Fusicoccin. Part V. The biosynthesis of fusicoccin from 1-13C- and 2-13C-acetate

摘要

1975Fusicoccin. Part V.l The Biosynthesis of Fusicoccin from [I -13C]- and[2J3C] -AcetateBy Kevin D. Barrow,* Robert B. Jones, Phillip W. Pemberton, and Lawrence Phillips,' DepartmentsThe positions of labelled atoms in fusicoccin biosynthesised from [I -13C]- and [2-l3C]-acetate have been deter-mined by 13C n.m.r. spectroscopy. The results are consistent with fusicoccin being formed by direct cyclisationof a precursor such as geranylgeraniol pyrophosphate.of Chemistry and Biochemistry, Imperial College of Science and Technology, London SW7 2AYWE have previously shown that the minor metabolitefusicoccin H (I) can act as a biosynthetic precursor offusicoccin (IIa), the major wilting toxin of the plantpathogen fungus Fz~sicoccztm amydali DeZ., and haveargued that this provides evidence that fusicoccin (IIa) isa diterpene, not a degraded sesterterpene.2 We nowpresent evidence that fusicoccin i s formed by cyclisationof a terpenoid precursor such as a geranylgeraniol pyro-phosphate.The terpenoid nature of the fusicoccin aglycone andside chain was confirmed in our early work by feeding[2-14C]mevalonic A considerable amount of newchemistry would have to be undertaken to locate andverify the expected positions of the labelled carbon atoms.A simpler approach is to use the non-radioactive 13Cisotope and 13C n.m.r.to locate the positions enriched in13c.T3ie success of this type of experiment depends uponobtaining incorporations high enough to be detectedspectroscopically. This is the major limitation of such a1 Part IV, K.D. Barrow, D. H. R. Barton, Sir Ernst Chain,D. Bageenda-Kasujja. and G. Mellows, J.C.S. Perkiiz I , 1976,877.a K. D. Barrow, D. H. R. Barton, Sir Ernst Chain, U. F. W.Ohnsorge, and R. P. Sharma, J.C.S. Perkin I , 1973, 1690.K. D. Barrow, D. H. R. Barton, Sir Ernst Cham, C. Conlay,T. C. Smale, R. Thomas, and E. S. Waight, J . Chem. SOC. (C),19711, 1269.study, as the massive amounts of precursors that must beadded to give high incorporations can perturb or inter-rupt the delicate biological system which may have beencarefully set up to give maximum yield of the metaboliteunder study. In the case of rifamycin biosynthesis5adequate incorporations could only be obtained byadding small amounts of 13C precursor at regular inter-vals.Fungal metabolites are generally produced moreslowly, taking days or weeks to reach maximum yieldsand so are quite amenable to this type of experiment.Secondary metabolites are usually produced by micro-organisms in the late exponential phase of growth, i.e.after most of the growth of the organism has occurred,6 sowe used an alternative procedure and added our precur-sors just as fusicoccin production was starting, and hopedto avoid serious interference with the growth. The totalyield of fusicoccins in the various acetylated forms wasreduced 3-4 fold, but was still adequate for our purpose.For experimental convenience we isolated fusicoccin (IIa)as its triacetate (IIb), which crystallised directly from thecrude reaction product. The results of the spectral4 K.D. Barrow, D. H. R. Barton, Sir Ernst Chain, U. F. W.R. J. White, E. Martinelli, G. G. Gallo, G. Lancini, and6 J. D. Bu'Lock, D. Shepherd, and D. J . Winstanley, Canad.Ohnsorge, and R. Thomas, J . Chem. Soc., (C) 1971, 1265.P. J. Beynon, Nature, 1973, 243, 273.J . Micmbiol., 1967, 15, 2791406 J.C.S. Perkin Iassignments and the feeding experiments withand [2-13C]-acetate are shown in Table 4. Thisclearly shows ten signals enhanced &fold after feeding[l-l3C]acetate, and fifteen signals enhanced %fold afterfeeding [2-13C]acetate. This is consistent with fusicoccinbeing formed by cyclisation of a precursor such as ger-anylgeraniol pyrophosphate (111) (Scheme 1). This pro-cess bears resemblance to that producing the sesterter-pene ophiobolin F (VI) from geranylfarnesyl pyrophos-phate (IV) (Scheme 2), but the unsaturation and oxygenOPPcm,C)Ac MeAA LSCHEME 1pattern of fusicoccin can also be explained by alternativeroutes from a bicyclic carbocation analogous to (V).7 L.Canonica, A. Fiecchi, M. Galli Kienle, B. M. Ranzi,A. Scala, T. Salvatori, and E. Pella, Tetrahedron Letters, 1967,3371.The assignment of the 13C n.m.r. spectrum of the tri-acetate (IIb), achieved by using the derivatives (11c-e)for comparisons, is detailed below. We were unable toassign the quaternary olefinic carbon signals [C-2, C-10,and C-14, 80 144.1, 140.1, and 139.1 (pap.m. to low field ofMe,Si)], but this does not affect the biosynthetic result asall three are labelled by [2-13C]acetate.The situation iscomplicated because these are all bridgehead positions infused rings, and the chemical shifts appear to be verysusceptible to steric distortions. No attempt was madeto assign the sugar carbon atoms C-2'-5' as these are notinvolved in the terpenoid biosynthesis. The C-4 andC-5 signals (80 28.5 and 36.3) could not be assigned indivi-dually by spectral methods or correlation. However,one of C-4 and C-5 is labelled by [lJ3C]acetate and theother by [2-13C]acetate, so if we accept that fusicoccin is anormally cyclised terpene (i.e. without backbone re-arrangements), C-5 resonates at 80 36.3 and C-4 at 28.5.These are the only two of the 25 terpenoid carbon atomsin fusicoccin for which biosynthetic assumptions were-P nuv 1 1 6H (Yl (YIISCHEME 2made to enable spectral assignments. Carbon atoms 25and 26 could not be assigned, but as again they are bothlabelled by [2-13C]acetate and not by [l-13C]acetate, theassignment does not affect the biosynthetic argument.Fztsicoccin Aglycone Tetrabenzoate (IIe) .-The mainfeatures of the lH n.m.r.spectrum of (IIe) have beenassigned and the 13C n.m.r. spectrum may be related tothis by the incremented heteronuclear decoupling tech-nique.8 A coherent secondary irradiating field at lHresonance frequencies was moved, in increments of 0.50p.p.m., from 0.50 to 6.50 p.p.m. to high frequency (lowfield) of the Me,Si resonance while successive 13C spectrawere recorded.A plot of the residual l J 0 ~ upon a given13C resonance 'us. the lH irradiation frequency passesthrough a minimum corresponding to the chemical shiftof the directly bonded proton; the carbon may then beassigned.The lH noise-decoupled 13C n.m.r. spectrum shows the21 expected resonances from the ' backbone ' plus par-tially resolved lines due to the benzoate functions (carb-onyl resonances appear at 80 165.2,165.9,166.1, and 166.2and the ring carbon atoms absorb in the range 60 128.0-133.1). High-power single-frequency off -resonance(proton) decoupling (s.f .o.r.d.) enabled the number ofprotons bonded to each carbon atom to be deduced, and8 B. Birdsall, N. J. M. Birdsall, and J. Feeney, J.C.S. Chem.Coinm., 1972, 3161975 1407in addition to the benzoate resonances the spectra showthe following details.OZeJinic resonaace region.This showed four signals,three of which arise from the quaternary C-2, C-10, andC,14 (6c 138.0, 143.6, and 144.2) and one from C-1 (127.4,doublet in the s.f .o.r.d. spectrum; optimum decouplingoccurs at a proton frequency corresponding to 6 4-5).' Carbon bonded to oxygen ' region. There are sixresonances in this region, and that due to C-21 is easilyrecognised (So 58.7, quartet); optimum decoupling at 63.35 [6(H-21) is 3.325. Two lines which are triplets ins.f.0.r.d. occur at SC 67.4 and 76.5. The latteris assignedto C-16 because of its similar chemical shift in the spectraof compounds (IIc and d) ; this is supported by the incre-mented decoupling experiment, although poorly definedline-shapes necessitated a visual estimation of the opti-mum decoupling frequency in each case (6(C-16) 76.5,optimum decoupling at 6 ca.3.4 [6(H-16) 2.9-3.5 (multi-plet)]; S(C-19) 67.4, optimum decoupling a t 6 cn. 4.0[6(H-19) not assigned]}.The three remaining signals appear as residual doubletson s.f.0.r.d. The lowest field of these (SC 80.7) is rela-tively sharply defined, and since optimum decouplingoccurred at 6 5.2 [S(H-12) is 5.33 is assigned to C-12. Theresonances due to C-8 and C-9 appear as characteristic' doublets of doublets ' from which very poor plots of lJcH(residual) VS. proton decoupling frequency were obtained.This type of band-shape has been reported previously foroff -resonance decoupling experiments in which the carbonnucleus is the X part of an ABX spin system,g and is aconsequence of the strong proton coupling between A andB.Visual estimation of the optimum decoupling fre-quency suggests that the resonance due to C-8 occurs atSc 77.2, and that due to C-9 a t 66.9.' Uwsubstit.uted aliphatic ' region. Many of the signalshere show considerable distortion in s.f .o.r.d. spectra,because of the second-order nature of the proton systemto which they are coupled. We have noted three degreesof line definition as follows. (a) When the 13C nucleus iscoupled to proton(s) which are part of a tightly coupledspin system a great distortion (unresolved broadening orfine structure) is observed. (b) When the 13C nucleus iscoupled to proton(s) which form part of a loosely coupledspin system, line-broadening occurs, but the multiplet iswell defined.(c) When the I3C nucleus is coupled toproton(s) which are not further coupled to other protons,the narrowest lines occur.These line-shapes proved of great diagnostic value, bothfor estimating the nature of the inter-proton spin systemto which the carbon nucleus is coupled (as with C-8 andC-9 above) and as an aid to relating spectra of differentmolecules ( e g . the C-12 signal may immediately be dis-tinguished from those of c-8 and c-9 in all spectra by thebetter definition of its residual doublet).The difference between situations (b) and (c) is subtle,and best seen by comparison within a given spectrum (seeFigure 1).By way of confirmation, we have demon-s R. A. Newmark and J. R. Hill, J . Amev. Chem. SOC., 1973,95, 4435.strated such an effect in the s.f.0.r.d. spectrum of ethylacetate; here the residual quartet due to CH3*C0 isappreciably narrower than that due to CH,CH,.Three residual quartets are observed, corresponding tothe methyl carbon atoms C-17, C-18, and C-20. Thesignal at 6c 11.4 showed optimum decoupling at 6 1.15,and since 6(H-17) is 1.20 and S(H-20) 1.11, may beassigned to either C-17 or C-20. The lines at 6~ 15.3 and24.9 showed optimum decoupling at 6 1.40-1.43 and1.30- 1.35, respectively. Previous assignment of the lHspectrum gave the chemical shift of H-18 as 1.38, that ofH-17 as 1.20, and that of H-20 as 1.11. This is inconsis-tent with the present results and the lH spectrum wasre-examined.In the lH spectrum, H-7 absorbs at SH 2.22; irradiationhere with a (homonuclear) decoupling field collapses thehighest field doublet (6 1.12, 3JHE:7 Hz), and this thereforecorresponds to the C-17 protons.The H-15 signal wasC-18 c-I 7 xFIGURE 1 Fusicoccin aglycone tetrabenzoate n.m.r.spectrum, single frequency (lH) off-resonance decoupled ;high-field methyl region, t o illustrate different line-shapesnot identified in the spectrum, but the most probableabsorption occurs at 6 4.25 and irradiation here causes apartially hidden doublet ( SH 1.42, J H H 7 Hz) to collapse to asinglet. This is therefore assigned to the C-20 protons.The C-18 protons absorb as a singlet at BE 1.40.When these results are related to the 13C spectrum, theC-17 signal (at Sc11.4) may be assigned, but those of C-18and C-20 may not because of the close proximity of theirproton resonances.In the s.f.0.r.d. spectrum, however,the quartet at Sc 15.3 p.p.m. is significantly narrower andtaller than that at 24.9 (Figure 1); the former may thenbe assigned to C-18 and the latter to C-20. Similar line-shape differences were observed for these resonances inthe s.f.0.r.d. spectra of (IIb-d) and this provides afurther example of the importance of the subtle differ-ence between cases (b) and (c) above. However thechemical shifts of C-18 and C-20 are probably more inaccord with the alternative assignment.Three residual triplets are observed, corresponding toC-4, C-5, and C-13.That at a0 35.2 possessed the betterdefined line-shape, and since optimum decoupling occur-red at 6 2.70-2.75 it was assigned to C-13 [6(H-l3a1408 J.C.S. Perkin IH-13p) 2.83 and 2.531. The other triplets were too dis-torted for a successful plot of l J g ~ residual us. decouplingfrequency to be niade, but a visual estimate of the opti-mum decoupling frequency was 6 1.5. This is consistentwith their assignment to C-4 and C-5 [6(H-4a, H-4p,H-5a, H-5@) all in the range 1-21; the relative invari-ance of these signals to the substituent on C-12 (unlike theC-13 signal, which moves upfield by the expected amount)confirms this. The C-4 and C-5 signals may not be dis-tinguished from each other at this or any later stage, andthe assignment was only achieved by consideration of thepattern of labelled acetate incorporation.Four residual doublets are observed on s.f .o.r.d., corres-ponding to C-3, C-6, C-7, and C-15.The lH n.m.r.assignment of H-7 and H-15 has been described above,and this enables the assignment of a signal at a0 32.6 toC-15 [optimum decoupling at 6 4.3; 6(H-15) 4.251, andthat at 40.7 to C-7 [optimum decoupling a t 6 2.2,6(H-7) 2.221. The H-3 and H-6 signals may be identi-fied, but not distinguished, by their 4 J ~ ~ (allylic)coupling to H-1, and in the lH n.m.r. spectrum they occurat 6 3.00 and 2.64; optimum decoupling of the 13C doub-let at 6c 41.4 occurred at 6 3.1 and of that at 47.6 at 62.45. This indicates that the lower field proton is bondedto the higher field carbon atom, and vice versa.Of the pair of doublets corresponding to C-3 and C-6,the higher-field doublet appears the better defined (lesssecond-order effects).Inspection of the lH spectrumindicates that H-6 is part of a more weakly coupledhomonuclear spin-system than is H-3; the 60 41.4 linemay thus be assigned to C-6, and that at 47.6 to C-3.The signal due to the single quaternary carbon atom,C-11, is easily identified from the s.f.0.r.d. spectrum, andappears at 6,5623.With the exception of the olefinic carbon atoms C-2,C-10, and C-14, and the differentiation between C-4 andC-5, the assignment of the 13C n.m.r. spectrum of (IIe) iscomplete, and details are given in Table 4.Fusicoccin Aglycone (IIc) and Aglycone Tetra-acetate(IId) .-The spectra were compared with that of (IIc), andcorrelations were drawn on the basis of (a) expected sub-stituent effects and (b) the line-shapes (residual second-order effects) in s.f.0.r.d.spectra. These were entirelyconsistent with the assignments made for (IIe).Comparison between (IIc) and (IId) showed that acetyl-ation of the primary alcohol function (C-19) produced theanticipated downfield shift of the a-carbon (C-19) signaland upfield shift of the p-carbon (C-15) signal. Acetyl-ation of the secondary alcoholic function at C-12 producesa smaller a-shift and correspondingly smaller @-shiftsupon C-11 and C-13. The effect upon C-11 is significantlysmaller than upon C-13 (0.5 cj. 1.4 p.p.m.) and thisappears to be a consequence of the fact that C-11 is' quaternary ' in nature whereas C-13 is ' secondary '; wehave characterised this phenomenon in other systems,and this work will be reported elsewhere.Esterificationof the secondary alcoholic functions at C-8 and C-9 haslittle effect upon the chemical shifts of these nuclei,because the a- and p-effects tend to balance out. TheC-7 signal shows the expected upfield p-shift. Table 4demonstrates the inter-relationships between the spectra.In the spectrum of (IIc), the assignment of the C-6 andC-7 signals was based upon (a) the smaller shift expectedfor C-6 upon esterification of the C-8 hydroxy-group, and(b) the relative magnitudes of the residual splittings in thes.f.0.r.d.spectra of (IIc) and (IIe). In (IIe), the H-6signal occurs at 6 3.00, and should not change positionsignificantly in (IIc). The H-7 signal occurs at 6 2.20 in(IIe), and should be in a comparable position in (IIc). In(IIc) the residual splittings in the s.f.0.r.d. spectrum were14.8 (So 42.0 line) and 27.0 Hz (60 41.8 line), respectively,in accord with the above assignment.The olefinic carbon atoms C-2, C-10, and C-14 allshowed significant shifts on going from (IIc) to (IId) or(IIe), i.e. upon esterification, and it was not possible tocorrelate the data. In an attempt to resolve this prob-lem, small quantities of the lanthanide shift reagentEu(fod), were added to a solution of (IId) in CDCl,. Thetotal induced shifts after addition of five 5 mg portionsare shown in Figure 2, for those carbon atoms whoseFIGURE 2 Pattern of lanthanide-induced shifts in (IId)assignment is certain.unassigned lines was as shown in Table 1.In addition, the behaviour of theTABLE 1Lanthanide-induced&! shift (Hz)143.1 13.3142.8 56.0137.6 43.625.4 11.014.8 7.9It may be deduced that the major site of complexationis the acetate function on C-12, followed by that on C-19.Complexation at the C-8 and C-9 acetate groups seems ofonly secondary importance, and co-ordination of theOCH? group is negligible.Examination of the shifts ofthe lines assigned collectively to C-2, C-10, and C-14suggests that the a0 143.1 resonance may be assignedspecifically to C-2. Comparison of (IId) with (IIc) sug-gests that there should be but little effect of acetylationupon C-14, but that C-10 should show the usual upfield(p) shift.The following assignments then result: C-14,60 142.8 in (IId) and 142.3 in (IIc); C-10, 137.6 in (IId)and 140.1 in (IIc). This means that in (IIc), the C-2signal occurs at 145.9 and upon acetylation undergoes a1975 1409upfield shift to 143.1 [in (11d)l. This is a surprisinglylarge effect when it is realised that C-2 is four bondsremoved from the functions on C-12 and C-8; modelssuggest, however, that the conformation of the ring issuch as to bring C-8 close to the 1,2-double bond.Extrapolation of these results to (IIe) indicates that inthis compound C-2 corresponds to the SC 144.2 line, C-14TABLE 26 , andoff -resonancemultiplicity Assignment99.3 (d) c-1'Unidentifiedsugar resonancesC-6'c-2170.5 (d) (two lines)69.8 (d)68.9 (d)61.1 (t)58.7 (9)to 143.6 and C-10 to 138.0; the assignments of C-2 andC-14 may, however, be reversed.Comparison of the lines assigned to C-18 and C-20 inthese spectra gives no further information which canresolve the uncertainty between them.However, in allcases the s.f .o.r.d. spectra showed the quartet centredaround Sc 15 to be much sharper than that at 6,325; theAssignmentcriteria tAA, BA, B, CA, B, CA BAA , BA , RA, BA, B,IAABABA4I3Carbon1236891011121314" I15161718192021222324252611'4'5'6'(W127.4144.2 or143.6 or138.047.641.440.777.2138.056.880.7138.032.676.511.415.367.424.958.8former probably corresponds, then, to C-18, and the latterto C-20, as indicated in the previous discussion of (IIe).Assignmentc- 1 c-3 3C-6c-7c-11c-12C-13C-15C-16C-17c-18c-19c-2 1TABLE 3Fusicoccin Atriacetate (IIb)6.2126.548.036.3128.5141.440.955.180.634.932.876.39.815.868.058.7Fusicoccin aglyconeacetate (IId)6C126.747.935.8128.1;41.639.954.980.534.632.376.211.014.866.858.7It was not possible to distinguish between the signalsdue to C-4 and C-5.Fusicoccin A Triacetats (IIb) .-Excluding acetatefunctions, the spectrum of (IIb) should consist of 32TABLE 4(IW125.8145.9 or142.3 or140.148.141.842.078.6140.155.479.9140.135.576.59.614.364.424.358.7(W126.7143.1 or142.8 or137.647.941.539.976.8137.654.980.5137.632.376.211.014.866.824.558.7(IIb)126.5144.1 or140.1 or139.148.028.541.440.977.8139.155.180.6139.132.876.39.815.868.024.958.7114.5143.175.699.361.1[ 1-13c: -+[ 2-'3C] -Acetate * Acetate *+i- +t+I f1 -tI c+ft.+++ + + + +++ +* + Indicates enrichment observed (five-fold enhancement of signal for [1-13C] acetate, three-fold for [2J3C] acetate.) t Assign-ments are all based initially upon the observed multiplicity in s.f.0.r.d.spectra.Where additional criteria are necessary, these areindicated by the code letters A, incremented heteronuclear decoupling ; B, line-shape in s.f.0.r.d. spectra; C, biosynthetic arguments1410 J.C.S. Perkin Iresonances, and 31 of these are observed. Thirteenresonances would be expected in the region which ischaracteristic of 13C directly bonded to an oxygen func-tion, and two of these appear to be coincident at 6070.5. This region is very complex because of the sixcarbon atoms in the sugar residue which absorb here;these are not labelled during biosynthesis in the presenceof either [1-13C]- or [2-13C]-acetate, since they are of non-acetate origin, and it is convenient to eliminate them fromconsideration. The methoxy-carbon atom C-21 is notenriched either, and these seven resonances are identifiedin Table 2.The resonances due to the C-6' side-chain wereassigned from s.f .o.r.d.and undecoupled spectra. TheC-22, C-23, and C-24 signals are instantly recognisablefrom their multiplicities and characteristic chemicalshifts : the C-22 (80 114.5) and C-23 (143.1) signals appearas a poorly defined triplet and doublet respectively, thelatter being distinguished from the C-1 signal which ismuch more sharply defined. The C-24 signal is the loweraliphatic singlet, at SC 75.6.The majority of resonances due to the aglycone carbonatoms were assigned by comparison with the resultsobtained for the aglycone derivatives. This is illustratedin Table 3.The methyl carbon signals (C-20, C-25, and C-26) occurat 6~ 25.8, 24.9, and 24.7, respectively.In the s.f.0.r.d.spectrum, the quartet corresponding to the 80 24.9 linewas significantly broader than the others, and is thereforeassigned to C-20.Individual assignment of tl ie quaternary olefinic car-bon signals is difficult, since considerable variations occuramongst the compounds studied; these probably arisefrom conformational differences between fusicoccin andits aglycone derivatives.Detailed assignments are presented in Table 4, whichalso indicates the sites of enrichment in the two biosyn-thetic studies using [1-l3C]- and [2-13C]-acetate.EXPERIMENTALProton homonuclear decoupling was performed at 100MHz with a Varian HA-100 spectrometer, for a sample of(IIe) (30 mg) in CDCl, (0.4 ml).Carbon-13 spectra were recorded at 25.16 MHz on aVarian XL- 100- 12IVarian Data Machines 16K 620-L spectro-meter system; 8 K sampling points were used to give 4 Kplotted data points after Fourier transformation.Sampleswere dissolved in 2 ml of CDC1, in a 10 mm n.m.r. tube, thesolvent deuterium being used to provide a field-frequencylock. Interference from the CDCI, carbon signal made i tpreferable to record spectra of the two enriched samples offusicoccin A triacetate in CCl,, with the deuterium lockprovided by an external D,O sample contained in a concen-tric 5 mm 1i.m.r. tube.Incorporation of [l-13C]- and [2-13C]-A cetate into Fusicoccin.-Fusicoccum amydali was grown in submerged culture inshaken flasks (4 1 capacity; 1 1 medium) as previouslydescribed., After 48 h sodium [l-13C]acetate (250 mg, 90atom yo excess and 254 mg, 88.2 atom yo excess, respec-tively) was added aseptically to each of 2 flasks. The flaskswere harvested after 5 days and the combined culture filtrateextracted with chloroform (3 x 750 ml). The combinedextracts were evaporated and the residue acetylated a t roomtemperature with pyridine-acetic anhydride. The acetyl-ating reagents were removed under high vacuum and theresidue was crystallised from light petroleum (b.p. 60-80'yielding tri-O-acetylfusicoccin (IIb) (40 mg) as needles, m.p.116-117'. The same procedure was followed with sodium[2-13C]acetate (2 x 260 mg, 60 atom yo excess K).We thank Professors Sir Derek Barton and Sir ErnstChain for their interest and help in this work, and the S.R.C.for financial support towards the purchase of the VarianXL- 100-12 spectrometer, and maintenance awards (toP. W. P. and R. B. J.).Received, 1 lth December, 19741 [4/268