Part I: Pyrrolidine derivatives were attached a methoxy group on a-C position of pyrrolidine-ring via anodic oxidation. Followed with alkylation and series of transformation under Lewis acids to obtain tropane alkaloids. Part II: Application of intramolecular radical cyclization toward the synthesis of aporphine alkaloids. Tributyltinhydride and AIBN were used to generate aryl radicals. Trapping of aryl radicals with unsaturated alkenes led to products.

No description available.

Made available in DSpace on 2015-05-14T12:59:34Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3371563 bytes, checksum: 0f72f1a088caa63b4cd2bf4057752d08 (MD5) Previous issue date: 2012-02-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This work describes the results of phytochemical studies from three species of the Erythroxylum genus: Erythroxylum caatingae Plowman, Erythroxylum subrotundum A. St.-Hil and Erythroxylum revolutum Mart, which were identified by the botany sector of the Laboratory of Pharmaceutical Technology, UFPB. The species were submitted to extraction process, followed by partition with hexane, chloroform and ethyl acetate, resulting in their respective phases. Four Tropane alkaloids were isolated by chromatography methods from the chloroformic phase of Erythroxylum caatingae, in which two were already isolated [3α, 6β dibenzoyloxytropane and 3α-(3 ,4 ,5 - trimethoxybenzoiloxi)-6β-benzoyloxytropane (Catuabine B)] and the other two were reported for the first time in the literature [3-(3 ,4 -dimethoxy)-6-hydroxytropane and 3α-(trans-3 ,4 ,5 trimethoxycinnamoyloxy)-6β-benzoyloxytropane]. From the ethyl acetate phase of Erythroxylum subrotundum, two flavonoids were isolated: Quercetin-3-O-α-L-rhamnoside and 5,7,4 -trihydroxyflavone-3-O-α-L-rhamnoside. The study of the Erythroxylum revolutum hexanic phase resulted in the isolation of six diterpenes: ent-kauran-16-ene, 13-hydroxy-8(17),14-labdadien (Manool), ent-kaur-16- en-3β-ol, 3-oxo-13-hydroxi-8(17),14-labdadien, 3,13,19-trihydroxy-8(17),14-labdadien and ent-kauran-16β, 17-diol. All the species had their chemical constituents identified through data analysis obtained from spectroscopic methods such as Infrared and Nuclear Magnetic Resonance of 1H and 13C with uni-bidimensional techniques, besides comparison with literature data. Therefore, the given results of this work contributed to the chemical study of the species from Erythroxylaceae family. / Este trabalho descreve os resultados dos estudos fitoquímicos de três espécies do gênero Erythroxylum: Erythroxylum caatingae Plowman, Erythroxylum subrotundum A. St.- Hil e Erythroxylum revolutum Mart, identificadas pelo setor de botânica do Laboratório de Tecnologia Farmacêutica da UFPB. As espécies foram submetidas a processos de extração e posterior particionamento de seus extratos resultando nas fases hexânica, clorofórmica e acetato de etila. Do estudo da fase clorofórmica de Erythroxylum caatingae foram isolados, através de métodos cromatográficos, quatro alcaloides tropânicos, sendo dois destes alcaloides [3α,6β dibenzoiloxitropano e 3α-(3 ,4 ,5 - trimetoxibenzoiloxi)-6β-benzoiloxitropano (Catuabina B)], já isolados anteriormente e dois alcalóides inéditos na literatura [3-(3 ,4 -dimetoxi)-6-hidroxitropano e 3α- (trans-3 ,4 ,5 trimetoxicinamoiloxi)-6β-benzoiloxitropano]. Da fase acetato de etila de Erythroxylum subrotundum foram isolados dois flavonoides: a Quercetina-3-O-α-Lraminosídeo e 5,7,4 -trihidroxiflavona-3-O-α-L-raminosídeo. Do estudo da fase hexânica de Erythroxylum revolutum foram isolados seis diterpenos: ent-cauran-16-eno, 13-hidroxi-8(17),14-labdadieno (Manool), ent-caur-16-en-3β-ol, 3-oxo-13-hidroxi- 8(17),14-labdadieno, 3,13,19-trihidroxi-8(17),14-labdadieno e ent-cauran-16β, 17-diol. As espécies tiveram seus constituintes químicos identificados através da análise de dados obtidos por métodos espectroscópicos como Infravermelho e Ressonância Magnética Nuclear de 1H e 13C uni-bidimensional, além de comparação com dados obtidos na literatura. Assim, os resultados obtidos neste trabalho contribuíram para o estudo químico de espécies da família Erythroxylaceae.

Made available in DSpace on 2014-08-20T13:42:06Z (GMT). No. of bitstreams: 1 Dissertacao_Gabrie_Olle_ Dalmazo.pdf: 526235 bytes, checksum: 5e78165179f8f9e3164e0269bd617ab9 (MD5) Previous issue date: 2011-02-28 / The tropane alkaloids (TA) pathway has a branch-point controlled by the enzymes tropinone reductase 1 and 2 (TR1 and TR2). Tropinone is the common substrate for these enzymes and is reduced either by TR1 to form tropine, hyoscyamine and scopolamine or by TR2 to form pseudotropine and calystegines. Hyoscyamine and scopolamine are largely used in medicine as anticholinergic, antiemetic, parasympatholytic and anaesthetic. Calystegines mimic different sugars and are potent and specific inhibitors of glucosidases. The function of hyoscyamine, scopolamine and calystegines in the plants is not fully understood. Recent studies suggest that they are involved in the plant defense against pathogens. Regulation of TA biosynthesis in planta has attracted interest not only in view of its applications in the pharmaceutical industry, but also in respect to human nutrition and plant physiology. In the present study a virus-based transgenic approach devised to induce PTGS of tr1 and tr2 in whole transformed Hyoscyamus muticus plants is evaluated. It was observed that a significant reduction in transcript accumulation for tr2 caused a tremendous increase in transcript accumulation for tr1. / Um ponto de bifurcação na rota metabólica de tropano alcalóides (TA) é controlado pelas enzimas tropinona redutase 1 e 2 (TR1 e TR2). Tropinona, o substrato comum para estas enzimas, é reduzido por TR1 para formar tropina, hiosciamina e escopolamina ou por TR2 para formar pseudotropine e calisteginas. Hiosciamina e escopolamina são largamente utilizadas em medicina como anticolinérgicos, antieméticos, parassimpatolíticos e anestésicos. Calisteginas têm estrutura molecular semelhante a açúcares e são potentes inibidores específicos de glucosidases. A função dos alcalóides hiosciamina, escopolamina e calisteginas em plantas não é completamente entendida. Estudos recentes sugerem o envolvimento destes alcalóides na defesa da planta contra patógenos. A regulação da biossíntese de TA na planta tem atraído interesse não apenas quanto à aplicação na indústria farmacêutica, mas também com respeito à nutrição humana e fisiologia de plantas. No presente estudo avaliou-se uma estratégia transgênica, baseada em vírus, para induzir o silenciamento gênico pós-transcricional de tr1 e tr2 em plantas de Hyoscyamus muticus L. Observou-se que reduções significativas no acúmulo de transcritos para tr2 causaram um tremendo aumento no acúmulo de transcritos para tr1.

No description available.

This thesis is concerned with the development of transannular iodoamination methodology for the synthesis of pyrrolizidine, indolizidine and tropane alkaloids. Chapter 1 introduces the concept of a ‘transannular cyclisation’ and outlines the utility of such cyclisations in the synthesis of a range of [x.y.z]-azabicyclic alkaloids. Chapter 2 describes the development of a three step lithium amide conjugate addition, ring-closing metathesis and transannular iodoamination protocol for the preparation of the pyrrolizidine scaffold ([3.3.0]-azabicycle). Cyclisation of a hexahydroazocine occurs with concomitant N-debenzylation to give a single diastereoisomer of the corresponding C(7)-iodopyrrolizidine product, which is then elaborated to the known pyrrolizidine, (−)-7a-epi-hyacinthacine A1. Chapter 3 delineates an extension of the methodology described in Chapter 2, and an investigation into accessing alternate diastereoisomeric pyrrolizidine scaffolds via the transannular iodoamination process. These studies culminate in the synthesis of two pyrrolizidine alkaloids, (−)-hyacinthacine A1 and (−)-hyacinthacine A2. Chapter 4 details investigations into the further elaboration of the C(7)-iodopyrrolizidine scaffold synthesised in Chapter 2. A nucleophilic displacement reaction with azide leads to the synthesis of novel 7-deoxy-7-aminoalexine analogues, whilst radical-mediated substitution of the iodide by oxygen allows the synthesis and isolation of the pyrrolizidine alkaloid (−)-1-epi-alexine. Chapter 5 outlines the development of the transannular iodoamination reaction to facilitate the synthesis of the tropane architecture ([3.2.1]-azabicycle). A tandem lithium amide conjugate addition and aldol reaction sequence is followed by ring-closing metathesis to give the required aminocycloheptene. Subsequent treatment with iodine results in transannular cyclisation to give a single iodotropane product which, following elaboration culminates in the synthesis of (+)-pseudococaine. Chapter 6 contains full experimental procedures and characterisation data for all compounds synthesised in Chapters 2, 3, 4 and 5.

Datura innoxia Mill., une plante de la famille des Solanacées synthétisant des métabolites secondaires, dont les alcaloïdes tropaniques, a été utilisée comme modèle pour cette étude portant sur les cultures en hydroponie. Nous avons déterminé et hiérarchisé les paramètres environnementaux (biotiques et abiotiques) permettant d’améliorer croissance et production de métabolites. La réponse de la plante dépend de l’approvisionnement en oxygène de la solution nutritive, de la température perçue tant niveau des parties aériennes qu’au niveau racinaire. Une augmentation de l’intensité d’éclairage n’améliore pas la teneur en alcaloïdes de la plante mais l’utilisation de lumière orange engendre un changement des paramètres de croissance et des teneurs en molécules recherchées. Nous avons également montré que la présence de microflore dans le milieu de culture provoque une dégradation des molécules d’intérêt et altère leur accumulation dans la plante. Par contre, A. rhizogenes a un effet favorable sur la croissance de la biomasse et la biosynthèse d’alcaloïdes tropaniques dans la plante. L’état transgénique des racines, issues de co-culture hydroponique de Datura innoxia avec A. rhizogenes TR7, a été estimé par une vérification précoce et confirmé par des analyses moléculaires. Nous avons pu mettre en évidence les modifications du phénotype racinaire et les mécanismes d'interaction plante-microorganisme. L’inoculation des agrobactéries peut donner lieu à des transferts naturels de gènes induisant ainsi l’obtention d’un pool racinaire chimérique regroupant des racines normales et des racines transformées. Ces travaux ont ouvert de nouvelles perspectives en vue de déterminer la capacité de production de métabolites secondaires par des plantes en culture hors sol, de manière à obtenir une biomasse riche en molécules recherchées / Datura innoxia Mill. a plant species belonging to Solanaceous family, produces hyoscyamine and scopolamine as two main tropane alkaloids. It has been studied in the present work for the development of hydroponic based secondary metabolites bioproduction. More specifically, biotic and abiotic environmental factors have been analysed in order to improve growth and alkaloid accumulation in plant tissues. Oxygen concentration in the nutrient solution is proved to be important for both biomass and compound production in temporary immersion culture conditions occur as one optimum. Temperature has also a major effect, directly and most probably through oxygen solubility and root needs. We showed that artificial light may positively complete natural one but the kind of spectrum must be chosen carefully. From this point of view, orange light from High Pressure Natrium Vapour light showed better results than classical white light. Nitrogen (NO3-15mM) and pH (5 to 6) management may lead to culture optimization. We also show that well chosen wild Agrobacterium rhizogenes strains may be added to the hydroponic nutrient solution in order to improve growth and alkaloid bioproduction. This result is due to the multi occurrence of genetic transformation events. Our results lead to a first classification for impacts of biotic and abiotic factors. It opens new window for plant milking technology industrial development

<p>U okviru doktorske disertacije su razvijeni i optimizovani postupci pripreme uzoraka hrane (žitarica, sirovina, poluproizvoda i prehrambenih proizvoda na bazi žitarica, voća i povrća) i stočne hrane, kao i simultano određivanje tropanskih i glikoalkaloida primenom gasne i tečne hromatografije uz maseno-spektrometrijsku detekciju. Razdvajanje alkaloida primenom gasne hromatografije je izvedeno na kapilarnoj koloni (HP-5MS UI), uz detekciju sa kvadrupolnom masenom spektrometrijom SIM tehnikom, u intervalu m/z 50&ndash;550 a.m.u. Pri optimizovanim uslovima postignuto je razdvajanje homatropina, atropina i skopolamina u vremenu od 12.75 min, pri čemu<br />tropin i anisodamin nisu mogli biti detektovani. Glikoalkaloidi nisu direktno mogli biti određivani gasnom hromatografijom usled njihove visoke molekulske mase i male isparljivosti. Za simultano određivanje tropanskih alkaloida (tropin, l-hiosciamin, atropin, skopolamin, homatropin i anisodamin) i glikoalkaloida (&alpha;-solanin i &alpha;-kakonin) validirana je brza i osetljiva multirezidualna LC&ndash;MS/MS metoda. Kori&scaron;ćena je disperzivna čvrsto-fazna ekstrakcija sa 0.5% mravlje kiseline u acotonitrilu/vodi (75:25, v/v), solima magnezijum sulfat, natrijum hlorid i natrijum citrat kao i C₁₈ sorbent. Analiti su razdvojeni na Chirobiotic V koloni, sa mobilnom fazom sastavljenom od 10 mM amonijum formata u voda/acetonitril (90:10, v/v) i metanol/acetonitril (50:50, v/v), me&scaron;anim u odnosu 20:80 (v/v). Detekcija komponenti je izvedena u pozitivnom elektrosprej modu (ESI+), kori&scaron;ćenjem reakcionog vi&scaron;estrukog monitoring akvizacionog moda (MRM). Optimizovana tečno hromatografska metoda je podrazumevala brz i jednostavan proces ekstrakcije, sa prinosom od 61-111% za pojedine alkaloide, dobru linearnost u &scaron;irokom opsegu (5-80 &mu;g/kg, r²=0.998), dobru selektivnost, robusnost i preciznost (CV &lt; 5%). Granica detekcije (LOD) za sve alkaloide je bila u opsegu od 0.74 do 0.79 ng/g dok se granica kvantifikacije (LOQ) kretala od 2.17 do 2.46 ng/g u svim žitaricama, izuzev za tropin u soji i lanenom semenu (LOD = 1.55 i LOQ = 4.91 ng/g). Pri analizi uzoraka sa trži&scaron;ta alkaloidi nisu detektovani u analiziranim uzorcima hrane, kao &scaron;to su: p&scaron;enica, raž, kukuruz, bra&scaron;no od me&scaron;anih žitarica, kukuruzno bra&scaron;no, biskvit i krekeri. Stočna hrana namenjena ishrani svinja i peradi je sadržala atropin, skopolamin, &alpha;-solanin i &alpha;-kakonin u koncentracionom opsegu od 3.6 do 21.7 ng/g. Atropin i skopolamin su detektovani u koncentracionom opsegu od 2.8 do 9.8 ng/g u hrani namenjenoj ishrani krava i zečeva, dok u hrani nemenjenoj ishrani konja i divljih životinja alkaloidi nisu detektovani. U analiziranim uzorcima voća i povrća (jabuka, kru&scaron;ka, avokado, mrkva, krastvac i paprika) po prvi put u ovom radu su detektovani glikoalkaloidi, &alpha;-solanin i &alpha;-kakonin. Koncentracija oba glikoalkaloida se kretala u opsegu od 0.3 do 15 ng/g. Sadržaj glikoalkaloida u krompiru i čipsu (8708.9-18794.7 ng/g) se nalazio u okviru preporučenog ukupnog sadržaja glikoalkaloida od 200 mg/kg (sirova odvaga) (FAO/WHO, 1999). Apsorpcija i translokacija alkaloida od strane p&scaron;enice u koren, listove i stabljiku je ustanovljena upotrebom atropina ¹⁴C. Utvrđeno je da biljka nakon 15 dana apsorbuje 4.30% i 2.28% atropina ¹⁴C za niži (13676 dpm/g soil) i vi&scaron;i (37203 dpm/g soil) sadržaj standarda sa kojim je zemlji&scaron;te spajkovano, redom. Količina apsorbovanog atropina se smanjivala tokom rasta biljke. Biljka starosti 90 dana je apsorbovala 0.38% i 0.21% atropina ¹⁴C za niži i vi&scaron;i sadržaj standarda, redom. Povećanje koncentracije supstance u zemlji&scaron;tu nije uticalo na dalje povećanje apsorpcije. Do nagomilavanja supstance je do&scaron;lo u listovima (83%) dok su niže koncentracije zabilježene u korenu (8.6%), stabljici (4.2%) i semenu (4.1%). U analiziranim uzorcima vode, koja je poticala od ispiranja zemlji&scaron;ta obogaćenog atropinom ¹⁴C, nakon 30 dana supstanca je detektovana u koncentraciji od 0.5%. Tokom vremena je ustanovljeno smanjenje ispiranja ove supstance (0.01% nakon 90 dana). Analiza uzoraka zemlji&scaron;ta nakon 30 i 60 dana je pokazala značajne adsorpcione osobine atropina, tako da je količina čvrsto adsorbovanog (neekstrahovanog) atropina iznosila 60% i 70%, redom. Zaostali atropin je bio stabilan i nakon 90 dana.</p> / <p>The methods of sample preparation have been developed and optimized for food (grains, raw materials, food products based on grains and fruits and vegetables) and feed analyses for the purpose of simultaneous determination of tropane and glycoalkaloids by gas and liquid chromatography hyphenated to mass spectrometry. Separation of alkaloids by gas chromatography was achieved by using capillary column HP-5MS UI. The alkaloids have been detected by using a quadrupole mass spectrometer inSIM mode for the range of m/z 50&ndash;550 a.m.u. Under optimised conditions, good separation of homatropine, atropine and scopolamine was achieved after 12.75 min, while tropine and anisodamine couldn`t be detected. Glycoalkaloids could not be analysed directly by gas chromatography due to their high molecular weight and low volatility. For simultaneous determination of tropane alkaloids (tropine, atropine, scopolamine, homatropine, anisodamine) and glycoalkaloids (&alpha;-solanine, &alpha;-chaconine) fast and sensitive multiresidue LC&ndash;MS/MS method was validated. In sample preparation dispersive solid phase extraction (DSPE) was performed with 0.5% formic acid in acetonitrile/water (75:25, v/v) and a mixture of magnesium sulphate, sodium chloride, sodium citrate and C18 sorbent. The analytes were separated by isocratic HPLC on a Chirobiotic V column with the mobile phase that consisted of 10 mM ammonium formate in water/acetonitrile (90:10, v/v) and methanol/acetonitrile (50:50, v/v), mixed in the ratio of 20:80 (v/v).<br />Compounds were detected in positive electrospray ionization mode (ESI+), using multi reaction monitoring (MRM). Optimised liquid-chromatographic method exhibited good linearity in the range 5-80 ng/g (r²=0.998). The method has shown to be specific, selective, accurate (recoveries from 61- 111%), precise (CV &lt; 5%) and rugged. Calculated limits of detection (LOD) for all alkaloids were in the range 0.74-0.79 ng/g, while the limits of quantitation (LOQ) were in the range 2.2-4.9 ng/g in all grains, except for tropine in soybean and linseed (LOD = 1.55 and LOQ = 4.91 ng/g). The proposed method was applied in the analyses of samples obtained from local supermarkets. The alkaloids were not detected in following food samples:<br />wheat, rye, maize, mixed grain flour, corn flour, biscuits and crackers. The feeds for pigs and chicken were the most contaminated with atropine, scopolamine, &alpha;-solanine and &alpha;-chaconine with the contents of alkaloids in the range of 3.6 to 21.7 ng/g. Atropine and scopolamine were detected in cattle and rabbit feed in concentrations ranging from 2.8 to 9.8 ng/g, while in feed for horses and wild animals none of the target alkaloids were detected. Conducted research reports for the first time the presence of glycoalkaloids, &alpha;-solanine and &alpha;-chaconine in fruit and vegetable samples (apple, pear, avocado, carrot, cucumber and paprika). The concentrations of these alkaloids were in the range of 0.3 to 15 ng/g. The content of glycoalkaloids in potato and chips complied with the recommended content of total glycoalkaloids concentration of 200 mg/kg (raw weight) (FAO/WHO, 1999). Absorption and translocation of alkaloids in wheat (roots, leaves, stem) was determined by using atropine ¹⁴C. It was found that young wheat after 15 days absorbed approximately 4.30% and 2.28% of atropine ¹⁴C for low (13676 dpm/g soil) and high (37203 dpm/g soil) spiked levels in soil, respectively. Increase in the spiked concentration in the soil did not affect higher absorption of atropine ¹⁴C in wheat. The highest accumulation of atropine ¹⁴C was observed in the leaves (83%) while lower<br />concentrations were detected in the roots (8.6%), stems (4.2%) and seeds (4.1%). In collected water samples during the soil leaching, atropine ¹⁴C was detected in the concentration of 0.5%. Leaching decreased with the time (0.01% after 90 days). Analyses of soil samples after 30 and 60 days showed strong adsorption of atropine to the soil and quantity of adsorbed atropine was 60% and 70%, respectively. Adsorbed atropine in the soil was stable after 90 days.</p>

Datura metel is a herbaceous plant found in almost all tropical parts of the world. It belongs to the family Solanaceae whose members, viz. Duboisia, Atropa, Hyoscyamus and Datura plants are known to produce tropane alkaloids- hyoscyamine and scopolamine which are most noted for their therapeutic use as anti-cholinergic agents. Since these alkaloids are produced in very low amounts in plants, alternative sources and methods of production for these alkaloids have been crucial in meeting the demands for these drugs. Endophytic fungi inhabiting a plant may have the potential to produce the same compounds as the host plants. The aim of the present study was to search for tropane alkaloid producing endophytic fungal isolates from Datura metel. Eighteen endophytic fungi were isolated from various tissues of Datura metel and screened for the presence of three tropane alkaloid biosynthetic genes- putrescine N-methyltransferase (PMT), tropinone reductase I (TRI) and hyoscyamine 6β-hydroxylase (H6H) using PCR-based screening approach. Six endophytic fungal isolates were found to possess the PMT, TR1 and H6H genes. The fungi were identified using molecular taxonomy as Col letotrichum boninense, Phomopsis sp., Fusarium solani, Col letotrichum incarnatum, Col letotrichum siamense and Col letotrichum gloeosporioides and the identity was confirmed using colony and spore morphology. The production of tropane alkaloids hyoscyamine and scopolamine by the fungi has been ascertained using various techniques like TLC, HPLC and ESI-MS/MS by comparison with the authentic reference standards. The amount of tropane alkaloids produced by all six fungi in liquid cultures was quantified using HPLC analysis. Among the six tropane alkaloid-producing fungi Col letotrichum incarnatum gave the highest yields of hyoscyamine and scopolamine which were 3.906 mg/L and 4.13 mg/L, respectively. With an aim to characterize the tropane alkaloid biosynthetic genes in these fungi, the PMT gene was isolated from five of the endophytic fungi- Col letotrichum boni-nense, Fusarium solani, Col letotrichum incarnatum, Col letotrichum siamense and Col-letotrichum gloeosporioides for the first time and the sequence analysis showed high ho-mology (98%) to the Datura metel PMT cDNA. The gene was found to be devoid of introns in the fungi. Further phylogenetic analysis of the full length PMT sequence from the fungi strongly supports the hypothesis of horizontal gene transfer between the host plant and endophytic fungi. For further in detail characterization of fungal PMT, the Col letotrichum boninense PMT gene was taken as a representative. CbPMT gene was cloned in pRSET A expres-sion vector and heterologously expressed in E. coli and biochemically characterized. For optimal yield of soluble protein upon heterologous expression different conditions such as IPTG concentration, temperature and time post induction were optimized. Optimal yield was obtained by inducing the culture by 0.25 mM IPTG once it had reached and O.D. of 0.6 and incubating at 37◦ C for 3 h. The recombinant CbPMT enzyme expressed as histidine tagged fusion protein was purified using Ni-NTA affinity chromatography. Gel elution studies were carried out to determine molecular weight of the protein and it was found that the protein exists as a homodimer in solution with some amount also present as a monomer. Catalytic activity of the purified recombinant enzyme was studied for its dependence on both substrates putrescine as well as S-adenosylmethionine (SAM). The Km and Vmax values for putrescine were found to be 464 µM and 18.55 nkat/mg, respectively, while those for S-adenosylmethionine were found to be 628 µM and 18.63 nkat/mg, respectively. Optimum temperature for activity was found to be 37◦ C and optimum pH range was found to be 8-9. Fluorescence spectroscopy was used to study the binding affinity of both the sub-strates to the enzyme. Fluorescence quenching data for each substrate was analysed by using a nonlinear regression curve fit and Kd values were found to be 0.309 mM for pu-trescine and 0.118 mM for SAM, respectively. Circular dichroism spectrum of the enzyme indicated a pattern typical for alpha helix in the secondary structure. Binding of either substrate led to increase in ellipticity of the protein. Fluorescence quenching studies with collisional quenchers- acrylamide, potassium iodide, and cesium chloride indicated that the native protein is folded in a conformation that allows tryptophan residues to be acces-sible for quenching. The fraction of tryptophan residues (fa ) accessible for quenching by acrylamide (1.06) was found to be higher than that for potassium iodide (0.54) while that cesium ions was the least (0.38). The neutral quencher acrylamide could access all the tryptophans meaning that none of tryptophans are completely buried inside hydrophobic cores. the differential accessibility to the charged quenchers, however, indicates that more of the tryptophans are surrounded by positively charged amino acids. The unfolding of the protein was studied with the aid of chaotropic agents guanidine-HCl and urea and thermodynamic parameters were determined. The denaturant m-values were found to be 2.313 kcal/mol/M for Gdn-HCl and 2.345 kcal/mol/M for urea respectively. The free energy of unfolding was estimated to be 2.635 kcal/mol for Gdn-HCl and 4.630 kcal/mol for urea. Since no reports are available about the thermodynamics of folding and unfolding of PMT from any plant source, this study contributes towards the understanding of protein stability. Although a lot of reports are available on the biochemical characterization of PMT from different plant sources, the crystal structure of PMT is not yet available. In the current work, homology based modelling studies on CbPMT were carried out to get some idea about the protein tertiary structure. Homology based modelling studies showed that a significant amount of protein is present as α-helices which are present on the surface while the β-sheets are present in the interior of the protein. Each monomer of the protein is capable of binding both the substrates and hence the dimerization property of the enzyme could be a purely structural one leading to more stability and solubility of the protein. In conclusion, this study has shown for the first time that endophytic fungi have significant potential to be used for tropane alkaloid production and six such fungal strains have been identified. Although the production of tropane alkaloids by endophytic fungi is not very high, it can be scaled up by over-expressing the biosynthetic gene putrescine N-methyltransferase in the highest producer- Col letotrichum incarnatum to further increase the yield. These endophytic fungi have significant potential to be applied in fermentation technology to meet the demands for these drugs economically.