Carbon-bound Hydrogen Research Articles

Sources of hydrogen abstraction by activated neocarzinostatin chromophore.

Activation of the enediyne neocarzinostatin chromophore (NCS-Chrom) by thiol addition at C-12 generates a diradical species with radical centers at C-2 and C-6, which abstract hydrogens from deoxyribose in the minor groove of DNA. Since hydrogen abstraction from DNA accounts for only part of the hydrogen incorporated at these sites, it is important to determine the other possible sources. At low concentration of thiol, a condition resembling that during NCS-Chrom-induced DNA damage, the major non-DNA hydrogen donation source was found to be the carbon-bound hydrogen of the aqueous methanol solvent, rather than the expected sulfur-bound hydrogen of the thiol. Further, experiments with the gamma-L-glutamyl-DL-cysteinylglycine labeled with deuterium on the alpha- or beta-carbons to the sulfur showed small amounts of internal transfer of hydrogen into C-2 of the drug from the naturally occurring L,L diastereomer only. Quantitation of the hydrogen transfer was accomplished by separation of the L,DL diastereomeric mixtures of the thiol-NCS-Chrom adducts. In all, these various hydrogen donation sources can account for at least 70-80% of the hydrogen incorporated at C-2 of the drug under DNA damage conditions. Selective quenching of the radical at C-2 could account for the predominance of single-stranded over double-stranded DNA lesions.

Electrons trapped in single crystals of sucrose: Induced spin densities

Electrons are trapped at intermolecular sites in single crystals of sucrose X irradiated at 4.2 K. The coupling tensors for the hyperfine couplings between the electron and surrounding protons have been deduced from electron-nuclear double resonance (ENDOR) data. Electron spin densities at nearby hydroxy protons are positive, whereas spin densities at the more remote protons of carbon-bound hydrogen atoms are negative. The origin of these negative spin densities is discussed.

Oxygen and Hydrogen Isotope Fractionation during Cellulose Metabolism in Lemna gibba L.

Lemna gibba L. B3 was grown under heterotrophic, photoheterotrophic, and autotrophic conditions in water having a variety of hydrogen and oxygen isotopic compositions. The slopes of the linear regression lines between the isotopic composition of water and leaf cellulose indicated that under the three growth conditions about 40, 70, and 100% of oxygens and carbon-bound hydrogens of cellulose exchanged with those of water prior to cellulose formation. Using the equations of the linear relationships, we estimated the overall fractionation factors between water and the exchanged oxygen and carbon bound-hydrogen of cellulose. At least two very different isotope effects must determine the hydrogen isotopic composition of Lemna cellulose. One reflects the photosynthetic reduction of NADP, while the second reflects exchange reactions that occur subsequent to NADP reduction. Oxygen isotopic composition of cellulose apparently is determined by a single type of exchange reaction with water. Under different growth conditions, variations in metabolic fluxes affect the hydrogen isotopic composition of cellulose by influencing the extent to which the two isotope effects mentioned above are recorded. The oxygen isotopic composition of cellulose is not affected by such changes in growth conditions.

Studies of Electrons Trapped in X-Irradiated Rhamnose Crystals

The electrons trapped in single crystals of rhamnose X-irradiated at low temperature were studied by ENDOR spectroscopy. Hyperfine couplings of protons in the environs of the electron have been determined from ENDOR measurements, including those of some of the more remote carbon-bound hydrogen atoms. The likely site of electron trapping in the crystal structure of rhamnose was inferred from calculations of the electric potential generated by the dipoles of hydroxy groups about preexisting void spaces. Electron-proton distances for nonexchangeable hydrogen atoms from points within the void were calculated from the crystal structure and compared with distances obtained from hyperfine couplings. Good agreement was obtained between experimental and calculated values.

Peptide Free-Radicals: The Reactions of OH Radicals with Glycine Anhydride and its Methyl Derivatives Sarcosine and Alanine Anhydride. A Pulse Radiolysis and Product Study

The reactions of radiolytically generated OH radicals and H atoms with the cyclic dipeptides of glycine, alanine and sarcosine in deoxygenated aqueous solutions and the subsequent reactions of the transient peptide radicals were studied in the absence and presence of K3Fe(CN)6 as oxidant by pulse radiolysis and product analysis. Hydroxyl radicals and H atoms react with glycine anhydride and alanine anhydride by abstracting an H atom bound at C-3; there is no evidence for any other site of attack at these two peptides. The resulting radicals have pKa values of 9.8 and 10.6, respectively. In the absence of an oxidant the radicals decay by second order (2k = 7.0×108 dm3 mol-1 s-1 and 2k = 4.4×108 dm3 mol-1 s-1, resp.), the main fraction (94% of the glycine anhydride-derived radicals, 90% of the alanine anhydride-derived radicals) yielding dehydrodimers (G = 0.58 μmol J-1 and 0.56 µmol J-1 (in monomer units), resp.). A small portion however disproportionates via abstraction of a C-6-bound Η atom followed by isomerization to 2,5-dihydroxypyrazines (pKa values of the parent 2,5-dihydroxypyrazine at about 7.9 and 10.1) and subsequent addition of water to 2,5-diketo-3-hydroxypiperazines, thus indicating that the transfer of a carbon-bound hydrogen atom is prefered to the transfer of a nitrogen-bound hydrogen atom. No disproportionation products but three different dehydrodimers (G = 0.36, 0.18 and 0.04 µmol J-1 (in monomer units)) were found after irradiation of sarcosine anhydride. In this case a dose rate and solute concentration dependence of dehydrodimer formation indicates a radical-solute reaction converting part of the N-methyl radicals (21% of ‘initial’ attack) into the C-3-yl radicals. A rate constant of k = 600 ± 50 dm3 mol-1 s-1 was obtained for this reaction by measuring and computing the dehydrodimer yields as a function of dose rate and solute concentration. Thus the observed transient spectrum accounts only for about 79% of the radicals from the ‘initial’ attack at C-3. The rate of oxidation of the glycine anhydride-derived radicals by Fe(CN)6 3- reflects the pKa of the transient radical. The rate constant for oxidation of the (protonated) radical derived from glycine anhydride is: k = 1.0x 108 dm3 mol-1, the corresponding radical anion is oxidized with k = 3.1 × 108 dm3 mol-1 s-1. No change with pH was observed in the case of the alanine anhydridederived radicals (k = 7.9x 108 dm3 mol-1 s-1). In contrast to the disproportionation, oxidation by Fe(CN)6 3- leads to the removal of a proton from the heteroatom, a carbocation being the intermediate. The resulting dehydropiperazines rapidly add water to yield the corresponding 2,5-diketo-3-hydroxypiperazines (G = 0.61 μmol J-1 after oxidation of the glycine anhydride-derived radicals, G = 0.58 µmol J-1 after oxidation of the alanine anhydride-derived radicals). The radicals derived from sarcosine anhydride are readily oxidized with k = 4.0×108 dm3 mol-1 s-1, independent of pH. 1H and 13C{1H} NMR-spectroscopic and mass-spectroscopic data of the products are given.

Climatic significance of δD variations in a tropical tree species from India

The climatic significance of the stable-isotope ratio of hydrogen (δD) in tree cellulose has been demonstrated using data mainly from coniferous trees that grew in temperate climates1–4. In such regions, the mean annual δD of precipitation is linearly related to the mean annual temperature5, and trees thriving on precipita-tion preserve this temperature signal in the δD of the carbon-bound hydrogen of cellulose in their growth rings. Similar studies on tropical deciduous trees could yield useful information on climate, such as the monsoon variability over the past few centuries. However, because of the small summer-to-winter temperature con-trast, tropical trees grow throughout the year and hence do not lay down well-defined growth rings (even in the few species with clear rings, it is difficult to ascertain if they are annual6). Also, throughout the year tropical mean surface air temperatures are >20 °C, above which the linear relation between δD of precipita-tion and temperature breaks down7. Thus there is no temperature signal in the δD of precipitation for the trees to record. In spite of these problems, we report here a significant correlation (0.7) between δD variations in two individual teak trees from the west coast of India with the amount of rainfall and mean maximum temperature, as in the case of temperate trees4. We propose a mechanism whereby δD of teak tree cellulose is correlated with amount of rainfall.

2] Deuteration in protein proton magnetic resonance

Publisher Summary This chapter discusses the deuteration in protein proton magnetic resonance. There are three general patterns of deuterium labeling that in practice serve rather different functions: selective protonation in a deutero background, selective deuteration in a protio background, and random fractional deuteration, in which all carbon-bound hydrogen positions have been uniformly enriched to an intermediate level with deuterium. The chemical means of producing deuterated amino acids can be separated into the generally more facile exchange procedures, which start with the natural abundance amino acid sample, and de novo chemical synthesis. In addition, for amino acids possessing methylene positions, deuteration procedures can be separated according to whether the labeling is stereospecific. A large number of deuteration patterns are accessible for all of the standard amino acids. When combined with the metabolic genetics and protein expression systems as available for E. coli , a truly extraordinary range of deuteration experiments is practical and can allow for detailed nuclear magnetic resonance (NMR) studies that are not feasible using natural abundance samples.

Protein NMR resonance assignment by isotropic mixing experiments on random fractionally deuterated samples

The 108-residue protein E. coli thioredoxin has been uniformly enriched to 50% with deuterium at all carbon-bound hydrogen positions. Isotropic mixing (i.e. TOCSY) experiments have been conducted for both the deuterated and natural-abundance samples. Using a 54 ms mixing time correlation peaks can be seen for all four protons on the benzenoid ring of tryptophan in both samples. The deuteration results in an average decrease in cross-sectional area of a factor of 2–3 for the TOCSY cross-peaks. The cross-peak intensities for the deuterated sample systematically decrease as a function of the number of protons involved in the transfer process thus overcoming a common ambiguity in the TOCSY experiment.

NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation.

All non-proline residues except for the N-terminal dipeptide have been assigned in the 108-residue protein Escherichia coli thioredoxin. Central to these experiments has been the use of protein samples in which all carbon-bound hydrogen positions are substituted to 75% with deuterium by bacterial growth on partially deuteriated carbon sources and media. The dilution of the local proton density gives rise to narrower line widths with little loss in sensitivity. In addition, passive or secondary coupling to protons not directly involved in the coherence transfer process of correlation experiments is largely suppressed, thus significantly improving the resolution for side-chain couplings. Simultaneous multiresidue-type assignments have been obtained by incorporation of several amino acids with differing selective alpha- and/or beta-deuteriation into a fractionally deuteriated background. Combined with several single residue type labeling experiments, these selective labelings have yielded direct residue type assignments for two-thirds of the protein. In addition to improved resolution, the amide to carbon-bound proton NOESY spectra offered equivalent sensitivity while the amide to amide NOESY spectra offered superior sensitivity to that observed for natural abundance samples. The resultant sequential assignment has an average number of nearest-neighbor NOE connectivities of 2.35 out of the possible 3 alpha-amide, beta-amide, and amide-amide connectivities.

Assessing meteoric water composition and relative humidity from 18O and 2H in wood cellulose: paleoclimatic implications for southern Ontario, Canada

An empirical model is presented that derives the initial isotopic composition of water used by terrestrial plants and the relative humidity that prevailed during photosynthesis from the isotopic composition of oxygen and carbon-bound hydrogen in the plant cellulose. The model uses fixed values for the fractionation factors that describe the evapotranspirative isotopic enrichment of leaf waters and the biochemical fractionations occurring during cellulose synthesis. When applied to cellulose extracted from fossil plant matter, the model can be used to generate quantitative estimates of the temperature and moisture regimes of the past. The application of the model is demonstrated by consideration of δ 18O and δ 2H results on cellulose from fossil wood collected at Brampton, Ontario. The Brampton data yield records of systematic changes in the temperature and moisture regimes of the eastern Great Lakes region over the past 11 500 a. Both mean annual temperature and photosynthetic humidity increased substantially from values much lower than the present at the end of the Wisconsin Stage, to values higher than the present in the mid-Holocene. The phase relation of the paleotemperature and paleohumidity curves engenders differentiation of the climatic history at Brampton into four characteristic zones: (I) a period of ameliorating climate predominantly much colder and drier than now, lasting until about 7400 B.P.; (II) an “early Hypsithermal” period of warm and dry conditions, that transformed after 6000 a B.P. into (III) the “main Hypsithermal” time of warmth and pronounced moistness. Subsequent climatic detrioration led to establishment of (IV) cool, moist conditions like the present sometime within the last few thousand years.

Stable isotopic studies on chitin. III. The D/H and 18O/ 16O ratios in arthropod chitin

Stable hydrogen and oxygen isotope ratios are presented for carbon-bound hydrogen and for oxygen in chitin-derived substrates from 57 arthropod species collected in 50 different locations or grown under controlled conditions in the laboratory. No systematic isotopic differences were found among Insecta, Crustacea, and Merostomata. The determination of infra- and interindividual isotopic variabilities in a lobster and among individuals of crustacean populations yielded small variances of about ±3 per mil for δD values and ±0.3 per mil for δ 18O values. Molting stage and sex of crustaceans showed no systematic effects on isotopic composition. The δD and δ 18O values of ambient water showed only weak correlations with the respective δ values of chitin-derived substrates. Positive correlation was observed between δD values and trophic level. No temperature effects on δ 18O and δD values from marine crustaceans were found that exceed the natural isotopic noise level. Taken together, these observations indicate that reconstruction of water isotopic composition from arthropod chitin δD and δ 18O values will require specific information about the habits and habitats of the species involved in the analysis.

Climatic implications of the natural variations of D/H ratios in tree ring cellulose

Natural variations in the deuterium composition of carbon-bound hydrogen of cellulose nitrate from tree rings have been measured. Trees from Edmonton have been analysed in detail and the δD values of 5-year groups of rings show a strong temporal correlation with mean annual temperature at the growth site. This is shown to result from the cellulose isotopic composition being related to the isotopic composition of soil water used during photosynthesis. The soil water is in turn related to meteoric water which at this growth site appears to correlate temporally with mean annual temperature. Similar measurements from trees which grew in various areas of North America show a strong spatial correlation between δD of cellulose and mean annual temperature. The spatial functional relationship between δD values and mean annual temperature at the growth site ( t) is found to be δD = 5.5t - 130‰ swhich compares with the spatial relationship between δD of meteoric water and mean annual air temperature which is δD = 5.6t - 100‰.

A reexamination of cellulose carbon-bound hydrogen δD measurements and some factors affecting plant-water D/H relationships

The method of Epstein et al. (1976) for analysis of D/H ratios of cellulose carbon-bound hydrogen has been modified. This modified “renitration” method yields δD values which are in agreement with those obtained by the sodium chlorite delignification method. Comparison of results obtained by the renitration method with the published results of Epstein et al. (1976) indicate some differences in the δD values of individual samples. However, the overall plant-water δD relationship determined by Epstein et al., is not greatly changed upon redetermination by the renitration method. Additional data from a variety of plants representing a wide geographical range reveal that relative humidity is an important variable in determining the δD value of cellulose C-H hydrogen on this inter-regional scale. The role of relative humidity can be reasonably explained by a leaf water model that assumes an isotopic steady-state during transpiration. These results reaffirm the conclusion of Epstein et al., that the δD variations of the source water are the dominant control of the δD variations of cellulose C-H hydrogen from naturally grown plants. Thus, there is an expectation that these cellulose δD variations can have climate significance.

Isotopic composition of cellulose from aquatic organisms

The stable isotopic ratios of oxygen, carbon and the non-exchangeable carbon-bound hydrogen of cellulose from marine plants (algae and higher vascular forms) and animals (tunicates) collected in their natural habitats and from freshwater vascular plants grown in the laboratory under controlled conditions were determined. The δ 18 Ovalues of cellulose from all the plants and animals were 27 ±3% more positive than the δ 18 O values of the waters in which the organisms grew. Temperature had little or no influence on this relationship for three species of freshwater vascular plants that were analyzed. The relationship between the δ 18 O values of cellulose and the water used in its synthesis is probably established by the isotopic fractionation that occurs during the hydration of carbonyl groups of the intermediates involved in cellulose synthesis. The δD values of the non-exchangeable hydrogen of cellulose (determined by analyzing cellulose nitrate) from different organisms that grew in the same environment differed by large amounts. This difference ranged up to 200‰ for different species of algae collected at a single site: the corresponding difference for different species of tunicates and vascular plants was 60 and 20‰ respectively. The δD values of cellulose nitrate from different species of freshwater vascular plants grown in water of constant temperature and isotopic composition differed by as much as 60‰ The relationship between the δD values of the carbon-bound hydrogen of cellulose and the water used in its synthesis displayed a significant temperature dependence for four species of freshwater vascular plants that were analyzed. The δD values of cellulose nitrate prepared from different parts of one of the plants grown under constant conditions differed by 40‰ Hydrogen isotopic fractionation during cellulose synthesis appears to be more variable among different species and displays a larger temperature dependence than was suggested by previous studies.

The synthesis of per-C-deuterated D-glucose

A new, isotopic hydrogen-exchange technique that allows the introduction of deuterium by catalytic exchange with carbon-bound hydrogen was employed for the preparation of per-C-deuterated D-glucose.

The biosynthesis of long-chain fatty acids. Incorporation of radioactivity from stereospecifically tritiated malonyl thiol esters, and the stereochemistry of the acetyl-CoA carboxylase reaction.

1 Fatty acid synthetase was purified to homogeneity from bakers' yeast. 2 (2S,3R)-[3-3H1]Malic acid was prepared by incubation of fumarate with fumarase in tritiated water. 3 [2-3H1,3-3H1]Fumarate was synthesised via a published procedure and used for the preparation of (2S,3S)-[2-3H1,3-3H1]malate by incubation with fumarase in water. 4 The above malates after being mixed with [U-14C]malate were converted into the 4-nitrophenyl hydrogen malates esterified at the C-4 carboxyl, the C-1 carboxyl and the 2-hydroxyl group being protected at intermediate stages as a 1,3-dioxolanone. 5 The individual 4-nitrophenyl hydrogen malates were oxidised by zinc permanganate in carefully defined conditions to give 4-nitrophenyl hydrogen 2R-[U-14C, 2-3H1]- and 2S-[U-14C, 2-3H1]malonates. 6 These 4-nitrophenyl hydrogen malonates were converted to the corresponding malonyl thiol esters by transesterification with coenzyme A or N-acetylcysteamine under conditions of minimum tritium exchange and racemization. 7 The malonyl thiol esters were immediately incubated with purified fatty acid synthetase and the cofactors necessary for the biosynthesis of fatty acids. 8 The fatty acids, mainly palmitate and stearate, formed in these incubations were extracted and the 3H/14C ratio determined. Individual acids were recrystallised with carrier material or, after methylation, were separated by gas-liquid chromatography and the isotope ratio again determined. 9 In this way it was shown that palmitate or stearate derived from the 2S-[U-14C, 2-3H1] malonyl thiol ester retained 51% of the original tritium of the substrate whereas the acids derived from the 2R-[U-14C, 2-3H1]malonyl thiolester retained only 23% of the original tritium. 2RS-[U-14C, 2-3H1] substrates gave an intermediate result. 10 From a comparison of these results with those obtained previously using chiral acetate substrates it is deduced that carboxylation of acetyl-CoA catalysed by the chicken liver acetyl-CoA carboxylase occurs with retention of configuration at C-2. The results are shown to be in quantitative agreement with the theory, deduced from experiments with chiral acetates, that partial exchange of carbon-bound hydrogen occurs on the synthetase at some stage between malonate and fatty acid subsequent to the stereospecific elimination of hydrogen. 11 The stereochemical course of individual reactions in the biosynthesis of fatty acids is discussed.

The Biosynthesis of Long-Chain Fatty Acids. Stereochemical Differentiation in the Enzymic Incorporation of Chiral Acetates

1 Acetyl-CoA carboxylase and fatty acid synthetase were purified from chicken liver. Fatty acid synthetase was also purified from bakers' yeast. 2 The corresponding CoA thiol esters were prepared enzymically from R-[2H1, 3H1]acetate, S-[2H1, 3H1]acetate and [3H1]acetate, using a linked acetate kinase/phosphotransacetylase system. [2-14C]Acetate was added to the tritiated specimens to provide doubly-labelled products, which were purified by column chromatography on DEAE-cellulose. 3 The three acetyl-CoA specimens thus obtained were incubated separately in a combined acetyl-CoA carboxylase/fatty acid synthetase system containing the cofactors necessary for synthesis of long-chain fatty acids. 4 The fatty acids formed were extracted into n-pentane, methylated, separated by gas-liquid chromatography and the 3H/14C isotope ratio determined. 5 In this manner it was shown, using the chicken liver synthetase, that in palmitic acid a higher proportion of tritium was retained from S-[2-14C, 2H1, 3H1]acetyl-CoA than from the R-(2-14C, 2H1, 3H1)-labelled thiol ester, the non-chiral (2-14C, 3H1)-labelled substrate giving an intermediate result. 6 This result was confirmed using the fatty acid synthetase preparation purified from bakers' yeast in place of the corresponding chicken liver enzyme. 7 In a separate experiment it was shown that the intramolecular tritium isotope effect in the carboxylation of [3H1]acetyl-CoA is normal, but small. The slightly smaller, normal deuterium isotope effect calculated from this was shown to be in good agreement with the value expected, based on the experiments on tritium incorporation from chiral acetyl-CoA. 8 It is demonstrated that partial exchange of carbon-bound hydrogen must occur on the synthetase during the enzymic synthesis of fatty acids from malonyl thiol esters and that the extent of this exchange differs in synthetases of different origin. 9 A theoretical treatment of the effect of partial hydrogen exchange on the chirality of asymmetrically labelled methylene groups is given. 10 The results indicate the existence of an overall stereospecificity in the reactions involved in the de novo biosynthesis of long-chain fatty acids in different cell types.

The determination of the D/H ratio of non-exchangeable hydrogen in cellulose extracted from aquatic and land plants

A method has been developed for the analysis of D/H ratios of non-exchangeable hydrogen in plant cellulose. Plant samples are nitrated at low temperature and pure cellulose nitrate is extracted by acetone dissolution. Tests of this nitrated product have demonstrated that the nitration-extraction procedure eliminates the OH hydrogen and does not alter the D/H ratio of the cellulose carbon-bound hydrogen. Significant differences exist between δD values of plant total hydrogen and δD values of cellulose nitrate hydrogen. This difference is due to the effect of chemical heterogeneity of the δD value of plant material. Plant-extracted cellulose nitrate D/H ratios are systematically related to the D/H ratios of the associated environmental water. The overall relationship is linear with slope of one and intercept of −22%. Five aquatic plants which grew at 16–17°C are related isotopically to the water by a linear curve with a slope of 1 and intercept of −36%. Three plants which grew at 28–29°C have an intercept of −11%. The general dependence of plant cellulose non-exchangeable hydrogen D/H ratios on the D/H ratios of the associated environmental water suggests that variations of the extracted cellulose nitrate δD values of plants can be used as indicators of climatic change.

Kinetics of hydrogen isotope exchange reactions - XXVIII. Steric course of radiation-induced tritium exchange between water and cyclohexane-1, 2-diols

Under the influence of tritium β-radiation, dissolved trans -cyclohexane- 1, 2-diol exchanges carbon-bound hydrogen with the solvent (tritiated water). The exchange occurs not only at the hydroxyl-substituted (α) carbon atoms but also at non-α positions, which are, on the average, less reactive. The overall G -value for exchange in all positions is ca . 0.33. With γ-irradiation, exchange takes place with a slightly lower overall G -value. Exchange at α-positions occurs with predominant retention of con­figuration (76%). In the corresponding α-exchange of cis -cyclohexane-1, 2-diol, 79% of the reaction proceeds with inversion, i. e. the labelled products are in both reactions formed in practically the same ratio ( trans -diol: cis -diol ⋍ 3.5:1). It is concluded that the reactions go through a common intermediate (or rapidly equilibrating common set of inter­mediates), for both diols as substrates. A chemically reasonable mechanism in accordance with this requirement involves as the first step hydrogen abstraction (mainly by hydroxyl radicals) from a C—H grouping, followed by conversion of the organic radical produced to the carbanion by electron capture. The carbanion then reverts to a diol by protonation (tritonation). Equilibration of diastereomers can occur in the radical and/or in the carbanion. The involvement of both the hydroxyl radical and the solvated electron is qualitatively in accord with the action of electron and radical scavengers on the velocity of the exchange reaction.

Radiolysis of estrone and estradiol.

Gamma irradiation of estrone and estradiol in 1 Naqueous sodium hydroxide results in the formation of 2-hydroxyestrone and 2-hydroxyestradiol, respectively. Estrolactone (16a-oxa-D-homoestrone) was not encountered.