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1.
In this paper, poly(MMA-M12-BPMA)/TiO 2 composite particles were prepared by the copolymerization of a reactive surfactant sodium sulfopropyl-laurylmaleate (M12) and a reactive UV-stabilizer 2-hydroxy-4-(3-methacryloxy-2-hydroxylpropoxy) benzophenone (BPMA) with methyl methacrylate (MMA) in the presence of TiO 2. The structure and performance of composite particles were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray photoelectron spectroscopy, gel permeation chromatography, ultraviolet–visible absorption spectroscopy, differential scanning calorimeter, and scanning electron microscope.The measurement results indicate that the percentage of grafting and the grafting efficiency can reach 336.87% and 36.38%, respectively, and the glass transition temperature of poly(MMA-M12-BPMA)/TiO 2 composite particles is higher than that of poly(MMA-M12-BPMA); the size of the composite particles is about 130–200 nm. The poly(MMA-M12-BPMA) grafted from the surface of TiO 2 keeps the excellent characteristic of BPMA that possesses high absorbance of ultraviolet light, which is very important for improving UV-resistant performance of the polymethyl methacrylate. These research results are very useful for preparing polymethyl methacrylate with resistance to UV light. 相似文献
2.
Serum proteins associated with acute myocardial infarction (AMI) have been monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high resolution two-dimensional electrophoresis (2-DE) under nonreducing conditions. Proteins a, b, c (Mr 13,000; pI6.2, 6.7 and 7.5, respectively) and e(Mr27,000; pI5.2) appear simultaneously approximately 30 h after infarction, reach maximum intensity after 48 h and progressively decline thereafter. Protein d (Mr15,000; pI7-8.5; identified as hemoglobin) sometimes appears within 18 h of infarction. Proteins a-c are not detected in the 2-DE patterns of healthy myocardium, infarcted myocardium, pectoral muscle or tongue, but e is present in all and tentatively identified as myosin light chain. Other myocardial proteins which are either reduced in amount following infarction or more specifically associated with myocardium than pectoral muscle are not detected in the serum of AMI patients. Analysis of unconcentrated urine by SDS-PAGE and silver staining does not reveal proteins specific to AMI. 相似文献
3.
Iodinations of the ortho, meta, and para fluorous arenes (R(f8)CH(2)CH(2)CH(2))(2)C(6)H(4) (R(f8)=(CF(2))(7)CF(3)) with I(2)/H(5)IO(6) in AcOH/H(2)SO(4)/H(2)O give 3,4-(R(f8)CH(2)CH(2)CH(2))(2)C(6)H(3)I (5) and the analogous 2,4- (6) and 2,5- (7) isomers, respectively. Spectroscopic yields are >90 %, but 5 and 7 must be separated by chromatography from by-products (yields isolated: 70 %, 97 %, 61 %). Reaction of 1,3,5-(R(f8)CH(2)CH(2)CH(2))(3)C(6)H(3) with PhI(OAc)(2)/I(2) gives 2,4,6-(R(f8)CH(2)CH(2)CH(2))(3)C(6)H(2)I (8) on multigram scales in 97 % yield. The CF(3)C(6)F(11)/toluene partition coefficients of 5-8 (24 degrees C: 69.5:30.5 (5), 74.7:25.3 (6), 73.9:26.1 (7), 98.0:2.0 (8)) are lower than those of the precursors, but CF(3)C(6)F(11)/MeOH gives higher values (97.0:3.0 (5), 98.6:1.4 (6), 98.0:2.0 (7), >99.3:<0.3 (8)). Reactions of 5-8 with excess NaBO(3) in AcOH yield the corresponding ArI(OAc)(2) species 9-12 (9, 85 % as a 90:10 9/5 mixture; 10, 97 %; 11, 95 %; 12, 93 % as a 95:5 12/8 mixture). These rapidly oxidize 1,4-hydroquinones in MeOH. Subsequent additions of CF(3)C(6)F(11) give liquid biphase systems. Solvent removal from the CF(3)C(6)F(11) phases gives 5-8 in >99-98 % yields, and solvent removal from the MeOH phases gives the quinone products, normally in >99-95 % yields. The recovered compounds 5-8 are easily reoxidized to 9-12 and used again. 相似文献
4.
The proton-pumping H+,K+-adenosinetriphosphatase (H,K-ATPase), responsible for acid secretion by the gastric parietal cell, faces a harshly acidic environment, with some pepsin from neighboring chief cells, at its luminal surface. Its large catalytic alpha-subunit is mostly oriented cytoplasmically. The smaller beta-subunit (HKbeta), is mainly extracellular, with one transmembrane domain and a small cytoplasmic domain. Seven N-linked oligosaccharides in the extracellular domain of HKbeta are thought to contribute to protection of the H,K-ATPase, since previous work has shown that their complete removal, by peptide N-glycosidase F (PNGase F), greatly increased susceptibility of HKbeta to proteolysis. The possibility of graded protection by different numbers of oligosaccharides was investigated here with the use of mutant HKbeta cDNA, having various N-glycosylation sites mutated (Asn to Gln), transfected into HEK-293 cells. Membrane preparations, two days after transfection, were solubilized in 1% Triton X-100 and subjected to trypsinolysis (pH 8, 37 degrees C, trypsin:protein 1:10-1:25). Relative amounts of HKbeta remaining after 20 min trypsin were determined, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probing of Western blots with an antibody to the HKbeta extracellular domain, by chemiluminescent development of blots and densitometry of resulting films. Maturely glycosylated HKbeta was made significantly more susceptible to trypsin than wild type when at least five oligosaccharides were deleted, while the high-mannose form (pre-beta), from the endoplasmic reticulum, became significantly more susceptible than wild-type pre-beta with removal of only two or more oligosaccharides. For each mutant, and wild type, pre-beta was consistently more susceptible than the mature form. While the number, and kind, of oligosaccharides seem to affect protection for HKbeta against trypsinolysis, other aspects of protein maturation, including proper folding of peptide domains and possible subtle alterations of conformation during Golgi processing, are also likely to contribute to this protection. 相似文献
5.
The purification of uricase from Candida sp. was carried out by precipitation with ammonium sulfate then further proceeded with Sephadex G200, and DEAE-cellulose
DE52 chromatographies. The specific activity of the enzyme was enhanced from 0.05-12 (U/mg protein). The purity of the enzyme
was judged to be homogeneous by SDS-PAGE. Some of the general properties of enzyme were investigated. The optimum reaction
pH and temperature were 8.5 and 30‡C, respectively. The enzyme was stable at a pH range from 8.5-9.5 and at temperatures lower
than 35‡C. The apparent K
m
value of the enzyme was calculated to be about 5.26 x 10 -6 mol/L. The molecular weight was determined to be 70,000-76,000 by the gel filtration and SDS-PAGE techniques. The isoelectric
point was determined to be pH 5.6. The effects of some metallic ions on enzyme activity and stability were discussed. The
partial purified uricase was used in serum uric acid determination. The within-batch imprecision percentage ranged from 2.16-2.63
and the between-batch imprecision percentage ranged from 2.4-3.6. The recovery ratio were from 96–101%. The correlation among
this method and Boehringer, Roche, or Biotrol enzymatic kits were Y = 1.086x - 0.50 (r = 0.981), Ya = 0.959x - 0.29 (r = 0.97), and Yb = l.ll0x - 0.45 (r = 0.956), respectively. A linear calibration curve was obtained at 2.5-15 mg/dL uric acid. The stability of reagents and the
effects of some substances in serum were also surveyed. 相似文献
6.
The response criteria for complete remission (CR) in acute myeloid leukemia (AML) are currently based on morphology and blood cell counts. However, these criteria are insufficient to establish a diagnosis in cases with poor quality bone marrow (BM) samples demonstrating a loss of cellular morphology. We investigated whether the sera of patients contained biomarkers that indicate disease response status. First, we performed multidimensional liquid chromatography-differential gel electrophoresis (MDLC-DIGE) to generate protein profiles of two pooled, paired serum samples from patients who had achieved CR; one collected at diagnosis (PreCR) and the other collected after chemotherapy (CR). Then, with the biomarker candidates found, ELISA was carried out for individual PreCR and CR samples, and for other verification sets including nonremission (NR) patients and normal samples. We selected two proteins, complement factor H (CFH) and apolipoprotein H (ApoH), with dye (Cy) ratios showing greater than 2.0-fold differences between the pooled samples. ELISA showed that CFH and ApoH are useful for distinguishing between the recovered (CR and normal) and nonrecovered (PreCR, PreNR, and NR) states in AML (p <0.001). We successfully applied a protein profiling technology of MDLC-DIGE and LC-MS/MS to discover two biomarkers for CR which needs further validation for a clinical setting. 相似文献
7.
New methacrylate based monomers 2-(4-benzoylphenoxy)-2-oxoethyl-2-methylacrylate (BOEMA), 2-(4-acetylphenoxy)-2-oxoethyl-2-methylacrylate (AOEMA), and 2-[(4-fluorophenyl)amino]-2-oxoethyl-2-methylacrylate (FPAMA), were synthesized first time. The free-radical-initiated copolymerization of AOEMA and BOEMA with FPAMA were carried out in 1,4-dioxane solution at 65 °C using 2,2′-azobisisobutyronitrile (AIBN) as an initiator with different monomer-to-monomer ratios in the feed. The monomers and copolymers were characterized by FTIR, 1H NMR and 13C NMR spectral studies. The copolymer compositions were evaluated by nitrogen content in polymers. The reactivity ratios of the monomers were determined by the application of Fineman-Ross and Kelen-Tudos methods. The analysis of reactivity ratios revealed that BOEMA and AOEMA are less reactive than FPAMA, and copolymers formed are statistically in nature. The molecular weights ( and ) and polydispersity index of the polymers were determined using gel permeation chromatography. Thermogravimetric analysis of the polymers reveals that the thermal stability of the copolymers increases with an increase in the mole fraction of FPAMA in the copolymers. Glass transition temperatures of the copolymers were found to decrease with an increase in the mole fraction of FPAMA in the copolymers. The prepared homo and copolymers were tested for their antimicrobial activity against bacteria, fungi and yeast. 相似文献
8.
Molecular imprinting is an attractive biomimetic approach that creates specific recognition sites for the shape and functional group arrangement to template molecules. The purpose of this study is to prepare cytochrome c-imprinted poly(hydroxyethyl methacrylate) (PHEMA)-based supermacroporous cryogel which can be used for the separation of cytochrome c from protein mixtures. N-Methacryloyl-(L)-histidinemethylester (MAH) was used as the metal-coordinating monomer. In the first step, Cu(2+) was complexed with MAH, and the cytochrome c imprinted PHEMA (MIP) cryogel was prepared by free radical cryopolymerization initiated by N,N,N',N'-tetramethylene diamine at -12°C. After polymerization is completed, the template cytochrome c molecules were removed from the MIP cryogel using 0.5 M NaCl solution. The maximum cytochrome c binding amount was 126 mg/g polymer. Selective binding studies were performed in the presence of lysozyme and bovine serum albumin. The relative selectivity coefficients of MIP cryogel for cytochrome c/lysozyme and cytochrome c/bovine serum albumin were 1.7 and 5.2 times greater than those of the non-imprinted PHEMA cryogel, respectively. The selectivity of MIP cryogel for cytochrome c was also confirmed with fast protein liquid chromatography. The MIP cryogel could be used many times with no remarkable decrease in cytochrome c binding capacity. 相似文献
9.
Poly(2-aminoethyl methacrylate) (PAMA) has a p Ka of approximately 7.6 and is chemically stable in acidic or neutral aqueous solution in its protonated form. However, chemical degradation of PAMA is known to occur in alkaline media as its primary amine groups become deprotonated (He L et al. Macromolecules 2007; 40: 4429-38). In the present work, the effect of temperature, pH and polymer concentration on the rate of PAMA degradation in dilute aqueous solution has been examined. 1H NMR spectroscopy indicates that both elimination of 2-aminoethanol and formation of 2-hydroxyethyl methacrylamide repeat units occur above pH 9; elimination is observed first and occurs to a greater extent. FT-IR studies of aqueous PAMA solutions aged at pH 12 and 50 °C confirm the presence of anionic carboxylate groups, which suggests that such elimination is simply due to ester hydrolysis. A control experiment suggests that methacrylamide formation occurs via internal rearrangement, rather than by amidation of the remaining ester groups by the eliminated 2-aminoethanol. 相似文献
10.
The reactivity of a range of Keggin and Wells–Dawson type heteropolyacids (HPAs): H 3PW 12O 40 H 4SiW 12O 40, H 3PMo 12O 40, K 6P 2W 18O 62, and NaH 2W 12O 4, towards the heavily glycosylated α-1-acid glycoprotein (AGP) is reported. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) show that after incubation of the protein with HPAs at 80 °C and pH 2.8 complete hydrolysis of terminal glycosidic bond has been achieved, resulting in the removal of sialic acids with no observed destruction of the protein core or the residual glycan chains. The 1H NMR spectroscopy confirmed that the released sialic acids preserve intact structure upon their excision from the protein, which makes the reported method suitable for the analysis of sialic acid modifications which play an important role in numerous biological processes. The presence of other sugars was not detected by 1H NMR and HPAEC-PAD, suggesting that HPAs hydrolyze only the terminal glycosidic bond in the glycoprotein, resulting in the selective release of sialic acid from AGP. The kinetic results have shown that under equal temperature and pH conditions, the hydrolysis of the terminal glucosidic bond occurred faster in the presence of HPAs compared to conventional mineral acids. The observed rate constants were in the range 6,7×10 −2 −11,9×10 −2 min −1 and the complete and selective excision of sialic acids could be achieved within 60 min of incubation. The Trp fluorescence and CD spectroscopy show that non-covalent interaction between HPA and protein takes place in solution which could lead to stabilization of the sialosyl cation that is formed during the glycosidic bond hydrolysis by anionic HPA cluster. 相似文献
11.
Nitrogen trifluoride, NF(3), a trace gas of purely anthropogenic origin with a large global warming potential is accumulating in the Earth's atmosphere. Large uncertainties are however associated with its atmospheric removal rate. In this work, experimental and theoretical kinetic tools were used to study the reactions of NF(3) with three of the principal gas-phase atmospheric oxidants: O((1)D), OH and O(3). For reaction (R2) with O((1)D), rate coefficients of k(2)(212-356 K) = (2.0 ± 0.3) × 10(-11) cm(3) molecule(-1) s(-1) were obtained in direct competitive kinetics experiments, and experimental and theoretical evidence was obtained for F-atom product formation. These results indicate that whilst photolysis in the stratosphere remains the principal fate of NF(3), reaction with O((1)D) is significant and was previously underestimated in atmospheric lifetime calculations. Experimental evidence of F-atom production from 248 nm photolysis of NF(3) was also obtained, indicating that quantum yields for NF(3) destruction remain significant throughout the UV. No evidence was found for reaction (R3) of NF(3) with OH indicating that this process makes little or no contribution to NF(3) removal from the atmosphere. An upper-limit of k(3)(298 K) < 4 × 10(-16) cm(3) molecule(-1) s(-1) was obtained experimentally; theoretical analysis suggests that the true rate coefficient is more than ten orders of magnitude smaller. An upper-limit of k(4)(296 K) < 3 × 10(-25) cm(3) molecule(-1) s(-1) was obtained in experiments to investigate O(3) + NF(3) (R4). Altogether these results underpin calculations of a long (several hundred year) lifetime for NF(3). In the course of this work rate coefficients (in units of 10(-11) cm(3) molecule(-1) s(-1)) for removal of O((1)D) by n-C(5)H(12), k(6) = (50 ± 5) and by N(2), k(7) = (3.1 ± 0.2) were obtained. Uncertainties quoted throughout are 2σ precision only. 相似文献
12.
In this work, small-angle neutron scattering (SANS) is used to probe the structural transformations that accompany temperature-induced gelation of emulsions stabilized by a temperature-responsive polymer. The latter is poly(NIPAM-co-PEGMa) (N-isopropylacrylamide and poly(ethyleneglycol) methacrylate) and contains 86 mol% NIPAM. Turbidity measurements revealed that poly(NIPAM-co-PEGMa) has a lower critical solution temperature (T(LCST)) of 36.5 degrees C in D(2)O. Aqueous polymer solutions were used to prepare perfluorodecalin-in-water emulsions (average droplet size of 6.9 mum). These emulsions formed gels at 50 degrees C. SANS measurements were performed on the poly(NIPAM-co-PEGMa) solutions and emulsions as a function of temperature. The emulsion was also prepared using a D2O/H2O mixture containing 72 vol% D2O in order to make scattering from the droplets negligible (on-contrast). The SANS data were analyzed using a combination of Porod and Ornstein-Zernike form factors. The results showed that the correlation length (xi) of the polymer scaled as xi approximately phi(p)(-0.68) at 32 degrees C, where phi(p) is the polymer volume fraction. The xi value increased for all systems as the temperature increased, which was attributed to a spinodal transition. At temperatures greater than T(LCST), the polymer solution changed to a polymer dispersion of poly(NIPAM-co-PEGMa) aggregates. The aggregates have features that are similar to microgel particles. The average size of these particles was estimated as 160-170 nm. The particles are "sticky" and are gel-forming. The on-contrast experiments performed using the emulsion indicated that the interfacial polymer chains condensed to give a relatively thick polymer layer at the perfluorodecalin-water interface at 50 degrees C. The gelled emulsions appear to consist of perfluorodecalin droplets with an encapsulating layer of collapsed polymer to which sticky microgel particles are adsorbed. The latter act as a "glue" between coated droplets in the emulsion gel. 相似文献
13.
The nickel(II) hexaazamacrocyclic complex (1) containing pendant pyridine groups has been synthesized by the one-pot template condensation reaction of amine and formaldehyde. From the self-assembly of 1 with deprotonated cis,cis-1,3,5-cyclohexanetricarboxylic acid, H2CTC- and CTC3-, three-dimensional supramolecular open-frameworks of [Ni(C20H32N8)][C6H9(COOH)2(COO)]2 x 4H2O (2) and [Ni(C20H32N8)]3[C6H9(COO)3]2 x 16H2O (3), respectively, have been constructed. The solids 2 and 3 are insoluble in all solvents. X-ray crystal structure of 2 indicates that each nickel(II) macrocyclic complex binds two H2CTC- ions in trans position and two pendant pyridine groups of the macrocyclic complex are involved in hydrogen-bonding interactions with the hydroxy groups of H2CTC- belonging to the neighboring macrocyclic complexes, which provides the beltlike one-dimensional chain composed of rectangular synthons. The one-dimensional chains are linked together through lattice water molecules by the hydrogen-bonding interactions to generate two-dimensional networks, which are again connected to each other by the offset pi-pi stacking interactions between the pendant pyridine rings to give rise to a three-dimensional structure in which channels are present. The X-ray crystal structure of 3 indicates that each nickel(II) macrocyclic unit binds two CTC3- ions in trans position and each CTC3- ion coordinates three nickel(II) macrocyclic complexes to form a two-dimensional layer, in which pendant pyridine rings are involved in the hydrogen bonding and the herringbone pi-pi interaction. Between the layers, the pendant pyridine rings belonging to the neighboring layers participate in the offset pi-pi stacking interactions, which gives rise to a three-dimensional network structure. The network creates channels running parallel to the a, b, and c axes, which are filled with guest water molecules. The X-ray powder diffraction patterns indicate that the frameworks of 2 and 3 are deformed upon removal of water guests but restored upon rebinding of water. The host solids 2 and 3 bind [Cu(NH3)4](ClO4)2 in MeCN with a binding constant (Kf) of 210 M(-1) and 710 M(-1), respectively, while they do not bind [Cu(en)2](ClO4)2 (en = ethylenediamine). The dried solids of 2 and 3 do not interact with benzene and toluene, but they differentiate methanol, ethanol, and phenol in toluene solvent with the Kf values of 42, 14, and 12 M(-1), respectively, for 2, and 13, 8.2, and 8.9 M(-1), respectively, for 3. In terms of binding sites for guest molecules, the solid 3 has greater capacity than the solid 2. 相似文献
14.
A novel “on–off” Al3+ ions fluorescence-enhanced sensor (E)-1-(((2-hydroxyphenyl) imino)methyl)naphthalen-2-ol (AH-2) and its hydrogel hybrid (PAMN) were synthesized. AH-2 showed excellent selectivity and ultrasensitive to Al3+ ions; the detection limit was 2.36?×?10–9 M. The most plausible complexation mechanism was studied by 1H NMR, FT-IR, HR-MS, Job’s plot and theoretical calculation. And, it was interesting that PAMN could adsorb Al3+ ions with a removal rate of over 99%, which also could easily be distinguished by the naked eye in UV lamp (365 nm). Before and after adsorption of Al3+ ions, the microstructures of PAMN were analyzed by scanning electron microscope and X-ray energy spectrometer. The silica gel detect plates prepared in this work could rapidly and conveniently detect Al3+ ions with a concentration greater than 5?×?10–6 M (0.13 mg/L) in aqueous solution, and the detection concentration (0.13 mg/L) was lower than the national standard concentration of Al3+ ions (0.2 mg/L) in city tap water of china. 相似文献
15.
Abstract— Growing wheat seedlings in the presence of BASF 13.338 [4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone], a PS II inhibitor of the pyridazinone group, brought about notable changes in the structure and functioning of photosynthetic apparatus. In BASF 13.338 treated plants, there was a decrease in the ratio of Chi a/Chl b, an increase in xanthophyll/carotene ratio and an increase in the content of Cyt b 559 (HP + LP). Chl/p700 ratio increased when measured with the isolated chloroplasts but not with the isolated PS I particles of the treated plants. The SDS-PAGE pattern of chloroplast preparations showed an increase in the CPII/CP I ratio. The F685/F740 ratio in the emission spectrum of chloroplasts at -196°C increased. The difference absorption spectrum of chloroplasts between the control and the treated plants showed a relative increase of a chlorophyll component with a peak absorption at 676 nm and a relative decrease of a chlorophyll component with a peak absorption at 692 nm for the treated plants. The excitation spectra of these chloroplast preparations were similar. Chloroplasts from the treated plants exhibited a greater degree of grana stacking as measured by the chlorophyll content in the 10 K pellet. The rate of electron transfer through photosystem II at saturating light intensity in chloroplast thylakoids isolated from the treated plants increased (by 50%) optimally at treatment of 125 μ M BASF 13.338 as compared to the control. This increase was accompanied by an increase in (a) I50 value of DCMU inhibition of photosystem II electron transfer; (b) the relative quantum yield of photosystem II electron transfer; (c) the magnitude of C550 absorbance change; and (d) the rate of carotenoid photobleaching. These observations were interpreted in terms of preferential synthesis of photosystem II in the treated plants. The rate of electron transfer through photosystems I and through the whole chain (H 2O → methyl viologen) also increased, due to an additional effect of BASF 13.338, namely, an increase in the rate of electron transfer through the rate limiting step (between plastoquinol and cytochrome f). This was linked to an enhanced level of functional cytochrome f. The increase in the overall rate of electron transfer occurred in spite of a decrease in the content of photosystem I relative to photosystem II. Treatment with higher concentrations (> 125 μ M) of BASF 13.338 caused a further increase in the level of cytochrome f, but the rate of electron transfer was no greater than in the control. This was due to an inhibition of electron transfer at several sites in the chain. 相似文献
16.
In time-of-flight secondary ion mass spectrometry (ToF-SIMS), the choice of primary ion used for analysis can influence the resulting mass spectrum. This is because different primary ion types can produce different fragmentation pathways. In this study, analysis of single-component protein monolayers were performed using monatomic, tri-atomic, and polyatomic primary ion sources. Eight primary ions (Cs(+), Au(+), Au(3) (+), Bi(+), Bi(3) (+), Bi(3) (++), C(60) (+)) were used to examine to the low mass (m/z < 200) fragmentation patterns from five different proteins (bovine serum albumin, bovine serum fibrinogen, bovine immunoglobulin G and chicken egg white lysozyme) adsorbed onto mica surfaces. Principal component analysis (PCA) processing of the ToF-SIMS data showed that variation in peak intensity caused by the primary ions was greater than differences in protein composition. The spectra generated by Cs(+), Au(+) and Bi(+) primary ions were similar, but the spectra generated by monatomic, tri-atomic and polyatomic primary ion ions varied significantly. C(60) primary ions increased fragmentation of the adsorbed proteins in the m/z < 200 region, resulting in more intense low m/z peaks. Thus, comparison of data obtained by one primary ion species with that obtained by another primary ion species should be done with caution. However, for the spectra generated using a given primary ion beam, discrimination between the spectra of different proteins followed similar trends. Therefore, a PCA model of proteins created with a given ion source should only be applied to datasets obtained using the same ion source. The type of information obtained from PCA depended on the peak set used. When only amino acid peaks were used, PCA was able to identify the relationship between proteins by their amino acid composition. When all peaks from m/z 12-200 were used, PCA separated proteins based on a ratio of C(4)H(8)N(+) to K(+) peak intensities. This ratio correlated with the thickness of the protein films and Bi(1) (+) primary ions produced the most surface sensitive spectra. 相似文献
17.
Site-selective fluorination of aliphatic C–H bonds remains synthetically challenging. While directed C–H fluorination represents the most promising approach, the limited work conducted to date has enabled just a few functional groups as the arbiters of direction. Leveraging insights gained from both computations and experimentation, we enabled the use of the ubiquitous amine functional group as a handle for the directed C–H fluorination of Csp 3–H bonds. By converting primary amines to adamantoyl-based fluoroamides, site-selective C–H fluorination proceeds under the influence of a simple iron catalyst in 20 minutes. Computational studies revealed a unique reaction coordinate for the catalytic process and offer an explanation for the high site selectivity.By converting primary amines to adamantoyl-based fluoroamides, site-selective C–H fluorination proceeds under the influence of a simple iron catalyst in 20 minutes. Due to the pervasiveness of fluorine atoms in industrially relevant small molecules, all practicing organic chemists appreciate the importance of this element. As a result of its unusual size and electronegativity, fluorine imparts unique physicochemical properties to pendant organic molecules. 1 For example, the strong C–F bond can prevent biological oxidation pathways, thereby thwarting rapid clearance and potentially improving pharmacokinetics of molecules. 2 Moreover, the installation of fluorine or trifluoromethyl groups, with their strong inductive effects, 2 can have a profound effect on the p Ka of nearby hydrogen atoms. 3 These attributes, among others, have solidified the importance of fluorinated molecules in the medicinal, 1–4 material, 5 and agrochemical 6 industries. Yet, the same unique properties that make fluorine atoms attractive chemical modifiers also make their installation difficult. Consequently, new methods for site-selective fluorine incorporation remain highly desirable. 7Methods to construct Csp 2–F bonds traditionally make use of the Balz–Schiemann fluorodediazonization 8 and halogen exchange (“Halex” process). 9 Advances in transition metal-mediated fluorination have broadened access to Csp 2–F-containing molecules, 10 but methods to access aliphatic fluorides remain limited. Conventional methods to make Csp 3–F bonds—such as nucleophilic displacement of alkyl halides 11 and deoxyfluorination 12—can have limited functional group compatibility and unwanted side reactions. A more efficient route to form aliphatic C–F bonds would target the direct fluorination of Csp 3–H bonds ( Scheme 1). 13Open in a separate window(a) Previous work on functional-group directed Csp 3–H fluorination; (b) our approach to N-directed fluorination.Recent efforts with palladium catalysis employ conventional C–H-metallation strategies to target Csp 3–H bonds for fluorination. 14 Alternatively, radical H-atom abstraction can remove the transition metal from the C–H-cleavage step, thereby offering a promising approach for Csp 3–H-bond functionalization. 15 With undirected C–H fluorination, 16 however, selectivity remains a challenge in molecules without strength-differentiated Csp 3–H bonds. 17 To overcome this, our group pioneered the directed fluorination of benzylic Csp 3–H bonds through an iron-catalyzed process that involves 1,5 hydrogen-atom transfer (HAT) to cleave the desired Csp 3–H bond. 18 Since this work, other groups have demonstrated directed Csp 3–H fluorination based on radical propagation that proceeds through an interrupted Hofmann–Löffler–Freytag (HLF) 19 reaction ( Scheme 1a). These examples employ various radical precursors such as enones, 20 ketones, 21 hydroperoxides, 22 and carboxamides 23 to direct fluorination to specific Csp 3–H bonds. Since amines are ubiquitous in natural products and drugs, we sought to use amines as the building block of our directing group to achieve fluorination of unactivated Csp 3–H bonds ( Scheme 1b). By using amines as the starting point, one could use the approach in straightforward synthetic planning for the late-stage functionalization of remote C–H bonds.In the design phase of the project, we needed to devise a synthetically tractable N–F system that would enable 1,5-HAT and allow for fluorine transfer ( Scheme 1b). To begin, we decided to examine common amine activating groups that would support 1,5-HAT while avoiding undesired radical reactions. The chosen activating group would provide the ideal steric and electronic properties to enable both N–F synthesis and N–F scission for 1,5-HAT. We first examined common acyl groups ( e.g., acetyl-, benzoyl, and tosyl-based amides), but these proved unsatisfactory. For example, fluoroamide synthesis was either not achieved or low yielding, and the desired fluorine transfer proceeded with significant side reactions or returned starting material. We then turned our attention to more sterically hindered amides—which allow for higher yielding fluoroamide synthesis. For fluorine transfer, we hypothesized that the increased steric bulk could slow intermolecular H-atom transfer, thereby leading more efficient intramolecular 1,5-HAT. To that end, we were delighted that pivaloyl-based fluoroamide 1a proceeded in 64% yield to form product 2a ( Scheme 2a). Interestingly, 7% of 1a underwent fluorination at the tert-butyl group of the pivaloyl—presumably through a 1,4-HAT reaction (2aa, Scheme 2a). 24 The problem is further exacerbated when the pivaloyl group is homologated by one methylene—providing only 7% yield of desired 2b with 32% of the fluorination taking place on the iso-pentyl group (2bb, Scheme 2a). In an attempt to “tie back” the pivaloyl group and prevent the undesired fluorination, we employed a cyclopropylmethyl-based fluoroamide but observed no improvement. Open in a separate window(a) The targeted 1,5-fluorination of unactivated aliphatic C–H bonds results in partial fluorination of the amine activating group; (b) DFT studies (uM06/cc-pVTZ(-f)-LACV3P**//uM06/LACVP** level of theory) identified the competing pathways responsible for alternate fluorination; (c) DFT (uM06/cc-pVTZ(-f)-LACV3P**//uM06/LACVP** level of theory) evaluation of adamantoylamides revealed higher transition state energy for 1,4-HAT due to restricted vibrational scissoring (d) adamantoyl-activated octylamine shows no fluorination of the activating group. a 1H-NMR yield using 1,3,5-trimethoxybenzene as an internal standard. b 19F-NMR yield using 4-fluorotoluene as an internal standard.At this point, 1a proved most promising for efficient fluorine transfer, as well as being the most synthetically accessible fluoroamide. The increased steric hindrance minimizes N-sulfonylation during fluorination with NFSI, a problem that plagued the synthesis of our previously targeted fluoroamides. 18 Therefore, to further investigate how to improve fluorine transfer from 1a, we decided to model H-abstraction computationally.We hypothesized that the fluorinated side product 2aa was formed after 1,4-HAT. Since 1,4-HAT is rare, 24 we employed DFT (see ESI † for details) to calculate the 5-membered and 6-memebered transition-states for 1,4- and 1,5-HAT, respectively. Surprisingly, we found that the barrier for 1,4 C–H abstraction in 1a was 18.7 kcal mol −1, which was only 2.6 kcal mol −1 higher in energy than the barrier calculated for 1,5 C–H abstraction in the same system ( Scheme 2b). This suggested that both processes were competing at room temperature. We attributed the comparable barriers to the flexibility of the tert-butyl group, which undergoes vibrational scissoring to accommodate the C–H abstraction. The transition state distortion is modest and allows the molecule to maintain bond angles close to the ideal 109.5° ( Scheme 2b). Based on this insight, we sought to limit the scissoring of the tert-butyl group and prevent the 1,4-HAT that leads to the undesired side product. After investigating several possible candidates, the underutilized adamantoyl group appeared promising. To evaluate the rigidity of adamantane, we calculated the barriers for 1,4- and 1,5-HAT for the adamantoyl-capped octylamine 1c ( Scheme 2c). As expected, the barriers for 1,4- and 1,5-HAT differed significantly—with 1,4 C–H abstraction proceeding with a barrier of 25.1 kcal mol −1 and the 1,5-HAT barely changed at 16.4 kcal mol −1—an 8.7 kcal mol −1 difference. Consequently, we synthesized 1c and subjected it to the reaction conditions. Excitingly, the adamantoyl-capped system produced desired product 2c in 75% yield with no fluorination of the adamantyl group ( Scheme 2d).Using the newly devised adamantoyl-based fluoroamides, the reaction conditions were optimized. While a range of metal salts, ligands, and radical initiators were evaluated, Fe(OTf) 2 proved unique in catalyzing fluorine transfer with fluoroamides. 18 Catalyst loading of 10 mol% allowed convenient setup and minor deviations above or below this loading had little effect on yield (see ESI †). Increasing the temperature to 40 °C produced a slight increase in yield (entry 2, ). Likewise, raising the temperature to 80 °C resulted in full conversion of the starting material in 20 minutes with 81% yield of the desired product (entry 3, ). It should be noted that fluorine transfer occurs efficiently at a variety of temperatures with adjustments in reaction time (see ESI †). Increasing the reaction concentration or changing the solvent resulted in decreased yield (entries 4 and 5, ). Furthermore, the absence of Fe(OTf) 2 leads to no reaction and quantitative recovery of starting material, attesting to the stability of fluoroamides and the effectiveness of Fe(OTf) 2 (entry 6, ).Optimization of pertinent reaction parameters |
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Entry | Solvent | Temp (°C) | Conc (M) | Time | Yielda (%) |
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1b | DME | rt | 0.05 | 15 h | 75 | 2 | DME | 40 | 0.05 | 18 h | 79 | 3 | DME | 80 | 0.05 | 20 min | 81 | 4 | DME | 80 | 0.1 | 20 min | 73 | 5 | THE | 80 | 0.05 | 20 min | 38 | 6c | DME | 80 | 0.05 | 20 min | 0 | Open in a separate windowaDetermined by 1H-NMR with 1,3,5-trimethoxybenzene as an internal standard. bReaction ran inside of glovebox. cReaction ran without Fe(OTf) 2.With the optimized conditions established, we evaluated the substrate scope of the reaction (). The reaction proved quite general for the fluorination of primary and secondary Csp 3–H bonds (2c–l, ), while tertiary Csp 3–H abstraction led to greater side reactions and lower yields (2m). While all reactions resulted in complete consumption of the fluoroamide, only a singly fluorinated product is produced with the parent amide being the major side product (see ESI †). The reaction proved selective for δ-fluorination even in the presence of tertiary Csp 3–H bonds ( e.g., 2h, 2j, and 2k), thereby demonstrating selectivity counter to C–H-bond strength. Interestingly, transannular fluorine transfer occurs with complete regioselectivity to produce 2l as the sole product. Additionally, benzylic C–H bonds can be fluorinated under these conditions (2n). The reaction also exhibits good functional group compatibility, allowing access to a variety of fluorinated motifs. In particular, the reaction proceeds in the presence of either free or protected alcohols (2o and 2p). Moreover, esters and halides are both tolerated to give fluorinated products 2q and 2r in good yield. Notably, the reaction provides access to fluorohydrin 2s—highlighting the unique ability of this methodology to access both fluorohydrins and γ-fluoroalcohols such as 2o. In addition to these examples, terminal alkene 1t works quite well giving 2t in 67% yield. Furthermore, alkene functionalizations of 2t would provide access to a diverse range of fluorinated motifs. To target difluoromethylene units with this methodology, fluoroamide 1u was prepared and subjected to the reaction conditions. Pleasingly, 2u was observed in 20% yield.Substrate scope for fluorine transfer Open in a separate windowaIsolated yields. All reactions were run on 0.3 mmol scale unless otherwise noted. bYield reported as an average of two trials. c35 min reaction time. ddr = 1 : 3.2 when ran at room temperature for 24 h. e0.25 mmol scale. f0.18 mmol scale. g0.1 mmol scale, yield determined by 19F-NMR with 4-fluorotoluene as an internal standard.While exploring the substrate scope, we were surprised to discover that the fluoroamide N–F bond is unusually stable to a variety of common reactions. For example, fluoroamide 1o was carried through an Appel reaction, PCC oxidation, and Wittig reaction with minimal loss of the fluoroamide. With such robustness, it becomes obvious that fluoroamides could act as secondary amide protecting group—being installed and carried through a multi-step synthesis until fluorine transfer is desired. Moreover, the greater rigidity of adamantoyl-based amides relative to pivalamides offers greater stability to acid and base hydrolysis—another feature of this system. Fortunately, the amide can be cleaved using conditions reported by Charette et al. with no evidence of elimination or loss of the alkyl fluoride (see ESI †). 25To evaluate the differences between C–H bonds, we calculated the hypothesized minima and maxima en route to C–F bond formation for primary, secondary, and tertiary substrates (). To begin, we defined the start of the pathway with the fluoroamides as octahedral, high-spin Fe(OTf) 2-DME complex (I). 18 Ligand dissociation results in the loss of DME to form II which is 7.2 kcal mol −1 higher in energy relative to I. This ligand loss opens a coordination site that allows Fe to enter the catalytic cycle via F-abstraction from the fluoroamides. This proceeds with a barrier (II-TS) of ∼25 kcal mol −1 for all systems to form the corresponding N-based radical (III). This new N-based radical is generally about −10 kcal mol −1 from the starting materials. The 1,5-HAT proceeds through a six-membered transition state (III-TS) with 16.4, 12.6, and 9.7 kcal mol −1 barriers for primary, secondary, and tertiary substrates, respectively. This abstraction forms the corresponding C-based radicals (IV) that were −15.0, −19.9 and −22.4 kcal mol −1 relative to the starting materials for primary, secondary, and tertiary substrates, respectively. A barrierless transition allows for the abstraction of fluorine from Fe( iii)-fluoride to simultaneously furnish the products (V) and regenerate catalyst II. Interestingly, this transition seems to proceed with an intermolecular electron-transfer from the alkyl radicals to the Fe( iii) center. The overall process is highly exergonic at −53.7, −58.6, and −61.9 kcal mol −1 for primary, secondary, and tertiary substrates, respectively. We attribute the low yields for the tertiary example to rapid oxidation of the carbon radical, likely by Fe( iii), that forms a tertiary carbocation and leads to unwanted side reactions. The turnover-limiting step is the N–F abstraction by Fe (II-TS). Open in a separate windowComputed relative Gibb''s free energies for intermediates and transition-states along the reaction pathway (uM06/cc-pVTZ(-f)-LACV3P**//uM06/LACVP** level of theory).An alternative pathway, related to the classic HLF reaction, 19a,b would involve radical chain propagation. Although unlikely, we also evaluated this pathway computationally (). Consistent with our previous report, 18 this process proceeds with an unfavorably high barrier of 30.0, 28.1, and 26.8 kcal mol −1 for primary, secondary, and tertiary substrates, respectively. Hence, this process cannot compete with the barrierless delivery of fluorine from the Fe( iii) fluoride species.In conclusion, we leveraged critical computational insights to enable the use of simple amines as a building block for the directed fluorination of C–H bonds. The reaction targets unactivated Csp 3–H bonds site selectively regardless of bond strength. The reaction proceeds under mild iron catalysis that allows broad functional-group compatibility and provides access to unique fluorinated motifs. Moreover, we identified fluoroamides as surprisingly stable functional groups with likely implications for biology and materials. Mechanistic evaluation of fluorine transfer with DFT provided a detailed reaction coordinate that explains the observed reactivity. The overall reaction and mechanistic insights should provide chemists a more predictable approach to site-selective fluorination of C–H bonds. 相似文献
18.
Indolmycin is an antibiotic from Streptomyces griseus ATCC 12648 with activity against Helicobacter pylori, Plasmodium falciparum, and methicillin-resistant Staphylococcus aureus. Here we describe the use of the indolmycin biosynthetic genes in E. coli to make indolmycenic acid, a chiral intermediate in indolmycin biosynthesis, which can then be converted to indolmycin through a three-step synthesis. To expand indolmycin structural diversity, we introduce a promiscuous tryptophanyl-tRNA synthetase gene ( trpS) into our E. coli production system and feed halogenated indoles to generate the corresponding indolmycenic acids, ultimately allowing us to access indolmycin derivatives through synthesis. Bioactivity testing against methicillin-resistant Staphylococcus aureus showed modest antibiotic activity for 5-, 6-, and 7-fluoro-indolmycin.A semi-synthetic system for producing indolmycin, an antibiotic, was developed and used to make indole-substituted, halogenated derivatives of indolmycin, some with modest bioactivity against methicillin-resistant Staphylococcus aureus. Antibiotic-resistant bacteria pose a great threat to human health, 1–4 and the rates of new antibiotic discoveries and clinical approvals have been in a steep decline since the 1980s. 1 Without the discovery and development of new antibiotics, drug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), will become increasingly prevalent. 2–4 One strategy to increase antibiotic development has been to “rediscover” known, but underdeveloped, antibiotics. 1 One such example is indolmycin, which was originally discovered in 1960 from Streptomyces griseus ATCC 12648 5 but was not originally developed for clinical use because of its narrow spectrum of activity 6–10 and its interference with tryptophan catabolism in the liver. 9,10 However, reignited interest in this old antibiotic led to the discovery of its activity against Helicobacter pylori, 6Plasmodium falciparum, 11 and MRSA. 12 For MRSA, indolmycin was found to be active against mupirocin- and fuscidic acid-resistant MRSA strains, with strains resistant to indolmycin emerging infrequently and with reduced fitness compared to sensitive strains. 12 In addition, indolmycin has been shown to have minimal activity against common members of the human microbiota, suggesting that its narrow spectrum of activity is an asset. 6 The first indole-substituted derivatives, 5-hydroxy and 5-methoxyindolmycin, were made by precursor-directed feeding of the indolmycin producer, Streptomyces griseus ATCC 12648, and they showed modest improvements in bioactivity against S. aureus and Escherichia coli. 13 Two practical synthetic routes to indolmycin and some indole-substituted derivatives have been reported more recently, 14,15 which enabled access to a small variety of indole-substituted derivatives. Additionally, a previous patent has described synthetic methods to produce a variety of derivatives; however, these methods do not appear to offer stereochemical control, and some require tailoring steps specific to each analog. 16 Therefore, further development of indolmycin would benefit from a simpler diversification method that could be applied to produce a wider variety of analogs with stereochemical control.Inspired by early biosynthetic studies, 17,18 our group previously identified the indolmycin gene cluster and elucidated the biosynthetic pathway, demonstrating that indolmycin (1) is assembled from tryptophan, arginine and S-adenosylmethionine (SAM) in a three-part process (). 19 In the first part, l-arginine is oxidized by Ind4 in an oxygen- and PLP-dependent reaction to 4,5-dehydro-2-iminoarginine, which is then enantioselectively reduced by imine reductase Ind5 and its chaperone Ind6 to 4,5-dehydro- d-arginine. In the second part, tryptophan (2) is deaminated by PLP-dependent transaminases, giving indole pyruvate (3). Compound 3 is then methylated by SAM-dependent C-methyltransferase Ind1 to 3-methyl-indolepyruvate (4) which is reduced by NADH-dependent ketone reductase Ind2 to form indolmycenic acid (5). Then, in the third part, 4,5-dehydro- d-arginine and 5 are coupled in an ATP-dependent fashion by Ind3 and Ind6, resulting in an oxazolinone-cyclized molecule, N-desmethyl-indolmycin, which is finally N-methylated by Ind7, a SAM-dependent N-methyltransferase, to form 1. Open in a separate windowIndolmycin biosynthesis from Streptomyces griseus ATCC 12648. (a) Indolmycin biosynthetic gene cluster. (b) Indolmycin biosynthetic pathway from Streptomyces griseus ATCC 12648. (c) Semi-synthetic scheme towards indolmycin and derivatives using indolmycin biosynthetic genes. The dashed arrow indicates a predicted side-product based on LC-MS analysis.Armed with the elucidated biosynthetic pathway for 1, we set out to create an in vivo system to make 1 in E. coli. We first cloned all necessary genes into four plasmids and co-expressed the genes in E. coli (Fig. S1 †), a strain which we named E. coli I1234670P5 (Table S1 †). We found that the genes needed to produce indolmycin in E. coli were ind1, ind2, ind3, ind4, ind6, ind7, ind0 and pel5, a homologous gene of ind5 from Paenibacillus elgii B69 showing better production of active protein in E. coli. 20,21 We also relied on the activity of endogenous E. coli aminotransferases to catalyze the initial tryptophan deamination step. However, only a small amount of 1 was produced (∼170 μg L −1 of bacterial culture) and the yield could not be improved despite our best efforts (). However, we found that this construct produced substantial amounts of 5 ([M + H] + = 220 m/ z) at 40–50 mg L −1 of culture, along with a shunt product, C-desmethyl-indolmycenic acid (6; [M + H] + = 206 m/ z). Open in a separate windowBiosynthetic production of 5 and semi-synthetic production of 1. (a) Extracted ion chromatograms show production of 5 with minimal production of 1 from E. coli I1234670P5. (b) Synthetic scheme to 1 from 5, adapted from literature methods. 14,15 (c) Total ion chromatogram of compound 1 isolated after semi-synthesis and final purification by semi-preparative HPLC. Compounds are indicated with coloured boxes and numbered.Compound 5 itself has been a focus of total synthetic efforts toward 1, as it is the key chiral precursor. 14,15,22–26 Since production of 5 was much higher than that of 1 from E. coli I1234670P5, we pursued a semi-synthetic method of obtaining 1 using our biosynthetic platform to access 5, combined with a three-step chemical transformation (). We attempted to remove extraneous genes from our biosynthetic platform by only including ind0, ind1, and ind2, but the changes resulted in reduced amounts of 5 (Fig. S2 †). At this time, it is unclear which of the other genes may be contributing to the production of compound 5. Therefore, we employed the full eight-gene construct toward synthesis of 5. We then adapted the three-step synthesis to make indolmycin, 14,15 in which purified 5 was esterified to make the ethyl ester (7; [M + H] + = 248 m/ z; Fig. S3a †), cyclized to give N-desmethyl-indolmycin (8; [M + H] + = 244 m/ z; Fig. S3b and S4a †), and methylated at the exocyclic nitrogen to give 1 ([M + H] + = 258 m/ z; , S4b and Table S4 †).Then, we wanted to make derivatives of 1 from indole derivatives, which are more widely accessible than derivatives of 2. The tryptophan synthase (TrpS) from Salmonella enterica has been previously shown to couple a wide variety of indole derivatives to l-serine to generate derivatives of 2. 27 We were able to replace pel5 with trpS in our biosynthetic platform without a reduction in the amount of 5 produced (Fig. S1b †), and we named the resulting strain E. coli I1234670TS. When we fed 5-fluoroindole to E. coli I1234670TS, we observed increased production of fluorinated metabolites, 5-fluoro-indolmycenic acid (5F-5; [M + H] + = 238 m/ z) and 5-fluoro- C-desmethyl-indolmycenic acid (5F-6; [M + H] + = 224 m/ z) (). We then optimized the feeding conditions, finding that 5F-5 amounts were optimal when we fed E. coli I1234670TS with 0.5 mM 5-fluoroindole per day over two days (Fig. S5 †). Open in a separate windowAddition of the trpS gene to the biosynthetic platform allows incorporation of substituted indoles into 5. (a) LC-MS analysis of strains with and without trpS ( E. coli I1234670TS and E. coli I1234670P5, respectively) when fed 5-fluoroindole. Chemical structures of compounds with the corresponding extracted ion chromatogram are shown to the right of the traces. (b) Indole derivatives tested. Dark blue shows indoles that were incorporated into an analog of 5 (>48% of underivatized 5 by LC-MS analysis; Table S5 †) and further purified; medium blue indicates indoles that were incorporated into an analog of 5 at lower levels (6–22% of underivatized 5 by LC-MS analysis; Table S5 †) but were not further verified by purification; and light blue indicates indoles that did not show detectable incorporation into 5.To determine the scope of indole derivatives accepted by our biosynthetic platform, we fed a variety of indoles to E. coli I1234670TS and monitored the production of 5 and its derivatives by LC-MS. Out of the indoles tested, we found that fluorinated and chlorinated indoles substituted at the 5-, 6- and 7-positions were the best accepted by the biosynthetic platform (, S6 and S7 †). We predict that lower acceptance of indoles substituted at the 4-position may be due to steric hindrance, as 4-fluoroindole was moderately accepted, while 4-chloroindole was not observed at all. Although we observed LC-MS peaks consistent with conversion of some of the azaindoles and hydroxyindoles into 5 derivatives, further work is required to confirm, optimize and scale up the purification of these compounds (Fig. S7 †). Cultures producing derivatives of 5, substituted at 5-, 6- and 7-positions, were further scaled up for purification of the 5-derivatives and downstream synthesis of 1-derivatives (Fig. S8–S10 and Table S4 †). Each purified derivative of 5 and 1 was characterized by HR-MS and NMR (Table S3 and ESI Methods †). Overall, cultures fed with the fluorinated indoles produced a higher amount of 5-derivatives than the cultures fed with the chlorinated indoles. 1 and its derivatives were tested against MRSA (Fig. S11 †). While the fluorinated derivatives showed bioactivity, the chlorinated derivatives of 1 did not show bioactivity at the maximum amount tested in the disk diffusion assay (30 μg). We determined MIC 50 values for each fluorinated compound (). The MIC 50 values demonstrate that 1 is a more potent inhibitor of MRSA than its derivatives, while 6F-1 showed the most potent inhibition of MRSA compared to any of the derivatives, followed by 7F-1 and 5F-1. The lack of bioactivity of the chlorinated compounds may be due to the bulky chlorinated substituent hindering the compounds'' abilities to bind to the tryptophanyl-tRNA synthetase (TrpRS) target, which is supported by docking studies of the analogs into a bacterial TrpRS structure (Fig. S12 †).MIC 50 values determined for 1 and its derivatives against MRSA. Values represent the average of three replicates ± the standard deviation. For 1, 5F-1, 6F-1 and 7F-1, concentrations ranging from 50 μg mL −1 to 0.128 ng mL −1 were tested, and for 5Cl-1, 6Cl-1 and 7Cl-1, concentrations ranging from 200 μg mL −1 to 0.512 ng mL −1 were tested. The reported MIC 50 value for indolmycin is 0.5 μg mL −1 (range: 0.125–2 μg mL −1) against MRSA 12 | MIC50 (μg mL−1) |
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1 | 1.21 ± 0.04 | 5F-1 | 32.5 ± 19.6 | 6F-1 | 6.49 ± 0.03 | 7F-1 | 16.7 ± 4.2 | 5Cl-1 | >200 | 6Cl-1 | >200 | 7Cl-1 | >200 | Open in a separate windowHere we have shown that 5-, 6- and 7-fluorinated and chlorinated indole derivatives are accepted as substrates by S. enterica TrpS, E. coli tryptophan aminotransferase, Ind1 and Ind2. The fluorinated indoles, particularly 5-fluoroindole, showed greater incorporation into 5 than the chlorinated indoles, perhaps because the larger chlorine atom may have steric hindrance in some of the enzyme active sites, reducing the efficiency of turnover. This result is consistent with previous studies on TrpS from S. enterica, which shows the lowest acceptance for 4-chloro, 5-chloro and 5-bromo indoles. 27 Other tested indoles, 5- and 6-hydroxyindoles and 7-azaindole, were only minimally converted to derivatives of 5 by this semi-synthetic platform, and 4-hydroxyindole and 4-, 5-, and 6-azaindole were not converted at all. Azatryptophans have been previously made using TrpS but required longer reaction times due to reduced nucleophilicity, 28–30 which may explain our results. For 4-hydroxyindole, previous studies of hydroxytryptophan production by S. enterica TrpS are limited; 29 however, E. coli TrpS was shown to accept 4-hydroxyindole during the production of psilocybin. 31 It is unclear at this time why the 4-hydroxylated derivative of 5 was not observed. The substrate acceptance and 5-derivative production could be further improved with optimized reaction and purification conditions and engineering of strains and enzymes. For instance, TrpS has already been the target of many enzyme engineering studies focused on improving the substrate scope. 32–38Previous work by Demain and co-workers demonstrated successful production of 5-hydroxy and 5-methoxy derivatives of 1 from S. griseus fed with the corresponding derivatives of indole or 2; however, 6-substituted derivatives were not converted to derivatives of 1. 13 By contrast, our system incorporated halogenated substituted indoles at the 6-position, meaning that the semi-synthetic method overcomes one limitation of the purely biosynthetic method. Development of this semi-synthetic method has also helped to identify some key hurdles to creating a purely biosynthetic method. These hurdles, such as accumulation of 5, an apparent role of proteins in Parts I or III () in the formation of 5, and substrate scope bottlenecks, could be addressed in future attempts to produce 1 from E. coli. Furthermore, the biosynthetic portion of our system also overcomes challenges associated with synthetic methods. Synthetic studies were typically associated with making 5 through methods such as using a chiral auxiliary, 25 epoxide ring-opening, 14,24 lipase-assisted enantioselective acetylation, 15 or a combination of these methods, 26 and few attempts to synthesize halogenated derivatives have been reported, as the methods employed are likely to be incompatible with halogenated indole precursors. A patent has described methods to produce several derivatives of 1, including 4Cl-1 and 6F-1, but stereocenters were not controlled, to the best of our knowledge, and some analogs utilized additional tailoring steps or entirely unique routes. 16 Therefore, these patented methods, individually, are not robust enough to enable production of a wide variety of derivatives, making a broad search for potentially bioactive candidates challenging. Our semi-synthetic method of obtaining 1 and its derivatives offers a simple and halogen-compatible alternative to purely synthetic methods that could be used to expand and diversify the accessible indolmycin molecules for broad bioactivity searches. Broad bioactivity searches could be followed by optimization of this platform, or creation of another, to produce enough amounts of the desirable compounds to continue work beyond the initial bioactivity testing. We note that this type of semi-synthetic method could also be applied to the diversification of other tryptophan-derived molecules. Moreover, the fluorinated derivatives of 1 showed bioactivity against MRSA, demonstrating that these compounds may serve as useful molecules for development of new antibiotics. Overall, this work lays a foundation for making derivatives of 1, while demonstrating the value of combining synthetic and biosynthetic methods to make and diversify natural product-derived compounds. 相似文献
19.
CF 2H groups are unique due to the combination of their lipophilic and hydrogen bonding properties. The strength of H-bonding is determined by the group to which it is appended. Several functional groups have been explored in this context including O, S, SO and SO 2 to tune the intermolecular interaction. Difluoromethyl ketones are under-studied in this context, without a broadly accessible method for their preparation. Herein, we describe the development of an electrochemical hydrodefluorination of readily accessible trifluoromethylketones. The single-step reaction at deeply reductive potentials is uniquely amenable to challenging electron-rich substrates and reductively sensitive functionality. Key to this success is the use of non-protic conditions enabled by an ammonium salt that serves as a reductively stable, masked proton source. Analysis of their H-bonding has revealed difluoromethyl ketones to be potentially highly useful dual H-bond donor/acceptor moieties.The electrochemical hydrodefluorination of trifluoromethylketones under non-protic conditions make this single-step reaction at deeply reductive potentials uniquely amenable to challenging electron-rich substrates and reductively sensitive functionalities. The difluoromethyl group (CF 2H) has attracted significant recent attention in medicinal chemistry, 1,2 which complements the well-documented importance and growing use of fluorine in small molecule pharmaceuticals. 3–6 The CF 2H group is an H-bond donor 7,8 that is also lipophilic, 9,10 a unique combination that positions it as an increasingly valuable tool within drug-discovery. 11 CF 2H has been used as a bioisostere of OH and SH in serine and cystine moieties, respectively, as well as NH 2 groups, where greater lipophilicity and rigidity provide advantages to pharmacokinetics and potency. 12–14The hydrogen-bond acidity of CF 2H groups is exceptionally dependent on the atom or group to which it is appended (). 1,2 The H-bond acidity of alkyl-CF 2H groups is half that of O–CF 2H and even a quarter of SO 2–CF 2H groups. 1 This mode of control allows the H-bonding strength and, therefore its function, to be finely tuned. While much research has focused on the synthesis, behaviour and use of XCF 2H groups, where X = O, S, SO, SO 2, Ar, it is surprising that the corresponding carbonyl containing moiety (X = CO) has remained relatively elusive in these contexts. Not only would difluoromethyl ketones (DFMK) be expected to provide a relatively strong H-bond, but the carbonyl unit provides a complementary, yet proximal mode of intermolecular interaction (). Indeed, the dual action of neighbouring H-bond donor and acceptor functionalities provides the fundamental basis for many biological systems, including in the secondary structure assembly mechanisms for proteins and DNA/RNA nucleobase pairing, as well as in enzyme/substrate complexes. Indeed, the DFMK functionality has demonstrated important utility in biological applications, including anti-malarial and -coronaviral properties. 15 Finally, the carbonyl provides a useful synthetic handle for further derivatization. Open in a separate windowH-Bonding in DFMKs and their synthesis via hydrodefluorination.While some progress has been made on the synthesis of DFMKs, 16 there still remains a need for a general and more broadly accessible route to their preparation. Current strategies for DFMK preparation require multi-step processes, expensive reagents, installation of activating groups, or are inherently low yielding. 15a,16–25 The hydrodefluorination of trifluoromethyl ketones (1) potentially represents the most accessible strategy, as the starting materials are most readily prepared through a high-yielding trifluoroacetylation of C–H or C–X bonds. 26–29 In 2001, Prakash demonstrated the viability of this approach using 2 equivalents of magnesium metal as stoichiometric reductant to drive the defluorination, with a second hydrolysis step (HCl (3–5 M) or fluoride, overnight stirring) to reveal the product. 30 The scope in this 2-step process (6 substrates) reflects the limitations of using a reductant, such as Mg, that has a fixed reduction potential, as well as incompatibilities arising from Mg/halide exchange with aryl halides. Similar limitations with the use of electron-rich substrates were revealed in related contributions from Uneyama. 31In order to access more electron-rich and reductively challenging substrates, such as those containing medicinally relevant heterocycles, we postulated that electrochemical reduction could be employed (). Electrosynthesis is becoming an increasingly valuable enabling technology and has seen a recent resurgence due to the precise control, unique selectivity, and the potential scalability and sustainability benefits that it offers. 32–36 This strategy would avoid the undesirable use of stoichiometric metals and the ‘deep-reduction’ potentials required are readily accessed by simply selecting the applied potential. Pioneering early work from Uneyama on the cathodic formation of silylenol ether intermediate 2, suggested this approach could be viable. 37,38 The fundamental challenge in designing a practical, single-step process under highly reducing potentials (<−2.0 V vs. Fc/Fc +), is to avoid the reduction of the proton source, which would otherwise compete to generate H 2 gas and leave the starting material untouched. Uneyama does not demonstrate hydrodefluorination, presumably due to this problem. Additional challenges posed by ‘deep-reduction’ include a lack of tolerance for reduction-sensitive functionality (alkene, C–X bonds etc.), low mass balance due to substrate decomposition and the undesirable use of sacrificial metal anodes. 39 Solving these problems should provide generally applicable, safe and scalable conditions for the hydrodefluorination of readily accessible trifluoromethyl ketones (1).Given the electron-rich nature of indoles, their ubiquity in bioactive compounds, and their ease of functionalisation, we chose indole 1a as the model substrate for optimisation. The highly reductive potentials required will render it a challenging substrate, which should lead to more general conditions suitable for other important substrate classes. Indeed, when we applied the Mg conditions of Prakash to this substrate, no silyl enol ether intermediate (2a) was observed, nor product 3a, and the starting material remained completely untouched ( |