Caffeine Synthesis Research Articles

Caffeine and Purine Derivatives: A Comprehensive Review on the Chemistry, Biosynthetic Pathways, Synthesis-Related Reactions, Biomedical Prospectives and Clinical Applications.

Caffeine and purine derivatives represent interesting chemical moieties, which show various biological activities. Caffeine is an alkaloid that belongs to the family of methylxanthine alkaloids and it is present in food, beverages, and drugs. Coffee, tea, and some other beverages are a major source of caffeine in the human diet. Caffeine can be extracted from tea or coffee using hot water with dichloromethane or chloroform and the leftover is known as decaffeinated coffee or tea. Caffeine and its derivatives were synthesized via different procedures on small and large scales. It competitively antagonizes the adenosine receptors (ARs), which are G protein-coupled receptors largely distributed in the human body, including the heart, vessels, brain, and kidneys. Recently, many reports showed the effect of caffeine derivatives in the treatment of many diseases such as Alzheimer's, asthma, parkinsonism, and cancer. Also, it is used as an antioxidant, anti-inflammatory, analgesic, and hypocholesterolemic agent. The present review article discusses the synthesis, reactivity, and biological and pharmacological properties of caffeine and its derivatives. The biosynthesis and biotransformation of caffeine in coffee and tea leaves and the human body were summarized in the review.

Caffeine Synthesis and Its Mechanism and Application by Microbial Degradation, A Review.

Caffeine is a metabolite derived from purine nucleotides, typically accounting for 2-5% of the dry weight of tea and 1-2% of the dry weight of coffee. In the tea and coffee plants, the main synthesis pathway of caffeine is a four-step sequence consisting of three methylation reactions and one nucleosidase reaction using xanthine as a precursor. In bacteria, caffeine degradation occurs mainly through the pathways of N-demethylation and C-8 oxidation. However, a study fully and systematically summarizing the metabolism and application of caffeine in microorganisms has not been established elsewhere. In the present study, we provide a review of the biosynthesis, microbial degradation, gene expression, and application of caffeine microbial degradation. The present review aims to further elaborate the mechanism of caffeine metabolism by microorganisms and explore the development prospects in this field.

Genome-Wide Investigation and Functional Analysis Reveal That CsKCS3 and CsKCS18 Are Required for Tea Cuticle Wax Formation.

Cuticular wax is a complex mixture of very long-chain fatty acids (VLCFAs) and their derivatives that constitute a natural barrier against biotic and abiotic stresses on the aerial surface of terrestrial plants. In tea plants, leaf cuticular wax also contributes to the unique flavor and quality of tea products. However, the mechanism of wax formation in tea cuticles is still unclear. The cuticular wax content of 108 germplasms (Niaowang species) was investigated in this study. The transcriptome analysis of germplasms with high, medium, and low cuticular wax content revealed that the expression levels of CsKCS3 and CsKCS18 were strongly associated with the high content of cuticular wax in leaves. Hence, silencing CsKCS3 and CsKCS18 using virus-induced gene silencing (VIGS) inhibited the synthesis of cuticular wax and caffeine in tea leaves, indicating that expression of these genes is necessary for the synthesis of cuticular wax in tea leaves. The findings contribute to a better understanding of the molecular mechanism of cuticular wax formation in tea leaves. The study also revealed new candidate target genes for further improving tea quality and flavor and cultivating high-stress-resistant tea germplasms.

The habit evolution of 6-amino-1,3-dimethyl-5-nitrosouracil hydrate: Effect of acid and polymer additives

6-amino-1,3-dimethyl-5-nitrosouracil (NAU) is an important intermediate for the synthesis of caffeine and theophylline, usually in the form of monohydrate. However, NAU·H2O is aggregated and has a plate-like habit, which is very unfavorable for downstream process such as filtration and transportation. In this work, carboxymethylcellulose (CMC), a very common polymer, was selected as an additive to manipulate the habit of NAU·H2O. It was found that under acidic conditions, the addition of CMC can change the habit from plate-like to block-like and can significantly improve the aggregation. On the contrary, the additive cannot change the habit under neutral or weakly alkaline conditions. The underlying mechanism was analyzed by experiment and molecular dynamics simulation. It is found that, under neutral or alkaline conditions, CMC will dissociate into negatively charged polymer chains, which have a stronger interaction with water. But CMC will combine with hydrogen ions under acidic conditions, weakening the interaction with water, allowing it to adhere to the (001) face and inhibiting the growth of this face. The results of this study should be helpful in the selection and mechanism research of polymer additives for crystal habit.

Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates.

The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity toward typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolized to a greater extent by certain younger ancestors and extant forms, suggesting that activity toward FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the hemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.

CsMYB184 regulates caffeine biosynthesis in tea plants.

CsMYB184 regulates caffeine biosynthesis in tea plants.

Metabolite Profiling and Transcriptome Analysis Revealed the Conserved Transcriptional Regulation Mechanism of Caffeine Biosynthesis in Tea and Coffee Plants.

Caffeine is a characteristic bioactive compound in tea and coffee plants, which is synthesized and accumulated extensively in leaves and seeds. However, little is known about the regulatory mechanism of caffeine synthesis in plants. This study compared the caffeine metabolite between tea and coffee plants. We found that tea leaves contained significantly higher caffeine than coffee leaves, which is perhaps due to more members of N-methyltransferase (NMT) genes as well as higher expression levels in tea plants. Substantial numbers of transcription factors were predicted to be involved in caffeine biosynthesis regulation, combining weighted gene co-expression network analysis and the cis-element of NMT promoter analysis in tea and coffee plants. Furthermore, analysis of the transcription factors from the caffeine-related modules suggested that the regulatory mechanism of caffeine biosynthesis was probably partly conservative in tea and coffee plants. This study provides an essential resource for the regulatory mechanism of caffeine biosynthesis in plants.

Transcriptomics analysis reveals the signal transduction mechanism of brassinolides in tea leaves and its regulation on the growth and development of Camellia sinensis

BackgroundBrassinosteroids (BRs) are a type of sterol plant hormone that play an important role in various biochemical and physiological reactions such as promoting cell growth, increasing biomass, and improving stress resistance.ResultsTo investigate the regulatory and molecular mechanism of BRs on the growth and development of tea plants (Camellia sinensis L.), changes in cell structure and gene expression levels of tea leaves treated with exogenous BRs were analyzed by electron microscopy and high-throughput Illumina RNA-Seq technology. The results showed that the number of starch granules in the chloroplasts and lipid globules increased and thylakoids expanded after BR treatment compared with the control. Transcriptome analysis showed that in the four BR treatments (CAA: BR treatment for 3 h, CAB: BR treatment for 9 h, CAC: BR treatment for 24 h, and CAD: BR treatment for 48 h), 3861 (1867 upregulated and 1994 downregulated), 5030 (2461 upregulated and 2569 downregulated), 1626 (815 upregulated and 811 downregulated), and 2050 (1004 upregulated and 1046 downregulated) differentially expressed genes were detected, respectively, compared with CAK (BR treatment for 0 h). Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, metabolic pathway enrichment analysis showed that the differentially expressed genes of CAA vs. CAK, CAB vs. CAK, CAC vs. CAK, and CAD vs. CAK significantly enriched the functional categories of signal transduction, cell cycle regulation, and starch, sucrose, and flavonoid biosynthesis and metabolism pathways. We also found that after spraying BR, the key genes for caffeine synthesis were downregulated. The results of qRT-PCR coincided with the findings of transcriptomic analysis.ConclusionsThe present study improved our understanding of the effects of BRs on the growth and development of tea leaves and laid the foundation for the in-depth analysis of signal transduction pathways of BRs in tea leaves.

The NAC-like transcription factor CsNAC7 positively regulates the caffeine biosynthesis-related gene yhNMT1 in Camellia sinensis.

Caffeine is an important functional substance and is abundant in tea plant, but little is known about how its biosynthesis is regulated by transcription factors. In this study, the NAC-like transcription factor-encoding gene CsNAC7, which is involved in caffeine synthesis, was isolated from a Yinghong 9 cDNA library using a yeast one-hybrid assay; this gene comprises 1371bp nucleotides and is predicted to encode 456 amino acids. The expression of CsNAC7 at the transcriptional level in tea shoots shared a similar pattern with that of the caffeine synthase gene yhNMT1 in the spring and summer, and its expressed protein was localized in the nucleus. Assays of gene activity showed that CsNAC7 has self-activation activity in yeast, that the active region is at the N-terminus, and that the transient expression of CsNAC7 could significantly promote the expression of yhNMT1 in tobacco leaves. In addition, overexpression or silencing of CsNAC7 significantly increased or decreased the expression of yhNMT1 and the accumulation of caffeine in transgenic tea calli, respectively. Our data suggest that the isolated transcription factor CsNAC7 positively regulates the caffeine synthase gene yhNMT1 and promotes caffeine accumulation in tea plant.

Purine alkaloids in tea plants: component, biosynthetic mechanism and genetic variation

The purine alkaloids, including caffeine, theobromine, and theacrine, are one of the most important quality and functional components of tea plants and commercial teas. In this paper, we review the component, biosynthetic mechanism and genetic variation of purine alkaloids in tea plants. The content of caffeine and theobromine in 403 accessions Chinese tea germplasms core collection preserved in the National Germplasm Hangzhou Tea Repository were analyzed using High Performance Liquid Chromatography (HPLC). The purine alkaloid profiles of different tea varieties, germplasm types, geographical origin, seasons, were highlighted. Some naturally low caffeine or caffeine-free, high theobromine or high theacrine germplasms were identified and the possible biosynthetic mechanism of hypernormal purine alkaloid content in tea plants were partially revealed. Some Cleaved Amplified Polymorphic Sequences (CAPS) DNA markers were developed for the identification of purine alkaloid content. The potential application of genetic engineering and DNA markers developed based on the low caffeine <italic>TCS1</italic> (tea caffeine synthesis gene) alleles for marker assisted selection (MAS), and cocoa tea and kucha in low caffeine/high theacrine tea cultivar breeding programs were also reviewed.

Identification of Co-Expressed Genes Related to Theacrine Synthesis in Tea Flowers at Different Developmental Stages.

Jiaocheng kucha is the first reported tea germplasm resource which contains theacrine founded in Fujian Province. Currently, the anabolic mechanism of theacrine within tea leaves is clear, but there are few studies focused on its flowers. In order to further explore the mechanism of theacrine synthesis and related genes in flowers, current study applied Jiaocheng kucha flowers (JC) as test materials and Fuding Dabaicha flowers (FD) as control materials to make transcriptome sequencing, and determination of purine alkaloid content in three different developmental periods (flower bud stage, whitening stage and full opening stage). The results showed that the flower in all stages of JC contained theacrine. The theacrine in the flower bud stage was significantly higher than in the other stages. The differentially expressed genes (DEGs) at three different developmental stages were screened from the transcriptome data, and were in a total of 5642, 8640 and 8465. These DEGs related to the synthesis of theacrine were primarily annotated to the pathways of purine alkaloids. Among them, the number of DEGs in xanthine synthesis pathway was the largest and upregulated in JC, while it was the smallest in caffeine synthesis pathway and downregulated in JC. Further weighted gene co-expression network (WGCNA) indicated that ADSL (CsTGY03G0002327), ADSL (CsTGY09G0001824) and UAZ (CsTGY06G0002694) may be a hub gene for the regulation of theacrine metabolism in JC. Our results will contribute to the identification of candidate genes related to the synthesis of theacrine in tea flowers, and explore the molecular mechanism of theacrine synthesis in JC at different developmental stages.

Q-Tube®-Assisted Alkylation and Arylation of Xanthines and Other N-H-Containing Heterocycles in Water

In this paper, a simple and clean process for the alkylation and arylation of nitrogen-containing heterocycles is reported. The reactions were conducted using the Q-tube® as a non-conventional technology, in water as a green solvent, at overboiling temperature. The developed strategy was used to improve two steps in the total synthesis of caffeine, as reported by Narayan, and then extended to the preparation of N-decorated xanthines. Finally, piperidine, methyl piperazine, and isatine were proven to be suitable substrates for the protocol proposed herein.

Биохимический состав чая и его изменения под влиянием различных факторов

Introduction. The present research featured the antioxidant complex of Russian tea varieties. The research objective was to study the formation patterns of the components responsible for the quality characteristics of tea as a raw material and finished product. The paper describes the changes that the biochemical composition of tea undergoes during the growing season and during processing. The study focused on pigments, vitamins, caffeine, and amino acids.&#x0D; Study objects and methods. The research featured the following varieties of tea grown in the Krasnodar region of Russia: “Kolkhida” (control) and “Sochi”; forms – No. 582, 3823, 855, and 2264. The research was performed on the foundation plantation of collection tea in the village of Uch-Dere (Lazarevsky district of Sochi, Krasnodar region, Russia) and in the laboratory of plant physiology and biochemistry. The study involved traditional and advanced research methods.&#x0D; Results and discussion. The paper introduces some results of a comprehensive analysis of the biochemical composition of various tea sorts. Shoots No. 2264, 3823, and the “Sochi” variety demonstrated high values of ascorbic acid. Form No. 3823 and the “Sochi” variety proved to have a relatively stable content of ascorbic acid. All experimental plants had the highest caffeine synthesis in July (from 24.633 to 28.614 μg/100 g). Processing destroyed caffeine and reduced its amount. The experimental samples of raw materials had eleven amino acids, the largest number being synthesized in May. Processing triggered both general changes in their quantity and varietal differences in the metabolic reactions of amino acid conversion.&#x0D; Conclusion. The tea flushes and finished products differed in all biologically active substances, which is associated with both varietal characteristics and the effect of weather conditions during vegetation.

Secondary metabolites of tea leaves and their role in protecting against environmental stressors

The dynamics of a number of secondary metabolites and the activity of guaiacol peroxidase in 3-leaf sprout of tea varieties and forms were studied. In the course of 3-year research we noted the presence of declines and peaks in the activity of the enzyme and the accumulation of flavonoids, carotenoids, caffeine and amino acids associated with the meteorological conditions of each month. It is shown that there is a close inverse relationship between increased enzyme activity, carotenoid content and hydrothermal factors. The most significant correlation was found between the activity of guaiacol peroxidase and carotenoids in a 3-leaf tea flush with precipitation (r = -0.86 and -0.68). At the beginning of vegetation the enzyme activity is low (ranging from 0.363 to 0.607 units/g·s). The decrease in the activity of guaiacol peroxidase in June is due to the biological peculiarities of the tea culture associated with a period of rest, during which the metabolic processes are somewhat slowed down. Increased activity of guaiacol peroxidase is accompanied by rapid synthesis of phenolic compounds, such as flavonoids. We have traced the dynamics of the formation of thearubigins and theaflavins in tea sprout. It is shown that their amount varies depending on the season of collection from 0.075 mg/g (theaflavins) and 1.178 mg/g (thearubigins) to 0.115 mg/g and 1.625 mg/g (theaflavins and thearubigins, respectively) and their dynamics is similar. The content of the routine varies depending on the season of tea leaf collection. The synthesis of caffeine also depends on the growing conditions, the active accumulation of which in experimental plants was observed in July (on average 26.6±1.4 mg/g). The largest amount of amino acids (16 665 mg/g) is synthesized in May, and then their synthesis is halved. Genotypic peculiarities are manifested in the content of secondary metabolites and the activity of guaiacol peroxidase. The revealed patterns are common for all varieties and forms of tea.

Metabolite and Transcriptome Profiling on Xanthine Alkaloids-Fed Tea Plant (Camellia sinensis) Shoot Tips and Roots Reveal the Complex Metabolic Network for Caffeine Biosynthesis and Degradation.

While caffeine is one of the most important bioactive metabolites for tea as the most consumed non-alcohol beverage, its biosynthesis and catabolism in tea plants are still not fully understood. Here, we integrated purine alkaloid profiling and transcriptome analysis on shoot tips and roots fed with caffeine, theophylline, or theobromine to gain further understanding of caffeine biosynthesis and degradation. Shoot tips and roots easily took up and accumulated high concentrations of alkaloids, but roots showed much faster caffeine and theophylline degradation rates than shoot tips, which only degraded theophylline significantly but almost did not degrade caffeine. Clearly feedback inhibition on caffeine synthesis or inter-conversion between caffeine, theophylline, and theobromine, and 3-methylxanthine had been observed in alkaloids-fed shoot tips and roots, and these were also evidenced by significant repression of TCS and MXMT genes critical for caffeine biosynthesis. Among these responsively repressed genes, two highly expressed genes TCS-4 and TCS-8 were characterized for their enzyme activity. While we failed to detect TCS-4 activity, TCS-8 displayed N-methyltransferase activities towards multiple substrates, supporting the complex metabolic network in caffeine biosynthesis in tea plants since at least 13 TCS-like N-methyltransferase genes may function redundantly. This study provides new insight into complex metabolic networks of purine alkaloids in tea plants.

Differential regulatory mechanisms of secondary metabolites revealed at different leaf positions in two related tea cultivars

In this study, ‘Zhongcha108′ (ZC) and its offspring cultivar ‘Zhongming7′ (ZM) were used to investigate the variation in characteristic compounds at different leaf positions. The results showed that there were differences between the two cultivars beyond the leaf position. The concentrations of theanine and caffeine were significantly higher in ZM, whereas more catechins accumulated in ZC. The expression levels of genes related to the theanine, caffeine and catechin pathways were determined by quantitative real time PCR. Compared with ZC, ZM showed higher expression levels of genes involved in theanine biosynthesis and transport, including GOGAT, GS(TS), AlaAT, AspAT, AS and AAP, suggesting that effective biosynthesis and translocation facilitate theanine accumulation in ZM. The caffeine content in ZC was positively correlated with the gene expression of TCS. However, such a correlation was not observed in ZM, which might be related to highly expressed genes involved in caffeine degradation, including CYP1A2, ALN and XO. Furthermore, catechin biosynthesis was regulated by different structural genes in the two cultivars. Higher catechins contents in ZC were related to higher genes expression levels, particularly for PAL, 4CL, F3H, FLS and LAR. A correlation analysis among key genes involved in theanine, caffeine, and catechin biosynthesis also showed consistent results. In ZC, catechins biosynthesis was more active, which inhibited the biosynthesis of nitrogen-rich metabolites, particularly for theanine. The balance of secondary metabolisms in ZM was shifted toward increasing the synthesis of nitrogen-containing compounds, i.e., theanine and caffeine. Taken together, these data reveal different regulatory mechanisms of theanine, caffeine, and catechins within two genetically similar tea cultivars. This work provides an important basis for further research on the characteristic metabolites of tea plants.

Green synthesis of caffeine based on methylating reagent dimethyl carbonate and environmental friendly separating method

AbstractIn this paper, a green process for the synthesis and separation of caffeine was reported. Theophylline sodium is used as the raw material, diazabicyclo[5.4.0]undec‐7‐ene as the catalyst, and Turkey red oil as the solvent, particularly, dimethyl carbonate was adopted in place of high toxic dimethyl sulfate to form a mixture of caffeine and by‐product sodium bicarbonate. After converting sodium bicarbonate to sodium carbonate, the solubility difference between caffeine and sodium carbonate at 40°C was for the first time utilized to achieve the purpose of separating caffeine with an excellent yield of 98.4% and a purity of greater than 99.0%. Furthermore, both the reaction mother liquor and separation mother liquor can be recycled and reused during the reaction and separation processes, respectively, with little caffeine loss. No industrial waste was discharged in the process of the improved synthesis and separation, which is therefore environmental friendly.

Guanine deaminase provides evidence of the increased caffeine content during the piling process of pu'erh tea.

Wet piling is a key process for producing pu'erh tea because various components change under the action of microorganisms. Among these components, caffeine content is increased. Evidence has indicated a salvage pathway for caffeine biosynthesis in microbes, in which xanthine is methylated in the order of N-3 → N-1 → N-7. In addition, guanine can be used to synthesize xanthine through guanine deaminase (EC: 3.5.4.3). In this study, we investigated the variation in caffeine content during piling fermentation with supplementary guanine, 15N-labeled guanine and xanthine. We cloned the guanine deaminase gene (GUD1) from Saccharomyces cerevisiae (one dominant strain in piling fermentation). The results revealed that [15N]xanthine could be synthesized from [15N]guanine, and [15N]caffeine was also detected during piling with supplementary [15N]xanthine. Furthermore, ScGUD1 could catalyze the conversion of guanine to xanthine, which is likely to be methylated for caffeine synthesis under microorganism action. The obtained results revealed the mechanism underlying the increased caffeine content during piling of pu'erh tea.

The Tea Tree Genome Provides Insights into Tea Flavor and Independent Evolution of Caffeine Biosynthesis

Tea is the world's oldest and most popular caffeine-containing beverage with immense economic, medicinal, and cultural importance. Here, we present the first high-quality nucleotide sequence of the repeat-rich (80.9%), 3.02-Gb genome of the cultivated tea tree Camellia sinensis. We show that an extraordinarily large genome size of tea tree is resulted from the slow, steady, and long-term amplification of a few LTR retrotransposon families. In addition to a recent whole-genome duplication event, lineage-specific expansions of genes associated with flavonoid metabolic biosynthesis were discovered, which enhance catechin production, terpene enzyme activation, and stress tolerance, important features for tea flavor and adaptation. We demonstrate an independent and rapid evolution of the tea caffeine synthesis pathway relative to cacao and coffee. A comparative study among 25 Camellia species revealed that higher expression levels of most flavonoid- and caffeine- but not theanine-related genes contribute to the increased production of catechins and caffeine and thus enhance tea-processing suitability and tea quality. These novel findings pave the way for further metabolomic and functional genomic refinement of characteristic biosynthesis pathways and will help develop a more diversified set of tea flavors that would eventually satisfy and attract more tea drinkers worldwide.

Assisted-selection of naturally caffeine-free coffee cultivars—characterization of SNPs from a methyltransferase gene

Breeding of caffeine-free coffee cultivars require tools for an early selection of progenies bearing this later trait. Genes from caffeine synthesis and degradation represent major targets for the development of molecular markers for assisted selection. In this study, we characterized SNPs identified on the caffeine synthase gene from AC1 mutant, a naturally caffeine-free arabica coffee plant. Molecular analysis of normal and mutant sequences indicates the occurrence of SNPs in protein domains, potentially associated with caffeine synthesis in coffee. Progenies F2, F1BC1 and BC from crosses of AC mutants and elite cultivars were evaluated regarding caffeine content in grains and genomic segregation profile of selected SNPs. Genotyping analysis allowed the discrimination between homozygous and heterozygous plants. Quantification of caffeine content indicated a significant variability among progenies and a low frequency of caffeine-free plants. Statistical analyses of genotyping and phenotyping results showed significant association between presence of selected SNPs and reduced caffeine content. Moreover, this association occurs through all evaluated genetic backgrounds and generations, indicating an inheritance stability of both trait and markers. The molecular markers described here represent a successful case of assisted-selection in coffee, indicating their potential use for breeding of caffeine-free cultivars.