Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins

An unnatural amino acid, 4‐(2‐azidoethoxy)‐L ‐phenylalanine (AePhe, 1 ), was designed and synthesized in three steps from known compounds in 54 % overall yield. The sensitivity of the IR absorption of the azide of AePhe was established by comparison of the frequency of the azide asymmetric stretch vibration in water and dimethyl sulfoxide. AePhe was successfully incorporated into superfolder green fluorescent protein (sfGFP) at the 133 and 149 sites by using the amber codon suppression method. The IR spectra of these sfGFP constructs indicated that the azide group at the 149 site was not fully solvated despite the location in sfGFP and the three‐atom linker between the azido group and the aromatic ring of AePhe. An X‐ray crystal structure of sfGFP‐149‐AePhe was solved at 1.45 Å resolution and provides an explanation for the IR data as the flexible linker adopts a conformation which partially buries the azide on the protein surface. Both sfGFP‐AePhe constructs efficiently undergo a bioorthogonal strain‐promoted click cycloaddition with a dibenzocyclooctyne derivative.     

Alpha-oximono-esters as precursors to heterocycles--generation of oxazinone N-oxides and cycloaddition to alkene dipolarophiles

Preparation of a series of terminally and internally substituted delta-alkenyl and delta-alkynyl esters 6, 7 and 9, potential precursors to oxazin-2-one nitrones, has been attempted. Condensation between pyruvic or benzoylformic acid and the appropriate alcohol proceeded smoothly in some cases whilst allylic transposition was a major feature in other cases--most especially during reactions with alpha-vinylbenzyl alcohol. Oximation of pyruvic acid derivatives furnished E-oxime isomers whilst benzoylformic acid derivatives afforded mixed geometrical isomers. The E-oxime of 4a1 carrying an internal Me group undergoes facile thermal cyclisation affording nitrones 1c and 1d in good yield. Oximes E-5a,b with a terminal methyl substituent on the alkene moiety furnish nitrone only under the influence of an external electrophile [PhSeBr/AgBF4]. A terminal Ph substituent on 5c,d prohibits formation of the cyclic dipole irrespective of reaction conditions, and whilst 5d reacts to afford a bicyclic isoxazolofuranone 13 by an IOOC reaction (intramolecular oxime olefin cyclisation) 5c remains thermally inert. Finally delta-alkynyl oximes 9c,d also failed to cyclise. The regio- and stereochemical characteristics of the cycloadditions between the new dipoles and electron poor olefinic dipolarophiles have been investigated. The conditions needed for reaction were rather forcing since the dipoles are somewhat stabilised by the adjacent alkoxycarbonyl group. All reactions proceeded regiospecifically to give adducts with 5-substituted isoxazolidine rings whilst diastereoselectivity varied with the choice of dipolarophile and the steric demands of the nitrone substituents. The phenylselenyl dipole 10a could not be trapped by any dipolarophiles bar dimethyl acetylenedicarboxylate.     

Vibrational stark effect probes for nucleic acids

The vibrational Stark effect (VSE) has proven to be an effective method for the study of electric fields in proteins via the use of infrared probes. To explore the use of VSE in nucleic acids, we investigated the Stark spectroscopy of nine structurally diverse nucleosides. These nucleosides contained nitrile or azide probes in positions that correspond to both the major and minor grooves of DNA. The nitrile probes showed better characteristics and exhibited absorption frequencies over a broad range; that is, from 2253 cm-1 for 2'-O-cyanoethyl ribonucleosides 8 and 9 to 2102 cm(-1) for a 13C-labeled 5-thiocyanatomethyl-2'-deoxyuridine 3c. The largest Stark tuning rate observed was |Deltamu| = 1.1 cm(-1)/(MV/cm) for both 5-cyano-2'-deoxyuridine 1 and N2-nitrile-2'-deoxyguanosine 7. The latter is a particularly attractive probe because of its high extinction coefficient (epsilon = 412 M-1cm-1) and ease of incorporation into oligomers.     

A direct comparison of azide and nitrile vibrational probes

The synthesis of 2'-azido-5-cyano-2'-deoxyuridine, N(3)CNdU (1), from trityl-protected 2'-amino-2'-deoxyuridine was accomplished in four steps with a 12.5% overall yield. The IR absorption positions and profiles of the azide and nitrile group of N(3)CNdU were investigated in 14 different solvents and water/DMSO solvent mixtures. The azide probe was superior to the nitrile probe in terms of its extinction coefficient, which is 2-4 times larger. However, the nitrile IR absorbance profile is generally less complicated by accidental Fermi resonance. The IR frequencies of both probes undergo a substantial red shift upon going from water to aprotic solvents such as THF or DMSO. DFT calculations supported the hypothesis that the molecular origin of the higher observed frequency in water is primarily due to hydrogen bonds between the probes and water molecules.     

Syntheses of silatranyl- and germatranyluridines

[reaction: see text] Silatranyluridine 1 and germatranyluridine 2 have been prepared in five steps from oxazolinouridine 3 in 27 and 29% yields, respectively. These compounds are novel transition-state analogues (TSAs) for RNA hydrolysis and offer a number of advantages over traditional vanadium- or rhenium-based TSAs. Germatrane 2 is completely stable in D(2)O at room temperature, and the half-life of silatrane 1 in D(2)O was found to be >7 days by (1)H NMR spectroscopy.     

Gas–liquid chromatography–tandem mass spectrometry methodology for the quantitation of estrogenic contaminants in bile of fish exposed to wastewater treatment works effluents and from wild populations

Fish can be exposed to a complex mixture of chemical contaminants arising from the exposure to wastewater treatment works (WwTWs) effluents. Some of these contaminants are estrogenic and have been associated with feminisation of male fish and the presence of populations containing intersex individuals. However the detection of trace levels (ng/L) of estrogenic chemicals surface waters can be difficult and does not give information on the exposure of aquatic organisms to these contaminants. In this study we assessed whether the analysis of estrogenic substances that bioconcentrate in fish bile can be used to detect the exposure of fish to feminising contaminants in receiving waters and effluents, and thus facilitate their monitoring of these substances in aquatic environments. Estrogenic metabolites in bile were deconjugated using enzymatic hydrolysis and partially purified by solid phase extraction. Steroidal and xenoestrogens were derivatized to their trimethylsilyl ethers and quantified by gas–liquid chromatography–mass spectrometry (GC–MS/MS) using multiple reaction monitoring. The method was validated using spiked bile samples from immature female rainbow trout (Oncorhynchus mykiss) as well as bile from sexually mature roach (Rutilus rutilus) that had been exposed to either tap water or an undiluted estrogenic effluent for 10 days or captured from a river site downstream of a WwTWs effluent discharge. The mean recovery of target analytes from spiked bile was between 86 and 99% and the limit of detection was between 0.1 and 0.7 ng/mL bile for bisphenol A (BPA), 17β-estradiol (E2), estrone (E1) and 17α-ethinylestradiol (EE2), and 11, 60 and 327 ng/mL bile for branched nonyl chain isomeric mixtures of 4-nonylphenolethoxylate (NP1EO), 4-nonylphenol (NP) and 4-nonylphenoldiethoxylate (NP2EO), respectively. All target analytes were detected in bile from roach exposed directly to a WwTWs effluent, with concentrations between 6–13 μg/mL bile for NP, 18–21 μg/mL for NP1EO, 75–135 μg/mL for NP2EO, 0.7–2.5 μg/mL for BPA, E2 and E1 and 17–29 ng/mL for EE2. With the exception of NP2EO, all analytes were detected in at least 2 out of the 5 fish sampled from the River Thames. BPA and NP1EO were detected in all three reference fish held in tap water indicating possible contamination from laboratory plastics. The work shows that analysis of 20–100 μL quantities of bile could be a useful approach in detecting exposure to mixtures of estrogenic contaminants taken up by fish from WwTW effluents and has the potential for monitoring the efficacy of remediation strategies that may be adopted for reduction of these endocrine disrupting chemicals in the aquatic environment.     

2D IR photon echo of azido-probes for biomolecular dynamics

The vibrations in the azido-, N(3), asymmetric stretching region of 2'-azido-2'-deoxyuridine (N(3)dU) are examined by two-dimensional infrared spectroscopy. In water and tetrahydrofuran (THF), the spectra display a single sharp diagonal peak that shows solvent sensitivity. The frequency-frequency correlation time in water is 1.5 ps, consistent with H-bond making and breaking dynamics. The 2D IR spectrum is reproduced for N(3)dU in water based on a model correlation function and known linear response functions. Its large extinction coefficient, vibrational frequency outside the protein and nucleic acid IR absorption, and sensitivity to water dynamics render -N(3) a very useful probe for 2D IR and other nonlinear IR studies: its signal is ca. 100 times that of nitriles.     

alpha-Keto amides as precursors to heterocycles--generation and cycloaddition reactions of piperazin-5-one nitrones

alpha-Keto amides 10a,b, formed from reaction of pyruvic or benzoylformic acid with allyl amine are found to present as single rotameric forms whilst their tertiary amido analogues 10c, d present as two rotamers in solution at rt. The hydroxyimino derivatives 8 share the conformational characteristics of their parents. The geometrical make-up of the new alpha-amidooximes is seen to depend on the structure of the starting acid and on the degree of substitution of the amido group. The oxime 8a derived from pyruvic acid and allyl amine is formed solely as the (E)-isomer whilst its tertiary amido analogue 8c is formed as both (E)- and (Z)-isomers. Oximes derived from benzoylformic acid have the opposite selectivity with both geometrical isomers forming from the secondary amide 8b and only the (Z)-isomer from the tertiary amide 8d. With the exception of 8b all oximes were configurationally stable with (Z)-isomers reacting to form isoxazolopyrrolidinones 11--compounds with a relatively rare bicyclic nucleus and (E)-isomers cyclising to piperazin-5-one nitrones 1--ketopiperazine N-oxides have to date only appeared once in the literature. New nitrones were trapped with phenyl vinyl sulfone, dimethyl acetylenedicarboxylate and methyl propiolate yielding isoxazolidine and isoxazoline fused piperazinones 13, 15, 21 and 22. Cycloadducts from dimethyl acetylenedicarboxylate and 8a, b are thermally labile and their rearrangement provides a novel route to pyrrolopiperazinones 16. The structure of a representative isoxazolopyrrolidinone, 11c, and a 2,3-dihydroisoxazoline fused piperazinone, 21b, are unambiguously solved following x-ray structural analysis.     

A vibrational probe for local nucleic acid environments: 5-cyano-2'-deoxyuridine

Nitriles have been shown to be effective vibrational probes of local environments in proteins but have yet to be fully utilized for the study of nucleic acids. The potential utility of 5-cyano-2'-deoxyuridine ( 1) as a probe of local nucleic acid environment was investigated by measuring the dependence of the IR nitrile stretching frequency (nu CN), line shape, and absorbance on solvent and temperature. The nu CN was found to be sensitive to solvent with an observed blue shift of 9.2 cm (-1) in going from THF to water. The dependence of the nitrile IR absorbance band was further investigated in water-THF mixtures. Global line shape analysis, difference FTIR spectroscopy, and singular value decomposition (SVD) were used to show the presence of three distinct local environments around the nitrile group of 1 in these mixtures. A modest blue shift in nu CN was observed upon a hydrogen-bond-mediated heterodimer formation between 2 (a silyl ether analogue of 1) and 2,6-diheptanamido-pyridine ( 3a) in chloroform. The intrinsic temperature dependence of the nu CN was found to be minimal and linear over the temperature range studied. The experimental studies were complemented by density functional theory (DFT) calculations on the dependence of the nitrile stretching frequency on solute-solvent interactions and upon heterodimer formation with model systems.