Theacrine (1,3,7,9-tetramethyluric acid) synthesis in leaves of a Chinese tea,kucha (Camellia assamica var. kucha)

Theacrine (1,3,7,9-tetramethyluric acid) and caffeine were the major purine alkaloids in the leaves of an unusual Chinese tea known as kucha (Camellia assamica var. kucha). Endogenous levels of theacrine and caffeine in expanding buds and young leaves were ca. 2.8 and 0.6-2.7% of the dry wt, respectively, but the concentrations were lower in the mature leaves. Radioactivity from S-adenosyl-L-[methyl-14C]methionine was incorporated into theacrine as well as theobromine and caffeine by leaf disks of kucha, indicating that S-adenosyl-L-methionine acts as the methyl donor not only for caffeine biosynthesis but also for theacrine production. [8-14C]Caffeine was converted to theacrine by kucha leaves with highest incorporation occurring in expanding buds. When [8-14C]adenosine, the most effective purine precursor for caffeine biosynthesis in tea (Camellia sinensis), was incubated with young kucha leaves for 24 h, up to 1% of total radioactivity was recovered in theacrine. However, pulse-chase experiments with [8-14C]adenosine demonstrated much more extensive incorporation of label into caffeine than theacrine, possibly because of dilution of [14C]caffeine produced by the large endogenous caffeine pool. These results indicate that in kucha leaves theacrine is synthesized from caffeine in what is probably a three-step pathway with 1,3,7-methyluric acid acting an intermediate. This is a first demonstration that theacrine is synthesized from adenosine via caffeine.     

Metabolism of purine bases, nucleosides and alkaloids in theobromine-forming Theobroma cacao leaves

We examined the purine alkaloid content and purine metabolism in cacao (Theobroma cacao L.) plant leaves at various ages: young small leaves (stage I), developing intermediate size leaves (stage II), fully developed leaves (stage III) from flush shoots, and aged leaves (stage IV) from 1-year-old shoots. The major purine alkaloid in stage I leaves was theobromine (4.5 μmol g^–1 fresh weight), followed by caffeine (0.75 μmol g^–1 fresh weight). More than 75% of purine alkaloids disappeared with subsequent leaf development (stages II–IV). In stage I leaves, ¹⁴C-labelled adenine, adenosine, guanine, guanosine, hypoxanthine and inosine were converted to salvage products (nucleotides and nucleic acids), to degradation products (ureides and CO₂) and to purine alkaloids (3- and 7-methylxanthine, 7-methylxanthosine and theobromine). In contrast, ¹⁴C-labelled xanthine and xanthosine were not used for nucleotide synthesis. They were completely degraded, but nearly 20% of [8-¹⁴C]Xanthosine was converted in stage I leaves to purine alkaloids. These observations are consistent with the following biosynthetic pathways for theobromine: (a) AMP → IMP → 5′-xanthosine monophosphate → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (b) GMP → guanosine → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (c) xanthine → 3-methylxanthine → theobromine. Although no caffeine biosynthesis from ¹⁴C-labelled purine bases and nucleosides was observed during 18 h incubations, exogenously supplied [8-¹⁴C]Theobromine was converted to caffeine in young leaves. Conversion of theobromine to caffeine may, therefore, be slow in cacao leaves. No purine alkaloid synthesis was observed in the subsequent growth stages (stages II–IV). Significant degradation of purine alkaloids was found in leaves of stages II and III, in which [8-¹⁴C]Theobromine was degraded to CO₂ via 3-methylxanthine, xanthine and allantoic acid. [8-¹⁴C]Caffeine was catabolised to CO₂ via theophylline (1,3-dimethylxanthine) or theobromine.     

The biosynthesis of caffeine in the coffee plant

Leaf disks of Coffea arabica were infiltrated simultaneously with L-methionine-(methyl-¹⁴C) and with various possible precursors of caffeine biosynthesis. The results permit the identification of theobromine, 7-methylxanthine and 7-methylxanthosine as precursors of caffeine. 7-methylguanosine seems not to be an intermediate in caffeine formation.     

Biosynthesis and metabolism of purine alkaloids in leaves of cocoa tea (Camellia ptilophylla)

The biosynthesis and metabolism of purine alkaloids in leaves ofCamellia ptilophylla (cocoa tea), a new tea resource in China, have been investigated. The major purine alkaloid was theobromine, with theophylline also being present as a minor component. Caffeine was not accumulated in detectable quantities. Theobromine was synthesized from [8-¹⁴C] adenine and the rate of its biosynthesis in the segments from young and mature leaves from flush shoots was approximately 10 times higher than that from aged leaves from 1-year old shoots. Neither cellfree extracts nor segments fromC. ptilophylla leaves could convert theobromine to caffeine. A large quantity of [2-¹⁴C] xanthine taken up by the leaf segments was degraded to¹⁴CO₂ via the conventional purine catabolic pathway that includes allantoin as an intermediate. However, small amounts of [2-¹⁴C] xanthine were also converted to theobromine. Considerable amounts of [8-¹⁴C] caffeine exogenously supplied to the leaf segments ofC. ptilophylla was changed to theobromine. These results indicate that leaves ofC. ptilophylla exhibit unusual purine alkaloid metabolism as i) they have the capacity to synthesize theobromine from adenine nucleotides, but they lack adequate methyltransferase activity to convert of theobromine to caffeine in detectable quantities, ii) the leaves have a capacity to convert xanthine to theobromine, probably via 3-methylxanthine.     

Caffeine biosynthesis in Camellia sinensis

The metabolism of adenine and guanine, relating to the biosynthesis of caffeine, in excised shoot tips of tea was studied with micromolar amounts of adenine-[8-¹⁴C] or guanine-[8-¹⁴C]. Among the presumed precursors of caffeine biosynthesis, adenine was the most effective, whereas guanine was the least effective. After administration of a ‘pulse’ of adenine-[8-¹⁴C], almost all of the adenine-[¹⁴C] supplied disappeared by 30 hr, and ¹⁴C-labelled caffeine and RNA purine nucleotide (AMP and GMP) synthesis increased throughout the experimental period, whereas the radioactivities of free purine nucleotides, 7-methylxanthine and theobromine increased during the first 10 hr incubation period, followed by a steady decrease. By contrast, more than 45% of the guanine-[8-¹⁴C] supplied remained unchanged even after a 120 hr period. The main products of guanine-[8-¹⁴C] metabolism in tea shoot tips were guanine nucleotides, theobromine, caffeine and the GMP of RNA. The results support the hypothesis that the purine nucleotides are synthesized from adenine and guanine via the pathway of purine salvage. Adenylate is readily converted into other purine nucleotides, whereas the conversion rate of guanylate into other purine nucleotides is very low.The results also support the view that 7-methylxanthine and theobromine are precursors of caffeine. For the origin of the purine ring in caffeine, purine nucleotides in the nucleotide pool rather than in nucleic acids are suggested.     

N-methyltransferases and 7-methyl-N9-nucleoside hydrolase activity in Coffea arabica and the biosynthesis of caffeine

The incorporation of radioactivity from L-[¹⁴CH₃]-methionine into caffeine by coffee fruits was enhanced by additions of theobromine and paraxanthine but was reduced by additions of theophylline and caffeine. Cell-free extracts prepared from seedlings, partially ripe and unripe coffee fruits showed that only the unripe green fruits contained significant methyltransferase and 7-methyl-N⁹-nucleoside hydrolase activity. The cell-free extracts catalysed the transfer of methyl groups fromS-adenosyl-L-[¹⁴CH₃]-methionine to 7-methylxanthine, and 7-methylxanthosine, producing theobromine and to theobromine producing caffeine. The two enzymic methylations exhibited a sharp pH max at 8.5 and a similar pattern of effects with metal chelators, thiol reagents and Mg²⁺ ions, which were slightly stimulating though not essential to enzyme activity. Paraxanthine (1,7-dimethylxanthine) was sh own to be the most active among methylxanthines as methyl acceptors; however its formation from 1-methylxanthine and 7-methylxanthine was not detectable, and biosynthesis from paraxanthine in the intact plant would therefore appear not to occur. The apparent K_m values are as follows: 7-methylxanthine 0.2 mM, theobromine 0.2 mM, paraxanthine 0.07 mM and S-adenosyl-L-methionine with each substrate 0.01 mM. The results suggest the pathway for caffeine biosynthesis in Coffea arabica is: 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine.     

Catabolism of caffeine and related purine alkaloids in leaves ofCoffea arabica L.

In a study of purine alkaloid catabolism pathways in coffee,¹⁴C-labelled theobromine, caffeine, theophylline and xanthine were incubated with leaves ofCoffea arabica. Incorporation of label into¹⁴CO₂ was determined and methanol-soluble metabolites were analysed by high-performance liquid chromatography-radiocounting. The data obtained demonstrate catabolism of caffeine theophylline 3-methylxanthine xanthine. Xanthine is degraded further by the conventional purine catabolism pathway to CO₂ and NH₃ via uric acid, allantoin and allantoic acid. The conversion of caffeine to theophylline is the rate-limiting step in purine alkaloid catabolism and provides a ready explanation for the high concentration of endogenous caffeine found inC. arabica leaves. Although theobromine is converted primarily to caffeine, a small portion of the theobromine pool appears to be degraded to xanthine by a caffeine-independent pathway. In addition to being broken down to CO₂, via the purine catabolism pathway, xanthine is metabolised to 7-methylxanthine. Metabolism of [2-¹⁴C]xanthine byC. arabica leaves in the presence of 5 mM allopurinol results in very large increases in incorporation of radioactivity into 7-methylxanthine as degradation of the substrate via the purine catabolism pathway is blocked. The identity of 7-methylxanthine in these studies was confirmed by gas chromatography-mass spectrometry analysis.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting This work was supported by the British Council which provided H.A. with Japan-UK travel grants. F.M.G. was supported by a Biotechnology and Biological Sciences Research Council grant to A.C.     

Caffeine biosynthesis and adenine metabolism in transgenic Coffea canephora plants with reduced expression of N-methyltransferase genes

In anti-sense and RNA interference transgenic plants of Coffea canephora in which the expression of CaMXMT1 was suppressed, caffeine biosynthesis from [8-(14)C]adenine was investigated, together with the overall metabolism of [8-(14)C]adenine. Compared with wild type control plants, total purine alkaloid biosynthesis from adenine and conversion of theobromine to caffeine were both reduced in the transgenic plants. As found previously, [8-(14)C]adenine was metabolised to salvage products (nucleotides and RNA), to degradation products (ureides and CO(2)) and to purine alkaloids (theobromine and caffeine). In the transgenic plants, metabolism of [8-(14)C]adenine shifted from purine alkaloid synthesis to purine catabolism or salvage for nucleotides. HPLC analysis revealed a significantly reduced caffeine content in the transgenic plants. A small quantity (less than 20 nmol g(-1) fresh weight) of xanthosine had accumulated in at least one of the transgenic plants.     

Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production

Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g. tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed.     

O(2),1,9-trimethyluric acid and 1,3,7,9-tetramethyluric acid in leaves of different Coffea species

O(2),1,9-Trimethyluric acid and 1,3,7,9-tetramethyluric acid were isolated from young leaves of Coffea liberica, C. arnoldiana, C. dewevrei var. excelsa and var. aruwimiensis. The first purine has not been found before in nature; its identification required the synthesis of nearly all of its possible isomers.     

Biosynthesis, accumulation and degradation of theobromine in developing Theobroma cacao fruits

We have studied the purine alkaloid content and purine metabolism in Theobroma cacao fruits at differing growth stages: Stage A (young small fruit, fresh weight, ca. 2 g); stage B (medium size fruit, fresh weight, ca. 100 g) and stage C (large size, fresh weight, ca. 500 g). The major purine alkaloid in stage A fruits (mainly pericarp) was theobromine (0.7 micromol g(-1) fresh weight), followed by caffeine (0.09 micromol g(-1) fresh weight). The theobromine content of the pericarp decreased sharply with tissue age, and the caffeine content decreased gradually. A large amount of theobromine (22 micromol g(-1) fresh weight) had accumulated in seeds (mainly cotyledons) of stage C fruits. Theobromine was found also in the seed coat and placenta. Tracer experiments with [8-(14)C]adenine show that the major sites of theobromine synthesis are the young pericarp and cotyledons of T. cacao fruits. Limited amounts of purine alkaloids may be transported from the pericarp to seed tissue, but most purine alkaloids that accumulated in seeds appeared to be synthesised in cotyledons. Degradation of [8-(14)C]theobromine and [8-(14)C]caffeine to CO2 via 3-methylxanthine and ureides (allantoin and allantoic acid) was detected only in the pericarp of stage C fruits.     

Purine alkaloid formation and CO2 gas exchange in dependence of development and of environmental factors in leaves of Coffea arabica L.

In the leaves of Coffea arabica L., purine alkaloid formation was estimated by analyzing the theobromine and caffeine content and by measuring the methylation rate of [2-¹⁴C]theobromine to [2-¹⁴C]caffeine in short-term experiments (6–24 h). At the same time, growth (in terms of dry weight and area), net photosynthesis (NPS), and dark respiration were determined. During leaf development, which was considered to be terminated when NPS was at a maximum (60–80 mol g^-1 s^-1) and dark respiration at a minimum (5–7.5 mol g^-1 s^-1), the content of theobromine and the velocity of caffeine formation were both found to decrease by a factor of more than 100. The close correlation between the theobromine content and the methylation rate is suspended when purine alkaloid formation is influenced by factors other than leaf development. Among these factors, temperature is the most effective: the velocity of caffeine biosynthesis is increased by raising the temperature and vice versa. Although the plants were well irrigated, a drastic decrease of NPS in the afternoon was observed under all environmental conditions tested. Light saturation was reached between 170–360 mol m^-2 s^-1. The temperature optimum of NPS was shown to be very broad (24–33°C)m provided the adaptation time was sufficiently long.Abbreviations MR methylation rate - NPS net photosynthesis - RMC relative methylation coefficient Dedicated to Professor Hans Wanner, as promoter of these investigations, on occasion of his 65th birthday     

Biosynthesis of Caffeine in Leaves of Coffee

The levels of endogenous caffeine and theobromine were much higher in buds and young leaves of Coffea arabica L. cv Kent than in fully developed leaves. Biosynthesis of caffeine from 14C-labeled adenine, guanine, xanthosine, and theobromine was observed, whereas other studies (H. Ashihara, A.M. Monteiro, T. Moritz, F.M. Gillies, A. Crozier [1996] Planta 198: 334-339) have indicated that there is no detectable incorporation of label into caffeine when theophylline and xanthine are used as substrates for in vivo feeds with leaves of C. arabica. The capacity for caffeine biosynthesis, especially from guanine and xanthosine, was reduced markedly in both fully developed mature and aged leaves. Data obtained in pulse-chase experiments with young leaves indicate the operation of an AMP -> IMP -> xanthosine 5[prime]-monophosphate (or GMP -> guanosine) -> xanthosine -> 7-methylxanthosine -> 7-methylxanthine -> theobromine -> caffeine pathway. The data obtained provide strong evidence against proposals by G.M. Nazario and C.J. Lovatt ([1993] Plant Physiol 103: 1203-1210) concerning the independence of caffeine and theobromine biosynthesis pathways and the role of xanthine as a key intermediate in caffeine biosynthesis.     

Lipid Composition and Metabolism of Volvox carteri

The membrane structural lipids of somatic cells and gonidia isolated from Volvox carteri f. nagariensis spheroids have been characterized. The principal polar lipid components of both cell types are sulfoquinovosyl diglyceride, mono- and digalactosyl diglyceride, phosphatidylglycerol, phosphatidylethanolamine, and 1(3), 2-diacylglyceryl-(3)-O-4′-(N,N,N,-trimethyl)homoserine. Light-synchronized cultures of spheroids were shown to incorporate [¹⁴C]bicarbonate, [³⁵S]sulfate, [¹⁴C]palmitic acid, and [¹⁴C]lauric acid into complex lipids. [¹⁴C]Palmitic acid was incorporated mainly into diacylglyceryltrimethylhomoserine and was not significantly modified by elongation or desaturation. In contrast, [¹⁴C]lauric acid was incorporated into a wider variety of complex lipids and was also converted into longer chain saturated and unsaturated fatty acids. Volvox is a promising system for studying the role of membranes in algal cellular differentiation.     

Intracellular formation of analogues of cyclic AMP: Studies with brain slices labeled with radioactive derivatives of adenine and adenosine

A variety of radioactive analogs of adenine and adenosine were incubated with guinea pig cerebral cortical slices. Neither 1,N⁶-ethano[¹⁴C]adenosine nor 1,N⁶-ethanol[¹⁴C]adenine were significantly incorporated into intracellular nucleotides. 2-chloro[8-³H]adenine was incorporated, but at a very low rate and conclusive evidence for the formation of intracellular radioactive 2-chlorocyclic AMP was not obtained. N⁶-Benzyl[¹⁴C]adenosine was converted only to intracellular monophosphates and significant formation of radioactive N⁶-benzylcyclic AMP was not detected during a subsequent incubation. 2′-Deoxy-[8-¹⁴C] adenosine was converted to both intracellular radioactive 2′-deoxyadenine nucleotides and radioactive adenine nucleotides. Stimulation of these labeled slices with a variety of agents resulted in formation of both radioactive 2′-deoxycyclic AMP and cyclic AMP. Investigation of the effect of various other compounds on uptake of adenine or adenosine suggested that certain other adenosine analogs might serve as precursors of abnormal cyclic nucleotides in intact cells.     

Biosynthesis of Caffeine in Flower Buds of Camellia sinensis

The biosynthesis of purine alkaloids in flower buds of tea plantswas investigated. More than 25% of total radioactivity of [8-¹⁴C]adeninetaken up by stamens isolated from tea flower buds was foundto have been incorporated into purine alkaloids, namely, theobromineand caffeine, 24 h after administration of the labelled compound.Pulse-chase experiments indicated that [8-¹⁴C]adenine takenup by the stamens was converted to adenine nucleotides and subsequentlyincorporated into theobromine and caffeine. Since 5 µMcoformycin, an inhibitor of AMP deaminase, inhibited the incorporationof radioactivity into the purine alkaloids, synthesis of caffeinefrom adenine nucleotides seems to be initiated by the reactionof AMP deaminase. Although most of the radioactivity from [8-¹⁴C]inosinewas recovered as CO₂ and ureides, considerable amounts of radioactivitywere recovered as purine alkaloids. The incorporation of radioactivityfrom [8-¹⁴C]inosine into the purine alkaloids was not affectedby coformycin. The five enzymes involved in synthesis of 5-phosphoribosyl-1-pyrophosphatefrom glucose were present in the stamens and petals of tea flowerbuds. From present and previous results, the pathway for thebiosynthesis of caffeine from adenine nucleotides in flowerbuds of tea is discussed.Copyright 1993, 1999 Academic Press Camellia sinensis, tea, stamen, flower, biosynthesis, purine alkaloids, caffeine, theobromine, adenine nucleotides, nucleotide biosynthesis     

Metabolic fate of guanosine in higher plants

The aim of the present study was to investigate the metabolic fate of guanine nucleotides in higher plants. The rate of uptake of [8-¹⁴C]guanosine by suspension-cultured Catharanthus roseus cells was more than 20 times higher than that of [8-¹⁴C]guanine. The rate of uptake of [8-¹⁴C]guanosine increased with the age of the culture. Pulse-chase experiments with [8-¹⁴C]guanosine revealed that some of the guanosine that had been taken up by the cells was converted to guanine nucleotides and incorporated into nucleic acids. A significant amount of [8-¹⁴C]guanosine was degraded directly to xanthine, allantoin and allantoic acid, with the generation of ¹⁴CO₂ as the final product. The rate of salvage of [8-¹⁴C]guanosine for the synthesis of nucleic acids was highest in young cells, while the rate of degradation increased with the age of the cells. In segments of roots from Vigna mungo seedlings, nearly 50% of the [8-¹⁴C]guanosine that had been absorbed over the course of 15 min was recovered in guanine nucleotides. A significant amount of the radioactivity in nucleotides became associated with nucleic acids and ureides during ‘chase’ periods. In segments of young leaves of Camellia sinensis, [8-¹⁴C]guanosine was initially incorporated into guanine nucleotides, nucleic acids, theobromine and ureides, and the radioactivity in these compounds was transferred to caffeine and CO₂ during a 24-h incubation. Our results suggest that guanosine is an intermediate in the catabolism of guanine nucleotides and that it is re-utilised for nucleotide synthesis by ‘salvage’ reactions. Guanosine was catabolised by the conventional degradation pathway via xanthine and allantoin. In some plants, guanosine is also utilised for the formation of ureide or the biosynthesis of caffeine.     

Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.).

1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage.     

Acylated Steryl Glycoside Synthesis in Seedlings of Nicotiana tabacum L

In tobacco seedlings (Nicotiana tabacum L.), glucose from supplied uridine diphosphate-[U-¹⁴C]glucose was first incorporated into steryl glycosides and later into acylated steryl glycosides. However, when [¹⁴C]cholesterol was used as substrate, the acylated steryl glycosides became labeled earlier than the steryl glycosides. With [¹⁴C]cholesteryl glucoside as substrate, most of the radioactive label was recovered as free sterol, and the acylated steryl glycosides were not readily labeled; however, palmitoyl [¹⁴C]cholesteryl glucoside was rapidly converted to steryl glycoside. In feeding experiments with free sterol, an unknown, highly radioactive steroid component was isolated. Incorporation of radioactivity into the unknown occurred before the acylated steryl glycosides were labeled.     

Biosynthesis of tropic acid: Feeding experiments with cinnamoyltropine and littorine

The administration of cinnamoyl-[2-¹⁴C]-tropine-[N-methyl-¹⁴C] to Datura stramonium plants resulted in the formation of labeled atropine and scopolamine. However the atropine was found to have almost all its radioactivity located on the N-methyl group of the alkaloid, indicating that the administered ester had undergone hydrolysis in the plant affording tropine and cinnamic acid, the latter not being utilized for the biosynthesis of tropic acid. Dual labeled RS-littorine (3β-(2-hydroxy-3-phenylpropionyloxy-[1-¹⁴C]-tropane-[3β-³H]) was also fed to D. stramonium and radioactive atropine was obtained. However the drastic change in the ³H:¹⁴C ratio found in the atropine indicated that the littorine was not converted directly to the alkaloid, and it is suggested that the littorine is hydrolysed _{in vivo} to tropine and phenyl-lactic acid, the latter undergoing rearrangement to tropic acid prior to esterification with tropine.