! -on
`! (HOCH,cH,),NH
`(_!'.) ~
`It..
`¢fo
`l~~k}.:. ·aary center not involving this special structural feature. We
`J1;/G? ~"\-Qi) :c:
`~~{.·
`(91:~~~:nty) . -
`L·.,~ ,.-;
`~;; ; . ~~(~A) NaOH
`~~t~~'.;~;,~ '
`l:(J i ~ [ar n + u.9°, s
`;l~1~~:'.:lf.}i:-'' Treatment of the bOrane with i~7~ ::~:~ :::::ce of
`··~···· cr.(-Q). -~'.rn~
`
`CH,<;:H
`
`CHarCH,CH,
`
`(6)
`
`{i;;f_;;:_:;.: .S9<fium methoxide-methanol yields 2-iodobutane (R) with
`{!\'}:;;·;. [a]20o -26.9° (84% optical purity) (eq 7).
`
`CffaCHCHaCH3 .
`. I
`.
`I
`[a]20D -26.9°, R
`(84% optical purity)16
`
`(7)
`
`~------------------··------
`
`1291
`
`C,/0
`
`Il
`
`w (6)
`
`\\Pir:i:\··volve reaction of iodine at a bicyclic center. It was impor(cid:173)
`~§W+>·:iant to establish whether inversion would occur at a secon-
`
`:~~:-~::._.,selected diisopinocampheyl-2-butylborane15 for study (eq
`~?..'>,,. ·6). Note that the 2-butanol produced from (-)-a-pinene
`V'.~i~:' :: possesses the S configuration:
`
`References aDd Notes
`(1) H. C. Brown, "llofilnes In Organic Chemistry", CorneU Unlvefsity Press,
`llhaca. N.Y., 1972.
`.
`12) H. C. Brownancl C. F, Lane, Chem. Commun.. 521 (1971}.
`(3) 0. S. Mattsson and J. 0. Waldbillig, J. Am. Chem. Soc .• 85, 1019
`{1983).
`(4) M. Glelen and R. Fosty. Bull Soc. Chim. &lg.. 83, 333 {1974).
`(6) W. A. Thaler, Methods Free Pv&al Chem., 2, 121 (1969).
`(8) C. F. ~and H. C. Brown, J. Am. Chem. Soc., 92, 7212 (1970).
`(7) C. Walling. "Free Radlcals in Solution", Wlley;New York, N.Y., 1957.
`(8) N. R. De Lue and H. C. Brown, Synthesis, In press.
`(9) H. C. Brown, G. W. Kramer, A. B. Levy, and M. M. Midland, "Organic
`Syn1heses via Bonnes", Wlley-tnt9"":lance. New Yortt, N.Y., 1975.
`(10) The eJd>.lodonorbornane appears io lsomarlze to eJCl)o under the Intl~
`enoe of light.
`(11) The methlns proton of !he endo Iodide gave an absorption centered at 6
`4.20 and the exo lodlde 12 at 6 3.95.
`·
`(12) A.G.. Oallles and R. Tudor, J. Chem. Soc. 8, 1816 (1970).
`(13) H. C. Brown and Y. Yamamoto. J.·0rp. Chem., 39, 861 (1974).
`( 14) Exhibited phrslc:af and chemical properties consistent with !he asslgnett
`alructlA"s:
`·
`(15) H. C. Brown, N. R. Ayyailgar, and G. Zweifel, J. Am. Chem. Soc., 86,
`S97(1964).
`.
`.
`..
`( 16) R. H. Pickard and J. Kenyon, J. Chem. , Soc., 99, 45 I 1911). report
`(a)"o + 13.87° for S{+)-2-bulanol end (a)17o -31.98° for R.(-,.)-2-
`lodobutane.
`.
`. .
`. ..
`{17) B. A. Qiauctl, 0. G .. Goodwin, H. R. Hudson. L Bertlett, ·anc1 P. M.
`Sc:apea, J. Chem. Soc. c, 1329 {1970).
`(18) Graduate re116111'Ch essistailt on·MPS 73-05136 A01 from the Natlonal
`Science Foundation.
`.
`Herbert C. Brown,• Norman R. De J.Uel8
`Richard B. Wetherill Laboratory, Purdue U~ioersiry
`West Lafayelle, '/1idiana 47907
`
`George W. Kabalka. Herbert C. Hedgecock. Jr.
`Department of Chemistry, University of Tennessee
`Knoxville, Tennessee 37916·
`Received N~mber ii, 1975
`
`Application of Unreactive Aoaiogs of Terpenoid
`Pyrophosphates to Studie5 of Multistep Bio5ynthesis.
`Demonstration That
`.
`·
`"Presqualene Pyroph!)Sphate" Is an Essential .
`Iiatermediate on the Path to Squalene ·
`
`Sir:
`Pyrophosphate monoesters play a dominating role in the
`biosynthesis of terpenoids, especially with reference to
`chain extension and ring formation. 1 The head·to-tail join(cid:173)
`ing of isoprene units by carbon coupling, for example, in(cid:173)
`volves intermolecular nucleophilic attack by a carbon-car_.
`bon dou~le bond at a saturated carbon with displacement· of
`a pyrophosphate leaving group:
`
`.
`·•
`R'.._,
`CH"-..
`R/C=~ CH,-~PO,OPO;',_
`
`Analogues of pyrophosphates in which· the carbinol ox.ygen
`(O•, above) is replaCed by methylene can reasonably be ex(cid:173)
`pected both to resist such enzymic C-C coupling and to
`function as selective enzyme inhibitors ("substrate ana·
`logue" type). In this communication we.describe the syn·
`thesis of a series of these pyrophosphate analogues (C-sub(cid:173)
`stituted methylphosphonopbosphates), the demonstration
`that they do inhibit biosynthetic processes involving pyro(cid:173)
`phosphate substrates as postulated, and an illustration of
`bow this inhibition can be utilized to gain new information
`regardi.ng multistep biosynthetic pathways.
`Geranylmethylphosphonophosphate trilithium salt (4, R
`= geranyl) was synthesized starting with the reaction of
`geranyl bromide (1, R = geranyl) with I equiv of dimethyl
`lithiomethylphosphonate2
`in
`tetrahydrofuran (THF) at
`-78° to form phosphonic diester 23 (60-70%). Cleavage of
`
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`Inhibition ofSqualene Biosyntliesis from Mcvalonate (S10
`Table I.
`Liver Preparation)
`·
`·
`
`I/MEVAL • ratio x W
`
`'lb
`inhibili·
`on
`
`20
`so
`80
`90
`
`ID~A.
`
`2.S
`45
`262
`375
`
`l.1PT
`i
`33
`170
`325
`
`lo Ell
`
`. 0.2.5
`I
`4
`12
`
`IFAR
`
`0.25
`l.2S
`4.8
`35
`
`IJ'SQ
`
`6
`22
`ISO
`250
`
`° Concn of MEVAL = 2 mM in.all experiments.
`
`100
`90
`80
`70
`60
`
`c
`.g
`:0 :c c 40
`...
`,,e 0
`
`30
`20
`10
`
`2 to diacid 3 was accomplished (ca. 80% yield) in two steps
`·(base hydrolysis to monoe5ter and subsequent demethyl(cid:173)
`ation with sodiur.n iodide in dry methyl ethyl ketone at re·
`flux 4 ). Phosphorylation. of 3_ was effected via the phospho-
`RBr + I...iCH1PO(OClfa)2 -
`RCH,POCOCH3)2 --+
`1
`2
`RCH2P()(OHJ1. -+ RCH,POiOPQ,3-3Li+
`3
`4
`R~geranyl
`noinorpholidate,s and. the resulting pho8ph~nophosphate
`was isolated and purified chromatographically5•6 as the tri(cid:173)
`lithium salt 4 (~. 50% yield). In a similar way three other
`is~pren~id phosphonopho~phates of stru~tu~ RCH2P070-
`P03l- ~Li+. were prepared: S, .R ::= farnesyl; 6, R = 'Y.'Y·
`dimethylallyl; and 7, R = isopentenyl. Finally, "l?resqual(cid:173)
`ene alcohol'' (8) 7,B was converted to the phosphonophos(cid:173)
`phate 9 by a sequence starting with synthetic presqualene
`alcohol8 (RCH20H) involving: {I) RCH 20H - RCHO
`(Collins oxidation at_ 25° for I h in CH2Cl2, 88%); (2)
`RCHO - RCH=CHPO(OCHJh {2 equiv of Bu3P=CH(cid:173)
`PO(OCH3h9 in.1:1 THF-n-butyl alcohol for 3 h ~t 25°,
`8_~); (J) diimide. reducti~n to RCH2CH2PO(OCl:hh (ex(cid:173)
`c1:5s· diimide perioda~e. 85%); and (4) two-stage hydrolysis
`(82%) and phosphorylatioi:i to_9 (50%) as describe4 above.
`HOCH,
`.
`·
`
`. CH, . ff. \-H ~H.
`~HR.
`
`RCH1
`
`'
`
`5
`
`10
`
`15
`
`20
`
`25
`
`:
`
`I
`
`50
`
`...
`
`it 103
`lGER I MEVAL Ratio
`Figure I. Inhibition of squalene biosynlhesis from mevalonate
`(MEVAL) by IGER (4) with the Sm rat liver squalenc synthesase prep(cid:173)
`aration. (MEVAL] = 2mM.
`
`hibitor using an amount. of enzyme sufficient to ca_use.· I .0%
`conversion· of 2 µmol .of mevalonate to _squ_alene after 10
`min at 37° .in the absence of inhibitor: The squalene pro(cid:173)
`ducCd (no.inhibitor) increased linearly with "time up to at
`least 20 min of incubation. Percent inhibition of squalene
`biosynt.hesis in the presence or inhibitor was measured for
`· variotis ratios of inhibitor to mevalonate with. the re5ults
`summarized in Table I. Degree of inhibition as a function of
`inhibitor-mevalonate ·ratio is shown for the specific case of
`IGeR in Figure 1; similar curves were obs~riled with the
`other phosphonophosphates. The observed effeetiveness of
`inhibition follows the order IFAR °" IGER > lpsQ > loMA ~
`lJP'r. In each case complete inhibition of squalene biosyn(cid:173)
`thesis could be effected at an appropriate ratio of I/
`MEVAL.
`The inhibition of kaurene biosynthesis from mevalonate ·
`in· the cell-free kaurene synthefase from Ricinus commu(cid:173)
`nis12 was studied with the inhibitors IFAR, IGER, loMA, and
`l1PT with similar results. Again, stronger inhibitfon was ob(cid:173)
`served for IFAR orlGER than for loMA or l1P'f. but ica_urene
`biosynthesis could be completCly blocked by any of the four
`inhibitors..
`·
`·
`· ·
`In both squalene and kaurene synthetase systems only ex·
`tremely weak inhibition was observed by the phosplionates
`corresp<>ndin.g to the phosphonophosphates 4-7. Further,
`perhydro loeR {synthesized by hydrogenatiOn of ldER over
`Rh/C catalyst) was a very poor inhibitor of squalene or
`kaurene biosynthesis.
`The fact that the biosynthesis of squalene from mevalon·
`ate by the S 1o preparation can be completely inhibited by
`IPSo {at fpso/MEVAL ~ ca. 0.5) provides a st.rong indiea(cid:173)
`tion that there is no other pathway than. that via pr~qµal­
`ene pyrophosphat~. Because of the complexity of the pre(cid:173)
`squalene ·pyrophosphate route from farnesyl pyrophosphate
`to squalene .as compared with mechanistically similar but
`more direct routes, and beciiuse the formation of presqual,
`ene pyrop~osphate is obsel"Ved in the absence of NAPPH, a
`cofactor for squalene biosynthesis, there has been. some
`question as to whether presqualene pyrophosphate is essen(cid:173)
`tial to squalene biqsynthesis or is off th.e major·biosynthetic
`pathway (but still convertible to squalene). 13 It .seemed to
`us that this matter could. be resolved by further experiments·
`using IPSQ·
`·
`First it was establish~ by experiment that the conversiOn
`of.a mixture of C(2)-14C labeled mevalonale and C(l)-3H
`labeled presqualen~ pyrophosphate to 14C or lH labeled
`
`8
`~:oP0,~3Bu,NH'
`
`.RCH,
`
`CH1R
`
`9
`R-gemnyl
`
`These phosphonophosphates ·are referred · to herein
`mnemonically a<;cording to the group attached. to methyl(cid:173)
`phosphonyl carbon; i.e., loeR. lFAR, loMA. lwr and lpsQ
`correspond to 4, S; 6. 7 and 9, respectively. Thus loeR is the
`analog of geranyl pyrophosphate.
`. The inhibition of squalene biosynthesis by the various
`phosphonophosphates was studied using. C(5)-3H and
`C(2)- 14C labeled mevalonate (MEVAL) with the rat liver
`.S 10 squalene synthetase preparation10 (in 0.1 M Tris·HCI
`: buffer of pH 7.5). Parallel anaerobic ·incubations were per-
`formed11 .with and without added phosphonophosphate in-
`
`Journal.of the American Chemical Society / 98:5 / March 3, 1976
`
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`~-------------------·-·-·--······--·········-
`
`·' palene by the S10 preparation could be completely inhib(cid:173)
`-~<Jby IPSQ· In contrast, there was no inhibition of squalene
`:~i~ynthesis from presqualene pyrophosphate at c0mpara(cid:173)
`».l~:l/MEVAL ratios by laER. IFAR, loMA, or lJYr, evidence
`:J~~t .~ffective inhibition requires a close correspondence of
`,,$i:Jbstrate and inhibitor carbon structure. The same results
`H¥.~'i:e ~btained with the microsomal liver preparation 14 (re(cid:173)
`:t:i:f.C:!fed to herein as MLP) which effects squalene biosynthe(cid:173)
`FPn~i~drom farnesyl or presqualene pyrophosphates but not
`ifr;;~:i'fi~~·Cs or Cio precursors, both with regard to inhibition of
`'f\/'#11falene biosynthesis from presqualene pyrophosphate by
`{:fo.d~- ;lnd lack of inhibition by the other phosphonophos-
`1Bkp~ates ..
`':{)i:'.f:iSincubation of 50 'nmol of tritiated mevalonate 25 nmol of
`[ii('.~'i~TI.l.:ibi:led ~resqualene pyrophosphate, and :Soo ~mol of IPSQ
`::5'?:':·~1~b.suffic1ent S10 enzyme15 to convert 12% of the mevalon(cid:173)
`-,;·, .. je::to squalene.in the absence of.Irso yielded no tritiated
`~9~.a}c:ne ·but showed a 9% conversion (7 53 of expected
`. i/1.~fmum) o_f meval.onate to .tritiated presqualene pyro(cid:173)
`·p~~phate .. For ide?tification the labeled presqualene pyro(cid:173)
`. ,Jfuf?.Sphate was purified by thin layer chromatography (sili(cid:173)
`/ikS~,,gel ... n-propyl alcohol-11.N ammonium hydroxide 1.5:1,
`ibf'.!lAidentica1 with that or u_nlabeled presqualene pyrophos-
`fditW.~te) and reincubated separately with: both S 10 enzyme
`;\wrn:a,n~. MLP enzyme to afford in each case· tritium labeled
`WU/~Wllene. Labeled .squalene was identified unambiguously
`:::?:;,;l>y,;:chr()matograph1c data and. also. by conversion to the
`g%~~·#talline thiourea complex which could be recrystallized
`,.L'(i!(l:~ristant specific radioactivity. Further, characterization
`tm%:~N~lie tritiated presqualene pyrophosphate produced in the
`%/!;¥t.19,v~ 'eitp_Criment was obtained. by reduction with. lithium
`i'.f{:\~J.!-l.~inum hydride to labeled presqualene alcobol, chroma(cid:173)
`;'W;(.;;~g~i',phically identical with authentic material (Rf 0.27 on
`f?:(~,Hit1i:gel .plates using 2:1 pentane-.etherJor development).(cid:173)
`U'if:~~e experimental data indicate that IPSQ can completely
`=Zi/Wf# off squalene ·hiosynthesis from mevalonate or- presqual(cid:173)
`{fi);~.fi~ pyrophosphate and also that presqualene. pyrophosphate
`.,:%'{\Js::.fQrmed and accumulated under normal conditions of
`Xtif!;S'<f,ll,qle11e biosynthesis from ~evalonate if ff5Q is preserit.
`~~'+\:91~,e.n these facts and the specific inhibition of the presqual.(cid:173)
`!-\~tf:i~~!;:· pyrophosphate to squalene conversion by only .IPSQ.
`'DAbt;r~ seems. to be no way to avoid the conclusion that prcs-
`1:,;::;\~.#ii.I.ene pyrophosphate is an essential intermediate in
`· · ~~ualene biosyn.thesis in liver; that is, there is no pathway
`:·p;:fr:~m mevalonate to squalene .which does not go through this
`;);T'.i~·termediate. 16
`j·}\{;::'.kseeins apparent that the study of phosphonophosphate
`t:\'(:::~~~fogs can be helpful in tile elucidation of biosynthetic
`'..·;~j~~(,~[')~~;'#:i~1:e~~oa~::::s.11
`.
`.
`
`:
`.. :
`
`kHJ1~ See R. Bentley, "Molecular Asymmetry in Biology", Vol. II, Academic
`;:=-):\:-,:<,'.;_.P;i_ess, New YO<k, N.Y., 1970, Ch8pter 4.
`/(,::;:(~1·:~ by addition of 1;1 equiv of n-butyllllhlum to dlme1hyl melhyl(cid:173)
`.,{':';'i,\'.:: ·, phOSphonale In THF.at -78° under argon and further reaction e1 -78°
`'''/'.:.>: ... · ror·o.5 h;·see E. J. Corey and G. T. Kwlalltowskl, J. Am. Chem. Soc.,
`_;,":· ., .. ,,,,.:., _. '88, 5654 (1966).
`.
`.
`::'\.HLl~)' The sltuctures assigned to subslanees reported herein were confltmed
`:'.''·';;,:·:,;.' .: !JY Infrared and NMR spectroscopy ( 1H and 31PI using chromalographl-
`
`'·
`
`.,,I,,~!;{;·:·'::·,,::-;;;:::,:.·· umn ol 50 g ol EMS smca gel 60, 70-230 mesh using n-propyl alcohol-
`
`\{f t~li'~~:~~;~~'.:
`· 111~~,:'~~§~1{~~
`
`1293
`
`(8) Prepared according to L J. Altman.' R. C. Kowerski, and H. C. Rilling. J.
`Am. 'Chem. Soc., 93, 1782 (1971).
`.
`(9) Prepared from 81'3 +PCH,PO(OPh)aCr by sequential treatment with 1
`equiv of potassium fett-butoxlde and 2 equiv of sodium melhoxlde· -
`J. G. Moffatt and G. H. Jones, U.S. Patent 3 583 974· Chem. Abstr'. 75
`130091q 11971).
`•
`•
`•
`(10) ~ Popjflk, MBthods Enzymol., 15, 438 (1969).
`(11) See T. T. Tehan, Methods Enzymo/., 6, 509 (1963), lor method or Incu(cid:173)
`bation. No dispersant (e.g., Tween 80) was used; lnhlbltOr (or substrate
`In the case of presqualene pyrophosphate) was depostted a5 a film In
`the incubator lube by evaporation from benzene solution arid mixed
`with the enzyme SOiution by agitation using a vortex mixer. l..alieled
`s~lene was purified by preparative thin layer chromatography on 11
`slltca gel plate (0.25 mm thickness of layer, 15 em. fength) using 2%
`ether-98 % petroleum ether for development ( R1 0.60 for squalene).
`(12) C. A. WOllt, MsthodB Enzymol., 15, 48t (1969).
`.
`(13) Sea. lor example, J. W. Cornforth, Chem. Soc. Rev., 2, 1 (1973}; t
`Schechter and K. Bloch, J. Biol. a.em .. 246, 7890 (1971).
`(14) See rel 10, pp 450-453.
`_
`·
`(15) In this and all other experiments with the S,0 system. NADPH. Mfr+
`and all olhat ne<:esswy cofactors had bean added In the usual amounlS:
`( 16) For other. mcent papera relevilnt to the role of presqualene p}irophos(cid:173)
`phale In thablosynthesls of squalena, see (8) F. Musco, J.P. Cei'lson, L.
`Kuehl, and H. C. Rllltng,.J. Biol. Chem.; 249, 3746.(1974); (b) G. Popj8k,
`H. Ngan, and W. Agnew, Bloorg. Chem., 4, 279 (1975).
`.
`t 17) This research was assisted financially by the National Science Fciunda(cid:173)
`tion and the National. Institutes of Health. We thank Profas8or Konrad
`Bloch and the members· of his research group for numerous helpful c11•
`··cusslona.
`·
`·
`·
`·::
`E. J. Corey,* R. P; Volante
`Department of Chemistry, Harvard· University
`Cambridge. Ma!sachusetts 02.J 38
`Received November u.: 197 5
`
`Effect of Photoselectionon Fluorescence-Detected
`Orcular Dichroism
`
`Sir:
`Jn a recent study Turner et al.I have proposed that the
`circular dichroism, CD, of a fluorescent ·chromophore can
`~ n;ieasured by ~etecting its n.uorescence upon excit~tion
`by nght-handed and left-handed circularly polarized light.
`The underlying assumption is that .the excitation si>ectrum
`of a fl?orescent chromophore parallels its absorption spec>.
`trum, I.e., that the measured nµorescence intensity of the
`chromopf'!ore depends exclusively on .the amount. o(light
`absorbed by it. It was pointed out that such studies may be
`advantageous for the specific measurement of the CD of ihe
`fluoresi:ent chroinophores in biopolymers, thus eliminating ·
`contributions from nonfluorescent chromophores with.over(cid:173)
`lapping absorption bands, which are often also. present ·in
`the macromolecules. I
`.
`· ·
`.
`While the proposed method .for measuring CD via emit-'
`~ed fluorescence intensity is promising and of much interest,
`1t may be in serious error when applied to chroinophores
`when rotato.ry Brownian motion is frozen (or restricted)
`during the lifetime of the excited state of .the chromopbore.
`This restriction may apply, for .example, to a variety of na(cid:173)
`tive chromophores i!l biopolymeis. The physical reason be- ·
`hind the complication which arises in frozen systems is as
`follows. The light absor~d by the system under study does
`not excite equally molecules of different orientatio·llS, since
`the probability of light absorption by a specific 'moleeule de(cid:173)
`pends on the orientations of its electric.and magnetic dipole
`as well as electric quadrupole transition moments relative to
`th~ vector potential and direction of propagation of the light·
`wave.2• Jn the case of circularly polarized light, the proba(cid:173)
`bility of excitation of a specific molecule. thus depends. on
`the sense of polarization. If rotatory Brownian motion does
`not randomize molecular orientations before light emission,
`different anisotropic popµlations of excited molecules con(cid:173)
`.tribute lo the nuorescence upon' excitation with 'right-hand'(cid:173)
`cd or left-handed circularly polarized light. The observed
`intensity of.fluorescence depends not only on the number of
`excited molecules, but also on the distribution .in SJ>l!Ce of
`
`Communications to the Editor
`
`' "' ' •
`
`.
`
`40.5 MHz field).
`
`.. _ H'l'na) H. c. Rilll0g and w. w. Epstein, J. Am. Chem. Soc .. 111, 1041
`'·'·:(' .;'. - .11.969J; (bl H. C •. Rilling, C. D. Poulter. W. W. Epstein, and B. Larsen.
`··Ibid., 93, 1783 (19711; (c) G. PopJSk. J. Edmond, and S.·M. Wong, Ibid.,
`. "95, 2713 (1973).
`.
`
`.
`
`.
`
`.
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