Proc. Nail. Acad. Sci. USAVol. 85, pp. 5759-5763, August 1988Chemistry

Conformational analysis of 9,f,19-cyclopropyl sterols: Detection ofthe pseudoplanar conformer by nuclear Overhauser effects andits functional implications

(periplanar structure/cycloartenol/24,25-dehydropolhinastanol/membranes/sterols)

W. DAVID NES*, MABRY BENSONt, ROBERT E. LUNDINt, AND PHU H. LE**Plant and Fungal Lipid Group, Plant Physiology Research Unit, Russell Research Center, U.S. Department of Agriculture, Athens, GA 30605; and tPlantDevelopment Quality Research Group, Western Regional Research Center, U.S. Department of Agriculture, Albany, CA 94710

Communicated by E. E. van Tamelen, February 10, 1988

ABSTRACT Nuclear Overhauser difference spectroscopyand variable temperature studies of the 9.3,19-cyclopropylsterols 24,25-dehydropollinastanol (4,4-desmethyl-5a-cyclo-art-24-en-3fi-ol) and cyclolaudenol [(24S)-24-methyl-5a-cy-cloart-25(27)-en-3,3-ol] have shown the solution conformationofthe B/C rings to be twist-chair/twist-boat rather than boat/chair as suggested in the literature. This is very similar to theknown crystal structure conformation of 9,3,19-cyclopropylsterols. The effect of these conformations on the molecularshape is highly significant: the first conformation orients intoa pseudoplanar or flat shape analogous to lanosterol, whereasthe latter conformation exhibits a bent shape. The results areinterpreted to imply that, for conformational reasons, cyclo-propyl sterols can be expected to maintain the pseudoplanarshape in membrane bilayers.

For reasons that are still unknown, organisms having a photo-synthetic lineage cyclize squalene oxide to the 9f3,19-cy-clopropyl sterol (CS) cycloartenol, while organisms having anevolutionary history that is completely nonphotosynthetic cy-clize squalene oxide to the isomeric tetracycle lanosterol (1, 2).Cycloartenol has been shown to isomerize to lanosterol underacidic conditions (3) and during routine metabolism in plants (2).

In lanosterol the 8,9 double bond approximates a transstructure for the B/C ring junction, maintaining a flat confor-mation of the molecule. However in CSs the 9,10 cyclopropylbridgehead and the ,B oriented hydrogen at C-8 approximate asyn-cis configuration at the A/C and B/C ringjunctions. Someinvestigators believe (3-8) that this configurational dispositionbends the plane of the molecule at the B/C ring junctionthrough almost 90°. Dreiding models also show the moleculeis no longer flat in the bent shape but neither they norstereochemical considerations of polycyclic systems showthat CSs could orient into the exaggerated "curvilinear belt"described by Bloch (4). That the configuration of the ringjunctures influences the overall conformation of the moleculeis known (8) in the isomeric pair of saturated sterols cholesta-nol and coprostanol. Here, inversion of the configuration ofH-5 changes the A/B ring juncture stereochemistry from flat(5a:A/B trans) to a bent system (5p3:A/B cis) analogous to thatdescribed by Bloch and others for CSs (3-8). The differencesin stereochemistry for this pair of sterols is not accompaniedby a change in conformational isomerism in the rings fromchair to boat. In CSs, however, rotational isomerism about theC-ring carbons may permit CSs to occur in two conformations,one with the B/C rings in a boat/chair conformation giving themolecule a bent shape and one with the B/C rings in atwist-half-chair/twist-boat conformation with the molecule ina pseudoplanar or flat shape (Fig. 1). This may occur without

B

FIG. 1. Two conformations of cycloartenol. (A) Boat/chair(bent); (B) twist-half-chair/twist-boat (pseudoplanar).

chemical or biochemical modification. In fact CSs are knownto exist in the flat shape in the solid state (9-11). Nevertheless,based on their physiochemical and biological studies, Bloch (4)and Benveniste et al. (5) have argued the exclusiveness of thebent shape when CSs are functional in membranes. Further-more, these investigators go on to suggest that in plants CSsmay completely replace the natural end products of biosyn-thesis such as sitosterol, for cell vitality (4, 5). This suggestionimplies that plants lack the same functional strictures forelimination of the 14a-methyl group in CSs as those that existfor lanosterol metabolism in mammalian systems (4, 12).

In contrast to this suggestion, we (13) and others (14, 15)have demonstrated a low steady state concentration of CSsin plants during development, indicating that these com-pounds act as intermediates. Additionally, we have foundthat cycloartenol cannot support the anaerobic growth ofyeast (16), nor can CSs replace the natural 24-alkylated endproducts of cultured sunflower cells (17), unless a trace ofendogenously formed end product(s) is available to regulatecell proliferation (18).

In light of the conflicting data and its implications, weundertook a proton nuclear magnetic resonance ('H NMR)study of the solution properties of two naturally occurringCSs. We assumed that if a similarity existed in the solutionand crystal state geometries of CSs then it would be unlikelythat CSs would exist in the other conformer in the interme-diate liquid-crystalline state of lipid bilayers.

MATERIALS AND METHODSCyclopropyl sterols were isolated from various plant sourcesand purified by established protocols (13, 19). Sterols wereacetylated by using acetic anhydride in pyridine (1:1,

Abbreviations: CS, cyclopropyl sterol; NOE, nuclear Overhausereffect.

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S = irradiated nucleus

rSN

/ - N~~~~DND

D = observed nucleus

FIG. 2. Three-spin system.

vol/vol). The 3-epi compound was formed by oxidation ofthecorresponding alcohol, using the Jones reagent followed byreduction with lithium aluminum hydride. The LiAlH4 prod-ucts (mixture of 3a and 3,) were purified by TLC. Additionalcyclolaudenol was kindly provided by P. Benveniste (Uni-versite Louis Pasteur, Strasbourg, France).1H NMR experiments were done on a Nicolet WB-200

instrument equipped with a Nicolet-1280 computer and a293B pulse programmer, operating at 200 MHz at an ambienttemperature of 20°C. Nuclear Overhauser effect (NOE)experiments were performed as difference experiments (20).The irradiated peak was presaturated at a power level justsufficient to cover the resonance for 10 times the T, values.Free induction decays, acquired with the decoupler off, werealternately added and subtracted from memory as the irra-diation frequency was alternately switched on and off reso-nance. T, values were estimated by using an inversionrecovery sequence 180-T-90°-t and fitting the points with athree-parameter exponential (21). The T, values determinedfor 1 (see Table 2 for systematic names) are H-18, 0.47 s;H-32, 0.65 s; H-19(endo), 0.58 s; H-19(exo), 0.61 s; the valuesfor 2 are H-18, 0.45 s; H-32, 0.94 s; H-19(endo), 0.52 s;H-19(exo), 0.67 s.

BACKGROUNDThe 1H NMR-NOE method has proved to be a powerful anduseful tool in the determination of the structure of organicmolecules (22-28).A complete description of the theory of the NOE and its

applicability to chemical systems has beenf given by Noggleand Schirmer (29). More recent reviews (22, 30) also give adescription of the theory and describe its application and thepitfalls of its interpretation. In brief, it is assumed that forsmall molecules that tumble fast (typical oforganic moleculesdissolved in a nonviscous solvent) only direct dipole-dipoleinteractions between protons contribute to the proton relax-ation mechanisms and hence to the NOE, so that in athree-spin system (Fig. 2) the NOE observed for proton Dwhen proton S is irradiated (as shown in equation 3.6 givenin ref. 29), fD{S} can be written

fDis} = PSD/2RD PDNPSN/4RDRN [1]1 - PDNPDN/4RDRN

in which R is a total relaxation rate and p is the direct dipole-dipole relaxation between spins.Examination of the structure of CSs in both conformations

showed that, in the flat conformation, Me-18t is considerablycloser to the cyclopropyl protons than is Me-32, while in thebent conformation, Me-32 is closer (Table 1). Thus, obser-vation ofNOEs at the cyclopropyl protons when the methylsare irradiated might indicate which conformation CSs prefer.The CS system is a particularly good one to study, as one canirradiate the different methyls and always observe the samenucleus. The observed cyclopropyl protons are in a region of

Table 1. Internuclear distances, in A, measured on Dreidingmodels for flat and bent conformers

H-19- H-19-(endo) (exo) Me-18 Me-32 Me-4,8 H-8 H-5a

H-19(endo) 1.9 2.8 4.1 1.7 2.3 3.7H-19(exo) 1.9 3.2 4.0 2.3Me-18 4.0 4.2 4.1 - 1.1 5.5Me-32 3.5 3.8 4.0 3.4 2.8Me4f3 2.2 2.6 - - 3.5H-8 3.0 - 1.2 3.6H-5a 3.6 5.2 1.3 3.5

Data for the bent conformer are below and left of the diagonal, anddata for the flat conformer are above and right. The distances to themethyls are to the closest proton.

the spectrum where no other protons occur. The methyls tobe irradiated are singlets, and they can be irradiated with asmall amount of power, reducing the likelihood of simulta-neously irradiating other resonances.§Recent studies on steroids and triterpenoids (20, 23, 31)

have mainly used NOE to help locate and assign all thesignals in the spectrum. Knowledge of the assignments andthe couplings of the signals allowed use of the couplings todetermine ring conformation. However, these studies weredone at 400-500 MHz, where the signals can be resolved. At200 MHz, which was used in this study, the signals of thesteroid skeleton remain a tightly coupled, unresolvable sys-tem. But even if all the sigtals could be assigned and thecouplings were known, particularly for the B and C rings, itwould not be possible to use them to determine the B and Cring conformation for CSs. Because ofthe absence ofprotonsat positions 9 and 13, the protons at 11 and 12 in the C ringform an isolated four-spin system that cannot be used todetermine the conformation of the molecule as was done with11p-hydroxyprogesterone (31). Furthermore, the presence ofthe 9f3,19-cyclopropyl group distorts the B ring so thatknowledge of the couplings to H-18 cannot give the confor-mation. In both conformers H-8 would see a large and a smallcoupling to H-7:ax-ax and ax-eq for the flat conformationand eclipsed and eq-eq for the bent conformation (ax, axial;eq, equatorial).

RESULTSBefore the NOE difference spectroscopic studies could beperformed, the assignments for the methyl and geminalcyclopropyl protons had to be established. While the assign-ments of the cyclopropyl protons were well established (32,33), to our surprise, the literature was inconsistent in themethyl assignments of CSs. For instance, Salt and Adler (34)claimed that in cycloartenol Me-18 is at 8 = 0.81 ppm, dePascual Theresea et al. (35) assigned Me-18 at 0.95 ppm, andSmith et al. (36) assigned Me-18 in 31-norcycloartenol acetateat 0.84 ppm. Bladocha and Benveniste (37) examined a seriesof CSs as free alcohols and reported Me-18 at 0.90 ppm.However, Matsumoto and coworkers (38, 39) reported shiftreagent studies of cycloartenol and its acetate that convinc-ingly assign Me-18 at 0.95 and Me-32 at 0.89 ppm. We alsoperformed shift reagent studies on CSs (data not shown) and

§Because we irradiated only methyls, not methyls and methylenes ormethines, we have neglected the effect on the correlation timescaused by the rotation of the different methyls. If the methyls rotateeither slowly or quickly compared to the tumbling for the rest of themolecule, Tc does not depend on the rate of methyl rotation but isonly a constant factor times the tumbling time (29). Nor did weinclude a factor of 3 that should be included in Eq. 1 to account forthe NOE induced by each of the protons in the methyl group, againbecause only methyls were irradiated.

*The triterpenoid and steroid numbering systems are used inter-changeably in the literature. For tetracyclic isopentenoids the C-4a,C-4,3, and C-14 methyls are labeled C-28, C-29, and C-30, respec-tively, in triterpenoid nomenclature and C-30, C-31, and C-32 insteroid nomenclature. We use the steroid nomenclature here.

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Table 2. Chemical shifts for 9f3,19-cyclopropyl sterolsDehydro- Cyclo- 31-Nor- 24-Methylene- 24-Methylene- 3-Epi-24-methylene-

pollinastanol laudenol cycloartenol Cycloartenol cycloartanol cycloartanol acetate cycloartanolMethyl (1) (2) (3) (4) (5) (6) (7)

18 0.959 (s) 0.956 (s) 0.966 (s) 0.965 (s) 0.968 (s) 0.963 (s) 0.968 (s)19 Ha (endo) 0.433 (d) 0.550 (d) 0.387 (d) 0.555 (d) 0.555 (d) 0.555 (d) 0.515 (d)

Hb (exo) 0.070 (d) 0.330 (d) 0.146 (d) 0.333 (d) 0.333 (d) 0.341 (d) 0.346 (d)21 0.885 (d) 0.859 (d) 0.884 (d) 0.884 (d) 0.884 (d) 0.894 (d) 0.895 (d)26 1.687 (br.s) 1.640 (br.s) 1.685 (br.s) 1.685 (br.s) 1.025 (d) 1.026 (d) 1.044 (d)27 1.604 (br.s) 4.655 (br.s) 1.607 (br.s) 1.607 (br.s) 1.030 (d) 1.030 (d) 1.010 (d)28 - 0.995 (d) 4.663 (br.s)/ 4.663 (br.s)/ 4.663 (br.s)/

4.716 (br.s) 4.716 (br.s) 4.716 (br.s)30 (a) 0.967 (s) 0.981 (d) 0.965 (s) 0.965 (s) 0.846 (s) 0.883 (s)31 (X3) 0.810 (s) 0.810 (s) 0.810 (s) 0.901 (s) 0.907 (s)32 0.886 (s) 0.885 (s) 0.887 (s) 0.889 (s) 0.889 (s) 0.889 (s) 0.951 (s)The chemical shifts (8) in this table are given in ppm with tetramethylsilane as internal standard. Compounds were dissolved in C2HCI3 and

the spectra were recorded at ambient temperature. s, d, and br.s refer to singlet, doublet, and broad singlet, respectively. Systematic namesare as follows: 1 is 4,4-desmethyl,14a-methyl-9,8,19-cyclo-Sa-cholest-24-en-3f3-ol; 2 is (24S)-24-methyl-4,4,14a-trimethyl-9,8,19-cyclo-Sa-cholest-25(27)-en-3f3-ol; 3 is 4a,14a-dimethyl-9,8,19-cyclo-5a-cholest-24-en-3I3-ol; 4 is 4,4,14a-trimethyl-9l3,19-cyclo-Sa-cholest-24-en-3,B-ol; 5 is4,4,14a-trimethyl-9,8,19-cyclo-Sa-ergost-24(28)-en-33-ol; 6 is 4,4,14a-trimethyl-913,19-cyclo-Sa-ergost-24(28)-en-3,8-ol acetate; and 7 is 4,4,14a-trimethyl-9,l,19-cyclo-5a-ergost-24(28)-en-3a-ol.

confirmed the results of the Japanese group. Our 'H NMRassignments for a series of CSs are shown in Table 2.We performed the NOE studies on dehydropollinastanol

(1) and cyclolaudenol (2), where other methyl signals do notoverlap those of Me-18 and Me-32. The NOE results areshown in Figs. 3 and 4, respectively. No significant NOE isobserved at the endo cyclopropyl proton at 0.55 ppm whenMe-32 is irradiated, yet an NOE is readily observed whenMe-18 of 1 is irradiated (Fig. 3), and an even larger one isobserved when Me-30 of 2 is irradiated (Fig. 4).On the basis of these observed NOEs, is it possible to

conclude that Me-18 is closer to the cyclopropyl proton thanMe-32 is? Both Sanders and Mersh (22) and Noggle andSchirmer (29) caution about such conclusions and giveexamples for which they are not justified, yet most studiesthat use NOE to support a particular structure suppose thatobservation of a larger NOE implies proximity and observa-tion of a smaller one implies distance (25, 28, 39-42). Theexamples that Sanders and Mersh (22) and Noggle andSchirmer (29) give to warn against making such conclusionsand specific instances (39, 42) in which observation of asmaller NOE does not imply a greater distance occur when

1'Id'n

C

one nucleus is irradiated and NOEs are observed at severalothers. However, the recommended way to perform theexperiment (22, 29) and the way which these experimentswere performed, is to observe the NOE induced at onenucleus while irradiating different nuclei. In this case, is theconclusion that Me-18 is closer to the cyclopropyl protonthan Me-32 justified? By using Eq. 1, the relative NOEsobserved at D when S and N are irradiated, fD{N}, can becalculated for all possible geometries: if rDS = 1, for aparticular rDN > rDS (i.e., S is closer than N to D; see Fig. 2),fD{S} and fD{N} are computed for the range of rSN from oneextreme of the linear case, where S is between D and N (rSN= rDN - rDS), to the other extreme of the linear case, whereD is between S and N (rSN = rDN + rDs). Two suchcalculations are shown in Figs. 5 and 6, wherefD{S} andfD{N}are calculated for rDN = 1.1 and rDN= 1.5. From the rangeof these plots, it is clear that no matter what the geometry ofthe system, the NOE observed at D is always larger when thecloser nucleus S is irradiated. When S is between D and N,the indirect effects are the greatest in reducing the NOE at D.The closer N is to S, the more the NOE at D is reduced bythese indirect effects. When D is between S and N, theindirect effects are the greatest in reducing the NOE at D.

0

d IC 1'

ItI

"I-~b

b I

a aI

1.0 0.8 0.6 0.4 0.2 08, ppm

FIG. 3. NOE difference spectra of 1: a, normal spectrum; b,irradiation at C-18; c, irradiation at C-32; and d, irradiation at a singletransition of H-6,B.

0.68, ppm

0.2

FIG. 4. NOE difference spectra of 2: a, normal spectrum; b,irradiation at C-30; c, irradiation at C-18; d, irradiation at C-32; ande, irradiation at a single transition of H-6,B.

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D between S & N

0.4 S between D & N

0.3 H'

0.2

0.1

fD(S)

fD (N)

'SiI

4)

14)C4

V.V

-0.1 0.3 0.7 1.1 1.5 1.9

0.5

0.4

0.3

0.2

S between D & N D between S & N

= * * U=f IS)

0.1 _

0.0 _

-0.1- o-0.2 * . ,

I

0.3 0.7 1.1 1.5 1.9 2.3

FIG. 5. Relative NOE observed at D; relative distance rDS = 1.0,rDN = 1.1.

When D is between S and N, the indirect effects are the least,and the NOE observed at D is essentially the direct effect.However, regardless of the geometry, the NOE at theobserved nucleus D is always greater when the closer nucleusis irradiated. This confirms the advice that it is better toalways observe the same nucleus while irradiating the othernuclei.Thus, on the basis of the size of the observed NOES, it

appears that Me-18 is closer to H-19(endo) and the compoundis in the flat conformation, since a larger NOE is observed atH-19(endo) when Me-18 is irradiated. However, we mustexamine whether indirect effects caused by the relativegeometry of other protons in the molecule can produce a

larger NOE at H-19(endo) when Me-18 is irradiated even ifMe-32 might be closer. The methyls in the cyclopropyl sterolsare not very close together, so that they do not form a

three-spin system with significant indirect NOEs at H-19(endo). But examination ofmodels ofthe conformers ofthecyclopropyl sterols indicates that some ofthe ring protons arebetween the H-19(endo) and the irradiated methyls. Also, thegeminal partner, H-19(exo), is extremely close to H-19(endo). These protons are ones that may produce signifi-cant indirect effects. Using Eq. 1 and the internucleardistances (Table 1), the NOEs to be expected for suchsystems {Me} x H-19(endo) can be calculated and comparedwith the NOEs observed. The NOEs computed with Eq. 1 arevery sensitive to the internuclear distance used, so that theNOEs given below are the range of the calculated NOEswhen the internuclear distances are varied by 0.2 A. In thefollowing discussion, the irradiated and observed nuclei aredenoted irrad and obs.

In the flat conformer, H-8P comes between Me-18 andH-19(endo). The NOEs calculated for the system irrad{Me}/H-8,3/obsH-19(endo) in the flat conformer are in the range- 0.13 to 0.00 for fH 19(endO){Me-18} and - 0.01 to 0.00 forfH 19(endO){Me-32}. Since a significant positive NOE is seenfor fH 19endo){Me-18}, this model does not account for theobserved results. In the bent conformer Me-32 comes veryclose to H-5a. For that system, irrad{Me}/H-5a/obsH-19(endo), the calculated NOE is 0.15 to 0.23 forfH 19(endO){Me-18} and 0.09 to 0.22 forfH19(endO){Me-32}. Butwe do not observe approximately equal NOEs at H-19(endo),so that here the bent conformer is not likely. Though theH-19(exo) is not intermediate between one of the methyls andH-19(endo), it is significantly closer to H-19(endo) than are

any of the other protons in the tetracyclic nucleus, andbecause of the r-6 dependence, it is possible for H-19(exo) toinduce a considerable indirect NOE at H-19(endo). For thesystem irrad{Me}/H-19(exo)/obsH-19(endo) the calculatedNOE is 0.02 to 0.07 forfWjg9(,ndO){Me-18} and 0.00 to 0.01 forfH 19(endO){Me-32} in the flat conformer and is 0.00 to 0.01 forfH 19(endo){Me-18} and 0.01 to 0.02 forfHF19(endO){Me-32} in the

FIG. 6. Relative NOE observed at D; relative distance rDS = 1.0,rDN = 1.5.

bent conformer. The observed results of a small positiveNOE upon irradiating Me-18 and a near zero NOE upon

irradiating Me-32 approximate those calculated for this sys-

tem in the flat conformer. It should be noted that a consid-erably smaller NOE is observed at H-19(exo) when themethyls are irradiated. When the three-spin system is calcu-lated for irrad{Me}/H-19(endo)/obsH-19(exo) the calculatedNOE is in the range -0.01 to + 0.01 for fHl19(exo){4P-Me},0.00 to 0.01 for fH_19(exO){Me-18}, and - 0.04 to - 0.06 forfH 19(exo){Me-32} for the flat conformer and in the range 0.00

to 0.00 forfHi19(exo){4-Me}, 0.00 to 0.00 forfHl19(exo){Me-18},and 0.01 to -0.05 for fHI19(exOJ{Me-32} for the bent con-

former. Though these results do not support either con-former, they explain the lack of a signiflcant NOE observedat H-19(exo). It thus appears that the only three-spin systemthat gives a qualitative prediction of NOEs similar to thoseobserved is irrad{Me}/H-19(exo)/obsH-19(endo) in the flatconformer.Because the ring protons of these compounds have not

been assigned, the question arises whether the NOEs seen

are due to irradiation of other peaks in the spectrum. H-6,Boccurs at an unusually high field (0.77 ppm for 1 and 0.79 ppmfor 2; qd, J = 12, 3 for both), and it approaches thecyclopropyl group closely. Some of the transitions of thissignal can be seen just upfield of the methyls in thesecompounds. Other of these transitions come close to Me-32and may be irradiated along with Me-32. This was shown notto be the case, however: when one of the upfield transitionsof H-6f3 was irradiated, an NOE was observed at H-19(endo)and the other H-6/3 transitions were observed by saturationtransfer (Fig. 3, spectrum d and Fig. 4, spectrum e), whileessentially no NOE was observed at H-19(endo) and no H-6M3lines were observed by saturation transfer upon irradiation ofMe-32. In the case of irradiation of Me-18, if one transition ofanother multiplet was simultaneously irradiated with Me-18,other transitions of the multiplet would appear in the differ-ence spectrum through saturation transfer. As none were

seen, we conclude that no other signal was irradiated.From examination of possible direct and indirect NOEs, it

appears that the observed NOE results support the existenceof the flat conformation of the cyclopropyl sterol in solutionat probe temperature. To determine whether the bent con-former occurs at other than ambient temperature, we exam-

ined the NMR spectrum in the range - 50°C to + 500C. Had

the bent conformer existed at other temperatures, we wouldhave expected to see changes in the chemical shifts of themethyls, particularly Me-32, as it would approach H-5a so

closely. But no significant change in the NMR positions ofthese methyls was seen over this temperature range, so itseems unlikely that the bent conformer occurs in this range.

'sfD(N)

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DISCUSSIONThe results of this study and the x-ray crystallographicexamination (9, 11) of CSs clearly demonstrate that thepreferred conformation of CSs is pseudoplanar in solutionand in the solid state. Formation of the 9/3,19-cyclopropylgroup during squalene oxide cyclization to cycloartenolpresumably acts as the conformational driving force for theC ring to adopt the disfavored but somewhat more stabletwist-boat conformation. The steric strain imposed on the Cring by the cyclopropyl group and boat form rather than thebent shape must be the structural determinants that governthe transformation of cycloartenol to lanosterol. In two oftheother principal stereoisomers of cycloartenol-euphol andtirucallol-the C ring also adopts the boat form and themolecules similarly orient into a pseudoplanar molecule (43).It would not be surprising to find that stereoisomers such asthe cucurbitacins similarly orient the tetracyclic ring systeminto the flat shape by the C ring orienting into the boat formeven though they possess a Me-9,8 and H-8,8. In contrast theother group of stereoisomers-the dammaranes (which in-clude isoeuphenol)-exhibit a flat shape similar to lanosterolon the basis of their having an alternating all-trans-antistereochemistry of the ring systems. Thus all the tetracyclicproducts of squalene oxide cyclization appear to be flat, amolecular feature we assume has functional importance.There are cogent reasons to believe that the 14-substituted

methyl group must remain intact after cyclization of squaleneoxide (44, 45), but there is no stereochemical requirement forhaving either the biosynthetic inclusion of a 9/3,19-cyclo-propyl group or a A8'9 bond (44). The biomimetic approachdeveloped in the laboratory of van Tamelen clearly docu-ments that if the D ring is preformed, the isoeuphenol systemcan be obtained in good yield by treatment of the epoxideprecursor with a Lewis acid (ref. 45 and references citedtherein). Since isoeuphenol possesses a Me-8,8 and H-9a,cyclization of squalene oxide in vivo need not proceed to an8,(9) bond. When the main asymmetric backbones of themolecules are compared with each other (44), it becomesobvious there is something special about the A8t(9)14a-methyl system peculiar to lanosterol. From a variety ofstudies it has been determined that this system is biosynthet-ically essential to sterol formation but is deleterious to cellviability. CSs apparently proceed through this system ratherthan through a fully saturated nucleus. Mechanistic consid-erations do permit cycloartenol to proceed to the nuclearsaturated system 8(9)-dihydrolanosterol. However, while thesymmetry of the 14a-methyl grouping in CS and A8'(9)-dihydrolanosterol is very similar to that in the A8'(9)-14a-methyl system of lanosterol, the 14-demethylase apparentlyrecognizes and metabolizes only certain 14-substituted ste-rols.

Since no 14-desmethyl CSs occur in nature, it follows thatplants evolved an isomerase to specifically open the 9f3,19-cyclopropyl group to form the A8")9)bond to allow for thecontinued metabolism of CS to 4,4,14-trisdesmethyl sterolend products. When the cyclopropyl group is left intact in thesterol molecule and CSs accumulate in the cell, CSs no longerfunction as an intermediate. When this aberrant conditionpersists, a new function, that of a membrane component,occurs. As the supply of natural end products is diminishedand replaced by CSs, then CSs play a compensatory fluid-izing role as a structural insert. Instead of the idea that thebent conformer mediates membrane fluidity, the control ofbulk membrane properties can be rationalized in terms of theamphipathic properties of CSs, which are similar to choles-terol, and in terms of the steric repulsion exerted by thecyclopropyl group itself on the van der Waals attractiveforces. In support of this view, cyclopropyl groups intro-

duced into fatty acids mimic the function of cis-unsaturatedfatty acids in bacteria, presumably by acting as steric repuls-ing agents (46).1. Nes, W. D., Le, P. H., Berg, L. R., Patterson, G. W. & Kerwin, J. L.

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Muller, W., Schwartz, S., Shanker, D., deWolf, W. H. & Yang, F. (1978)J. Am. Chem. Soc. 100, 3235-3237.

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