique was used on llama V_HH fragments specific for the azo-dye reactive red-6. In the DNA shuffling process, three parental llama V_HH with high amino acid sequence identity with significant differences in production and functional characteristics were used. From these parental sequences, a S. cerevisiae library was created and 16 antigen specific shuffled V_HH fragments were selected. We found that these shuffled V_HH fragments were, (i) unique in sequence; (ii) composed of two or three parental sequences; (iii) in three V_HHs point mutations occurred; and (iv) antigen specificity was not changed. The four highest producers in the yeast S. cerevisiae were selected and production, affinity, and antigen binding at 90°C were compared with parental V_HHs. One shuffled V_HH was enhanced both in production (3.4-fold) and affinity (four-fold). A second shuffled V_HH displayed increased production (1.9-fold), and improved stability (2.4-fold) in antigen binding at 90°C. Structural analysis suggested that improved antigen binding is associated with the A24→V24 substitution, which reduces the size of the hydrophobic pit at the llama V_HH surface. We demonstrate that it is possible to improve desired characteristics of the same V_HH fragment simultaneously using DNA shuffling. Finally, this is one of the first examples of DNA shuffling improving temperature stability of an antibody fragment.     

Selective addressing of heteroditopic ligands by iron(II) and platinum(II)

The heteroditopic ligand 4′-(4,7,10-trioxadec-1-yn-10-yl)-2,2′:6′,2″-terpyridine, 2, contains an N,N′,N″-donor metal-binding domain that recognizes iron(II), and a terminal alkyne site that selectively couples to platinum(II). This selectivity has been used to investigate routes to the formation of heterometallic systems. The single crystal structures of ligand 2 and the complex [Fe(2)₂][PF₆]₂ are reported.     

Methylene bridged binuclear bis(imino)pyridyl iron(II) complexes and their use as catalysts together with Al(i-Bu)3 for ethylene polymerization

Three novel methylene bridged binuclear iron(II) complexes: (R,R′ = i-C₃H₇ (6); R = i-C₃H₇, R′ = CH₃ (7); R,R′ = CH₃ (8))} have been synthesized. Activated by Al(i-Bu)₃, complex 6 shows very poor activity for the polymerization of ethylene at one bar ethylene pressure, whereas, 7 and 8 exhibit much higher activity than mononuclear iron catalysts {[ArNC(Me)C₅H₃N(Me)CNAr′]FeCl₂ (Ar,Ar′ = 2,6-C₆H₃-i-Pr (9); Ar = 2,6-C₆H₃-i-Pr₂, Ar′ = 2,6-C₆H₃–Me₂ (10); Ar,Ar′ = 2,6-C₆H₃–Me₂ (11))}. The molecular weight (M_w) of PE produced by 7 and 8 are in the range 13.2–46.0 × 10⁴ and much higher than those produced by mononuclear iron catalysts 9 and 10. GPC results demonstrate that 7 and 8 yield PE with a broad/bimodal molecular weight distribution (MWD). In contrast, 9 and 10 yield PE with relatively narrow and unimodal MWD (4.26 and 3.55). Elevating the temperature and Al/Fe molar ratio will narrow the MWD of PE.     

Angiotensin II and angiotensin-(1-7) inhibit the inner cortex Na+ -ATPase activity through AT2 receptor

In the present paper, the modulation of the basolateral membrane (BLM) Na⁺-ATPase activity of inner cortex from pig kidney by angiotensin II (Ang II) and angiotensin-(1–7) (Ang-(1–7)) was evaluated. Ang II and Ang-(1–7) inhibit the Na⁺-ATPase activity in a dose-dependent manner (from 10⁻¹¹ to 10⁻⁵ M), with maximal effect obtained at 10⁻⁷ M for both peptides. Pharmacological evidences demonstrate that the inhibitory effects of Ang II and Ang-(1–7) are mediated by AT₂ receptor: The effect of both polypeptides is completely reversed by 10⁻⁸ M PD 123319, a selective AT₂ receptor antagonist, but is not affected by either (10⁻¹²–10⁻⁵ M) losartan or (10⁻¹⁰–10⁻⁷ M) A779, selective antagonists for AT₁ and AT_(1–7) receptors, respectively. The following results suggest that a PTX-insensitive, cholera toxin (CTX)-sensitive G protein/adenosine 3′,5′-cyclic monophosphate (cAMP)/PKA pathway is involved in this process: (1) the inhibitory effect of both peptides is completely reversed by 10⁻⁹ M guanosine 5′-O-(2-thiodiphosphate) (GDPβS; an inhibitor of the G protein activity), and mimicked by 10⁻¹⁰ M guanosine 5′-O-(3-thiotriphosphate) (GTPγS; an activator of the G protein activity); (2) the effects of both peptides are mimicked by CTX but are not affected by PTX; (3) Western blot analysis reveals the presence of the G_s protein in the isolated basolateral membrane fraction; (4) (10⁻¹⁰–10⁻⁶ M) cAMP has a similar and non-additive effect to Ang II and Ang-(1–7); (5) PKA inhibitory peptide abolishes the effects of Ang II and Ang-(1–7); and (6) both angiotensins stimulate PKA activity.     

Alkaloids from Heimia salicifolia

Two alkaloids, 9β,2′-dihydroxy-4′′,5′′-dimethoxy-lythran-12-one or 9β-hydroxyvertine (1) and (2S,4S,10R)-4-(3-hydroxy-4-methoxyphenyl)-quinolizidin-2-acetate (2), as well as seven known alkaloids, lythrine (3), dehydrodecodine (4), lythridine (5), vertine (6), heimidine (7), lyfoline (8) and epi-lyfoline (9), were isolated from Heimia salicifolia. The structures of these compounds were elucidated by extensive spectroscopic techniques. Furthermore, the structures of 2, 3, and 6 were confirmed by X-ray crystallography, including absolute configuration determination of 2 and 6. Compounds 6 and 9 showed moderate antimalarial activity.     

Synthesis and reactivity of coordinatively unsaturated osmium and ruthenium stannyl complexes

Treatment of MHCl(CO)(PPh₃)₃ (M=Ru, Os) with (CH₂=CH)SnR₃ is a good general route to the coordinatively unsaturated osmium and ruthenium stannyl complexes M(SnR₃)Cl(CO)(PPh₃)₂ (1: M=Ru, R=Me; 2: M=Ru, R = n-butyl; 3: M=Ru, R = p-tolyl; 4: M=Os, R=Me). These coordinatively unsaturated complexes readily add CO and CN-p-tolyl to form the coordinatively saturated compounds M(SnR₃)Cl(CO)L(PPh₃)₂ (5: M=Ru, R=Me, L=CO; 6: M=;Ru, R = n-butyl, L=CO; 7: M=Ru, R = p-tolyl, L=CO; 8: M=Os, R=Me, L=CO; 9: M=Ru, R=Me, L=CN-p-tolyl; 10: M=Ru, R = n-butyl, L=CN-p-tolyl; 11: M=Os, R=Me, L=CN-p-tolyl). In addition, the chloride ligand in Ru(SnR₃)Cl(CO)(PPh₃)₂ proves to be labile and treatment with the potentially bidentate anionic ligands, dimethyldithiocarbamate or diethyldithiocarbamate, affords the coordinatively saturated compounds Ru(SnR₃)(η²-S₂CNR′₂)(CO)(PPh₃)₂ (12: R=Me, R′ = Me; 13: R=Me, R′ = Et; 14: R = n-butyl, R′ = Me; 15: R = p-tolyl, R′ = Me; 16: R = p-tolyl, R′ = Et). Chloride is also displaced by carboxylates forming the six-coordinate compounds Ru(SnR₃)(η²-O₂CR′)(CO)(PPh₃)₂ (17: R=Me, R′ = H; 18: R=Me, R′ = Me; 19: R=Me, R′ = Ph; 20: R = n-butyl, R′ = Me; 21: R = p-tolyl, R′ = Me). IR and ¹H NMR spectral data for all the new compounds and ³¹P and ¹¹⁹Sn NMR spectral data for selected compounds are reported.     

Microbial and enzymatic production of 4,4′-dihydroxybiphenyl via phenol coupling

Phenol was coupled to produce 4,4′-dihydroxybiphenyl in an in vitro system containing phosphate buffer, H₂O₂, phenol, and horseradish peroxidase (EC 1.11.1.7). Other products were also detected. Preliminary experiments with an in vivo fungal fermentation system containing phenol as a substrate also resulted in the production of 4,4′-dihydroxybiphenyl. The results suggest that the biphenols are themselves substrates for the enzymes.     

Urea and amide-based inhibitors of the juvenile hormone epoxide hydrolase of the tobacco hornworm (Manduca sexta: Sphingidae)

A new class of inhibitors of juvenile hormone epoxide hydrolase (JHEH) of Manduca sexta and further in vitro characterization of the enzyme are reported. The compounds are based on urea and amide pharmacophores that were previously demonstrated as effective inhibitors of mammalian soluble and microsomal epoxide hydrolases. The best inhibitors against JHEH activity so far within this class are N-[(Z)-9-octadecenyl]-N′-propylurea and N-hexadecyl-N′-propylurea, which inhibited hydrolysis of a surrogate substrate (t-DPPO) with an IC₅₀ around 90 nM. The importance of substitution number and type was investigated and results indicated that N, N′-disubstitution with asymmetric alkyl groups was favored. Potencies of pharmacophores decreased as follows: amide>urea>carbamate>carbodiimide>thiourea and thiocarbamate for N, N′-disubstituted compounds with symmetric substituents, and urea>amide>carbamate for compounds with asymmetric N, N′-substituents. JHEH hydrolyzes t-DPPO with a K_m of 65.6 μM and a V_max of 59 nmol min⁻¹ mg⁻¹ and has a substantially lower K_m of 3.6 μM and higher V_max of 322 nmol min⁻¹ mg⁻¹ for JH III. Although none of these compounds were potent inhibitors of hydrolysis of JH III by JHEH, they are the first leads toward inhibitors of JHEH that are not potentially subject to metabolism through epoxide degradation.     

Synthesis and fungicidal activity of 2,2′-bipyridine derivatives

A series of substituted 2,2′-bipyridine derivatives was prepared using the Kröhnke reaction and alkylation of 4,4′-dimethyl-2,2′-bipyridine. These compounds were screened for fungicidal activity against 9 plant diseases. 5-Phenyl-2,2′-bipyridine exhibited strong preventative and curative fungicidal activity against wheat powdery mildew (Erisyphe graminis) and wheat leaf rust (Puccinia recondita).     

Reactions of tungsten phosphonium carbyne complexes with aryloxide nucleophiles to form aryloxycarbyne and η-ketenyl complexes

Tungsten phosphoranylideneketene complexes of the type Tp′(CO)(p-OC₆H₄R)W(η²-(C,C)---O=CC---PR′₂Ph) (R=NO₂, R′=Me (6a); R=NO₂, R′=Ph (6b); R=CN, R′=Me (7a); R=CN, R′=Ph (7b); R=Cl, R′=Ph (8b)) have been synthesized from phosphonium carbyne precursors in a reaction that reflects coupling of carbonyl and carbyne ligands. In addition to these products, aryloxycarbyne complexes Tp′(CO)₂WCO(p-C₆H₄NO₂) (9a), Tp′(CO)₂WCO(p-C₆H₄CN) (9b), and Tp′(CO)₂WCO(p-C₆H₄Cl) (9c)) have been prepared via substitution of the phosphonium carbyne phosphine with an aryloxide nucleophile. The product ratio of substitution at the carbyne carbon to carbonyl–carbyne coupling can be tuned by variation of the aryloxide para-substituent. Aryloxy carbyne complexes are the favored products with stronger nucleophiles, while weaker nucleophiles result in a mixture of aryloxy carbyne complexes and η²-ketenyl coupled complexes. Formation of η²-ketenyl complexes is favored for the least nucleophilic aryloxides. Ketenyl complexes 6a and 6b were methylated at the ketenyl oxygen to form cationic alkyne complexes [Tp′(CO)(p-OC₆H₄NO₂)W(η²-(C,C)---CH₃OCCPR₂Ph)][OTf] (R=Me (10a), R=Ph (10b)). The structures of η²-ketenyl complexes 6a and 7b and the structure of cationic alkyne complex 10a were determined by X-ray crystallography.     

Polychlorinated biphenyls as inducers of hepatic microsomal enzymes: Structure-activity rules

A number of highly purified polychlorinated biphenyl (PCB) isomers and congeners were synthesized and administered to male Wistar rats at dosage levels of 30 and 150 μmol · kg⁻¹. The effects of this in vivo treatment on the drug-metabolizing enzymes were determined by measuring the microsomal benzo[a]pyrene (B[a]P) hydroxylase, dimethylaminoantipyrine (DMAP) N-demethylase and NADPH-cytochrome c reductase enzyme activities, the cytochrome b₅ content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450: CO and ethylisocyanide (EIC) binding difference spectra. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC) and PB plus MC (coadministered) to the test animals. The synthetic PCB congeners used in this study included 3,4,4′,5-tetrachlorobiphenyl (TCBP-1), 2,3′,4,4′-tetrachlorobiphenyl (TCBP-2), 2,3′,4,4′,5′-pentachlorobiphenyl (PCBP-1), 2,3,4,4′,5-pentachlorobiphenyl (PCBP-2), 2,3,3′,4,4′,5-hexachlorobiphenyl (HCBP-1), 2,3,3′,4′,5,6-hexachlorobiphenyl (HCBP-2), 2,3,3′,5,5′,6-hexachlorobiphenyl (HCBP-3), 2,2′,3,5,5′,6-hexachlorobiphenyl (HCBP-4) and 2,3,3′,4,5,5′-hexachlorobiphenyl (HCBP-5) and were used to reappraise the structure-activity rules for PCBs as hepatic microsomal enzyme inducers. The results suggested that (a) PCBs which induce MC or mixed-type activity must be substituted at both para positions, at least two meta positions but not necessarily on the same phenyl ring and can also contain one ortho chloro substituent; (b) due to the considerable structural diversity of the PB-type inducers the rules for induction of this activity by PCB congeners are not readily defined.     

Molecular characterization and properties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa: metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid beta-oxidation

The use of (R)-specific enoyl-coenzyme A (CoA) hydratase (PhaJ) provides a powerful tool for polyhydroxyalkanoate (PHA) synthesis from fatty acids or plant oils in recombinant bacteria. PhaJ provides monomer units for PHA synthesis from the fatty acid ß-oxidation cycle. Previously, two phaJ genes (phaJ1_Pa and phaJ2_Pa) were identified in Pseudomonas aeruginosa. This report identifies two new phaJ genes (phaJ3_Pa and phaJ4_Pa) in P. aeruginosa through a genomic database search. The abilities of the four PhaJ_Pa proteins and the (R)-3-hydroxyacyl-acyl carrier protein [(R)-3HA-ACP] dehydrases, FabA_Pa and FabZ_Pa, to supply monomers from enoyl-CoA substrates for PHA synthesis were determined. The presence of either PhaJ1_Pa or PhaJ4_Pa in recombinant Escherichia coli led to the high levels of PHA accumulation (as high as 36–41 wt.% in dry cells) consisting of mainly short- (C4–C6) and medium-chain-length (C6–C10) 3HA units, respectively. Furthermore, detailed characterizations of PhaJ1_Pa and PhaJ4_Pa were performed using purified samples. Kinetic analysis revealed that only PhaJ4_Pa exhibits almost constant maximum reaction rates (V_max) irrespective of the chain length of the substrates. The assay for stereospecific hydration revealed that, unlike PhaJ1_Pa, PhaJ4_Pa has relatively low (R)-specificity. These hydratases may be very useful as monomer-suppliers for the synthesis of designed PHAs in recombinant bacteria.     

Multinucleating ligands from multiple palladium(0)-catalysed cross-coupling reactions — synthesis and characterisation of a trinuclear cyclometallated ruthenium(II) complex and a hexanuclear EPR-active molybdenum complex

Two multinucleating ligands have been prepared from 1,3,5-tris(3,5-dibromophenyl)benzene by multiple Pd(0)-catalysed cross-coupling reactions. 1,3,5-Tris[3,5-bis(4-pyridylethenyl)phenyl]benzene (L¹) has six remote pyridyl moieties, each of which can coordinate a 17 valence-electron Mo(tp^*)(NO)Cl fragment (tp^* = hydrotris(3,5-dimethylpyrazolyl)borate), affording the hexanuclear complex [Cl(NO)(tp^*)Mo₆(L¹) (1). 1,3,5-Tris[3,5-bis(2-pyridyl)phenyl]benzene (L²) incorporates three potentially terdentate, cyclometallating N,C,N-donor sets, and can coordinate three Ru(tpy)²⁺ fragments (tpy = 2,2′:6′,2″-terpyridine) giving the trinuclear complex [(tpy)Ru₃(L²)][PF₆]₃ (2). Complex 1 is EPR active, with nearest-neighbour pairs of molybdenum centres displaying magnetic exchange interactions. Electrochemical studies of the two complexes suggest that there is little ground-state interaction between the metal centres in either case.