Selective deuterium labeling of steroidal estrogens

Refluxing estrone ($\text{1}$) and equilenin ($\text{8}$) in methanol-OD under basic conditions places a deuterium atom at position 4 and 16, and for estradiol at position 4. The location of the label in ring A is confirmed by nmr examination of the aromatic protons. Milder procedures that can be used for labeling the $\text{ortho}$ and $\text{para}$ positions of phenol and the cresols were not successful among steroids. Using palladized charcoal and deuterium, the benzylic hydrogens of estrone can be exchanged. Following the same procedure, but using hydrogen for a back exchange, one deuterium atom remains at position 6 as shown by C¹³ nmr spectroscopy.     

Selective labeling of pre-synaptic receptors by 3H-dopamine, 3H-apomorphine and 3H-clonidine; labeling of post-synaptic sites by 3H-neuroleptics.

To test the hypothesis that nM concentrations of ³H-dopamine, ³H-apomorphine and ³H-clonidine prefer pre-synaptic sites, while ³H-neuroleptics and ³H-dihydroergocryptine prefer post-synaptic sites, we tested catecholaminergic agonists and antagonists on the binding of these radio-ligands to calf caudate tissue. 1) Dopamine agonists (apomorphine, NPA and bromocryptine) inhibited ³H-spiperone binding, but not ³H-dopamine binding, in direct correlation to their clinical potencies. 2) Dopamine agonists inhibited ³H-apomorphine binding at concentrations identical to those causing pre-synaptic cardio-inhibition. 3) The IC₅₀ values for ³H-dihydroergocryptine binding of alpha-adrenoceptor drugs did not correlate with the pre-synaptic IC₅₀ values for affecting noradrenaline release; those for ³H-clonidine did. The 3 findings are compatible with the working hypothesis.     

Facile syntheses of BODIPY derivatives for fluorescent labeling of the 3' and 5' ends of RNAs

As inexpensive and readily available fluorophores for 3′ and 5′ end labeling of RNA molecules, symmetrical BODIPY (boron dipyrromethene: 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) derivatives having a primary amino group were designed, and their facile synthetic route was established. Novel BODIPY derivatives exhibited photophysical properties comparable to commercially available BODIPY FL EDA (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine). To confirm utility of new derivatives, specific labeling of the 3′ and 5′ ends of in vitro transcribed RNAs was carried out. Furthermore, the 3′ end of the 5′ fragment of the bimolecular Tetrahymena ribozyme was labeled, and its catalytic activity was investigated.     

Oligonucleotides with bis-pyrene adduct in the backbone: syntheses and properties of intramolecular excimer forming probe.

2-(N-bis(2-pyrenylethyl)methyl-amino)ethanol (BPAE) was synthesized and its interaction with DNA was examined to explore the efficiency of intramolecular excimer forming probe. As a result the fluorescence of intramolecular excimer of BPAE disappeared with the addition of poly(dA)-poly(dT). This result suggests an unique type of probe with its introduction to oligonucleotide.     

Energy transfer cassettes for facile labeling of sequencing and PCR primers

Fluorescence energy transfer (ET) primers and terminators are the reagents of choice for multiplex DNA sequencing and analysis. We present here the design, synthesis and evaluation of a four-color set of ET cassettes, fluorescent labeling reagents that can be quantitatively coupled to a thiol-activated target through a disulfide exchange reaction. The ET cassette consists of a sugar-phosphate spacer with a FAM donor at the 3'-end, an acceptor linked to a modified T-base at the 5'-end of the spacer and a mixed disulfide for coupling to a thiol at the 5'-end. The acceptor dye emission intensities of ET labeled primers produced in this manner are comparable to commercial ET primers. The utility of our ET cassette-labeled primers is demonstrated by performing four-color capillary electrophoresis sequencing with the M13(-21)forward primer and by generating and analyzing a set of single-nucleotide-polymorphism-specific PCR amplicons.     

Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P?], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P? that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P? headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P? headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P?-binding proteins.     

Selective backbone labelling of ILV methyl labelled proteins

Adding the ¹³C labelled 2-keto-isovalerate and 2-oxobutanoate precursors to a minimal medium composed of ¹²C labelled glucose instead of the commonly used (²D, ¹³C) glucose leads not only to the ¹³C labelling of (I, L, V) methyls but also to the selective ¹³C labelling of the backbone C_α and CO carbons of the Ile and Val residues. As a result, the backbone (¹H, ¹⁵N) correlations of the Ile and Val residues and their next neighbours in the (i + 1) position can be selectively identified in HN(CA) and HN(CO) planes. The availability of a selective HSQC spectrum corresponding to the sole amide resonances of the Ile and Val residues allows connecting them to their corresponding methyls by the intra-residue NOE effect, and should therefore be applicable to larger systems.     

Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2-aminobutyl-1,3-propanediol backbone.

Novel CE-phosphoramidite (7a-e) and CPG (8a, c, d, e) reagents have been prepared from a unique 2-aminobutyl-1,3-propanediol backbone. The reagents have been used to directly label oligonucleotides with fluorescein, acridine, and biotin via automated DNA synthesis. The versatile 2-aminobutyl-1,3-propanediol backbone allows for labeling at any position (5', internal, and 3') during solid phase oligonucleotide synthesis. Multiple labels can be achieved by repetitive coupling cycles. Furthermore, the 3-carbon atom internucleotide phosphate distance is retained when inserted internally. Using this method, individual oligonucleotides possessing two and three different reporter molecules have been prepared.     

A facile method for the preparation of deuterium labeled salvinorin A: synthesis of [2,2,2-2H3]-salvinorin A

Salvinorin A is a novel hallucinogen isolated from the widely available leaves of Salvia divinorum. Based on its mechanism of action, salvinorin A has shown potential as a stimulant abuse therapeutic. However, there are no methods for the detection of salvinorin A or its metabolites in biological fluids. In order to begin developing salvinorin A as a potential therapeutic, an understanding of its metabolism is needed. Here, a straightforward synthesis of a deuterium labeled analog of salvinorin A and its utility as an internal standard for the detection of salvinorin A and its metabolites in biological fluids by LC-MS is described.     

Mechanism of urocanase as studied by deuterium isotope effects and labeling patterns

Nicotinamide adenine dinucleotide (NAD) dependent urocanase (4'-imidazolone-5'-propionate hydro-lyase, EC 4.2.1.49) from Pseudomonas putida was found to catalyze an exchange reaction between solvent and the 4'-hydrogen of urocanate or imidazolepropionate at a rate faster than that of overall deuterium was compared to unlabeled urocanate as a substrate, no isotope rate effect was noted. For examination of the possibility of an NAD+-mediated intramolecular hydride transfer of the 4'-hydrogen to a position on the side chain of oxoimidazolepropionate, the origins of hydrogen at positions 2 and 3 in the propionate chain were studied as a function of reaction time and extent of exchange of the 4'-hydrogen. No transfer of hydrogen from the 4' position to the side chain was observed, thereby eliminating mechanisms requiring hydride transfer via NADH between these positions. Catalytic rates in 1H2O vs. 2H2O revealed a 3-fold difference which was ascribed to a rate-limiting proton addition step. Similarly, a 5-fold decrease in Vmax was found for the reverse reaction when oxoimidazole[2,3-2H2]propionate was compared to unlabeled oxoimidazolepropionate. These data support a mechanism involving water addition across the conjugated double bond system of urocanate, rather than an internal oxidation--reduction process, yet NAD+ is required. A mechanism is proposed which uses electron delocalization in the imidazole nucleus, via an imidazole--NAD adduct, to facilitate water attack and subsequent formation of oxoimidazolepropionate.     

The effect of deuterium labeling on the genotoxicity of N-nitrosodimethylamine, epichlorohydrin and dimethyl sulfate

Deuterated and non-deuterated N-nitrosodimethylamine, epichlorohydrin and dimethyl sulfate were evaluated for the ability to induce DNA single-strand breaks in rat hepatocytes as measured by alkaline elution. Non-deuterated nitrosodimethylamine induced twice the amount of DNA-strand breaks as the deuterated form. No evidence of a deuterium isotope effect was seen for the direct-acting alkylating agents epichlorohydrin and dimethyl sulfate.     

The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data

Background  

The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange) experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues.     

Cassette labeling for facile construction of energy transfer fluorescent primers.

DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S6) formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attached to the 3' end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2-12-fold enhanced fluorescence intensity relative to that of the corresponding single dye-labeled primers. With single- stranded M13mp18DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.     

Oxygen-18 and deuterium labeling studies of choline oxidation by spinach and sugar beet

Chenopods synthesize betaine by a two-step oxidation of choline: choline → betaine aldehyde → betaine. The pathway is chloroplastic; the first step has been shown in isolated spinach (Spinacia oleracea L.) chloroplasts to be O₂- and light-dependent, the role of light being to provide reducing power (P Weigel, EA Weretilnyk, AD Hanson 1988 Plant Physiol 86: 54-60). Here, we report use of in vivo¹⁸O- and ²H-labeling in conjunction with fast atom bombardment mass spectrometry to test for two hypothetical choline-oxidizing reactions that would explain the observed requirements for O₂ and reductant: a desaturase or an oxygenase. Simple syntheses for ²H₃-choline, ²H₃, ¹⁸O-choline, and ²H₃, ¹⁸O-betaine are given. A desaturase mechanism was sought by giving choline deuterated at the 2-carbon, or choline unlabeled at this position together with ²H₂O and by analyzing newly synthesized betaine. About 15% of the ²H at C-2 was lost during oxidation of choline to betaine, and about 10% of the betaine made in the presence of 50% ²H₂O was monodeuterated. These small effects are more consistent with chemical exchange than with a desaturase, because 10 to 15% losses of ²H from the C-2 position also occurred if choline was converted to betaine by a purified bacterial choline oxidase. To test for an oxygenase, the incorporation of ¹⁸O from ¹⁸O₂ into newly synthesized betaine was compared with that from ¹⁸O-labeled choline, in light and darkness. Incorporation of ¹⁸O from ¹⁸O-choline was readily detectable and varied from about 15 to 50% of the theoretical maximum value; the ¹⁸O losses were attributable to exchange of the intermediate betaine aldehyde with water. In darkness, incorporation of ¹⁸O from ¹⁸O₂ approached that from ¹⁸O-choline, but in the light was severalfold lower, presumably due to isotopic dilution by photosynthetic ¹⁶O₂. These data indicate that the chloroplast choline-oxidizing enzyme is an oxygenase.     

Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine

In contrast to 5-methylcytosine (5-mC), which has been studied extensively, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types. Here we present a method for determining the genome-wide distribution of 5-hmC. We use the T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment and sequencing of 5-hmC-containing DNA fragments in mammalian genomes. Using this method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized. We also find a gene expression level-dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders.     

Selective chemical labeling of proteins in living cells

Labeling proteins with fluorophores, affinity labels or other chemically or optically active species is immensely useful for studying protein function in living cells or tissue. The use of genetically encoded green fluorescent protein and its variants has been particularly valuable in this regard. In an effort to increase the diversity of available protein labels, various efforts to append small molecules to selected proteins in vivo have been reported. This review discusses recent advances in selective, in vivo protein labeling based on small molecule ligand-receptor interactions, intein-mediated processes, and enzyme-catalyzed protein modifications.