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 ·.     

Nitrosothiol Formation and Protection against Fenton Chemistry by Nitric Oxide-induced Dinitrosyliron Complex Formation from Anoxia-initiated Cellular Chelatable Iron Increase

Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with ^•NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged ^•NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief ^•NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1–2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief ^•NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of ^•NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of ^•NO.     

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     

Chemical modification of active sites in relation to the catalytic mechanism of F1

Recent studies of chemically modified F₁-ATPases have provided new information that requires a revision of our thinking on their catalytic mechanism. One of the subunits in F₁-ATPase is distinguishable from the other two both structurally and functionally. The catalytic site and regulatory site of the same subunit are probably sufficiently close to each other, and the interaction between the various catalytic and regulatory sites are probably sufficiently strong to raise the uni-site rate of ATP hydrolysis by several orders of magnitude to that of promoted (multi-site) ATP hydrolysis. Although all three subunits in F₁ possess weak uni-site ATPase activity, only one of them () catalyzes promoted ATP hydrolysis. But all three subunits catalyze ATP synthesis driven by the proton flux. Internal rotation of the ₃₃ or ₃ moiety relative to the remainder of the F₀F₁ complex did not occur during oxidative phosphorylation by reconstituted submitochondrial particles.     

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.     

Assessment of the Role of the Glutathione and Pentose Phosphate Pathways in the Protection of Primary Cerebrocortical Cultures from Oxidative Stress

Abstract: Reactive oxygen species have been implicated in neuronal injury associated with various neuropathological disorders. However, little is known regarding the relationship between antioxidant enzyme capacity and resultant toxicity. The antioxidant pathways of primary cerebrocortical cultures were directly examined using a novel technique that measures pentose phosphate pathway (PPP) activity, which is enzymatically coupled to glutathione peroxidase (GPx) detoxification of hydrogen peroxide (H₂O₂). PPP activity was quantified from data obtained by gas chromatography/mass spectrometry analysis of released labeled lactate following metabolic degradation of [1,6-¹³C₂,6,6-²H₂]glucose by cerebrocortical cultures. The antioxidant capacity of these cultures was systematically evaluated using H₂O₂, and the resultant toxicity was quantified by lactate dehydrogenase release. Exposure of primary mixed and purified astrocytic cultures to H₂O₂ caused stimulation of PPP activity in a concentration-dependent fashion from 0.25 to 22.2% and from 6.9 to 66.7% of glucose metabolized to lactate through the PPP, respectively. In the mixed cultures, chelation of iron before H₂O₂ exposure was protective and resulted in a correlation between PPP saturation and toxicity. Conversely, addition of iron, inhibition of GPx, or depletion of glutathione decreased H₂O₂-induced PPP stimulation and increased toxicity. These results implicate the Fenton reaction, reflect the pivotal role of GPx in H₂O₂ detoxification, and contribute to our understanding of the etiological role of free radicals in neuropathological conditions.     

Kinetics of the Competitive Degradation of Deoxyribose and Other Molecules by Hydroxyl Radicals Produced by the Fenton Reaction in the Presence of Ascorbic Acid

The competition method in which the Fenton reaction is employed as an OH radical generator and deoxyribose as a detecting molecule, has been used to determine the rate constants for reactions of the OH radical with its scavengers. Nonlinear competition plots were obtained for those scavengers which reacted with the Fenton reagents (Fe²⁺ or H₂O₂). Ascorbic acid is believed to overcome this problem. We have investigated the kinetics of deoxyribose degradation by -OH radicals generated by the Fenton reaction in the presence of ascorbic acid, and observed that the inclusion of ascorbic acid in the Fenton system greatly increased the rate of OH radical generation. As a result, the interaction between some scavengers and the Fenton reagents became negligeable and linear competition plots of A7A vs scavenger concentrations were obtained. The effects of experimental conditions such as, the concentrations of ascorbic acid, deoxyribose, H₂O₂ and Fe²⁺-EDTA, the EDTA/Fe²⁺ ratio as well as the incubation time, on the deoxyribose degradation and the determination of the rate constant for mercaptoethanol chosen as a reference compound were studied. The small standard error, (6.76± 0.21) ±' 10⁹M^-1s^-1 observed for the rate constant values for mercaptoethanol determined under 13 different experimental conditions, indicates the latter did not influence the rate constant determination. This is in fact assured by introducing a term, k_x, into the kinetic equation. This term represents the rate of-OH reactions with other reagents such as ascorbic acid, Fe²⁺-EDTA, H₂O₂ etc. The agreement of the rate constants obtained in this work with that determined by pulse radiolysis techniques for cysteine, thiourea and many other scavengers, suggests that this simple competition method is applicable to a wide range of compounds, including those which react with the Fenton reagents and those whose solubility in water is low.     

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 .