Synthesis of novel acetylene-containing amino acids

Summary.  Novel synthetic procedures for the modification of non-proteinogenic acetylene-containing amino acids have been developed. The functionalization either proceeds via zinc/copper-mediated introduction of alkyl substituents, or via tungsten-catalyzed ring-closing alkyne metathesis reactions. Received March 28, 2002 Accepted October 3, 2002 Published online December 18, 2002 Acknowledgements These investigations are supported (in part) by the Netherlands Research Council for Chemical Sciences (CW) with financial aid from the Netherlands Technology Foundation (STW). Authors' address: Floris P. J. T. Rutjes, Prof. Dr., Department of Organic Chemistry, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands, E-mail: rutjes@sci.kun.nl  2, selected data: ¹H NMR (300 MHz, CDCl₃) δ 5.32 (d, J = 7.7 Hz, 1H), 4.44–4.40 (m, 1H), 3.76 (s, 3H), 2.75–2.73 (d, J = 5.0 Hz, 2H), 1.44 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.0, 155.0, 80.3, 74.6, 52.6, 51.9, 41.7, 28.3, 24.0; mp = 55°C.  Typical procedure for 5: zinc dust (116 mg, 1.408 mmol) was weighed into a 20 mL flask, which was repeatedly evacuated (with heating using a heat gun) and flushed with argon. Dry DMF (0.5 mL, distilled from CaH₂) and 1,2-dibromoethane (9.2 μL, 0.106 mmol) were added and the flask was heated at 80°C for 40 min. The reaction mixture was allowed to cool to room temperature, trimethylsilyl chloride (4 μL, 0.035 mmol) was added and the resulting mixture was stirred vigorously for a further 30 min under argon. Iodocyclohexane (69 μl, 0.528 mmol) was added and stirred at room temperature for 3 h more after which stirring was ceased to settle the zinc. CuCN (41 mg, 0.458 mmol) and LiCl (40 mg, 0.915 mmol) were heated to 150°C for 2 h and cooled to room temperature. Addition of DMF (1 mL) formed a soluble CuCN·2LiCl complex within 5 min. After cooling the Cu-complex to −15°C, the organozinc reagent was added dropwise followed by the bromoacetylene 2 (116 mg, 0.352 mmol). The mixture was allowed to stir overnight at room temperature. Water was added and the suspension was extracted using heptane, washed with brine, dried (MgSO₄) and concentrated. Purification using flash column chromatography (10% EtOAc in heptane) yielded 5 (100 mg, 81%) as a colorless oil. 5: IR ν 3355, 2929, 2852, 2359, 2337, 1749, 1717, 1498, 1447, 1365, 1251, 1181, 1060; ¹H NMR (300 MHz, CDCl₃) δ 5.28 (d, J = 7.7 Hz, 1H), 4.43–4.38 (m, 1H), 3.73 (s, 3H), 2.69–2.63 (m, 2H), 2.13 (m, 1H), 1.73–1.22 (m, 10H), 1.43 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.4, 155.0, 88.1, 79.9, 73.8, 52.3, 32.7, 32.7, 28.8, 28.2, 25.8, 24.6, 23.1; HRMS (EI): calculated for C₁₇H₂₇NO₄ 309.1940, found 309.1937.  A solution of the tungsten catalyst (7 mg, 10 mol%) in C₆H₅Cl (2 mL) was treated with a solution of 14 (49.0 mg, 0.120 mmol) in C₆H₅Cl (5.0 mL) under an argon atmosphere and the resulting mixture was heated at 80°C for 3 h. Evaporation followed by flash column chromatography (80% EtOAc in heptane) afforded 15 (21.0 mg, 50%; 64% after correction for starting material) and 14 (16 mg, 33%) as colorless oils. 15: [α]_D =–14.6 (c = 1, CH₂Cl₂); IR ν 3313, 2931, 2865, 2249, 1744, 1667, 1520, 1366, 1170; ¹H NMR (400 MHz, CDCl₃) δ 7.14 (d, J = 8.7 Hz, 1H), 6.08 (d, J = 8.3 Hz, 1H), 4.78 (q, J = 6.8 Hz, 1H), 4.27 (q, J = 7.9 Hz, 1H), 3.73 (s, 3H), 2.17–2.15 (m, 4H), 2.07–1.96 (m, 2H), 1.79–1.52 (m, 4H), 1.45 (s, 9H), 0.89–0.83 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 173.2, 171.8, 155.8, 80.4, 80.2, 79.3, 53.8, 52.5, 51.2, 32.8 (2×), 28.1, 24.6, 24.2, 18.3 (2×); HRMS (EI): calculated for C₁₈H₂₈N₂O₅     

The effect of buffers and chelators on the reaction of luminol with Fenton's reagent near neutral pH

The chemiluminescence of the system luminol +Fe²⁺ + H₂O₂ was measured in aqueous buffer at pH 7.2. In veronal (5,5-diethybarbiturate) buffer, the luminescence is strongly quenched by ethanol and mannitol, but only weakly by t-butanol, benzoate and superoxide dismutase (SOD); complexing Fe²⁺ with 1,10-phenanthroline or 2,2′-dipyridyl causes a decrease of light production that can be partially obviated by the simultaneous addition of SOD. In phosphate buffer, the luminescence is higher than in veronal and it is efficiently quenched by all four OH · quenchers and by SOD. In Tris buffer, no light production is observed as long as the Fe²⁺ is not complexed. When Fe²⁺ is complexed by pyrophosphate or phytate, there is a strong chemiluminescence in all three buffers, which is quenched by all four OH · quenchers and by SOD. When Fe²⁺ is complexed by EDTA or DTPA, very little luminescence is observed. The luminol analogue phthalhydrazide, which was suggested by Merényi and Lind as a reliable OH · detector, can replace luminol only in phosphate buffer, and thus turns out to be very specific indeed for free OH ·.     

Sulphate influx in wheat and barley roots becomes more sensitive to specific protein-binding reagents when plants are sulphate-deficient

When young wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) plants were deprived of an external sulphate supply (-S plants), the capacity of their roots to absorb sulphate, but not phosphate or potassium, increased rapidly (derepression) so that after 3–5 d it was more than tenfold that of sulphate-sufficient plants (+S plants). This increased capacity was lost rapidly (repression) over a 24-h period when the sulphate supply was restored. There was little effect on the uptake of L-methionine during de-repression of the sulphate-transport system, but S input from methionine during a 24-h pretreatment repressed sulphate influx in both+S and-S plants.Sulphate influx of both+S and-S plants was inhibited by pretreating roots for 1 h with 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) at concentrations > 0.1 mol · m^-3. This inhibition was substantially reversed by washing for 1 h in DIDS-free medium before measuring influx. Longer-term pretreatment of roots with 0.1 mol·m^-3 DIDS delayed de-repression of the sulphatetransport system in-S plants but had no influence on+S plants in 3 d.The sulphydryl-binding reagent, n-ethylmaleimide, was a very potent inhibitor of sulphate influx in-S roots, but was much less inhibitory in +S roots. Its effects were essentially irreversible and were proportionately the same at all sulphate concentrations within the range of operation of the high-affinity sulphate-transport system. Inhibition of influx was 85–96% by 300 s pretreatment by 0.3 mol·m^-3 n-ethylmaleimide. No protection of the transport system could be observed by including up to 50 mol·m^-3 sulphate in the n-ethylmaleimide pre-treatment solution. A similar differential sensitivity of-S and+S plants was seen with p-chloromercuriphenyl sulphonic acid.The arginyl-binding reagent, phenylglyoxal, supplied to roots at 0.25 or 1 mol·m^-3 strongly inhibited influx in-S wheat plants (by up to 95%) but reduced influx by only one-half in+S plants. The inhibition of sulphate influx in-S plants was much greater than that of phosphate influx and could not be prevented by relatively high (100 mol·m^-3 sulphate concentrations accompanying phenylglyoxal treatment. Effects of phenylglyoxal pretreatment were unchanged for at least 30 min after its removal from the solution but thereafter the capacity for sulphate influx was restored. The amount of new carrier appearing in-S roots was far greater than in+S roots over a 24-h period.The results indicate that, in the de-repressed state, the sulphate transporter is more sensitive to reagents binding sulphydryl and arginyl residues. This suggests a number of strategies for identifying the proteins involved in sulphate transport.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NEM n-ethylmaleimide - PCMBS p-chloromercuriphenyl sulphonic acid     

Ammoniacal silver staining of proteins: mechanism of glutaraldehyde enhancement

When the conditions for detecting proteins by ammoniacal silver staining (B. R. Oakley, D. R. Kirsch, and N. R. Morris (1980) Anal. Biochem. 105, 361-363.) following gel electrophoresis were varied, it was noted that glutaraldehyde pretreatment was necessary for maximal staining, which could not be explained simply as the result of "fixation." Further studies indicated that glutaraldehyde enhancement of protein staining with this silver reagent was probably due to oxidation of the aldehyde groups by silver ions, resulting in metallic silver depositions within the gel which act as nucleation sites for additional metallic silver localization in the protein bands upon the addition of formaldehyde developer. This proposed mechanism is consistent with the Tollen's reaction, as well as some aspects of the photographic process. Consistent with this notion, silver-staining intensities are directly related to mole percentage lysine of various standard proteins.     

Ultrafast protein determinations using microwave enhancement

We present here microwave-based modifications of standard protein assays that dramatically reduce the time required to determine protein concentrations. Typical protein determinations involve incubation times ranging from 15–60 min. Microwave irradiation of specimens reduces this time requirement to 10–20 s without compromising accuracy or reliability. The remarkable speed with which protein determinations may be carried out using microwave enhancement greatly simplifies general laboratory procedures that depend on the estimation of protein concentrations. An erratum to this article is available at .     

Superoxide dismutase activity of the captopril-iron complex

With an assay that generates superoxide anion radicals without the intervention of metal ions we investigated the antioxidant properties of captopril, an angiotensin-converting enzyme inhibitor with a sulfhydryl group. Under these conditions, increasing concentrations of the drug were seen not to scavenge O· ₂ ^– directly. However, a combination of captopril and iron could bring about the breakdown of the superoxide anion; a result that may help to understand the free radical-scavenging properties of captopril.     

Restoration of membrane potential in mitochondria deenergized with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)

The membrane potential (Δψ) of rat liver mitochondria dropped upon addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) but was gradually and fully restored to the original value by the subsequent addition of dithioerythritol. Concomitantly, Ca²⁺ released from mitochondria was reaccumulated and the oxidative phosphorylation process completely recoupled. Neither of these effects has been observed with dinitro-o-cresol or 2,4-dinitrophenol, uncouplers which, unlike FCCP, do not react with thiols. Δψ abolished by FCCP was also restored, though incompletely, by albumin; a prompt and complete restoration was however achieved upon subsequent addition of dithioerythritol. Dithioerythritol also completely and rapidly restored the Δψ decreased by addition of diazene dicarboxylic acid bisdimethylamide (diamide).     

Membrane surface properties of sheep erythrocytes,an immunological reagent,after different treatments as reflected by partition in two-polymer aqueous phases

Sheep erythrocytes (E) which, with or without certain treatments, are currently used as “immunological reagents” to detect cells with specific receptors (by rosette-formation) have been partitioned in two-polymer aqueousphase systems selected so as to reflect charge-associatedor lipid-related membrane surface properties. We have found that the partitioning behavior of E is not affected in these phases by reacting the cells with anti-E antibody (either IgG or IgM), forming EA. The additional binding of complement to the cell-antibody complex, forming EAC, results, however, in a marked decrease in the partition coefficient,K. Apparently both the charge-associated and hydrophobic properties reflected by partitioning remain accessible to the phase polymers when the cells are coated with antibody, but are not with the addition of complement. It is interesting that EA can still rosette with T-lymphocytes (14), a property of E, while the additional coating with complement results in EAC which does not appreciably do so (26). Neuraminidase or trypsin treatments of E, which yield Es having quite different rosetting properties with T-lymphocytes (14), cause increasedKs and unchangedKs, respectively, in phases reflecting lipid-related surface properties. Either treatment causes reducedKs of E in charged-phase systems. Neuraminidase treatment also results in a reduced electrophoretic mobility of E, while trypsin treatment is not detectable by cell electrophoresis (25). We are currently studying the possible usefulness of employing cell electrophoresis and cell partitioning in charged-phase systems jointly to obtain information on events occurring at the shear plane versus those occurring deeper in the membrane.     

体外诊断试剂防腐剂的选择策略

对体外诊断试剂中防腐剂的作用机理及选择原则进行了阐述,并对目前常用的防腐剂如叠氮钠、庆大霉素、硫柳汞、异噻唑啉酮类等的性能进行了比较,为体外诊断试剂的研究开发及生产中防腐剂的使用提供有益指导。     

酸、碱、盐胁迫下4种紫花苜蓿几项〖HJ*3〗〖HJ*2〗生理指标变化的比较研究

为了解4种进口紫花苜蓿在逆境条件下幼苗生理响应特点,并比较其对逆境因子的敏感程度以推测其抗逆性,便于指导在我国的因地制宜种植,发挥最大生产性能,特进行比较试验研究。用H2SO4、NaOH、土壤浸出液分别配制pH为3、4、5、6、7、8、9、10、11、12的试剂;当pH为7时,用土壤浸出液配制NaCl浓度分别为0.1%、0.2%、0.3%、0.4%、0.5%、0.6%的试剂;再配制pH分别为8、9、10、11、12,NaCl浓度分别相应为0.2%、0.3%、0.4%、0.5%、0.6%的试剂。分别种植4种苜蓿,检测生理指标,分析敏感性。结果:耐酸能力表现为:金皇后>飞马>阿尔冈金>维多利亚;耐碱能力表现为:飞马>阿尔冈金>金皇后>维多利亚;耐盐能力表现为:飞马>金皇后>阿尔冈金>维多利亚;耐盐碱能力表现为:飞马=阿尔冈金>金皇后>维多利亚。结论:根据4种紫花苜蓿的抗逆特点,分别选择合适的土壤种植。