The Gram-negative gliding bacteria Lysobacter represent a new and rich source for bioactive natur... more The Gram-negative gliding bacteria Lysobacter represent a new and rich source for bioactive natural products. In an effort to discover new antibiotics, we found a cryptic biosynthetic gene cluster (BGC) in Lysobacter sp. 3655 that shared a high similarity with the putative lysocin BGC identified in silico previously from Lysobacter sp. RH2180-5. Lysocins are cyclic lipodepsipeptides with potent activity against MRSA (methicillin-resistant Staphylococcus aureus) using a novel mode of action, but the lysocin BGC had not been experimentally verified so far. Using an activity-guided screening, we isolated the main antibiotic compound and confirmed it to be lysocin E. However, the putative lysocin BGC was barely transcribed in the wild type, in which lysocins were produced only in specific conditions and in a negligible amount. To activate the putative lysocin BGC, we screened for strongly transcribed housekeeping genes in strain 3655 and found several powerful promoters. Upon engineering the promoters into the BGC, the lysocin gene transcription was significantly enhanced and the lysocin yield was markedly increased. With readily detectable lysocins production in the engineered strains, we showed that lysocin production was abolished in the gene deletion mutant and then restored in the complementary strain, even when grown in conditions that did not support the wild type for lysocin production. Moreover, the engineered strain produced multiple new lysocin congeners. The determination of the lysocin BGC and the Lysobacter promoters will facilitate the ongoing efforts for yield improvement and new antibiotics biosynthesis using synthetic biology strategies.
Protein biochemistry protocols typically include disulfide bond reducing agents to guard against ... more Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, β-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Δ1-pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD+ and the reducing agents TCEP and THPP. The product of the reaction exhibits a maximum ultraviolet absorbance peak at 334 nm and forms with an apparent association rate constant of 231–491 M−1 s−1. The reaction is reversible, and nuclear magnetic resonance characterization (1H, 13C, and 31P) of the product revealed a covalent adduct between the phosphorus of the tris(alkyl)phosphine reducing agent and the C4 atom of the nicotinamide ring of NAD+. We also report a 1.45 Å resolution crystal structure of short-chain dehydrogenase/reductase with the NADP+–TCEP reaction product bound in the cofactor binding site, which shows that the adduct can potentially inhibit enzymes. These findings serve to caution researchers when using TCEP or THPP in experimental protocols with NAD(P)+. Because NAD(P)+-dependent oxidoreductases are widespread in nature, our results may be broadly relevant.
ABSTRACT The photophysical properties of a metal chelating pyridyl-based aromatic ureas and poly(... more ABSTRACT The photophysical properties of a metal chelating pyridyl-based aromatic ureas and poly(1,4-phenylene-2,6-pyridylurea) were investigated. In their solution state and upon exposure to 365 nm UV radiation, these low and high molecular weight compounds were found to cleave the urea linkage nearly quantitatively, generating the corresponding amine-terminated subunits and CO2. Through a series of model compounds along with nuclear magnetic resonance (NMR), UV/vis absorption, and fluorescence spectroscopy characterization, the photocleavage process was delineated as a function of UV dose, O2, and water. The presence of water was found crucial to this photocleavage process with O2 playing a role only in subsequent oxidation of resulting amine functionalities. The experimental results support a light-assisted hydrolysis of the urea bond as the most plausible photocleavage mechanism. The photocleavage of such pyridyl-based ureas can potentially find usages in light-assisted base generation and/or for releasing metal cations.
ABSTRACT We have prepared a series of indocyanine green dicarboxylic acid derivatives (9a,b, 4, 1... more ABSTRACT We have prepared a series of indocyanine green dicarboxylic acid derivatives (9a,b, 4, 10, 13) with modified polyene linkers in an attempt to increase the structural rigidity of the polyene linker and thereby the fluorescent yield. Incorporation of five- and six-membered rings into the polyene system led to lower florescent yield for 9a,b, 4 and 10, but shortening the chain by two carbon atoms led to an increase in fluorescent yield for 13.
In the present contribution, the intrinsic dynamics of the Alq3 molecule has been studied using t... more In the present contribution, the intrinsic dynamics of the Alq3 molecule has been studied using two dimensional exchange NMR spectroscopy. The understanding of the intrinsic internal dynamics of the Alq3 complex provides the basis for future studies of dynamic processes. This information will contribute towards understanding both the processing and degradation of Alq3 based organic LED devices.
The inhibition of sialyl Lewis X can prevent unwanted cellular extravasation that is associated w... more The inhibition of sialyl Lewis X can prevent unwanted cellular extravasation that is associated with inflammatory diseases and tumor metastasis. Described is an efficient methodology to prepare peracetylated GlcNAcβ3Gal-aglycone disaccharides subsequently used as metabolic decoys to inhibit sialyl Lewis X expression. Four glycosides were synthesized from one single parent disaccharide that in turn was prepared by large-scale enzymatic synthesis. The
Journal of Pharmaceutical and Biomedical Analysis, Sep 1, 1995
19F NMR spectroscopy of a model fluoroquinolone, lomefloxacin, in an erythrocyte suspension showe... more 19F NMR spectroscopy of a model fluoroquinolone, lomefloxacin, in an erythrocyte suspension showed separate resonances for the intra- and extra-cellular compartments. The intra-cellular peak revealed significant line broadening of the fluorine signals of lomefloxacin. Line broadening also occurred in the presence of oxyhemoglobin (HbO2), hematin, globin and iron. This evidence indicated that lomefloxacin interacted with these compounds; however, ultrafiltration experiments indicated that there was only weak binding (5%) of lomefloxacin to HbO2. 19F and 31P NMR spectroscopy revealed that lomefloxacin may compete with 2,3-diphosphoglycerate for its binding site on HbO2. An apparent partition coefficient of 1.90 +/- 0.15 was observed for lomefloxacin in human erythrocytes, utilizing LC analysis.
The Gram-negative gliding bacteria Lysobacter represent a new and rich source for bioactive natur... more The Gram-negative gliding bacteria Lysobacter represent a new and rich source for bioactive natural products. In an effort to discover new antibiotics, we found a cryptic biosynthetic gene cluster (BGC) in Lysobacter sp. 3655 that shared a high similarity with the putative lysocin BGC identified in silico previously from Lysobacter sp. RH2180-5. Lysocins are cyclic lipodepsipeptides with potent activity against MRSA (methicillin-resistant Staphylococcus aureus) using a novel mode of action, but the lysocin BGC had not been experimentally verified so far. Using an activity-guided screening, we isolated the main antibiotic compound and confirmed it to be lysocin E. However, the putative lysocin BGC was barely transcribed in the wild type, in which lysocins were produced only in specific conditions and in a negligible amount. To activate the putative lysocin BGC, we screened for strongly transcribed housekeeping genes in strain 3655 and found several powerful promoters. Upon engineering the promoters into the BGC, the lysocin gene transcription was significantly enhanced and the lysocin yield was markedly increased. With readily detectable lysocins production in the engineered strains, we showed that lysocin production was abolished in the gene deletion mutant and then restored in the complementary strain, even when grown in conditions that did not support the wild type for lysocin production. Moreover, the engineered strain produced multiple new lysocin congeners. The determination of the lysocin BGC and the Lysobacter promoters will facilitate the ongoing efforts for yield improvement and new antibiotics biosynthesis using synthetic biology strategies.
Protein biochemistry protocols typically include disulfide bond reducing agents to guard against ... more Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, β-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Δ1-pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD+ and the reducing agents TCEP and THPP. The product of the reaction exhibits a maximum ultraviolet absorbance peak at 334 nm and forms with an apparent association rate constant of 231–491 M−1 s−1. The reaction is reversible, and nuclear magnetic resonance characterization (1H, 13C, and 31P) of the product revealed a covalent adduct between the phosphorus of the tris(alkyl)phosphine reducing agent and the C4 atom of the nicotinamide ring of NAD+. We also report a 1.45 Å resolution crystal structure of short-chain dehydrogenase/reductase with the NADP+–TCEP reaction product bound in the cofactor binding site, which shows that the adduct can potentially inhibit enzymes. These findings serve to caution researchers when using TCEP or THPP in experimental protocols with NAD(P)+. Because NAD(P)+-dependent oxidoreductases are widespread in nature, our results may be broadly relevant.
ABSTRACT The photophysical properties of a metal chelating pyridyl-based aromatic ureas and poly(... more ABSTRACT The photophysical properties of a metal chelating pyridyl-based aromatic ureas and poly(1,4-phenylene-2,6-pyridylurea) were investigated. In their solution state and upon exposure to 365 nm UV radiation, these low and high molecular weight compounds were found to cleave the urea linkage nearly quantitatively, generating the corresponding amine-terminated subunits and CO2. Through a series of model compounds along with nuclear magnetic resonance (NMR), UV/vis absorption, and fluorescence spectroscopy characterization, the photocleavage process was delineated as a function of UV dose, O2, and water. The presence of water was found crucial to this photocleavage process with O2 playing a role only in subsequent oxidation of resulting amine functionalities. The experimental results support a light-assisted hydrolysis of the urea bond as the most plausible photocleavage mechanism. The photocleavage of such pyridyl-based ureas can potentially find usages in light-assisted base generation and/or for releasing metal cations.
ABSTRACT We have prepared a series of indocyanine green dicarboxylic acid derivatives (9a,b, 4, 1... more ABSTRACT We have prepared a series of indocyanine green dicarboxylic acid derivatives (9a,b, 4, 10, 13) with modified polyene linkers in an attempt to increase the structural rigidity of the polyene linker and thereby the fluorescent yield. Incorporation of five- and six-membered rings into the polyene system led to lower florescent yield for 9a,b, 4 and 10, but shortening the chain by two carbon atoms led to an increase in fluorescent yield for 13.
In the present contribution, the intrinsic dynamics of the Alq3 molecule has been studied using t... more In the present contribution, the intrinsic dynamics of the Alq3 molecule has been studied using two dimensional exchange NMR spectroscopy. The understanding of the intrinsic internal dynamics of the Alq3 complex provides the basis for future studies of dynamic processes. This information will contribute towards understanding both the processing and degradation of Alq3 based organic LED devices.
The inhibition of sialyl Lewis X can prevent unwanted cellular extravasation that is associated w... more The inhibition of sialyl Lewis X can prevent unwanted cellular extravasation that is associated with inflammatory diseases and tumor metastasis. Described is an efficient methodology to prepare peracetylated GlcNAcβ3Gal-aglycone disaccharides subsequently used as metabolic decoys to inhibit sialyl Lewis X expression. Four glycosides were synthesized from one single parent disaccharide that in turn was prepared by large-scale enzymatic synthesis. The
Journal of Pharmaceutical and Biomedical Analysis, Sep 1, 1995
19F NMR spectroscopy of a model fluoroquinolone, lomefloxacin, in an erythrocyte suspension showe... more 19F NMR spectroscopy of a model fluoroquinolone, lomefloxacin, in an erythrocyte suspension showed separate resonances for the intra- and extra-cellular compartments. The intra-cellular peak revealed significant line broadening of the fluorine signals of lomefloxacin. Line broadening also occurred in the presence of oxyhemoglobin (HbO2), hematin, globin and iron. This evidence indicated that lomefloxacin interacted with these compounds; however, ultrafiltration experiments indicated that there was only weak binding (5%) of lomefloxacin to HbO2. 19F and 31P NMR spectroscopy revealed that lomefloxacin may compete with 2,3-diphosphoglycerate for its binding site on HbO2. An apparent partition coefficient of 1.90 +/- 0.15 was observed for lomefloxacin in human erythrocytes, utilizing LC analysis.
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