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Naturersquos Chiral Catalyst and Anti-Malarial Agent Isolation andStructure Elucidation of Cinchonine and Quinine from CinchonacalisayaAnne-Marie Carroll David J Kavanagh Fiona P McGovern Joe W Reilly and John J Walsh
School of Pharmacy and Pharmaceutical Sciences University of Dublin Trinity College Dublin 2 Ireland
S Supporting Information
ABSTRACT Nature is a well-recognized source of compounds of interest but access is often an issue One pertinent example isthe cinchona alkaloids from the bark of Cinchona calisaya In this experiment students at the third-year undergraduate levelundertake the selective isolation and characterization of two of the four main alkaloids present in the bark Beginning withpowdered bark acidminusbase extraction followed by selective crystallization serves to yield cinchonine naturersquos chiral catalyst fromthe complex mixture Slow crystallization provides suitable quality crystals for X-ray analysis Students readily appreciate thethree-dimensional nature of this chiral catalyst which aids subsequent NMR spectroscopic analysis Manipulation of the motherliquor by thin-layer and flash column chromatographic techniques proves a simple but elegant method to furnish quininenaturersquos anti-malarial agent Both alkaloids are treated to an intensive structure elucidation workshop comprising 1- and 2-dimensional NMR infrared and mass spectrometry The method is economical polished and robust bringing the student on ajourney from crude plant material to medicinally important natural products in three 2-h laboratory sessions Moreoverquestions in the student handout and model answers in instructorrsquos notes respectively require that students engage further intopics associated with the context of this practical
KEYWORDS Upper-Division Undergraduate Laboratory Instruction Organic Chemistry Hands-On LearningManipulativesBiosynthesis Chromatography DrugsPharmaceuticals Natural Products NMR Spectroscopy X-ray Crystallography
The purpose of this experiment is first to provide thestudents with a historical overview of cinchona bark its
alkaloids (Figure 1) and their application Second the studentslearn the essential skills required to isolate and purifycinchonine and quinine from the bark of Cinchona calisayaThe method is low-cost refined and reproducible It relies on
an understanding of the principles behind the selective isolationof basic compounds from neutral and acidic natural productswhich includes knowing the correct choice of extracting solventfor the selective isolation of cinchonine from its closely relatedalkaloids cinchonidine quinine and quinidine An under-standing of the principles behind crystallization as a means ofpurification of natural drug substances is also highlighted As anadded bonus flash column chromatography of the motherliquor provides a simple but elegant method of quinineisolation from the complex mixture The use of 1- and 2-DNMR IR MS and X-ray spectroscopy as analytical tools toperform a complete structure elucidation study on the isolatedcompounds is then conducted The experiment complements
Published October 9 2012Figure 1 Cinchona alkaloids
Laboratory Experiment
pubsacsorgjchemeduc
copy 2012 American Chemical Society andDivision of Chemical Education Inc 1578 dxdoiorg101021ed200713p | J Chem Educ 2012 89 1578minus1581
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ber
20 2
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Se
e ht
tps
pub
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sor
gsh
arin
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es f
or o
ptio
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n ho
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les
the many other laboratory-based experiments on the isolationof naturersquos medicines including valtrate from Centranthusruber1 galantamine from Leucojum aestivum2 lovastatin fromred yeast rice3 parthenolide from Tanacetum parthenium4
betulin from birch bark5 curcumin from turmeric6 andthiarubrine A from Ambrosia artemisiifoia7
BACKGROUND
Cinchona bark is the dried bark from the stem and branches ofspecies of Cinchona (Rubiaceae)8 large trees indigenous toSouth America The various species including C calisayaCinchona legeriana and Cinchona succinubra vary in theirrelative alkaloid content8 This may range from 4 to 17 thelarger percentages coming from hybrid species The four mainalkaloids in cinchona bark are quinine and cinchonine and theirrespective diastereoisomers quinidine and cinchonidine whichtogether account for approximately 30minus60 of the totalalkaloid content8 These alkaloids have a rich history Already inuse to cure fever before 1600 by Peruvian native Indians theirscientific name originates from the myth that in the early 1600sLady Chinchon wife of the Spanish viceroy of Peru was curedfrom malaria using an ancient herbal remedy the ldquoquinquinardquobark Around 1630 Jesuits exported cinchona bark (thereafteralso known as ldquoJesuitrsquos barkrdquo) to Europe where it soon becamethe remedy of choice for malaria widespread at the time9 Witha growing demand for cinchona bark and the threat ofextinction by overharvesting European governments set aboutestablishing cinchona plantations in their tropical colonies Bythe 1930s the Dutch plantations of C ledgeriana in theIndonesian island of Java provided the bulk of the worldrsquosproduction The advent of World War II saw the allied forcescut off from cinchona trees in Java their sole source of quinineThis led to an increased impetus especially in the UnitedStates for developing a synthetic route to quinine9 the claimby Woodward in 1944 of having successfully accomplished thiswas hailed by The New York Times as ldquoone of the greatestscientific achievements in a centuryrdquo10 Interest in the cinchonaalkaloids continues to this day They are a versatile class ofnatural products serving both as medicinally importantcompounds (quinine is used in the treatment of malaria andleg cramps quinidine as an anti-arrhythmic agent) and asprivileged catalysts and ligands (the major use of cinchonineand cinchonidine) for asymmetric synthesis11 These uses arefurther explored in the accompanying Supporting Information
EXPERIMENTAL OVERVIEW
The experiment conducted over three 2-h laboratory sessionsis particularly suited to upper-level undergraduate students whohave a basic understanding of the chromatographic andspectroscopic techniques used in the identification of naturalor synthetic compounds Throughout the experiment studentsgain first-hand experience of various techniques involved in theextraction isolation and purification of medicinally importantnatural products from the crude plant material These includeacidminusbase extraction on the powdered bark to attain analkaloid-rich fraction from a complex matrix and subsequentextraction of cinchonine and related alkaloids into a suitablesolvent ethyl acetate Taking advantage of the low solubility ofcinchonine in ethyl acetate12 crystals of cinchonine slowly formfrom the mixture over several days and are of suitable qualityfor X-ray crystallography (week 1) The students then isolatethe crystals and determine their percentage yield and purity by
thin-layer chromatography (TLC) The mother liquor is notdiscarded It is reduced in vacuo and the residue subjected tothin-layer and flash column chromatographic techniques tofurnish quinine (week 2) In the third week of the experimentan extensive structure elucidation study of cinchonine andquinine is carried out in the form of a workshop using 1H HminusH 13C HMQC and HMBC spectra This gives students a firmunderstanding of the applications of 1- and 2-dimensionalNMR spectroscopy Moreover assignment of the exactresonance positions of the cinchonine peaks is greatly facilitatedby having its X-ray crystal structure (Figure 2) Students can
quickly understand from the 3-dimensional nature of themolecule why the methylene protons on the quinuclidine ringare chemically inequivalent The students can appreciate thischiral catalyst in 3 dimensions from the steric bulk of thequinuclidine moiety to the chiral pocket of the β-hydroxyaminefunctionality central to enantioselective catalysis Furthermorestudents learn how to interpret infrared and mass spectral datato complement the information gathered from NMR spectros-copy In particular the similarities and differences of cinchonineand quinine are emphasizedFrom a class of 58 students the average yield of cinchonine
isolated from 30 g of C calisaya was 35 mg whereas theaverage yield of quinine isolated was 50 mg Considering theminimal time effort and resources involved (student-friendlyin every sense) the end result compares favorably with catalyticasymmetric total synthetic approaches to quinine (19 steps withoverall yields of 096 by Stork et al10 16 steps with overallyields of ca 5 by Jacobsen et al11) The complete experimentrequired three 2-h sessions with a maximum of 15 studentsconducting the experiment in a given session
EXPERIMENTAL SECTION
Isolation of Cinchonine by AcidminusBase Extraction Followedby Selective Crystallization
In week one of the experiment powdered C calisaya (30 g)from the Herbal Apothecary13 was accurately weighed out andplaced directly into a 15 mL centrifuge tube A 10 aqueoussolution of trifluoroacetic acid (80 mL) was added to thepowdered material The screw cap lid was placed onto the tubeand tightened gently The contents of the tube were shakenvigorously by hand for 15 min centrifuged (3000 rpm roomtemperature 10 min) and 35 mL of the supernatant wastransferred to another 15 mL centrifuge tube containing ethylacetate (40 mL) and 10 M aqueous NaOH (50 mL) Thescrew cap lid was attached on the tube and tightened gentlyAgain the contents of the tube were shaken vigorously by handfor 5 min The sample was centrifuged (3000 rpm) for 10 minat room temperature Approximately 35 mL of the upperorganic layer was removed using a Pasteur pipet and placed into
Figure 2 X-ray crystal structure of cinchonine
Journal of Chemical Education Laboratory Experiment
dxdoiorg101021ed200713p | J Chem Educ 2012 89 1578minus15811579
a preweighed labeled sample vial Particular care was taken toavoid removal of the lower aqueous layer A lid was attachedonto the sample vial and the crystals of cinchonine wereallowed to form slowly over a period of one week In week 2 ofthe laboratory experiment the mother liquor was carefullyremoved using a Pasteur pipet The crystals that formed werewashed thoroughly with ethyl acetate (sim1 mL) by slowlyrotating the vial for 2 min Using a Pasteur pipet the washingswere then carefully removed taking care to avoid removal ofthe crystals The washing procedure was repeated The crystalswere dried under high vacuum for 10 min The sample vialcontaining the purified crystals was reweighed The mass ofcinchonine obtained from 30 g of cinchona bark was recorded
Isolation of Quinine by Flash Chromatography
In week two of the experiment the mother liquor and washingsfrom isolation of cinchonine were combined and reduced invacuo The residue was taken up in the minimum volume ofdichloromethanemethanol (101) to allow for easy transferonto the flash column The flash column was prepared asdescribed in the Supporting Information The mobile phase ofchoice was an isocratic system consisting of dichloromethaneethyl acetatepropan-2-oldiethylamine (207046) The vol-ume of eluent collected in each fraction was approximately 05mL The fractions containing pure quinine were identified usingsilica gel TLC employing the above mobile phase for whichquinine has an Rf of 016 These fractions were combinedreduced in vacuo in a preweighed round-bottomed flask thecontents dried under high vacuum and the yield of pure quininewas then calculated Overall this technique was reproduciblebut the student must use the correct quantity of silica and theminimum volume of solvent to allow for easy transfer of theresidue onto the column
Structure Elucidation Studies
In week three of the experiment IR mass spectrometry anddetailed NMR spectroscopic analyses confirmed that thesubstances isolated were cinchonine and quinine Bothmolecules have broadly similar IR spectra with the expectedcharacteristic absorption peaks for OminusH CminusH CC andalcoholic CminusO stretching An additional peak of interest in theIR spectrum of quininea strong absorption in the fingerprintregion (1228 cmminus1) for the CminusO stretch of the etherfunctionalitydistinguishes it from its demethoxy analoguecinchonineThe high-resolution mass spectrum (HRMS) reveals the
MH+ at mz 2951818 and 3251917 for cinchonine andquinine respectively as the most abundant ion with theexpected mass difference attributed to the methoxy function-ality of quinine In the 1H and 13C NMR spectra of quinine themethoxy protons resonate downfield as a singlet at 389 ppmAlthough the DEPT-135 spectrum still shows eleven peaks oneof these (at 556 ppm) is found to be a minusCH3 carbon by itsabsence from the DEPT-90 spectrum Although the aliphaticregion is comparable there is a notable difference in thearomatic region the six protons of cinchonine (four doubletsand two triplets) give way to the five of quinine (four doubletsand a double double doublet) and an extra quaternary carbon(to accommodate the methoxy) Complete assignment of allsignals is provided in the Supporting Information as iscomplementary information presented as questions andmodel answers Following completion of the experiment eachstudent submitted a detailed report Student feedback on theexperiment was positive They liked the relatively straightfor-
ward isolation procedures to isolate two important naturalproducts and in the case of cinchonine viewing the structure in3-dimensional form following X-ray analysis of the crystalsisolated from a representative student sample Students werepleased with the workshop session as they particularly liked thesmall group interactive format Moreover the manner in whichthe experiment blends into biosynthetic pharmacologicalpharmacokinetic and clinical material delivered in lectureswas particularly valued Thus the series of questions covered inthe Supporting Information was welcomed by the students togive them a more ldquobench to bedsiderdquo appreciation of theirsubject
HAZARDSSome potentially hazardous reagents and flammable solventsare used14 Ethyl acetate propan-2-ol diethylamine (all highlyflammable) trifluoroacetic acid and deuterated chloroform aretoxic by inhalation in contact with the skin and if swallowedDichloromethane and deuterated chloroform have limitedevidence of a carcinogenic effect Sodium hydroxide andtrifluoroacetic acid are corrosive causing severe burns Caremust be taken when handling these chemicals which must beused in the fume hood as must silica gel Contact with skin andeyes should be avoided and suitable personal protectiveequipment worn Protective gloves should be worn whenhandling the powdered C calisaya sample Ultraviolet (UV)radiation can cause severe damage to the eyes Do not lookdirectly into the light source
SUMMARYThe experiment has been performed with consistent reprodu-cibility by 58 third-year pharmacy students The method iseconomical polished and robust bringing the student on ajourney from crude plant material to isolation and character-ization of medicinally important natural products in three 2-hlaboratory sessions
ASSOCIATED CONTENTS Supporting Information
Student handout and instructorrsquos notes NMR spectra Thismaterial is available via the Internet at httppubsacsorg
AUTHOR INFORMATIONCorresponding Author
E-mail jjwalshtcdieNotes
The authors declare no competing financial interest
ACKNOWLEDGMENTSWe wish to thank John OrsquoBrien Brian Talbot and TomMcCabe for recording the NMR HRMS and X-ray datarespectively
REFERENCES(1) Doyle A M Reilly J Murphy N Kavanagh P V OrsquoBrien JE Walsh M S Walsh J J J Chem Educ 2004 81 1486minus1487(2) Halpin C M Reilly C Walsh J J J Chem Educ 2010 871242minus1243(3) Mohd Nazri M Samat F D Kavanagh P V Walsh J J JChem Educ 2012 89 138minus140(4) Walsh E L Ashe S Walsh J J J Chem Educ 2012 89 134minus137
Journal of Chemical Education Laboratory Experiment
dxdoiorg101021ed200713p | J Chem Educ 2012 89 1578minus15811580
(5) Green B Bentley M Chung B Lynch N Jensen B J ChemEduc 2007 84 1985minus1987(6) Anderson A M Mitchell M S Mohan R S J Chem Educ2000 77 359minus360(7) Reyes J Morton M Downum K OrsquoShea K J Chem Educ2001 78 781minus783(8) British Pharmacopoeia 2012 (Ph Eur monograph 0174) httpwwwpharmacopoeiacouk (accessed Oct 2012)(9) Bruce-Chwatt L J BMJ 1988 296 1486minus1487(10) Stork G Niu D Fujimoto A Koft E R Balkovec J MTata J R Dake G R J Am Chem Soc 2001 123 3239minus3242(11) Raheem I T Goodman S N Jacobsen E N J Am ChemSoc 2004 126 706minus707(12) Nyhlen J Eriksson L Backvell J E Chirality 2008 20 47minus50(13) Herba l Apotheca ry Home Page h t tp wwwherbalapothecaryukcom (accessed Sep 2012)(14) Sigma-Aldrich MSDS httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=270997 |SIALampN5=SEARCH_CONCAT_PNO|BRAND_KEYampF=SPEC (accessed Sep 2012)
Journal of Chemical Education Laboratory Experiment
dxdoiorg101021ed200713p | J Chem Educ 2012 89 1578minus15811581
the many other laboratory-based experiments on the isolationof naturersquos medicines including valtrate from Centranthusruber1 galantamine from Leucojum aestivum2 lovastatin fromred yeast rice3 parthenolide from Tanacetum parthenium4
betulin from birch bark5 curcumin from turmeric6 andthiarubrine A from Ambrosia artemisiifoia7
BACKGROUND
Cinchona bark is the dried bark from the stem and branches ofspecies of Cinchona (Rubiaceae)8 large trees indigenous toSouth America The various species including C calisayaCinchona legeriana and Cinchona succinubra vary in theirrelative alkaloid content8 This may range from 4 to 17 thelarger percentages coming from hybrid species The four mainalkaloids in cinchona bark are quinine and cinchonine and theirrespective diastereoisomers quinidine and cinchonidine whichtogether account for approximately 30minus60 of the totalalkaloid content8 These alkaloids have a rich history Already inuse to cure fever before 1600 by Peruvian native Indians theirscientific name originates from the myth that in the early 1600sLady Chinchon wife of the Spanish viceroy of Peru was curedfrom malaria using an ancient herbal remedy the ldquoquinquinardquobark Around 1630 Jesuits exported cinchona bark (thereafteralso known as ldquoJesuitrsquos barkrdquo) to Europe where it soon becamethe remedy of choice for malaria widespread at the time9 Witha growing demand for cinchona bark and the threat ofextinction by overharvesting European governments set aboutestablishing cinchona plantations in their tropical colonies Bythe 1930s the Dutch plantations of C ledgeriana in theIndonesian island of Java provided the bulk of the worldrsquosproduction The advent of World War II saw the allied forcescut off from cinchona trees in Java their sole source of quinineThis led to an increased impetus especially in the UnitedStates for developing a synthetic route to quinine9 the claimby Woodward in 1944 of having successfully accomplished thiswas hailed by The New York Times as ldquoone of the greatestscientific achievements in a centuryrdquo10 Interest in the cinchonaalkaloids continues to this day They are a versatile class ofnatural products serving both as medicinally importantcompounds (quinine is used in the treatment of malaria andleg cramps quinidine as an anti-arrhythmic agent) and asprivileged catalysts and ligands (the major use of cinchonineand cinchonidine) for asymmetric synthesis11 These uses arefurther explored in the accompanying Supporting Information
EXPERIMENTAL OVERVIEW
The experiment conducted over three 2-h laboratory sessionsis particularly suited to upper-level undergraduate students whohave a basic understanding of the chromatographic andspectroscopic techniques used in the identification of naturalor synthetic compounds Throughout the experiment studentsgain first-hand experience of various techniques involved in theextraction isolation and purification of medicinally importantnatural products from the crude plant material These includeacidminusbase extraction on the powdered bark to attain analkaloid-rich fraction from a complex matrix and subsequentextraction of cinchonine and related alkaloids into a suitablesolvent ethyl acetate Taking advantage of the low solubility ofcinchonine in ethyl acetate12 crystals of cinchonine slowly formfrom the mixture over several days and are of suitable qualityfor X-ray crystallography (week 1) The students then isolatethe crystals and determine their percentage yield and purity by
thin-layer chromatography (TLC) The mother liquor is notdiscarded It is reduced in vacuo and the residue subjected tothin-layer and flash column chromatographic techniques tofurnish quinine (week 2) In the third week of the experimentan extensive structure elucidation study of cinchonine andquinine is carried out in the form of a workshop using 1H HminusH 13C HMQC and HMBC spectra This gives students a firmunderstanding of the applications of 1- and 2-dimensionalNMR spectroscopy Moreover assignment of the exactresonance positions of the cinchonine peaks is greatly facilitatedby having its X-ray crystal structure (Figure 2) Students can
quickly understand from the 3-dimensional nature of themolecule why the methylene protons on the quinuclidine ringare chemically inequivalent The students can appreciate thischiral catalyst in 3 dimensions from the steric bulk of thequinuclidine moiety to the chiral pocket of the β-hydroxyaminefunctionality central to enantioselective catalysis Furthermorestudents learn how to interpret infrared and mass spectral datato complement the information gathered from NMR spectros-copy In particular the similarities and differences of cinchonineand quinine are emphasizedFrom a class of 58 students the average yield of cinchonine
isolated from 30 g of C calisaya was 35 mg whereas theaverage yield of quinine isolated was 50 mg Considering theminimal time effort and resources involved (student-friendlyin every sense) the end result compares favorably with catalyticasymmetric total synthetic approaches to quinine (19 steps withoverall yields of 096 by Stork et al10 16 steps with overallyields of ca 5 by Jacobsen et al11) The complete experimentrequired three 2-h sessions with a maximum of 15 studentsconducting the experiment in a given session
EXPERIMENTAL SECTION
Isolation of Cinchonine by AcidminusBase Extraction Followedby Selective Crystallization
In week one of the experiment powdered C calisaya (30 g)from the Herbal Apothecary13 was accurately weighed out andplaced directly into a 15 mL centrifuge tube A 10 aqueoussolution of trifluoroacetic acid (80 mL) was added to thepowdered material The screw cap lid was placed onto the tubeand tightened gently The contents of the tube were shakenvigorously by hand for 15 min centrifuged (3000 rpm roomtemperature 10 min) and 35 mL of the supernatant wastransferred to another 15 mL centrifuge tube containing ethylacetate (40 mL) and 10 M aqueous NaOH (50 mL) Thescrew cap lid was attached on the tube and tightened gentlyAgain the contents of the tube were shaken vigorously by handfor 5 min The sample was centrifuged (3000 rpm) for 10 minat room temperature Approximately 35 mL of the upperorganic layer was removed using a Pasteur pipet and placed into
Figure 2 X-ray crystal structure of cinchonine
Journal of Chemical Education Laboratory Experiment
dxdoiorg101021ed200713p | J Chem Educ 2012 89 1578minus15811579
a preweighed labeled sample vial Particular care was taken toavoid removal of the lower aqueous layer A lid was attachedonto the sample vial and the crystals of cinchonine wereallowed to form slowly over a period of one week In week 2 ofthe laboratory experiment the mother liquor was carefullyremoved using a Pasteur pipet The crystals that formed werewashed thoroughly with ethyl acetate (sim1 mL) by slowlyrotating the vial for 2 min Using a Pasteur pipet the washingswere then carefully removed taking care to avoid removal ofthe crystals The washing procedure was repeated The crystalswere dried under high vacuum for 10 min The sample vialcontaining the purified crystals was reweighed The mass ofcinchonine obtained from 30 g of cinchona bark was recorded
Isolation of Quinine by Flash Chromatography
In week two of the experiment the mother liquor and washingsfrom isolation of cinchonine were combined and reduced invacuo The residue was taken up in the minimum volume ofdichloromethanemethanol (101) to allow for easy transferonto the flash column The flash column was prepared asdescribed in the Supporting Information The mobile phase ofchoice was an isocratic system consisting of dichloromethaneethyl acetatepropan-2-oldiethylamine (207046) The vol-ume of eluent collected in each fraction was approximately 05mL The fractions containing pure quinine were identified usingsilica gel TLC employing the above mobile phase for whichquinine has an Rf of 016 These fractions were combinedreduced in vacuo in a preweighed round-bottomed flask thecontents dried under high vacuum and the yield of pure quininewas then calculated Overall this technique was reproduciblebut the student must use the correct quantity of silica and theminimum volume of solvent to allow for easy transfer of theresidue onto the column
Structure Elucidation Studies
In week three of the experiment IR mass spectrometry anddetailed NMR spectroscopic analyses confirmed that thesubstances isolated were cinchonine and quinine Bothmolecules have broadly similar IR spectra with the expectedcharacteristic absorption peaks for OminusH CminusH CC andalcoholic CminusO stretching An additional peak of interest in theIR spectrum of quininea strong absorption in the fingerprintregion (1228 cmminus1) for the CminusO stretch of the etherfunctionalitydistinguishes it from its demethoxy analoguecinchonineThe high-resolution mass spectrum (HRMS) reveals the
MH+ at mz 2951818 and 3251917 for cinchonine andquinine respectively as the most abundant ion with theexpected mass difference attributed to the methoxy function-ality of quinine In the 1H and 13C NMR spectra of quinine themethoxy protons resonate downfield as a singlet at 389 ppmAlthough the DEPT-135 spectrum still shows eleven peaks oneof these (at 556 ppm) is found to be a minusCH3 carbon by itsabsence from the DEPT-90 spectrum Although the aliphaticregion is comparable there is a notable difference in thearomatic region the six protons of cinchonine (four doubletsand two triplets) give way to the five of quinine (four doubletsand a double double doublet) and an extra quaternary carbon(to accommodate the methoxy) Complete assignment of allsignals is provided in the Supporting Information as iscomplementary information presented as questions andmodel answers Following completion of the experiment eachstudent submitted a detailed report Student feedback on theexperiment was positive They liked the relatively straightfor-
ward isolation procedures to isolate two important naturalproducts and in the case of cinchonine viewing the structure in3-dimensional form following X-ray analysis of the crystalsisolated from a representative student sample Students werepleased with the workshop session as they particularly liked thesmall group interactive format Moreover the manner in whichthe experiment blends into biosynthetic pharmacologicalpharmacokinetic and clinical material delivered in lectureswas particularly valued Thus the series of questions covered inthe Supporting Information was welcomed by the students togive them a more ldquobench to bedsiderdquo appreciation of theirsubject
HAZARDSSome potentially hazardous reagents and flammable solventsare used14 Ethyl acetate propan-2-ol diethylamine (all highlyflammable) trifluoroacetic acid and deuterated chloroform aretoxic by inhalation in contact with the skin and if swallowedDichloromethane and deuterated chloroform have limitedevidence of a carcinogenic effect Sodium hydroxide andtrifluoroacetic acid are corrosive causing severe burns Caremust be taken when handling these chemicals which must beused in the fume hood as must silica gel Contact with skin andeyes should be avoided and suitable personal protectiveequipment worn Protective gloves should be worn whenhandling the powdered C calisaya sample Ultraviolet (UV)radiation can cause severe damage to the eyes Do not lookdirectly into the light source
SUMMARYThe experiment has been performed with consistent reprodu-cibility by 58 third-year pharmacy students The method iseconomical polished and robust bringing the student on ajourney from crude plant material to isolation and character-ization of medicinally important natural products in three 2-hlaboratory sessions
ASSOCIATED CONTENTS Supporting Information
Student handout and instructorrsquos notes NMR spectra Thismaterial is available via the Internet at httppubsacsorg
AUTHOR INFORMATIONCorresponding Author
E-mail jjwalshtcdieNotes
The authors declare no competing financial interest
ACKNOWLEDGMENTSWe wish to thank John OrsquoBrien Brian Talbot and TomMcCabe for recording the NMR HRMS and X-ray datarespectively
REFERENCES(1) Doyle A M Reilly J Murphy N Kavanagh P V OrsquoBrien JE Walsh M S Walsh J J J Chem Educ 2004 81 1486minus1487(2) Halpin C M Reilly C Walsh J J J Chem Educ 2010 871242minus1243(3) Mohd Nazri M Samat F D Kavanagh P V Walsh J J JChem Educ 2012 89 138minus140(4) Walsh E L Ashe S Walsh J J J Chem Educ 2012 89 134minus137
Journal of Chemical Education Laboratory Experiment
dxdoiorg101021ed200713p | J Chem Educ 2012 89 1578minus15811580
(5) Green B Bentley M Chung B Lynch N Jensen B J ChemEduc 2007 84 1985minus1987(6) Anderson A M Mitchell M S Mohan R S J Chem Educ2000 77 359minus360(7) Reyes J Morton M Downum K OrsquoShea K J Chem Educ2001 78 781minus783(8) British Pharmacopoeia 2012 (Ph Eur monograph 0174) httpwwwpharmacopoeiacouk (accessed Oct 2012)(9) Bruce-Chwatt L J BMJ 1988 296 1486minus1487(10) Stork G Niu D Fujimoto A Koft E R Balkovec J MTata J R Dake G R J Am Chem Soc 2001 123 3239minus3242(11) Raheem I T Goodman S N Jacobsen E N J Am ChemSoc 2004 126 706minus707(12) Nyhlen J Eriksson L Backvell J E Chirality 2008 20 47minus50(13) Herba l Apotheca ry Home Page h t tp wwwherbalapothecaryukcom (accessed Sep 2012)(14) Sigma-Aldrich MSDS httpwwwsigmaaldrichcomcatalogProductDetaildolang=enampN4=270997 |SIALampN5=SEARCH_CONCAT_PNO|BRAND_KEYampF=SPEC (accessed Sep 2012)
Journal of Chemical Education Laboratory Experiment
dxdoiorg101021ed200713p | J Chem Educ 2012 89 1578minus15811581
a preweighed labeled sample vial Particular care was taken toavoid removal of the lower aqueous layer A lid was attachedonto the sample vial and the crystals of cinchonine wereallowed to form slowly over a period of one week In week 2 ofthe laboratory experiment the mother liquor was carefullyremoved using a Pasteur pipet The crystals that formed werewashed thoroughly with ethyl acetate (sim1 mL) by slowlyrotating the vial for 2 min Using a Pasteur pipet the washingswere then carefully removed taking care to avoid removal ofthe crystals The washing procedure was repeated The crystalswere dried under high vacuum for 10 min The sample vialcontaining the purified crystals was reweighed The mass ofcinchonine obtained from 30 g of cinchona bark was recorded
Isolation of Quinine by Flash Chromatography
In week two of the experiment the mother liquor and washingsfrom isolation of cinchonine were combined and reduced invacuo The residue was taken up in the minimum volume ofdichloromethanemethanol (101) to allow for easy transferonto the flash column The flash column was prepared asdescribed in the Supporting Information The mobile phase ofchoice was an isocratic system consisting of dichloromethaneethyl acetatepropan-2-oldiethylamine (207046) The vol-ume of eluent collected in each fraction was approximately 05mL The fractions containing pure quinine were identified usingsilica gel TLC employing the above mobile phase for whichquinine has an Rf of 016 These fractions were combinedreduced in vacuo in a preweighed round-bottomed flask thecontents dried under high vacuum and the yield of pure quininewas then calculated Overall this technique was reproduciblebut the student must use the correct quantity of silica and theminimum volume of solvent to allow for easy transfer of theresidue onto the column
Structure Elucidation Studies
In week three of the experiment IR mass spectrometry anddetailed NMR spectroscopic analyses confirmed that thesubstances isolated were cinchonine and quinine Bothmolecules have broadly similar IR spectra with the expectedcharacteristic absorption peaks for OminusH CminusH CC andalcoholic CminusO stretching An additional peak of interest in theIR spectrum of quininea strong absorption in the fingerprintregion (1228 cmminus1) for the CminusO stretch of the etherfunctionalitydistinguishes it from its demethoxy analoguecinchonineThe high-resolution mass spectrum (HRMS) reveals the
MH+ at mz 2951818 and 3251917 for cinchonine andquinine respectively as the most abundant ion with theexpected mass difference attributed to the methoxy function-ality of quinine In the 1H and 13C NMR spectra of quinine themethoxy protons resonate downfield as a singlet at 389 ppmAlthough the DEPT-135 spectrum still shows eleven peaks oneof these (at 556 ppm) is found to be a minusCH3 carbon by itsabsence from the DEPT-90 spectrum Although the aliphaticregion is comparable there is a notable difference in thearomatic region the six protons of cinchonine (four doubletsand two triplets) give way to the five of quinine (four doubletsand a double double doublet) and an extra quaternary carbon(to accommodate the methoxy) Complete assignment of allsignals is provided in the Supporting Information as iscomplementary information presented as questions andmodel answers Following completion of the experiment eachstudent submitted a detailed report Student feedback on theexperiment was positive They liked the relatively straightfor-
ward isolation procedures to isolate two important naturalproducts and in the case of cinchonine viewing the structure in3-dimensional form following X-ray analysis of the crystalsisolated from a representative student sample Students werepleased with the workshop session as they particularly liked thesmall group interactive format Moreover the manner in whichthe experiment blends into biosynthetic pharmacologicalpharmacokinetic and clinical material delivered in lectureswas particularly valued Thus the series of questions covered inthe Supporting Information was welcomed by the students togive them a more ldquobench to bedsiderdquo appreciation of theirsubject
HAZARDSSome potentially hazardous reagents and flammable solventsare used14 Ethyl acetate propan-2-ol diethylamine (all highlyflammable) trifluoroacetic acid and deuterated chloroform aretoxic by inhalation in contact with the skin and if swallowedDichloromethane and deuterated chloroform have limitedevidence of a carcinogenic effect Sodium hydroxide andtrifluoroacetic acid are corrosive causing severe burns Caremust be taken when handling these chemicals which must beused in the fume hood as must silica gel Contact with skin andeyes should be avoided and suitable personal protectiveequipment worn Protective gloves should be worn whenhandling the powdered C calisaya sample Ultraviolet (UV)radiation can cause severe damage to the eyes Do not lookdirectly into the light source
SUMMARYThe experiment has been performed with consistent reprodu-cibility by 58 third-year pharmacy students The method iseconomical polished and robust bringing the student on ajourney from crude plant material to isolation and character-ization of medicinally important natural products in three 2-hlaboratory sessions
ASSOCIATED CONTENTS Supporting Information
Student handout and instructorrsquos notes NMR spectra Thismaterial is available via the Internet at httppubsacsorg
AUTHOR INFORMATIONCorresponding Author
E-mail jjwalshtcdieNotes
The authors declare no competing financial interest
ACKNOWLEDGMENTSWe wish to thank John OrsquoBrien Brian Talbot and TomMcCabe for recording the NMR HRMS and X-ray datarespectively
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