Graham Eaton

Biosynthesis of podophyllotoxin in Linum album cell cultures

Abstract. Cell cultures of Linum album Kotschy ex Boiss. (Linaceae) showing high accumulation of the lignan podophyllotoxin (PTOX) were established. Enzymological studies revealed highest activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-hydroxycinnamate:CoA ligase and cinnamoyl-CoA:NADP oxidoreductase immediately prior to PTOX accumulation. To investigate PTOX biosynthesis, feeding experiments were performed with [2-13C]3′,4′-dimethoxycinnamic acid, [2-13C]3′,4′-methylenedioxycinnamic acid (MDCA), [2-13C]3′,4′,5′-trimethoxycinnamic acid, [2-13C]sinapic acid, [2-13C]- and [2,3-13C2]ferulic acid. Analysis of the metabolites by HPLC coupled to tandem mass spectrometry revealed incorporation of label from ferulic acid into PTOX and deoxypodophyllotoxin (DOP). In addition, MDCA was also unambiguously incorporated intact into PTOX. These observations suggest that in L. album both ferulic acid and methylenedioxy-substituted cinnamic acid can be incorporated into lignans. Furthermore, it appears that, in this species, the hydroxylation of DOP is a rate-limiting point in the pathway leading to PTOX.

Reassessment of the Reaction Mechanism in the Heme Dioxygenases

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are heme enzymes that catalyze the O(2)-dependent oxidation of L-tryptophan to N-formyl-kynurenine. Previous proposals for the mechanism of this reaction have suggested that deprotonation of the indole NH group, either by an active-site base or by oxygen bound to the heme iron, as the initial step. In this work, we have examined the activity of 1-Me-L-Trp with three different heme dioxygenases and their site-directed variants. We find, in contrast to previous work, that 1-Me-L-Trp is a substrate for the heme dioxygenase enzymes. These observations suggest that deprotonation of the indole N(1) is not essential for catalysis, and an alternative reaction mechanism, based on the known chemistry of indoles, is presented.

Spectroscopic studies of the solvation of amides with N—H groups. Part 1.—The carbonyl group

Infrared shifts of the CO stretch (νco) band, and n.m.r. shifts for the 13CO carbon have been studied for formamide, acetamide, N-methyl formamide and N-methyl acetamide for dilute solutions in a range of pure and mixed solvents. The results are compared with those previously reported for N,N-dimethylamides in the same systems. There are good linear relationships between Δν(13C) and νco for the pure solvent systems, provided allowance is made for the presence of two types of solvate for methanol. For mixed methanol-aprotic solvents (B) the low-frequency (νco) component for pure methanol was lost as the concentration of B was increased. The high-frequency band initially gained intensity, but this was ultimately replaced by a third band characteristic of the amide in pure B. These results suggest that the CO group forms both one and two hydrogen bonds in methanol. Aqueous solutions have a single νco band close to that for the disolvate in methanol. As [B] was increased, this gave way to a band close to that for the mono-solvate, which again was steadily replaced by the non-hydrogen-bonded form. Hence it is concluded that for all the amides, the di-hydrogen-bonded species dominates in water. Reasons for the different behaviour in methanol and water are discussed.In all cases, as well as the gain and loss of i.r. bands, those assigned to the hydrogen-bonded units shifted considerably as [B] increased. For aqueous systems these shifts are assigned to changes in secondary solvation.We have looked for specific differences between the results for the dimethyl derivatives and the present compounds, in the expectation that N—H solvation would have a discernible affect on CO solvation. There are small differences, but these are not systematic on going from RCONMe2via RCONHMe to RCONH2, and it is concluded that cooperativity effects involving CO and N—H solvation are small compared with those for water and alcohols. The 13C resonance (13CO) shifted systematically for mixed protic–aprotic solvent systems. Using the i.r.–n.m.r. correlation and the intensity changes for the νCO bands, reasonable predictions of these n.m.r. shifts were obtained.Marked changes in the amide II band were also observed, but these are less readily interpreted. Studies of the overtone infrared spectra for aqueous solutions reveal the presence of (OH)free and (NH)free bands, showing that the N—H groups are not fully hydrogen-bonded in water despite the effect of two bonds to the carbonyl group.

Spectroscopic studies of the solvation of N,N-dimethyl amides in pure and mixed solvents

The i.r. spectra of dilute solutions of N,N-dimethylformamide (DMF) and N,N-dimethylacetamide (DMA) have been studied in the >CO stretching region using a range of pure and binary mixed solvents in which the ‘probe’ amide concentration was kept as low as possible. Shifts of the 13C carbonyl resonance [Δv(13C)] were measured under identical conditions. A linear relationship between vmax(i.r.) and Δv(13C) exists for most pure solvents. For solvents in which two i.r. bands were observed, the correlation was obeyed when the weighted mean of these i.r. bands was used. For mixed protic–aprotic solvents the i.r. spectra displayed two discrete bands. By studying the complete mole-fraction range it was possible to assign specific bands to specific hydrogen-bonded amide units. Bulk water forms two hydrogen bonds to the carbonyl oxygen atom and addition of basic aprotic solvents such as dimethyl sulphoxide (DMSO) resulted in the sequential loss of these water molecules together with relatively minor shifts for the di- and mono-solvate bands. Using weighted averages of the mixed-solvent spectra together with the i.r.–n.m.r. correlation the 13C shifts for the mixed-solvent system water-cyanomethane over the whole mole-fraction range have been interpreted. For alcohol solvents two i.r. bands were detected, assigned to mono- and di-solvates on the basis of mixed-solvent studies. The frequencies for these bands are close to those for the mono- and di-hydrate bands, suggesting that the strengths of the corresponding hydrogen bonds are comparable. These results are compared with those from our previous study of the CO stretch and 13C(O) n.m.r. spectra for acetone. There is a linear correlation, but the shifts for both functions are greater for the amides. Bands assigned to individual solvates shift to low frequencies as the concentration of protic solvent increases. Limiting shifts, estimated for the units amide–––HOH–––B, where B is a basic solvent, are a function of the hydrogen-bond basicity of B, being at high frequencies for strong bases and low frequencies for weak bases. This is explained in terms of an ‘anti-cooperativity’ effect for water. Measurements of 2v(OH) and 3v(OH) for HOD in D2O on the addition of these amides showed a fall in intensity in the band assigned to (OH)free groups. By comparing this fall with that induced by triethylphosphine oxide (solvation number 3) we obtain a solvation number close to 2 for aqueous amides, in good agreement with the CO stretch and n.m.r. studies. These combined results strongly support the concept of dibonding to oxygen rather than bonding at oxygen and nitrogen, as has been recently suggested for matrix-isolated systems.

Solvation of cyanoalkanes [CH3CN and (CH3)3CCN]. An infrared and nuclear magnetic resonance study

The CN stretching band (ν2) has been studied for dilute solutions of CH3CN and (CH3)3CCN in a range of aprotic and protic solvents. The former induce a low-frequency shift, whereas the latter induce a high-frequency shift relative to dilute solutions in hexane. In both cases there is a large increase in oscillator strength with increasing shift. This is the first example of a solvent that has an absorption band displaying such a dichotomy, the normal behaviour being a progressive low-frequency shift on going via aprotic to protic media. In contrast such ‘normal’ behaviour is observed in the n.m.r. spectra for 14N shifts, but the 13C (CN) shifts are small and seem to be random. In contrast to our previous studies of ‘probe’ molecules the ν2 bands for solutions in water are almost identical to one of the bands in methanol; however, the band in water is a single feature, whilst that in methanol is a doublet, the low-frequency feature being close to the unsolvated region. The interpretation is that MeCN in water is fully monosolvated (hydrogen-bonded), whilst in MeOH it is only ca. 50% monosolvated. However, the effects of temperature changes and studies of mixed water–aprotic solvent systems suggest that this may not be correct, and the possibility that MeCN forms two very weak hydrogen bonds in water is also considered.The methanol doublets are well defined at low temperatures (–50 °C) but resolution is lost on warming. At ca. 50 °C there is only one symmetrical band. For mixed water–aprotic solvent systems, the band remains a narrow singlet throughout the whole mole fraction range, there being no indication of twin bands for hydrogen-bonded and non-hydrogen-bonded units, in contrast with the results for methanol at low temperatures, and our normal experience with other probe molecules. One explanation is that there is rapid equilibrium between hydrogen-bonded and non-hydrogen-bonded units which is fast on the i.r. timescale. Results for other mixed-solvent systems are also reported.We have attempted to use changes in the first and second overtone O—H stretch bands for HOD in D2O on adding MeCN to obtain a measure of the number of hydrogen bonds. This is at best only qualitative because of the proximity of the O—H band for solvated MeCN and the ‘(OH)free’ band for water. However, for MeCN in Me3COH the (OH)free and OH---(NCMe) bands are clearly resolved. The results support the concept that MeCN is dihydrated in water. A less extensive study has been made for 2-cyano-2-methylpropane (Me3CCN) for comparative purposes. The results are broadly similar, the major difference being a reduction in the extent of hydrogen bonding in methanolic solutions.

The Mechanism of Formation of N-Formylkynurenine by Heme Dioxygenases

Heme dioxygenases catalyze the oxidation of l-tryptophan to N-formylkynurenine (NFK), the first and rate-limiting step in tryptophan catabolism. Although recent progress has been made on early stages in the mechanism, there is currently no experimental data on the mechanism of product (NFK) formation. In this work, we have used mass spectrometry to examine product formation in a number of dioxygenases. In addition to NFK formation (m/z = 237), the data identify a species (m/z = 221) that is consistent with insertion of a single atom of oxygen into the substrate during O2-driven turnover. The fragmentation pattern for this m/z = 221 species is consistent with a cyclic amino acetal structure; independent chemical synthesis of the 3a-hydroxypyrroloindole-2-carboxylic acid compound is in agreement with this assignment. Labeling experiments with 18O2 confirm the origin of the oxygen atom as arising from O2-dependent turnover. These data suggest that the dioxygenases use a ring-opening mechanism during NFK formation, rather than Criegee or dioxetane mechanisms as previously proposed.

Spectroscopic and molecular dynamics studies of solvation of cyanomethane and cyanide ions

Infrared (C—N stretch) and n.m.r (13C, 14N) methods have been used to probe the solvation of MeCN molecules and CN– ions in water, methanol and a range of mixed protic-aprotic media. For MeCN, the i.r. results are unusual in that protic solvents induce a high-frequency shift in νmax(CN), whereas dipolar aprotic solvents give a low-frequency shift. The interpretation given of this is that both σ- and π-electron interactions play an important role in hydrogen-bond formation, whereas π interactions are dominant for dipolar solvents such as Me2SO. The 13C(CN) shifts are too small to give useful information, but the 14N shifts correlate well with the i.r. results. It is argued that in methanolic solution MeCN forms only one hydrogen bond and that ca. 50% of the molecules are not hydrogen bonded. In water, all MeCN molecules are hydrogen bonded. The results do not distinguish between mono- and di-bonding, but other evidence suggests that di-bonding is likely to occur. Similar experiments on CN– ions show again that protic solvents induce a high-frequency shift. Mixed-solvent studies suggest that at least four methanol or water molecules are normally hydrogen bonded to the ions; this is in accord with other spectroscopic results on the same solvents. The experiments have been complemented by a series of molecular dynamics simulations on dilute solutions of MeCN and CN– in water and methanol. The predicted solvation numbers from the simulations are in good agreement with those derived from the experimental data, particularly for MeCN. There are some discrepancies for CN–, but these may in part be attributable to difficulties involved in finding an acceptable definition of solvation number in cases where this quantity is large.

Polyoxygenated cyclohexene derivatives from Ellipeiopsis cherrevensis

Aerial parts of Ellipeiopsis cherrevensis contained the polyoxygenated cyclohexenes zeylenol, ferrudiol and three analogs, ellipeiopsols A, B and C. The C-1 stereochemistry of ferrudiol has been revised.

Micelle reorganisation characterised by differential scanning calorimetry for aqueous solutions containing hexadecyltrimethylammonium bromide and tetradecyltrimethylammonium bromide

Over a small concentration range in the 10–2 mol dm–3 region, the differential isobaric heat capacities δCp(sln; T) of aqueous solutions containing hexadecyltrimethylammonium bromide (CTAB) show extrema near 310 K when measured as a function of temperature over the range 209 ⩽T/K⩽ 360. The temperatures corresponding to maxima in δCp(sln; T) depend on the concentration of CTAB(aq). Similar extrema are observed for CTAB solutions in D2O, but in aqueous solutions the extrema become less intense when either hexanol or KBr is added, the maxima being completely lost in the latter case. A pattern in δCp(sln; T) similar to that shown by CTAB(aq) is observed for aqueous solutions containing tetradecyltrimethylammonium bromide (TDTAB). At fixed total concentration, 0.02 mol dm–3, the temperatures at maxima in δCp(sln; T) show a simple dependence on the molar ratio CTAB to TDTAB in mixed solutions of the two surfactants. The dependence on temperature of the relaxational (configurational) contribution of the solute Cpm(sln; config) to the molar heat capacity of the solution, ΔCpm(sln; T) is analysed in terms of both single and coupled equilibria. The results are discussed in terms of changes in the shape of the micelles and in the extent of counterion binding resulting from an increase in temperature.

Solvation of acetone in protic and aprotic solvents and binary solvent mixtures

The infrared spectra of dilute solutions of acetone in a range of protic and aprotic solvents have been measured in the C—O stretch region. Band maxima are shown to correlate linearly with 13C n.m.r. shifts for the carbonyl carbon and, after suitable corrections, with solvent acceptor numbers. In mixed solvent systems involving protic and aprotic solvents, bands are gained and lost, indicative of the presence of discrete solvates. We infer that acetone in methanol is ca. 90% monohydrogen-bonded and ca. 10% non-hydrogen bonded. As basic aprotic cosolvents are added, the former species is lost and the latter gained, the efficiencies of these changes being proportional to the donor numbers (base strengths) of the cosolvents (cyanomethane, dimethyl sulphoxide, hexamethylphosphoramide and triethylamine).For systems involving water + basic aprotic cosolvents, three distinct species were detected. We suggest that the species present in pure water is acetone with two hydrogen bonds to water, that dominating in the middle range, with νmax close to that for methanolic solutions, has one hydrogen bond and the third species forms no such bonds. The overall tendency to dehydrate is again proportional to the donor number of the cosolvent except in the water-rich region, where the solvation number of the cosolvent is thought to be the overriding factor.Small shifts in the band maxima (νmax) for these discrete species with change in solvent composition are discussed in terms of secondary solvation and specific interactions between acetone and aprotic solvent molecules. The latter is particularly marked for methanol + dimethyl sulphoxide systems.Attempts are made to justify our assignments of the species detected in protic solvents to specifically hydrogen-bonded units. In particular, our suggestion that acetone is completely present as doubly hydrogen-bonded units in dilute aqueous solutions despite the evidence that the resulting hydrogen bonds are weaker than the average water–water bonds is defended.