A photostable monomeric superfolder green fluorescent protein

The green fluorescent protein (GFP) from Aequorea victoria has been engineered extensively in the past to generate variants suitable for protein tagging. Early efforts produced the enhanced variant EGFP and its monomeric derivative mEGFP, which have useful photophysical properties, as well as superfolder GFP, which folds efficiently under adverse conditions. We previously generated msGFP, a monomeric superfolder derivative of EGFP. Unfortunately, compared to EGFP, msGFP and other superfolder GFP variants show faster photobleaching. We now describe msGFP2, which retains monomeric superfolder properties while being as photostable as EGFP. msGFP2 contains modified N‐ and C‐terminal peptides that are expected to reduce nonspecific interactions. Compared to EGFP and mEGFP, msGFP2 is less prone to disturbing the functions of certain partner proteins. For general‐purpose protein tagging, msGFP2 may be the best available derivative of A. victoria GFP.     

A new route to trihydroxamate-containing artificial siderophores and synthesis of a new fluorescent probe

A fluorescent labelled artificial siderophore 1 was synthesized by coupling a 7-nitrobenz-2-oxa-1,3-diazole (NBD) derivative to the terminal amino group of a new trihydroxamate-containing amine 2, a ferrichrome-type siderophore that was obtained from tris(hydroxymethyl)aminomethane. Compound 1 was shown to be a suitable tool for experiments on siderophore transport and uptake processes in various organisms cells and particularly in Candida albicans cells.     

1-Deoxynojirimycins with dansyl capped N-substituents as probes for Morbus Gaucher affected cell lines

Cyclization by double reductive amination of d-xylo-hexos-5-ulose with methyl 6-aminohexanoate gave (methoxycarbonyl)pentyl-1-deoxynojirimycin. Reaction of the terminal carboxylic acid with N-dansyl-1,6-diaminohexane provided the corresponding chain-extended fluorescent derivative. By reaction with bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Both compounds are strong inhibitors of d-glucosidases and could also be shown to distinctly improve, at sub-inhibitory concentrations, the activity of β-glucocerebrosidase in a Gaucher fibroblast (N370S) cell-line through chaperoning of the enzyme to the lysosome.     

Structure of pseudobactin 7SR1, a siderophore from a plant-deleterious Pseudomonas

When grown in iron-limiting culture medium, sugar beet deleterious Pseudomonas 7SR1 produced extra-cellularly the yellow-green, fluorescent siderophore pseudobactin 7SR1. Pseudobactin 7SR1 had a molecular formula of C46H63N13O23 and a molecular mass of 1166 g/mol. Pseudobactin 7SR1 contained a cyclic octapeptide with the amino acid sequence L-Ala-Gly-Ser-Ser-threo-beta-OH-Asp-Thr-Ser-N delta-OH-Orn. Since pseudobactin 7SR1 was not affected by nonspecific enzymes, it might contain D-amino acids. A yellow-green, fluorescent quinoline derivative is postulated to be attached via an ester bond to the serine residue following the glycine. A malamide group was attached to carbon 3 of the quinoline derivative. The three bidentate iron(III)-chelating groups consisted of an alpha-hydroxy acid group derived from beta-hydroxyaspartic acid, an omicron-dihydroxy aromatic group derived from the yellow-green, fluorescent chromophore, and a hydroxamate group derived from N delta-acetyl-N delta-hydroxyornithine. The chemical structure of pseudobactin 7SR1 is remarkably similar to that of pseudobactin, the siderophore of plant growth promoting Pseudomonas B10.     

New and more efficient multivalent glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes

New multi-valent, carbohydrate ligands that contain terminal N-acetylgalactosamine (GalNAc) or lactose (Lac) were prepared using a nitrilotriacetic acid (NTA) derivative of L-lysine as scaffold. Tri-valent structures were prepared by attaching an ω-amino glycoside of GalNAc or Lac to each of the three carboxyl groups of N(ε)-protected N(α)-dicarboxymethyl-L-lysine. In addition, a hexa-valent lactoside was synthesized by attaching N(ε)-deprotected trivalent lactoside to each of the carboxyl group of N(α)-(trifluoroacetamido)hexanoyl L-aspartic acid. Tri-valent GalNAc glycosides and the hexa-valent lactoside had high affinity (dissociation constants approaching nM) for rat hepatocytes. The hexa-valent lactoside, after de-N(ε)-protection, was modified with a chelator, diethylenetriaminepentaacetic acid (DTPA), through which a fluorescent or radioactive tag, such as europium or indium, can be firmly attached. Intravenous infusion of (111)Indium-tagged hexa-valent lactoside to rats and mice resulted in nearly exclusive accumulation of radioactivity in the liver.     

1-Deoxy-D-galactonojirimycins with dansyl capped N-substituents as β-galactosidase inhibitors and potential probes for GM1 gangliosidosis affected cell lines

Two simple and reliably accessible intermediates, N-carboxypentyl- and N-aminohexyl-1-deoxy-D-galactonojirimycin were employed for the synthesis of a set of terminally N-dansyl substituted derivatives. Reaction of the terminal carboxylic acid of N-carboxypentyl-1-deoxy-D-galactonojirimycin with N-dansyl-1,6-diaminohexane provided the chain-extended fluorescent derivative. Employing bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Partially protected N-aminohexyl-1-deoxy-D-galactonojirimycin served as intermediate for two additional chain-extended fluorescent 1-deoxy-D-galactonojirimycin (1-DGJ) derivatives featuring terminal dansyl groups in the N-alkyl substituent. These new compounds are strong inhibitors of d-galactosidases and may serve as leads en route to pharmacological chaperones for GM1-gangliosidosis.     

Structure of pseudobactin A214, a siderophore from a bean-deleterious Pseudomonas

Bean-deleterious Pseudomonas A214 produced the extracellular yellow-green, fluorescent siderophore [microbial iron(III) transport agent] pseudobactin A214 under iron-limiting conditions. Pseudobactin A214 has a molecular formula of C46H64N13O22 and a molecular mass of 1151 g/mol. Pseudobactin A214 contained an N-blocked linear octapeptide with the amino acid sequence Ser-Ala-Gly-Ser-Ala-threo-beta-OH-Asp-L-allo-Thr-N delta-OH-Orn with a yellow-green, fluorescent quinoline derivative attached via an amide bond to the amino terminus. A succinamide group was linked to carbon 3 of the quinoline derivative. Sequencing was accomplished by two-dimensional NMR spectroscopy and by Edman degradation of smaller peptides obtained from partial acid hydrolysis. Since pseudobactin A214 was not affected by nonspecific proteolytic enzymes, it might contain D-amino acids. The three bidentate iron-(III)-chelating groups consisted of a 1,2-dihydroxy aromatic group in the quinoline chromophore, an alpha-hydroxy acid group present as beta-hydroxyaspartic acid, and a hydroxamate group derived from N delta-acetyl-N delta-hydroxyornithine. The chemical structure of pseudobactin A214 is remarkably similar to those of pseudobactin and pseudobactin 7SR1, the siderophores of plant growth promoting and plant-deleterious Pseudomonas B10 and Pseudomonas 7SR1, respectively.     

A simple fluorometric method for cAMP: application to studies of brain adenylate cyclase activity

A simple fluorometric method for the determination of cAMP is presented. The fluorescent derivative is 1,N6-etheno cyclic 3,5-monophosphate (etheno-cAMP). Maximal formation of this derivative occurs after reacting cAMP with chloroacetaldehyde for 15 minutes at 100 degrees C. Fluorescent derivatives are also produced from compounds which contain a 6-amino purine. The specificity of the method resides in the use of a reverse phase/HPLC system. The derivatization as well as the fluorescent response of etheno-cAMP is linear between 2.5 and 700 picomoles of cAMP. Studies of brain adenylate cyclase by the fluorometric/HPLC method indicated that this method is comparable to the established radioenzymatic method. Thus, the present method provides a simple rapid nonradioactive means for the determination of adenylate cyclase activity.     

铽(Ⅲ)与人血清脱铁转铁蛋白结合的荧光光谱研究

在ｐＨ７．４０．１ｍｏｌ／ＬＨｅｐｅｓ及室温条件下,使用荧光光谱进行了Ｔｂ３＋对人血清脱铁转铁蛋白的滴定．结果表明Ｔｂ３＋与人血清脱铁转铁蛋白结合后,其５４９ｎｍ处的荧光强度增强约１０５倍．在５４９ｎｍ处Ｔｂ３＋－脱铁转铁蛋白络合物的摩尔荧光强度是（９．６５±０．０５）×１０４ｍｏｌ－１Ｌ,Ｔｂ３＋可占据脱铁转铁蛋白的两个金属离子结合部位,优先占据脱铁转铁蛋白的Ｃ端结合部位,条件平衡常数是ｌｇＫＣ＝９．９６±０．２０,ｌｇＫＮ＝６．３７±０．１６．Ｔｂ３＋与Ｒ３＋Ｅ（ＲＥ＝Ｎｄ、Ｓｍ、Ｅｕ和Ｇｄ）间的线性自由能关系表明稀土离子占据脱铁转铁蛋白的Ｃ端结合部位时受离子大小的影响     

Receptor-mediated endocytosis of tuftsin by macrophage cells

A fluorescent analog of the phagocytosis stimulating peptide tuftsin was prepared by coupling tetramethyl rhodamine isothiocyanate to a C-terminal elongated derivative of tuftsin. This analog, Thr-Lys-Pro-Arg-Gly-Lys(N epsilon-tetramethyl rhodamine)-OH, was used to visualize tuftsin receptors on mice macrophage cells by fluorescent image intensification. Fluorescent labelling was carried out at 37 degrees C, using a concentration of 200 nM and 2 microM of the fluorescent tuftsin derivative. The formation of peptide-receptor clusters and their subsequent internalization, as discerned by image intensification, were rapid processes, 5 min and 5-30 min, respectively. Preincubation of macrophages with tuftsin for various time intervals, followed by quantification of the tuftsin receptor using radiolabelled tuftsin, suggest that tuftsin receptors are initially increased in amount (5-7 min) and subsequently reduced (after 10-15 min) as judged by sites available for tritiated tuftsin. The binding studies are rather complementary to the fluorescence observations and support the assumption that the tuftsin receptor on the membrane of the mice macrophage cell is rapidly mobilized.     

Iminoalditol-amino acid hybrids: synthesis and evaluation as glycosidase inhibitors

Cyclization by double reductive amination of D-xylo-hexos-5-ulose with the terminal amino group of alpha-N-Boc-lysine methyl ester gave a 4:1-mixture of (1'R)-N-methoxycarbonyl-(1-N-Boc-amino)pentyl-1-deoxynojirimycin and the corresponding L-ido epimer whereas D-lyxo-hexos-5-ulose furnished the desired N-alkylated 1-deoxymannojirimycin derivative without any observable epimer formation at C-5. By subsequent modification of the lysine moiety, additional chain-extended derivatives as well as fluorescent compounds were obtained. All fluorescent iminoalditol-amino acid hybrids prepared in this study exhibited glycosidase inhibitory activities better than or comparable to the parent compounds'.     

A novel approach to the high-specific-activity labeling of small peptides with the technetium-99m fragment [99mTc(N)(PXP)]2+ (PXP = diphosphine ligand).

A new labeling approach for incorporating bioactive peptides into a technetium-99m coordination complex is described. This method exploits the chemical properties of the novel metal-nitrido fragment [99mTc(N)(PXP)]2+, composed of a terminal Tc[triple bond] N multiple bond bound to an ancillary diphosphine ligand (PXP). It will be shown that this basic, molecular building block easily forms in solution as the dichloride derivative [99mTc(N)(PXP)Cl2], and that this latter complex selectively reacts with monoanionic and dianionic, bidentate ligands (YZ) having soft, pi-donor coordinating atoms to afford asymmetrical nitrido heterocomplexes of the type [99mTc(N)(PXP)(YZ)]0/+ without removal of the basic motif [99mTc(N)(PXP)]2+. The reactions of the amino acid cysteine was studied in detail. It was found that cysteine readily coordinates to the metal fragment [99mTc(N)(PXP)]2+ either through the [NH2, S-] pair of donor atoms or, alternatively, through the [O-, S-] pair, to yield the corresponding asymmetrical complexes in very high specific activity. Thus, these results were conveniently employed to devise a new, efficient procedure for labeling short peptide sequences having a terminal cysteine group available for coordination to the [99mTc(N)(PXP)]2+ fragment. Examples of the application of this novel approach to the labeling of the short peptide ligand H-Arg-Gly-Asp-Cys-OH (H(2)1) and of the peptidomimetic derivative H-Cys-Val-2-Nal-Met-OH (H2) will be discussed.     

肽链及蛋白质N-末端喹喔啉类荧光衍生物的形成

蛋白质及肽链N-末端经Dixon转氨后的羰酰基与邻苯二胺作用可形成喹喔啉类荧光衍生物.形成这一发色团的过程较为缓慢;喹喔啉短肽的激发(Ex 293-305nm)与发射(Em 360-365nm)波长随肽链氨基酸残基组成不同而有一定变化;喹喔啉胰岛素荧光衍生物的激发光波长为Ex318nm,发射波长为Em 353nm.喹喔啉三肽的荧光在乙二醇溶液中的变化因其氨基酸残基组成的不同而有差异.以上结果提示:喹喔啉类荧光衍生物可能作为研究蛋白和肽结构与功能的一种手段.     

Synthesis and biological evaluation of a fluorescent analogue of folic acid

A fluorescein derivative of the lysine analogue of folic acid, N alpha-pteroyl-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (PLF), was synthesized as a probe for dihydrofolate reductase (DHFR) and a membrane folate binding protein (m-FBP). Excitation of PLF at 282 nm and at 497 nm gave a fluorescence emission maximum at 518 nm. Binding of PLF to human DHFR or human placental m-FBP results in approximately a 20-fold enhancement in the magnitude of the fluorescence emission, suggesting that the ligand interacts with a hydrophobic region on these proteins. Additional evidence suggests that an energy transfer may occur between the pteridine and the fluorescein moieties. PLF binds to the active site of human DHFR since methotrexate (MTX) competes stoichiometrically and the denatured enzyme in the presence of PLF did not exhibit fluorescent enhancement. The dissociation constant for the fluorescein derivative with respect to human DHFR is 115 nM as compared to 111 nM for folic acid. The Ki value for the competitive inhibition of human DHFR by the fluorescent analogue of folic acid is 2.0 microM compared to 0.48 microM for folic acid. PLF was reduced to N alpha-(7,8-dihydropteroyl)-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (H2PLF) and assayed by the enzymatic conversion to the tetrahydro derivative. The Km value for human DHFR for the dihydrofolate analogue is 2.0 microM. The KD value for H2PLF to human DHFR is 47 nM as compared to 44 nM for dihydrofolate. The KD values for both H2PLF and PLF indicate that the fluorescein moiety does not significantly affect folate binding in enzyme binary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

1-Deoxy-d-galactonojirimycins with dansyl capped N-substituents as β-galactosidase inhibitors and potential probes for GM1 gangliosidosis affected cell lines

Two simple and reliably accessible intermediates, N-carboxypentyl- and N-aminohexyl-1-deoxy-d-galactonojirimycin were employed for the synthesis of a set of terminally N-dansyl substituted derivatives. Reaction of the terminal carboxylic acid of N-carboxypentyl-1-deoxy-d-galactonojirimycin with N-dansyl-1,6-diaminohexane provided the chain-extended fluorescent derivative. Employing bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Partially protected N-aminohexyl-1-deoxy-d-galactonojirimycin served as intermediate for two additional chain-extended fluorescent 1-deoxy-d-galactonojirimycin (1-DGJ) derivatives featuring terminal dansyl groups in the N-alkyl substituent. These new compounds are strong inhibitors of d-galactosidases and may serve as leads en route to pharmacological chaperones for GM1-gangliosidosis.     

Syntheses and Biological Activities of Danicalipin A Derivatives

Synthesis of three derivatives of danicalipin A, tetrachloride, trisulfate and a fluorescent probe was achieved through Wittig reaction strategy. Toxicity of the derivatives against brine shrimp (Artemia salina) as also investigated to provide useful information for the biological activity; i) less chloride derivative showed similar toxicity to danicalipin A, ii) the amphiphilic property, a characteristic feature of danicalipin A, was crucial because trisulfate considerably decreased the toxicity and iii) fluorescent derivative kept brine shrimp toxicity of danicalipin A.     

Fluorescent derivatives of bile salts. II. Suitability of NBD-amino derivatives of bile salts for the study of biological transport.

Interaction of unconjugated and taurine-conjugated NBD-amino-dihydroxy-5 beta-cholan-24-oic acids bearing the fluorophor in the 3 alpha, 3 beta, 7 alpha, 7 beta, 12 alpha, or 12 beta position with albumin results in a small hypsochromic shift of the emission maximum and an increase in quantum yield, suggesting binding by hydrophobic interactions. The different unconjugated fluorescent bile salt derivatives are metabolized by intact rat liver in different ways. The unconjugated 3 beta-NBD-amino derivative is completely transformed to its taurine conjugate and secreted as such, whereas the 3 alpha-NBD-amino derivative is completely transformed to a polar fluorescent compound not identical with its taurine conjugate. The unconjugated 7 alpha- and 7 beta-NBD-amino derivatives are only partially conjugated with taurine and mainly secreted in unmetabolized form. The unconjugated 12 alpha- and 12 beta-NBD-amino derivatives are not at all transformed to their taurine conjugates, but are partially metabolized to unidentified compounds. They are predominantly secreted as the unmetabolized compounds. In contrast to the unconjugated derivatives, all taurine-conjugated fluorescent bile salt derivatives are secreted into bile unmetabolized. With the exception of the 3 alpha-compound, all synthesized taurine-conjugated fluorescent derivatives interfere with the secretion of cholyltaurine. Differential photoaffinity labeling studies using (7,7-azo-3 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2'-[2'-3H(N)]aminoethanesulfonate as a photolabile derivative revealed that in liver cells all fluorescent bile salt derivatives interact with the same polypeptides as the physiological bile salts. The hepatobiliary transport of taurine-conjugated NBD-amino bile salt derivatives is, due to hydrophobic interactions, accompanied by an increase in fluorescence intensity which is favorable for the study of biological bile salt transport by fluorescence microscopy.     

Fluorescent 2''(3'')-O-aminoacylnucleosides-acceptor substrates for ribosomal peptidyltransferase+.

2'(3')-O-L-Phenylalanylderivatives of fluorescent 1,N6-ethenoadenosine and 3,N4-ethenocytidine were prepared by chemical synthesis. Both compounds are good acceptor substrates in ribosomal peptidyltransferase reactions. Since these compounds cannot form Watson-Crick base pairs, the results indicate that the terminal aminoacyladenosine unit of AA-tRNA is bound to ribosomal protein on the acceptor site of peptidyltransferase and not to rRNA.     

Synthesis of N-4-butylamine acridone and its use as fluorescent probe for ctDNA

A novel N-4-butylamine acridone (BAA), which is an acridone derivative was synthesized and characterized by IR, MS and (1)H NMR. The fluorescent characteristics of BAA was investigated in detail and used as the fluorescent probe for detection of calf thymus DNA (ctDNA). It was found that DNA was able to quench the fluorescence of BAA at 426 nm with the excitation at 254 nm. Under optimal conditions, the corresponding linear response range was from 1.0 to 20.0mg/L and the limit of detections (LOD) was 0.020 mg/L (defined as S/N=3). Moreover, the interaction between BAA and ctDNA was investigated by fluorescence, absorption and viscosity measurements. The results suggested that the interaction between BAA and ctDNA is groove binding in nature.