Yen-Chywan Liaw

Directed Evolution of a Lysosomal Enzyme with Enhanced Activity at Neutral pH by Mammalian Cell-Surface Display

Human beta-glucuronidase, due to low intrinsic immunogenicity in humans, is an attractive enzyme for tumor-specific prodrug activation, but its utility is hindered by low activity at physiological pH. Here we describe the development of a high-throughput screening procedure for enzymatic activity based on the stable retention of fluorescent reaction product in mammalian cells expressing properly folded glycoproteins on their surface. We utilized this procedure on error-prone PCR and saturation mutagenesis libraries to isolate beta-glucuronidase tetramers that were up to 60-fold more active (k(cat)/K(m)) at pH 7.0 and were up to an order of magnitude more effective at catalyzing the conversion of two structurally disparate glucuronide prodrugs to anticancer agents. The screening procedure described here can facilitate investigation of eukaryotic enzymes requiring posttranslational modifications for biological activity.

Studies on 1,2,4-benzothiadiazine 1,1-dioxides VII1 and quinazolinones IV2: synthesis of novel built-in hydroxyguanidine tricycles as potential anticancer agents

Two representative built-in hydroxyguanidine tricycles containing 1,2,4-benzothiadiazine 1,1-dioxides (3) and quinazolinones (4) were prepared by reductive cyclization of 1-(2-nitrophenylsulfonyl)-2-benzylthio-2-imidazoline (9a), 1-(2-nitrophenylsulfonyl)-2-benzylthio-1,4,5,6-tetrahydropyrimidine (9b), 1-(2-nitrobenzoyl)-2-benzylthio-2-imidazolidine (10a) and 1-(2-nitrobenzoyl)-2-benzylthio-1,4,5,6-tetrahydropyrimidine hydrobromide respectively (10b) with zinc dust in acetic acid under ice-cooling. 2,10-Dihydro-10-hydroxy-3H-imidazo[1,2-b][1,2,4]benzothiadiazine 5,5-dioxide (3a) and 2,3,4,11-tetrahydro-11-hydroxypyrimido[1,2-b][1,2,4]benzothiadiazine 6,6-dioxide (3b) were found to be active against solid tumor cell lines such as KB, Colo 205, HeLa, and Hepa-2

Rhenium carbonyls containing pyridyl ligands incorporating an alkyne entity

Abstract Reactions of pyridyl ligands, 4,4′-dipyridylbutadiyne (DPB), 1,4-bis(4′-pyridylethynyl)benzene (BPEB), ferrocenyl-4-pyridylacetylene (FPA), 4-nitrophenyl-4′-pyridylacetylene (NPPA) and 4-aminophenyl-4′-pyridylacetylene (APPA), with Re(CO)5X (X  Cl,Br), cis-Re(CO)4(L)Cl (L  PPh3, P(OMe)3), and [Re(CO)3(2,2′-bipy)(MeCN)][PF6], provides fac-Re(CO)3(η1-DPB)2Cl (1), fac-Re(CO)3(PY)2Br (2, PY  FPA; 3, L  NPPA; 4, L  APPA), [fac-Re(CO)3(PPh3)Cl]2(μ-PY) (5, PY  BPEB; 6, PY  DPB), [fac-Re(CO)3(P(OMe)3)Cl]2(μ-DPB) (7, fac-Re(CO)3(PPh3(PY)Cl (8, PY = NPPA; 9, PY = FPA), [fac-Re(CO)3(2,2′-bipy)(PY)][PF6] (10, PY  NPPA; 11, PY  APPA; 12, PY  FPA), and [{fac-Re(CO)3(2,2′-bipy)}2(μ-PY)][PF6] (13, PY  DPB; 14, PY  BPEB). The energy of the metal to pyridyl π∗ charge-transfer (MLCT) is investigated by electronic absorption spectra and cyclic voltammetry. X-ray structural analyses for 2·CH2Cl2 and a12·2H2O were carried out. 2·CH2Cl2: C12H28BrClN2P2 Fe2: monoclinic; P21/n, Z=4; a = 15.188(2), b = 15.100(2), c = 16.254(1) A ; β = 102.22(1)°; R = 0.047; Rw = 0.040. 12 ·2h 2 O: C 30 H 21 F 6 N3O3PFeRe; monoclinic; P21/n, Z = 4; a = 19.022, b = 9.339(2), c = 21.111(3) =A; β = 116.1569(9)°; R = 0.043; rw = 0.061.

Structure determination of porcine haemoglobin

To investigate a potential candidate material for making artificial red blood cells to supplement blood transfusion, the X-ray structure of porcine haemoglobin at 1.8 A resolution was determined as part of research towards synthesizing human blood. Porcine haemoglobin was crystallized by the vapor-diffusion method, producing crystals of dimensions 0.3-0.5 mm after successive seeding. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 68.10, b = 72.27, c = 114.85 A. The initial phase was determined by the molecular-replacement method, using human oxyhaemoglobin as a model. The final R factor was 21.1% for 36 820 reflections after validation of 574 water molecules. The r.m.s. deviations of bond lengths, angles, torsion angles and improper angles from their ideal values are 0.017 A, 3.0, 20.6 and 1.8 degrees, respectively. The average B factor is 33.63 A(2) for the haemoglobin molecule and 50.53 A(2) for the water molecules. The structure could be superimposed on a 2.8 A resolution structure with an r.m.s. difference of 0.59 A in main-chain atomic positions and 1. 27 A in side-chain atomic positions. Porcine and human haemoglobins are compared. A tentative model for artificial blood is proposed based on the complementarity relationship of the surface charges between haemoglobin and the surrounding cell membrane.

Crystallization of agglutinin from the seeds of Abrus precatorius

Agglutinin protein purified from the seeds of Abrus precatorius has a high antitumour activity and was crystallized at room temperature with polyethylene glycol 8000 as the precipitant. The agglutinin crystal diffracted to 3.45 A and belongs to one of two possible tetragonal space groups, P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 141.91, c = 105.63 A. The asymmetric unit contains a heterotetrameric protein molecule of molecular weight 134 kDa and has a solvent content of approximately 38%.

Unusual conformational flexibility in N1‐substituted uncommon purine nucleosides Crystal structure of 1‐allyl‐isoguanosine and 1‐allyl‐xanthosine

Several new N1‐substituted uncommon purine nucleosides, including doridosine (1‐methyl‐isoguanosine; m‐iG), 1‐allyl‐isoguanosine (a‐iG) and 1‐allyl‐xanthosine (a‐X), have been synthesized and tested as agonists for the adenosine receptors. Some have smooth muscle relaxant or negative chronotropic activities. The X‐ray crystal structure of these compounds has been determined at atomic resolution in order to understand the structure‐activity relationship. The structures were solved by direct methods and refined by full‐matrix least‐squares refinement procedure. The crystallographic parameters are: a‐iG, space group P21, a=10.573 (1) Å, b=21.955 (2) Å, c=14.360 (1) Å, β=110.65 (1)°, no. of 3σ Fos=4585, R=0.047; a‐X, space group P212121, a=16.015 (2) Å, b=16.239 (1) Å, c=5.3723 (5) Å, no. of 3σ Fos= 1169, R=0.031. In the a‐iG crystal, there are 4 independent molecules (with different conformation) per asymmetric unit. While all 4 molecules adopt anti χCN glycosyl torsion angle, their riboses have 3 distinct puckers (C2′‐exo, C2′‐endo and C1′‐exo). In contrast, the a‐X structure adopts a syn χCN glycosyl torsion angle, which is stabilized by an intramolecular hydrogen bond between the N3 or purine base and the O5′ of the ribose (in C2′‐endo pucker). Both purine bases (a‐iG and a‐X) are mainly in the keto tautomer form. For the isoguanine base, the averaged N1–C2 bond distance (1.42 Å) is significantly longer than that (1.375 Å) of the guanine base. For the xanthine base, N3 nitrogen has an imino proton attached which is unambiguously located in the electron density map. The surprising flexibility in the ribose ring of these N1‐substituted uncommon purine nucleosides suggests flat the ribose moiety may not participate in the binding of nucleoside to the adenosine receptors.

Heterocyclic Compounds. I. Reactions of o-Amino-carboxamide with b-Diketones: Synthesis of Imidazo[1,5-a]pyrimidine and Pyrazolo[1,5-a]pyrimidine Derivatives

Treatment of 5-aminoimidazole-4-carboxamide hydrochloride (1) and 5-aminopyrazole-4-carboxamide hemisulfate (2) with β-diketones furnished 2,4-disubstituted imidazo[1,5-a]pyrimidine-8-carboxamide (7) and 5,7-disubstituted pyrazolo[1,5-a]pyrimidine-3-carboxamide (8) respectively. The 1 H nmr and 13 C nmr spectra of these compounds and the X-ray crystallography of compound 7a are discussed

A Lon-Like Protease with No ATP-Powered Unfolding Activity

Lon proteases are a family of ATP-dependent proteases involved in protein quality control, with a unique proteolytic domain and an AAA+ (ATPases associated with various cellular activities) module accommodated within a single polypeptide chain. They were classified into two types as either the ubiquitous soluble LonA or membrane-inserted archaeal LonB. In addition to the energy-dependent forms, a number of medically and ecologically important groups of bacteria encode a third type of Lon-like proteins in which the conserved proteolytic domain is fused to a large N-terminal fragment lacking canonical AAA+ motifs. Here we showed that these Lon-like proteases formed a clade distinct from LonA and LonB. Characterization of one such Lon-like protease from Meiothermus taiwanensis indicated that it formed a hexameric assembly with a hollow chamber similar to LonA/B. The enzyme was devoid of ATPase activity but retained an ability to bind symmetrically six nucleotides per hexamer; accordingly, structure-based alignment suggested possible existence of a non-functional AAA-like domain. The enzyme degraded unstructured or unfolded protein and peptide substrates, but not well-folded proteins, in ATP-independent manner. These results highlight a new type of Lon proteases that may be involved in breakdown of excessive damage or unfolded proteins during stress conditions without consumption of energy.

Crystallization and preliminary X‐ray analysis of volvatoxin A2 from Volvariella volvacea

Volvatoxin A2, an ion channel disturbed cardiotoxic and hemolytic protein from the edible mushroom, Volvarilla volvacea, has been crystallized by the vapor diffusion method using polyethylene glycol 4000 and ammonium sulfate in sodium acetate buffer pH 4.6. The best crystals belong to the monoclinic space group C2 with unit cell dimensions a = 155.25 Å, b = 58.06 Å, c = 116.92 Å, and β = 119.5°. These crystals diffract to at least 2.2 Å and there are four molecules of molecular weight 24 kDa per asymmetric unit with a solvent content of 48%.

Inhibitory effects of nontoxic protein volvatoxin A1 on pore‐forming cardiotoxic protein volvatoxin A2 by interaction with amphipathic α‐helix

Volvatoxin A2, a pore‐forming cardiotoxic protein, was isolated from the edible mushroom Volvariella volvacea. Previous studies have demonstrated that volvatoxin A consists of volvatoxin A2 and volvatoxin A1, and the hemolytic activity of volvatoxin A2 is completely abolished by volvatoxin A1 at a volvatoxin A2/volvatoxin A1 molar ratio of 2. In this study, we investigated the molecular mechanism by which volvatoxin A1 inhibits the cytotoxicity of volvatoxin A2. Volvatoxin A1 by itself was found to be nontoxic, and furthermore, it inhibited the hemolytic and cytotoxic activities of volvatoxin A2 at molar ratios of 2 or lower. Interestingly, volvatoxin A1 contains 393 amino acid residues that closely resemble a tandem repeat of volvatoxin A2. Volvatoxin A1 contains two pairs of amphipathic α‐helices but it lacks a heparin‐binding site. This suggests that volvatoxin A1 may interact with volvatoxin A2 but not with the cell membrane. By using confocal microscopy, it was demonstrated that volvatoxin A1 could not bind to the cell membrane; however, volvatoxin A1 could inhibit binding of volvatoxin A2 to the cell membrane at a molar ratio of 2. Via peptide competition assay and in conjunction with pull‐down and co‐pull‐down experiments, we demonstrated that volvatoxin A1 and volvatoxin A2 may form a complex. Our results suggest that this occurs via the interaction of one molecule of volvatoxin A1, which contains two amphipathic α‐helices, with two molecules of volvatoxin A2, each of which contains one amphipathic α‐helix. Taken together, the results of this study reveal a novel mechanism by which volvatoxin A1 regulates the cytotoxicity of volvatoxin A2 via direct interaction, and potentially provide an exciting new strategy for chemotherapy.