STEREOCHEMICAL STUDIES ON THE METABOLISM. OF STEROLS BY Saccharomyces. cerevisiae STRAIN GL7. by YANGHONG LI, B.S. A THESIS CHEMISTRY

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1 STEREOCHEMICAL STUDIES ON THE METABOLISM OF STEROLS BY Saccharmyces cerevisiae STRAIN GL7 by YANGHONG LI, B.S. A THESIS IN CHEMISTRY Submitted t the Graduate Faculty f Texas Tech University in Partial Fulfillment f the Requirements fr the Degree f MASTER OF SCIENCE Apprved May, 1996

2 I ^ ^^ L ACKNOWLEDGEMENTS I wuld like t express my sincere gratitude t my advisr, Prf. W. David Nes fr his unfailing supprt, guidance and patience. I am als grateful t my cmmittee member Prf. Rbert W. Shaw fr his assistance and suggesdns. My appreciatin als ges t Dr. Dean Gu and Zhnghua Jia and ther members f the research grup, wh have always been very supprtive and helpful t me fr the past few years. The accmplishment f this thesis wuld nt have been pssible withut the encuragement f my family: my parents, brther, sister and cusins. T them, I dedicate this wrk. 11

3 TABLE OF NTENTS ACKNOWLEDGEMENTS ABSTRACT ii v LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS ix CHAPTER I. INTRODUCTION General Review Nmenclature f Sterls lupac Rules Nes System Bisynthesis f Sterls Research Objective 6 IL ANALYTICAL METHODS Chrmatgraphy Gravity Chrmatgraphy Thin Layer Chrmatgraphy Reverse-Phase High Perfrmance Liquid Chrmatgraphy Gas-Liquid Chrmatgraphy Spectrscpy Ultravilet Spectrscpy Infrared Spectrscpy Nuclear Magnetic Resnance Spectrscpy Mass Spectrscpy Other Methds 36 iii

4 ni. ISOLATION AND PURIFICATION OF STEROLS FROM NATURAL SOURCES Intrductin Material and Methds General Extractin f Sterls Islatin f Sterl Subclass Fractins Results and Discussin 50 IV. MECHANISM STUDIES ON A22 DESATURATION OF THE STEROL SIDE CHAIN BY SACCHAROMYCES CEREVISIAE STRAIN GL Intrductin Material and Methds Sterl Substrates Yeast Strains Culture Cnditins Analysis f Sterls Results and Discussin 80 LIST OF REFERENCES Ill APPENDICES A. FIGURES 116 B. WOODWARD'S RULES FOR DIENE ABSORPTION 120 C. OPERATION INSTRUCTION OF BRUCKER AF-200 NMR SPECTROMETER 121 IV

5 ABSTRACT The relatinship between sterl structure and functin was estabushed using a sterl auxtrph Saccharmyces cerevisiae strain GL7. The 21 sterl substrates incubated with GL7 were btained thrugh natural prduct islatin and rganic synthesis. The majr surce f sterl substrates was Cereus giganteus (cactus) puen. Frm the pllen was islated 27 sterls. The sterls were characterized in sterechemical detail by chrmatgraphic (TLC, GLC, and HPLC) and spectral (MS, and ^H-NMR) methds. Several nvel sterls were identified frm cactus: desmsterl, 24(28)-methylenezymsterl, and 9(ll),24(25)-dehydrpllinastanl. 24(28)-Methylene chlesterl was fund t be the dminant sterl in the plant. It was used as the majr sterl substrate as well as the substrate fr the preparatin f several synthetic sterls. A key sterl, [24-2H]-24 3-methyl chlesterl prepared fr the metablism studies was synthesized by stereselective istpic labeling f the sterl side chain. The A^^-desaturase enzyme frm GL7 was fund t be stereselective and regispecific. Fr sterl transfrmatin by the A22 desaturase, we fund that the sterl side chain must be arranged in the staggered cnfrmatin at the time f catalysis. In this cnfrmatin the 22-Pr-S (bttm) and 23-Pr-S (tp) hydrgen atms may be remved by a syn mechanism t frm the trans-22,23-bnd; 24p alkyl sterls induce greater A22-desaturatin than 24a alkyl sterls. The literature reprts that ther ergsterl-synthesizing fungi Aspergillus fumigatus and Blackeslea trispra intrduced hydrgen at C22 and C23 by a 5>7z-mechanism. We interpret the results t imply that sterl specificity f the sterl A^^-desaturase enzyme in mre advancedascmycetus fungi is similar. In cntrast t the sterl specificity bserved in prtein binding, a lack f sterl specificity was fund in sterl-cntrued grwth f GL7.

6 LIST OF TABLES 3.1 Chrmatgraphic and spectral prperties f sterls examined in cactus plant ^H-NMR spectral values f cactus pllen sterls Anatmical distributin f sterls and pentacycuc triterpenids frm cactus plant Chrmatgraphic and spectral prperties f sterls examined in this study ^H-NMR spectral values f sterls incubated with and recvered frm GL l^c-nmr spectral values f substrate and metablites Mdificatin f the sterl side chain by GL7 110 VI

7 LIST OF FIGURES Dimensinal gemetry and cnfrmatin f a generic sterl Cmparisn f the cnventinal and revised cnventins fr numbering f the carbn atms f the sterl skeletn based n the lupac rules Nmenclature f structural features f the sterl skeletn using the Nes system.. 9 ^ -4 Cmparisn f the R/S and a/(5 ntatins fr describing side chain chlral ^ centers 1.5 Bisynthetic pathway fram acetate t squalene Bisynthetic bifurcatin f squalene xide t sterls Chrmatgraphic behavir f sterls and steryl acetate in adsrptin TLC and silver nitrate TLC Temperature dependent chrmatgraphic separatin f chlesterl and lansterl by RP-HPLC eluted with 6% aq. methanl Chrmatgraphic behavir f structurauy similar sterls n fur GLC clumns Typical ultravilet spectra f sterls: chlesterl (A^), 5a-8(9),14(15)- ergstadien-3p-l (A8(9),14(15)) and ergsterl (A5.7) 4Q 2.5 Infrared spectra f typical plant sterls in the "finger print regin" Spin cupling between tw prtns with very different chemical shifts Generic ^H-NMR regins f sterls ir-nmr (200 MHz) spectra f campesterl and epicampesterl Hypthetical bisynthetic pathway in cactus pllen A flw chart describing the extractin, islatin and characterizatin f sterls frm plants Thin-layer chrmatgram f cactus pllen NSF sterl fractins (#85-# 180) eluted frm aluminum xide clumn A plt f mass vs. fractin number frm the aluminum xide clumn used t islate sterl fractins frm cactus pllen NSF GLC and HPLC chrmatgrams f 4,4-dimethyl sterls frm cactus pllen GLC and HPLC chrmatgrams f 4-mnmethyl sterls frm cactus puen vii

8 3.7 GLC and HPLC chrmatgrams f 4,4-desmethyl sterls frm cactus pllen ^H-NMR spectrum f 24,25-dehydrpllinastanl ^H-NMR spectrum f 24(28)-methylenezymsterl ih-nmr spectrum f 24(28)-methylene chlesterl Mass spectrum f 24(25)9(1 l)-dehydrpllinastanl IR-NMR spectrum f desmsterl GLC f brwn algal NSF The bisynthetic pathway f ergsterl Side chain structures f the substrates and majr metablites The stereselective synthesis and sterechemical transfrmatin by GL7 t [24- ^H] brassicasterl A cmparisn f the ^H NMR and MS spectra f brassicasterl and [24-2H]- brassic as terl 9^ 4.5 Alternate cnfrmatins f sme sterls incubated with GL A HETR spectrum f 24,25-cyclprpyl lansterl A DEPT l^c spectrum f 24,25-cyclprpyl lansterl An UV spectrum f 24,25-cyclprpyl ergsterl Seventy-five hur incubatin with 5.0ppm chlesterl fed t yeast GL HPLC radichrmatgram f 4-desmethyl sterl mixture frm [3-3H]- chlesterl incubatin with GL7 102 A. 1 A bl(x:k diagram f a dual-clumn gas chrmatgraph shwing essential parts A.2 Three pieces f equipment used t extract sterls frm plants 118 A.3 Experimental set-ups fr the synthesis f 24,25-cyclprpyl lansterl 119 vm

9 LIST OF ABBREVIATIONS ttc DEPT FPP GC/MS GPP HETR HMGCA HPLC IPP IR MAU MVA NMR NOE NSF RP-HPLC Rrtc TLC TLP UV Retentin time relative t chlesterl n high perfrmance liquid chrmatgraphy Distrtinless enhancement by plarizatin transfer Famesyl diphsphate Gas Uquid chrmatgraphy/mass spectrscpy Geranyl diphsphate Heternuclear chemical shift crrelatin 3-Hydrxy 3-methyl glutaryl-ca High perfrmance liquid chrmatgraphy Ispentyl diphsphate Infrared MiU ampere unit Mevalnic acid Nuclear magnetic resnance Nuclear Overhauser Effect Nnspnifiable lipid fractin Reverse phase high perfrmance liquid chrmatgraphy Retentin time relative t chlesterl in gas liquid chrmatgraphy Thin layer chrmatgraphy Ttal Upid fractin Ultravilet IX

10 CHAPTER I INTRODUCTION 1.1 General Review Sterls are a class f natural prducts which have intrigued scientists fr 150 years. They are ubiquitus natural prducts fund thrughut the evlutinary hierarchy frm cyanphytic bacteria t trachephytes and man. In 1825, M.E. Chevereul, a 19th century French Chemist, discvered that a gallstne substance pssessed physical prperties different frm ther lipidal materials, ntably waxes. He named the substance "chlesterine" frm the Greek "chle" fr bile and "'steres" fr sud; frm chlesterine evlved chlesterl. Chlesterl mdulates the fluidity f eucarytic membranes, and "buffers" the transitin temperature f the plasma membrane, maintaining its fluidity. Many substances fund in animals and plants, including thse with such diverse bilgical functins as bile acids, sex hrmnes and the sapnins, are related structurally t chlesterl. ^'2 The term "sterls" can be defined as: any chiral tetracyclic ispentenid which may be frmed by the cyclizatin f squalene xide thrugh the transitin state pssessing sterechemistry similar t the trans-syn-trans-anti-trans-anti cnfiguratin, i.e., the prtsterid catin, and retains a plar grup at C-3 (hydrxyl r ket), an all trans-anti sterechemistry f the ring system and a side chain 20R-cnfiguratin. Mlecules that fail t incrprate these sterechemical features may be cnsidered sterl-like mlecules which are caued "triterpenids."^ The glbal shape f the sterl, including tp and bttm faces f the mlecule, may be defined when the sterl mlecule is viewed n the edges t assess its 3-dimensinal gemetry and cnfrmatin (Figure 1.1). With the nucleus as a reference pint it becmes pssible t establish cnnecting lines and planes f sterechemical demarcatin and 1

11 cnfluence between the nucleus and side chain and alng the side chain. As shwn in Figure 1.1, the sterl mlecule assumes a defined vlume which is determined by its length (riented frm prximal t distal end r frm C-3 t C-26 (27)), height (tp t bttm r C-5 t C-10, respectively). The magnitude f vlume is a functin f the ppulatin f side chain rtamers which assume the right-handed staggered cnfrmatin, the presence f angular methyl grups which prtrude frm the p and a faces and the degree t which die side chain sweeps ut a cne.4 In membranes, chlesterl interdigitates between pairs f phsphupid acyl grups anistrpicauy in either half f the bilayer where the plar end f the mlecule (3 p-hydrxyl) is situated near a ket functin f the phsphlipid acyl grups belw the plane f rtatin f the phsphatidylchline grup. Since sterls mainly serve this membrane insert rle, and the thickness f a membrane ranges frm 35 t 40 A, the sizes f the sterls (19.1A in length) are seemingly designed fr this functin. 1.2 Nmenclature f Sterls The trivial naming and systematic nmenclature fr sterls cntinue t be an area f much debate. Tw sets f rules are nw in use; ne fllws an archaic numbering system that estabushes pririties based n revised lupac recmmendatins;^ the ther ne is based n a cmbinatin f histrical, chemical, and bisynthetic reasning, the Nes system.3»6, njpac Rules The lupac rules discuss the sterechemical descriptin (R/S system) and numbering f the carbn atms in the ring and side chain based n the chlestane skeletn (Fig. 1.2). The revised nmenclature system numbers the nuclear methyl grups and incrprates a higher-rder labeling fr added methyls attached t C-24 rather than numbering them utright as they have been in the past. One prblem with this numbering system is that it fails t allw fr the numbering in sequence f germinal methyls attached t C-24, i.e., as

12 in 24-dimethyl chlesterl. In ther wrds which f the tw C-atms is t receive the lwer rdered prime number, the ne in thefi-ntr in the back f the plane? In lupac rules, C- 26 and C-27 are viewed as chemically equivalent even thugh they are magnetically (in NMR) and bichemically distinct carbn atms (ne is derived frm C-2 f mevalnic acid and the ther is derived frm C-6 f mevalnic acid). Regardless f which ever f the tw terminal methyl grups attached t C-25 is substituted, e.g., hydrxylated, that metiiyl is assigned by the lwer number (C-26) and the ther C-atm becmes C-27, n sterechemical r bichemical distinctin between the tw grups is t be implied. When ne f the tw terminal isprpyl grups becmes unsubstituted r unsaturated, the tw methyl grups attached at C-25 are then numbered with, the carbn bearing the duble bnd as C-26 as in 25(26)-duble bnd (Fig. 1.2). The revised lupac rules suggest tiiat when an unsubstituted 25(26) bnd is present the tw methyl grups attached at psitin 25 are numbered 26, 27 with the lwer number 26 fr the methyl grup trans t psitin C- 23. lupac rules als suggest using the R/S sequence rules t describe the sterechemistry f sterls. But this causes cnfusin because similar cmpunds which have different substituent patterns assciated with the same site may have reverse cnfiguratins-frm R t S (with n inversin t be implied). This numbering system als failed t fully realize a system which can number the cmplicated multiple bialkylated side chains. Djerassi and cwrkers have suggested an alternative apprach and that is t number the alkyl grups by the bisynthetic rder in which they were intrduced.^ This system is nt fully satisfactry at the present time but with additinal knwledge f alkylatin bisynthesis this prcedure may prve t have merit Nes System Nes system, mdified frm Fieser and Fieser,^ incrprates the "bisynthetic side chain rule" (Fig. 1.3). The side chain rule was develped by phytchemists studying plant

13 sterl metablism.^ In this rule, C-26 is defined as the side chain carbn atm which is derived ft-m C-2 f mevalnic acid, whereas C-27 is derived frm C-6 f mevalnic acid. When the side chain pssesses the 24,25-duble bnd, e.g., as in lansterl and cyclartenl, the (Z)-methyl grup is derived frm the cis-methyl (C-27 cis t C-23) in squalene and C-6 f mevalnic acid, whereas the (E)-methyl grup is derived frm the trans-methyl (C-26 trans t C-23) in squalene and C-2 f mevalnic acid. By this rule, C-2 f mevalnic acid may never becme C-27. The Nes system uses the a/p ntatins t describe the sterechemistry f the mlecule. The a/p nmenclature f the nucleus is different frm the ne used in the side chain. Grups r atms attached t the ring system are dented P if they lie abve the plane f the paper r a if they lie belw the plane f the paper. In the side chain, a dentes-in the frnt f the plane and p dentes-in the back f the plane. The advantage f this nmenclature system is that it is independent f substituents at neighbring atms (Fig. 1.4). The language f sterid nmenclature is based n the names used fr a small number f parent r stem cmpunds frm which all ther sterids are cnsidered t be described structurally. Unsaturatin is dented by changing the terminal "ane" f the parent hydrcarbn t "ene", "diene", "triene", etc., fr ne, tw, three, etc., duble bnds. The psitin f a duble bnd is indicated by inserting the number f the carbn atm with the lwer number f the pair linked by the duble bnd. If the duble bnd links tw carbn atms nt cnsecutively numbered, bth numbers are inserted. A is n lnger used in the nmenclature system fr duble bnd ntatins, but it can still be used t categrize sterls. 1.3 The Bisynthesis f Sterls Sterls are ultimately prduced in plants frm the phtsynthetic fixatin f carbn dixide t sugar. The sugar frmed in plastids is metablized t acetyl cenzyme A, the 4

14 basic C2 building blck f cellular bisynthesis. There are tw pathways f carbn flux t sterl, the ispentenid and the fatty acid pathways. Acetyl-CA may be directed int the ispentenid pathway r accumulate as a pl f cmpartmentalized carbn reserve. It can als be fed by sugar degradatin and p-xidatin f acyl Hpids (fatty acids). The acetyl-ca pl may als be increased by metablism f ispentenids and amin acids thrugh the. mevalnic acid (MVA) shunt. 10 The MVA shunt is respnsible fr channehng amin acids int sterls and triterpenids, s that carbn flux between prtein degradatin and plycychc ispentenidsi^-^^ may bypass the sugar/acyl-ca circuit. It was fund that the MVA shunt was respnsible fr 10% f the carbn flux int sterls and triterpenes in the germmatin f sme plants, i^ Since A^ sterls are majr metablites frmed in active cell grwth, the frmatin f the A5 bnd represents cmpletin f the sterl pathway. On the ther hand, A^ sterls may als serve as precursrs fr saturated sterls (stanls) and highly functinalized sterids which play different rles in plant physilgy. Tw sterl precursrs, lansterl and cyclartenl, are cnsidered t be the first tw tetracyclic prducts f squalene xide cyclizatin. A cyclartenl-based pathway is assciated with a phtsynthetic lineage, whereas a lansterl-based pathway is assciated with a nnphtsynthetic lineage. ^^ The bisynthetic bifurcatin is smetimes viewed, incrrectiy, as representing a plant-animal divisin (Fig. 1.6). The utiine f sterl bisynthesis is as fllws (Fig. 1.5): 1. Cnversin f acetyl-ca int acetacetyl C A. 2. Cnversin f acetacetyl-ca int (3S)-3-hydrxy-3-methyl glutaryl-ca (HMGCA). 3. Cnversin f HMGCA int (+)-(3R)-mevalnic acid (MVA). 4. Cnversin f MVA int 5-phsphmevalnic acid (5P-MVA). 5. Cnversin f 5P-MVA int 5-diphsphmevalnic acid (5PP-MVA).

15 6. Cnversin f 5PP-MVA int A^-ispentyl diphsphate (A^-IPP). 7. Ismerizatin f A^-IPP int A^-IPP. 8. Head t tail cndensatin f A^-IPP with A^-IPP t frm geranyl diphsphate (GPP). 9. Head t tail cndensatin f GPP with A^-IPP t frm famesyl diphsphate (FPP). 10. Tail t tail cndensatin f tw FPP mlecules t frm squalene. 11. Oxidatin f squalene t frm squalene epxide. 12. Frmatin f prtsterid catin. 13. Frmatin f lansterl (anunal) r cyclartenl (plant) thrugh the cyclizatin f squalene epxide and prtsterid catin. 14. Bisynthesis f A^ sterls frm lansterl and cyclartenl. 1.4 Research Objective My research gal was t establish the relatinship between sterl structure and functin using a strain f yeast, Saccharmyces cerevisiae strain GL7, which is auxtrphic fr sterls. This study includes: islatins and characterizatins f sterls using mdem chrmatgraphic and spectral methds, and mechanism studies n A^^ desaturatin f the sterl side chain using intact cells. In rder t cnduct the metablism studies, a large amunt f sterl substrates were required. Therefre, the first part f my research was t islate and purify sterl substrates frm natural surces, then t prepare several additinal substrates synthetically. The secnd part f my research was devted t stmcture-activity studies f sterls incubated with GL7.

16 distal end N. ^v 2^ I I I 2^ 00 c t>jd P3 ^ 1^ 00 Si. "». a 0\ C/3 u s -K G O C/5 C P3.=3 Q I i 4 pu9 [BlUjXOJJ in O) W) T3 <D ^-^ 03 U > O) ^4-> c C4^ u

17 Cnventinal numbering system Revisins t the cnventinal numbering system -4- ( N Prvisin fr identifying A^^^^^ sterls as required when cnsidering bisynthetic rigin f carbn atms) 26 (26-hm) (27a-hm) (26-hydrxy) NO CHANGE cis-a^"^^^^^^ Series trans-a^^^^^ Series 24a-Alkyl series 24P-Alkyl series Figure 1.2 Cmparisn f the cnversin and revised cnventins fr numbering f the carbn atms f the sterl skeletn based n the lupac rules (references 4 &44). g

18 Frm c-2 MVA P ind equiirikj. ' lies in pline f mlecule VJ :4ajJky)i$R. but S when A22 present a ind c^uatni]. lid in pline f mlecule P 2LJ)d ix\i}. lies ut f plane f mlecule P-alVylitS. back f side v^ ^^ but R vkbcn A22 chain ^O. present Figure 1.3 Nmenclature f stmctural features f the sterl skeletn using the Nes system (reference 6).

19 . CS r <s cs cs TI CS cs n / a: / sa r^ X-; ( V \ 24S cs (N cs 00 cs cs CJ G J3 00 t G c G O -r-t 4 > G. "B G O OH e O U 6J) 10

20 #C-Atms In Mlecu H3C--CA i HOOC-CH2-C(CH3)(OH)-CH2--CA I c-6 HOO' ACETYL-CA HMO CA MVA Cg -* MVA Shunt H pp- I pp- A^-IPP A^-IPP Squalene C 30 I %,^:^\x'*x,^^ Squalene Epxide Figure 1.5 Bithetic pathway frm acetate t squalene. 11

21 Prtsterid Catin HO / \ Lansterl A^-Sterls Figure 1.6 Bisynthetic bifurcatin f squalene xide t sterls (reference 1). 12

22 CHAPTERII ANALYTICAL METHODS 2.1 Chrmatgraphy Chrmatgraphy is a methd f separating a mixture f cmpunds int its cmpnent parts. The methd is used t separate trace impurities and/r majr fractins frm each ther. All chrmatgraphic prcedures in essence depend upn the phenmenn f partitin, which is the equilibratin f a substance between tw phases that are nt mutually miscible; ne is the mbile phase and the ther ne is the statinary phase. The cmpnents f the mixture have different adsrbtivity with respect t the tw phases invlved, and thus have different partitin cefficients and may be separated accrdingly. The efficiency f a chrmatgraphic clumn is expressed in the number f theretical plates in the clumn. Theretical plate is a term brrwed frm distillatin, which describes the distributin cycle. The mre theretical plates, the better the separatin. There are fur principal methdlgies used fr the chrmatgraphy f sterls, and related bigenetically derived sterids: ^^'^^ gravity-flw clumn liquid chrmatgraphy (GCC), thin-layer chrmatgraphy (TLC), gas-liquid chrmatgraphy (GLC) and highperfrmance liquid chrmatgraphy (HPLC). Each chrmatgraphic system serves a unique functin in the separatin, purificatin, quantitatin and structural elucidatin f these cmpunds. Their chrmatgraphic behavir is knwn t be influenced by the hydrgen-bnding character and ther electrnic attractins (e.g., Van Der Waals frces and diple-diple interactins) between the lipid and the adsrbent. The rate f mvement fr each cmpund will depend ni^^'^^ (1) the sterechemistry and lcatin f the plar substituents; (2) the slubility, partitin cefficient and equilibrium cnstants f the cmpund in the slvent (and its plarity) used t develp the system; (3) the size and the shape f the mlecule; and (4) the degree f hydratin and surface area f the adsrbent 13

23 which affects intermlecular attractin between slvent, lipid and statinary phase. When the fur systems are used in series; GCC -^ TLC -^ HPLC -> GLC, ne can achieve hmgeneity nearly 100% and btain imprtant diagnstic infrmatin abut the lcatin and gemetry f selected substituents and f the three-dimensinal shape f the mlecule as a whle. Prf f structure requires cnfirmatin by sme additinal analytical prcedure, such as NMR, and MS Gravity Clumn Chrmatgraphy Gravity clumn chrmatgraphy includes rdinary-phase (adsrptin) and argentatin chrmatgraphy. It is als knwn as liquid-slid chrmatgraphy, in which the liquid is the mbile phase, r a slvent, such as hexane, and the slid supprt is the statinary phase, r adsrbent, such as silica gel. The driving frce fr separatin is gravity Adsrptin Chrmatgraphy A wide variety f substances have been emplyed as the statinary phase in this technique. In practice, hwever, tw materials have becme dminant in this type f separatin chemistry. Finely grund aluminum xide ( mesh) and silica gel ( mesh) are by far the mst useful f the knwn adsrbents. The liquid, which acts as the mving (mbile) phase, and eluate (wash) which cntains the sample, are passed thrugh the clumn with several cmmn rganic slvents. The slvents may be eluted thrugh the clumn either iscratically r step-wise, i.e., f increasing plarity. Adsrptin chrmatgraphy is used t islate the sterl subclasses when the NSF (nnsapnifiable lipid fractin) is cmplex r high in weight (i.e., 200 mg r mre). Adsrptin systems are gd methds fr separating sterls accrding t the kind and number f their xygen functinal grups. ^^ Size can als be a cntributing factr in the separatin f sterls with adsrptin systems. ^^ Duble bnds have a small but definite effect n the mbility f sterls in this system. This chrmatgraphic methd invlves the 14

24 binding f a substrate t the surface f a statinary plar phase thrugh hydrgen-bnding and ther electrnic attractins, fllwed by elutin with mbile phases f increasing plarity. Fr silica gel clumns the series f slvents are added in the rder f increasing plarity: hexane< hexane-benzene< diethyl ether< methanl< methanl-chlrfrm. Fr elutin f nitrgen cmpunds, the last slvent emplyed is triethylamine-ethyl acetate. The step-wise gradient is diethyl ether graded in 10% increments int Skelly Slvent B (mixed hexanes). A typical elutin rder is as fllws: hydrcarbns (100% hexane) < ketnes = esters < 4,4-dimethyl sterids < 4-mnmethyl sterids = lng chain fatty alchls < 4- desmethyl sterids (30/70 = hexane/ether) < steryl glycsides < phsphlipids < nitrgen cntaining sterids Argentatin Clumn Chrmatgraphy Argentatin clumn chrmatgraphy can be used in the separatin f sterls differing in the number and psitin f duble bnds frm a variety f surces. In general, the mre a duble bnd can interact with the silver ins, usually assciated with decreased substitutin r steric hindrance abut the duble bnd, the greater the adsrptin and the slwer the rate f mvement; multiple duble bnds als retard mbility. Tetrasubstituted duble bnds have a small affinity fr cmplexing with the silver in while trisubstituted duble bnds have a strng affinity fr the silver in. Duble bnds in the nucleus are shielded t a greater extent than islated duble bnds in the acyclic side chain, therefre gd separatin is achieved with sterls in which ne duble bnd is lcated in the nucleus and the ther lcated in the side chain. The influence f the duble bnd is enhanced by acetylatin with pyridine/acetic anhydride (1:1), befre chrmatgraphy in argentain systems. 15

25 2.1.2 Thin Laver Chrmatgraphy (T\r.) Thin layer chrmatgraphy is anther slid-liquid partitin technique t separate and partially identify sterls. While the mbile phase descends in clumn chrmatgraphy; in TLC, the mbile phase ascends because f the capillary effect. Rf value is used t calculate the separatin factr. Specifically, the Rf is the rati f the flw rates f the sample and the slvent ver the plate. Fr a particular system f substrate, slvent, and sample, the Rf value is a cnstant. Cmpunds can be separated int varius classes based n their hydrgen bnding strength f the plar grup at C-3. Chlesterl is used as the reference marker. The structural feature which mst cntributes t the chrmatgraphic behavir f chlesterl in adsrptin TLC is the presence f a free 3p-0H grup. With additinal methyl grups t C-4, the hydrgen bnding strength f -OH at C-3 decreases, and thus the sample mves up the plate faster. By this means, samples can be characterized as cntaining mainly 4- desmethyl-, 4-mnmethyl-, r 4,4-dimethyl sterl cmpunds with the desmethyl sterls have the smallest Rf value and the dimethyl sterls have the largest Rf value (Fig. 2.1). A cmmn methd fr detectin f sterls and ther sterids n TLC is t spray the plate with 50% sulfuric acid; then, after cntinuus heating, lipids char black. During the initial phases f heating, sterls with duble bnds shw characteristic clrs: ^^ ^5 sterls, pink; A5'7 sterls, gray r green; A^ sterls, range. The advantages in the use f TLC systems are (1) speed, relative t pen clumn chrmatgraphy; (2) ability t chrmatgraph several samples simultaneusly; and (3) ease in system manipulatin. The drawbacks in using TLC separatin include: (1) lss during separatin; (2) generatin f artifacts frm bulk sterls r lss f trace sterls by interactin f the cmpunds with the supprt; (3) reductin in number f theretical plates which affects the efficiency f separatin. 16

26 2.1.3 Reverse-Phase High Perfrmance Liquid Chrmatgraphy HPLC is an example f liquid-liquid chrmatgraphy, in that the sample is partitined between a statinary and a mbile phase that travels dwn a clumn. The mbile phase, r slvent, is delivered t the system by a pump capable f pressures up t abut 6000 psi. The sample is intrduced by the injectin f a slutin int an injectin lp. The slid phase in HPLC clumns used fr rganic mnmers is usually based n silica gel. "Nrmal" HPLC refers t chrmatgraphy using a slid phase (usually silica gel) which is mre plar than the liquid phase (slvent) s that less plar cmpunds elute earlier. Typical slvents fr nrmal phase HPLC include ethyl acetate, hexane, acetne, etc. When the liquid phase is mre plar than the statinary phase, the mre plar cmpunds elute mre quickly. This system is knwn as reverse phase HPLC. In RP-HPLC, the statinary phase is usually a derivatized silica gel where the -OH grups f the silica gel have been replaced by -OSiOR grups where R is typically a linear C18 alkyl chain. These small diameter, highly prus particles give the statinary phases very large effective surface areas fr the interactin f the sterls with the bnded alkyl grups. This large area makes pssible rapid equilibratin f the sterl between the statinary phase and an apprpriate mbile phase, yielding clumns with as much as 104 theretical plates and thus gives better reslutin. On RP-HPLC, 24-alkyl sterls are nrmally eluted later cmpared t the sterls lack f 24-alkyl grups because f the steric hindrance caused by the alkyl grup. Sterls with duble bnds are mre plar than their crrespnding saturated sterls and thus cme ut early frm RP-HPLC clumn. As a result f this relatinship between stmcture and elutin time, crrelatin tables f stmcture versus OQ (relative retentin time t chlesterl) have been cnstmcted and used as references t predict and cnfirm sterl stmctures.^^'^^ RP-High Perfrmance Liquid Chrmatgraphy (HPLC) is becming the mst cmmnly used technique fr the separatin f individual sterls frm subclass fractins. 17

27 By adjusting the clumn temperature, slvent plarity, and increasing clumn length, stmcturally similar sterls can be separated t hmgeneity. Fr example, campesterl (24a-methyl chlesterl) and its epimer, 24P-methyl chlesterl can be separated cmpletely n TSK clumn (ODS-120A, 5 im, 4.6 mm i.d.x 250 mm) eluted witii methanl at 15 C. Chlesterl and lansterl, which cchrmatgraphed n a 25 cm Ci8 clumn eluted with 6% aqueus methanl at rm temperature, were partially separated n a 10 cm Ci8 clumn by elevating the clumn temperature t 40 C (Fig. 2.2). The plarity f the eluant will influence the hydrgen bnding character f sterls, i.e., sterl can act either as a hydrgen dnr r hydrgen acceptr. Hence, it is pssible t induce chrmatgraphic frameshifts n a given set f cmpunds based n the use f a nrmalphase and reversed-phase slvents. In fact, the rate f mvement f campesterl, stigmasterl, 7-dehydr-chlesterl, ergsterl, 24(28)-methylene chlesterl and chlesta- 5,22(E)-dienl relative t chlesterl is demnstrably different n Cig-clumns eluted with acetnitrile, acetnitrile-water, prpanl in hexane, methanl r methanl-water.l9 In cnjunctin with suitable detectrs, RP-HPLC can be used as a purely analytical tl in the primary characterizatin f sterls. When a multiple wavelength dide array detectr is emplyed in the use f RP-HPLC, the detectr can prduce a signal fr any sterl that passes thrugh the aperture. The respnse r peak height measured in mau is dependent n the amunt f sample laded nt the clumn, number and kind f chrmphres in the mlecule. Since all the sterls including stanls have an end absrptin spectmm, the UV mnitr is set at 205nm fr rutine analysis. Hwever, it shuld be kept in mind that similar peak heights may represent vastiy different levels f sterls. 18

28 2.1.4 Gas Liquid Chrmatgraphy General Principles Gas liquid chrmatgraphy (GLC) is a pwerful tl (Fig. A.l) in structure determinatin and quantitatin. It is a frm f partitin chrmatgraphy in which the vlatile cmpunds are separated. The cmpunds are passed in a stream f inert gas (the mbile phase) thrugh a clumn, which is packed with slid supprting material cated with a high biling pint liquid (the statinary phase). The substances are separated accrding t their partitin cefficients, which depend n their vlatilities and relative slubilities in the liquid phase Carrier Gas The carrier gas itself must be inert tward the sample and the substrate and must be thermally stable. The mst cmmnly used carrier gases are helium, argn, nitrgen, and hydrgen. Of these, helium is used mst ften because it is inert, can be perated at high flw rates, and is the mst cnvenient fr a number f detectrs, such as the thermal cnductivity detectr Sample Injectin System The sample is best injected in the frm f a liquid r a gas in rder t be intrduced nt the clumn in the frm f a cmpact "plug." Rapid sample vaprizatin is brught abut by heatmg the injectin cell t temperatures up t 350''C. When the sample is injected int the injectin cell, it immediately vaprizes, and the vaprs are swept nt the substrate by the carrier gas Detectrs The functin f the detectr is t detect and measure the different cmpnents f the sample as they emerge frm the clumn. The chice f detectr depends n the type f 19

29 analysis being perfrmed. High sensitivity can be achieved by using a flame detectr, but the sample is destryed in the prcess. High selectivity can be achieved with electrn capture detectrs. Other detectrs, such as the thermal cnductivity detectr, are nnselective and nndestmctive. In the flame inizatin detectr, the effluent frm the gas chrmatgraphic clumn is fed int an air-hydrgen flame. Tw wire electrdes are als placed in the flame and an electrical ptential is applied acrss them. When nly the inert carrier gas enters the flame, the current acrss the electrdes is small and cnstant. When an rganic cmpund frm the end f the chrmatgraphic clumn enters the flame, the cmpund is brken up int fragments that are highly cnductive. These are easily detected by the change in the current flwing acrss the electrdes which is capable f detecting lo'^^ g f rganic material. It is ne f the mst sensitive detectins available fr gas liquid chrmatgraphy and can be used fr cnventinal GC and capillary clumn GC. FID is sensitive t all rganic cmpunds, including hydrcarbns and heterrganic cmpunds. Hwever, it is nt sensitive t many inrganic cmpunds. The degree f change in cnductivity depends n the particular mlecules. Hence its respnse t a given quantity f an rganic cmpund depends n the particular cmpund. The electrn capture detectr is used when high sensivity and selectivity tward halide, phsphms, r nitrgen cmpunds are required. The surce f electrns, r P-rays, is 3H2 r ^^Ni fil. Many cmpunds, such as paraffins and simple hydrcarbns, are virtually transparent t the electrns, but rganic halide, phsphrus, and nitrgen cmpunds are nt, and they capture the electrns. There is an abmpt change in the number f electrns reaching the cllectr, and this prvides the detectin signal. Thermal cnductivity detectr is als called the katharmeter. As the carrier gas frm the gas chrmatgraph emerges frm the clumn, it flws tiirugh the detectr. The detectr cnsists f a hllw tube with a wire situated in the central axis. The wire is heated 20

30 electrically and reaches a steady temperature when the heat gained frm electrical energy is equal t the heat lst t the surrunding gas (i.e., the carrier gas). Under these cnditins f steady temperature, the electrical resistance f the wire is steady. When a different gas (e.g., a sample cmpnent) flws past the wire, there is a change in the thermal cnductivity f the surrunding gas, and therefre change the resistance f the wire. Heat is cnducted away frm the wire at a different rate. The temperature f the wire changes and therefre the electric resistance f the wire changes. This change in resistance is used t detect the presence f different gases flwing past the cnductr wire. The thermal cnductivity detectr detects all cmpnents with a thermal cnductivity different frm that f the carrier gas. Als, it is nndestmctive. This is a distinct advantage ver ther detectrs if the sample must be trapped after separatin and used fr ther purpses. It is cmmnly used in cnventinal GC, but it is nt sufficiently sensitive fr capillary clumn GC Liquid Phase The liquid phase in cmmn use are f tw types: (1) the nnplar silicnes (e.g., SE30, methyl silicne), in which silicn atms f the plymer bear alkyl grups and (2) plar phases f the silicne in which ne r mre f the alkyl grups are replaced by a plar grup (e.g., OV-17, phenyl-methyl silicne). The verall plarity f the phases is measured by McReynlds' cnstants,^^ which increase as the plarity increases. Since n liquid phase is capable f separating all sterls, the chice f phase will depend n the stmctural type t be encuntered. The separatin f an equal mass mixture f five 4,4- desmethylsterls n fur packed clumn is illustrated in Fig As shwn, chlesterl, campesterl, sitsterl, 24(28)-methylene chlesterl, and 24-ethylidene chlesterl are cmpletely separable nly n a very plar GLC clumn. Nrmally, when the plarity f a clumn increases, the cmpleteness f separatin increases as well. Hwever, the clumn 21

31 temperature f SP-1000 clumn is nrmally 255 C, much higher than that f SE-30 clumn's (235 C). Therefre, plar clumns have shrter half-lives cmpared t the less plar clumns, i.e., SE-30; because they tend t bleed very easily. SE-30 packed clumn is used by a lt f researchers because f its relatively lng half-life, and thus is csteffective. In additin, it has been prved that SE-30 packed clumn has very stable Rrtc (retentin time relative t chlesterl). The Rrtc value nly varies 0.05 frm ne injectin t anther when the flw rate f gases and temperature are kept cnstant Thus, a crrelatin table f sterl stmcture and Rrtc can be cnstmcted and used as a reference, and sterl stmctures can be predicted accurately 1^,16 (see applicatin f GLC n sterl analysis). The plar fatty acids are shifted t theft-ntf SE-30 clumn, and thus cme ut earlier than mst f the sterls Types f Clumns Packed-clumn gas chrmatgraphy, as the name implies, invlves the use f packed clumns with internal diameters f abut 0.25 inches. The tube may vary in length frm 3 t 20 feet. This methd, the wrkhrse f gas-liquid chrmatgraphy, is used fr all frms f cnventinal rganic mlecular analysis, including bth qualitative and quantitative analysis. Capillary clumns are particularly useful in the analytical separatin f stmcturally similar sterls. ^^ Lng plar capillary clumns where the number f theretical plates has been greatiy increased have been used t separate ismeric C-24 sterls which cannt be reslved n the packed clumns.24 There are tw types f capillary clumns which are generally used: thse in which the liquid phase has been bund t the inside surface (abut 0.3- im thick film) f a capillary clumn, called a wall-cated pen tubular clumn (WT); r thse in which the inert supprt (cated with liquid phase) has been bund t the inner surface, called a supprt-cated pen tubular clumns (ST). 22

32 Applicatin f GLC in Sterl Analysis Infrmatin frm the analysis f sterl fractins during their extractin, islatin, and separatin by GLC n packed (r capillary) clumns with a wide range f McReynlds' cnstants can be used t further characterize the cmpunds present. With a mixture f unknwns, the peaks wuld be assigned a retentin time relative t chlesterl (Rrtc) fr each clumn. Claytn^S recgnized that each alkyl grup, duble bnd, etc., in the sterl mlecule had its wn effect n retentin time. The retentin time f a sterl is the prduct f the retentin time f the basal sterl nucleus and the retentin factrs cntributed by each f the substituent grups. Therefre, it is pssible t predict the retentin time f the unknwn sterid by using the cntributin factr (O^) when a single feature is cmmn between pairs f stmcturally similar cmpunds. The relatinships are shwn by the fllwing series f equatins: O^ = Rrt with feature/rrt withut feature, RRTu = Rrtp x prduct f C^ values, Rrtu/Rrtp = prduct f G^ values. GLC is als frequently used t quantitate sterls. Dried sample is disslved in a knwn amunt f slvent t give a mass-t-vlume rati within the Imear range f the detectr, and the sample is injected int the clumn. Mdem cmputer-cntrlled integratrs can then relate detectr respnse t mass fr each peak. Based n a value determined fr the standard, the amunt f sterl in the sample can then be cmputed. Using FID (Flame Inizatin Detectin), the detectr respnse f the sterl mass is similar within the range f mlecular weights M+ frm 380 t 460 amu. Pyrlisis f xygenated and nitrgen cntaining sterls may ccur, but die pattern f pyrlisis prducts is ften diagnstic chrmatgraphically. 23

33 2.2 Spectrscpic Methds Ultravilet Spectrscpy General Principles Ultravilet spectrscpy (UV) is the ldest f the spectrscpic methds in the identificatin f sterls. Absrptin f radiatin in the UV regin ( nm) is due mainly t changes in the energy f electrns invlved in the frmatin f valence bnds. UV absrptin spectra are therefre f value in revealing the presence f duble bnds and, in particular, f cnjugated duble-bnd systems. Absrptins in the UV regin are usually assciated with n ^n* r n->7t* transitins. Bth the psitin and intensity f UV absrptin bands prvide stmctural infrmatin. The intensity f absrptin at a given wavelength is cnventinally expressed as the mlar extinctin cefficient r the mlar absrptivity (^) r as its lgarithm t base 10 Gg^)- ^ is calculated by the expressin 5 = A/(cxd) where A: ptical density; c: cncentratin f the slutin in mmiter^; d: the thickness f the cell that cntains the slutin in cm. Plts f absrptin intensity (lg^) versus wavelength (k) are presented semilgarithmically Applicatin f UV in Sterl Analysis Mst sterls, especially thse f plants, cntain islated duble bnds absrb in the range f nm. The absrptin is attributed t Tt^p* transitins and is knwn as end absrptin (peratinal definitin). Its very presence mdicates unsaturatin. There are tw principal types f chrmphric systems absrbing abve 220 nm which ccasinally ccur in plant sterls (Fig. 2.4). Ergsterl cntains 5,7-cnjugated duble bnd system, 24

34 and has Xj^ax at 262, 272, 283, and 293 nm. Its extinctin cefficient (^) at ^283 is 11,9007. Anther unusual diene system is the 22,24(28) chrmphre fund in yeast.26 It absrbs at 230 nm with an extinctin cefficient (^) f 21,400. Wdward's mles are used t predict the bathchrmic effect f alkyl substitutin in 1,3-butadiene systems (Appendix B) Infrared Spectrscpy General Principles The usefulness f IR spectrscpy fr the identificatin and analysis f sterids is limited t the determinatin f the presence f absrbing functinal grups and their sterechemical relatinship. When a beam f IR radiatin passes thrugh a mlecule, radiatins f certain frequencies are selectively absrbed, s that a series f peaks r bands appears in the spectmm f transmitted radiatins. The frequencies f the absrbed radiatins crrespnd t frequencies f stretching and bending vibratins f the varius bnds linking the atms in the mlecule. Certain stmctural features f the mlecule give rise t absrptin peaks which appear at certain regin f the spectrum respectively. The wavelength f IR radiatin is usually expressed in terms f wave-number, defined as the reciprcal f the wavelength in centimeter units('u, cm-l). The relatinship f the apprximate value f the atms Mi and M2 (in grams), the velcity f light (c), and the stretching cnstant f the bnd (k, in dyn/cm) is expressed in the frmula as fllwing: V = (1/27CC) (k/(mim2/(mi+m2)))l/2. The IR regin usually scanned in absrptin spectrscpy ranges frm 625 t 4000 cm-1. 25

35 Applicatin f IR in Sterl Analysis Infrared spectra are nt useful in distinguishing between sterls different nly in die number f side chain carbn atms.27 Because nly a few plant sterls cntain functinal grups ther than the 3p-0H, the primary use f IR is in identifying specific duble bnds. Hwever, in the regin between abut 650 and 1000 cm-1, die pattem f absrptin bands is very characteristic f the mlecule as a whle. This regin f the IR spectrum is therefre knwn as the "fingerprint" regin. By cmparing the IR fingerprint regin f an unknwn sterid with that f a reference cmpund, a psitive identificatin can ften be made r alternative pssibilities can be excluded. Figure 2.5 cmpares the infrared spectra f sitsterl, stigmasterl, fucsterl, and spinasterl. The single nuclear duble bnd f sitsterl gives trisubstituted ethylene absrptin in the cm-1 regin. Fucsterl shws absrptin in the same regin, but with an additinal peak at 820 cm^^ due t the E- 24(28) duble bnd. The IR spectmm f stigmasterl differs significantiy frm that f sitsterl due t the strng absrptin f the E-22 duble bnd at 967 cm-1. This strng absrptin peak is als diagnstic fr the E-22 duble bnd in ther sterls. Spinasterl, identical t stigmasterl in the side chain, has a 7-duble bnd rather than the 5-duble bnd f stigmasterl, giving a different "fingerprint" in the cm-l regin. Spinasterl als has a trans-a/b ring system which prduces a shift in the 1050 cm^^ regin in 5a, delta 7-sterls. Unfrtunately, IR fails t detect many imprtant stmctural features f sterls, such as the chirality f 24-alkyl grups, due t the absence f distinct functinal grups r neighbring functinal grups and the cmplexity f the finger print regin. 26

36 2.2.3 Nuclear Magnetic Resnance General Principles The nuclei f certain istpes are cntinuusly spinning with an angular mmentum, which can give rise t an assciated magnetic field. If a very pwerful external magnetic field is applied t the nucleus and made t scillate in the radi frequency range, the nucleus will resnate between different quantized energy levels at specific frequencies, absrbing sme f the applied energy. The variatin in the intensity f the resnance signal with increasing applied magnetic field, is knwn as Nuclear Magnetic Resnance (NMR) spectrscpy. Fr hydrgen nucleus, the angular mmentum (c), applied magnetic field (BQ), and irradiatin frequency (') are related as shwn in the accmpanying equatin: D = 0) /27i = BOY/27C where Y = nuclear magnetic mment/spin angular mmentum = i/i. There is a direct relatinship between the resnance frequency and the applied magnetic field BQ, and this is the mathematical basis f NMR. In practice, it is fund that if the applied field is 14,092 gauss, the frequency f radiatin (rf) absrbed is 60 MHz. The nmenclature 60 MHz NMR indicates the rf used and als defines the strength f the applied magnetic field. Similarly, a 100 MHz NMR uses 100 MHz and a magnetic field f 14,092 X 100/60 = 23,486 gauss. A 500 MHz NMR uses a magnetic field f 23,486 x 5 = 117,330 gauss. Such intense fields cannt be achieved with a permanent ferrmagnet, but use circular cils maintained at liquid He temperature (liquid N2 is used t prevent the evapratin f liquid He). When a strng rf field is used, fine stmcture due t spin-spin splitting is cllapsed and different infrmatin f the stmcture f the mlecule is btained. Thus, sterechemically different 24-alkyl sterl epimers are separable by strng field ^H- NMR. 27

37 Spin-Spin Cupling and Splitting Spin-spin splitting is quite strng between hydrgens n adjacent carbns, but is generally negligible between hydrgens farther remved than this. It causes a change in the fine stmcture f the adjacent hydrgens n the mlecule. The number f bands in fine stmcture due t spin-spin splitting is 2nl + 1. Fr hydrgen, I = 1/2 and the equatin simplifies t n + 1, in which n is the number f equivalent hydrgens n the adjacent grup. The number f bands is smetimes termed the multiplicity. The degree f splitting f a peak by adjacent hydrgens is a measure f the magnetic effect f that hydrgen. This magnetic effect is the cupling, r magnetic interactin, between the tw hydrgens. It is termed the J cupling cnstant and is measured by the distance between peaks in a given multiplet. The cupling cnstant is a measure f energy and is usually expressed in cycles per secnd. Since it is independent f the applied magnetic field, cupling cnstants between prtns rarely exceed 20 Hz, whereas chemical shifts usually range ver abut 1250 Hz at 100 MHz. The cupling cnstant prvides valuable infrmatin abut the mlecular interactin in the mlecule. Suppse that tw vicinal prtns are in very different ORCR3 RO CH CH CHR chemical envirnments frm ne anther as in the cmpund ^-^n-'-n-v.niv3 ^^^j^ prtn will give rise t an absrptin, and the absrptins will be quite widely separated, but the spin f each prtn is affected slightiy by the tw rientatins f the ther prtn thrugh the intervening electrns s that each absrptin appears as a dublet (Fig. 2.6a). As AvIJ becmes smaller iavlj is smaller than abut 10), the dublets apprach ne anther, the inner tw peaks increase in intensity, and the uter tw peaks decrease (Fig. 2.6b-f). The shift psitin f each prtn can be estimated witii fair accuracy by inspectin r determined precisely by the frmula (l-3)=(2-4)=((a')2-i-j2)l/2 j^i which the peak psitins (1,2,3,4 frm left t right) are given in hertz frm TMS. When A'0=jV3, the tw pairs culd be mistaken fr a quartet, which results frm splitting by three equivalent 28

38 lead t mistaking the tw large inner peaks fr a dublet (Fig When the chemical shift difference becmes zer, the middle peaks calesce t give a single peak, and the end peaks vanish; that is, the prtns are equivalent. The cupling cnstants fr a vinylic system i "A are characteristic; the trans cupling is larger than the cis, and geminal cupling is very small. -^"^ The prtns f any HaS R-^-(i:-L methylene grup in a mlecule cntaining chiral centers ^b ^ are nt chemical shift equivalent, and they cuple with each ther and each may have a different cupling t a vicinal prtn. But usually, the cupling cnstants are very small and easily be ignred when the prtn NMR is mn n lw field spectrmeter. When hydrgen is replaced by its istpe deuterium, neither spin-spin cupling nr splitting will happen. This istpe effect even affects the chemical shifts f the hydrgens n the adjacent carbns Chemical Shifts The difference in the absrptin psitin f a particular prtn frm the absrptin psitin f a reference prtn is called the chemical shift f the particular prtn. Prtns in "different" chemical envirnments have different chemical shifts. The mst cmmnly used reference cmpund is tetramethylsilane (TMS), and it is nrmally set at 0 ppm. CH3 H3C-Si-CH3 CH3 29

39 CH 3 H3C-Si-CH3 CH3 TMS has several advantages: it is chemically inert, symmetrical, vlatile (bp. 27^C), and sluble in mst rganic slvents; it gives a single sharp absrptin peak, and absrbs at higher field (shielded) than almst all rganic prtns (sme prtns will have negative chemical shifts when TMS is used as the reference). Deuterated chlrfrm cntaminated by 0.5% (v/v) TMS is the cmmnly used slvent. The cncentratin f TMS cannt be t high, r the spectmm will be limited by the dynamic range determined with respect t TMS. When the reference peak (TMS peak) cannt be determined crrectly, sme imprtant chemical shifts, i.e., negative values n the right side f TMS, will be verlked. Smetimes, chlrfrm is used as reference fr cnvenience. A slight difference f ppm has t be added t the chemical shifts using chlrfrm as reference when they are cmpared t the chemical shifts using TMS as reference. The symbl fr the chemical shift is 6. It is expressed as 5 = (BS-BTMS)/BTMS X 106 ppm where Bs = magnetic field at which the sample absrbs BTMS = magnetic field at which the reference TMS absrbs. The terms "dwnfield" r "lwer field" strength refer t larger 6 values, which are further left n the spectrum. Shielding by the drifting electrns is mdified by ther nuclei in their vicinity, which are affected by the chemistry, gemetry, and electrn density f the system. Cnsequentiy, sme deshielding takes place, and we are able t distinguish between different functinal grups in the neighbrhd f the nucleus. 30