Table Of Content.
INVERSE-DETECTED NMR OFCIGUATOXIN: QUATERNARY CARBON
LOCATIONS CONFIRMED IN CTX-1
RICHARD LEWIS ANDIAN M. BRERETON
J.
Lewis, R.J. & Brcrcton, I.M. 19940801; Inverse-delected NMR ofciguatoxin; quaternary
carbonlocationsconfirmedinCTX-1 MemoirsoftheQueenslandMuseum34(3),555-559.
.
Brisbane. ISSN0079-8835.
Short-range(HMQC,,Jch)andlong-range(HMBC,23Jch)2-dimensionaIinverse-detected
heieronuclear nuclear magnetic resonance spectra of0.45mg of ciguatoxin-1 are shown.
These spectra provide independent support forthe structure proposed forciguaioxin-1 and
confirmtheI3Cassignmentsandthelocationofthetwoquaternarycarbonsinciguatoxin-1
HMBC
Thepresenceoffouretherlinkages wasalsoconfirmed from the experiment.
RichardJ. Lewis,SouthernFisheriesCentre,QueenslandDepartmentofPrimaryIndustries,
PO Box 76, Deception Bay. Queensland 4508: km M. Brereum, Centrefor Magnetic
Resonance, UniversityofQueensland, StLucia, Queensland4072; 22November, 1993.
A majoradvance in ciguatera research was the detected spectraofCTX-1, determined usingthe
&
determination of the structure of ciguatoxin method ofMartin Crouch (1991), provide in-
(Murataet al.,1989,1990). Ciguatoxin-1 (=CTX- dependent support for this structure, including
1, Fig.1) is the most toxic ciguatoxin isolated confirmationofthelocationofthetwoquaternary
to-date (Lewis et al..l99I) and is dominant in carbons in the molecule.
ciguatericfishflesh(Lewis&Selling992).CTX-
METHODS
1 is probably responsible for the clinical
syndromethatfollowsconsumptionofciguatenc
fish; especially carnivorous species. CIGUATOXIN-1 (CTX-1)
The structure of CTX-1 was proposed on the CTX-1 was purified in 1991 (Lewis et al.,
NMR NMR
basis of one-dimensional 'H and nOe 1991). experiments were performed on a
spectroscopy, two-dimensional homonuclear 0.45mg sample ofCTX-1 in 0.45ml ofpyridine-
scalar coupled spectroscopy 267hh) and mass ds (99.96%, Cambridge Isotope Laboratories),
(
spectroscopy (Murata et al.,1989,1990). Short-
range (one bond) inverse-detected heteronuclcar Nuclear MagneticResonance(NMR)
experiments (HMQC) supported the structure Spectroscopy
proposed for CTX-1 (Murata et a!.,1992). The The formation ofheieronuclear multiple quan-
absolute stereochemistry of CTX-1 (Fig.l) has tum coherence between protons and C nuclei
been proposed (Suzuki etal.,1991). Short-range provides a powerful tool for molecular structure
(HMQC) and long-range (HMBC) inverse- determination. Two dimensional experiments
CTX - 1 R, = OH
CTX - 2 R = H
1
CTX - 3 Rt = H
Me H
60
HO'
FIHGM.B1.CStrsupcetcurteruomfcairgeusathooxwinn-b1ol(dRied=.OH)proposedby Murataetal. (1990). Connectivitiesconfirmedfromthe
556 MEMOIRS OFTHEQUEENSLANDMUSEUM
Mi
1.2 1.0 0.8 2.5 2.0
Protonchemicalshift(ppm) Protonchemicalshift(ppm)
r
4.0 3.5 6.2 6.0 5.8
Protonchemicalshift(ppm) Protonchemicalshift(ppm)
FIG.2. HMQC spectrum ofciguatoxin-1 (CTX-1) at 400 MHz in pyridine-ds (30°C). Vch clustered into four
regionsshownindetailinpanelsAtoD.Carbonnumberofthecarbonsgivingriseto'7charelabelledaccording
tothestructureofCTX-1 (Fig.l). Datawerezero filledtogive amatrixconsistingof4Kx 1 Kpoints.
provide extensive proton to carbon connec- short- or long-range correlations are determined
tivities, both direct via one bond scalarcoupling depends on the evolutionary manipulation of
HMQC
]Jcu) with the experiment and long heteronuclear coherences according to the value
(
range via 2 and 3 bond scalar couplings 2,37ch) of the respective heteronuclear coupling con-
(
with the HMBC. The long-range experiment al- stants (~140Hzforonebondcouplings and 5-10
lows correlations across and to quaternary car- Hz for 2 and 3 bond couplings). The sensitivity
bons (i.e. carbons without attached protons) and of these experiments can be maximised if the
across heteroatoms (e.g. oxygens). Whether generated heteronuclear multiple quantum
1
NMR OFCIGUATOX1N- 557
scansaveraged)overaspectral
width of43 0Hz) andwas op-
1
timised for 8.3Hz couplings.
The two- dimensional
homonuclear Hartman Hahn
experiment (HOHAHA) was
performed according to Bax &
I)
:, c Davis(1985) at 500MHzon an
AM
X-500 at 30CC (246 ti
I
values, each with 2048 points
(88 scans averaged) over a
P. At V Q Q spectral width of4505Hz) No
»!?• •' twhaetseerseuxppperreismseinotns.waTsheuseNdMfRor
no data were processed using
p o FTTOOLand spectra analysed
Ir JS t on a SUN SPARCstation-2
c also using this software. For
each 2-D experiment, a gaus-
1
sian deconvolution with line
broadening was applied to t?
data(datazerofilledtogive4K
points in F2) and a Hamming
filter applied lo ti data (zero-
3 filled to give IK points in Fl)
to obtain optimal signal to
noise in each dimension.
rhtniitiilshtfttpprri) Chemical shifts are given in
ppm downfield of the pyridine
1-1G.3.HOHAHAspectrumofciguatoxin-l (CTX-1)at>00MHzmpyridine- resonance ('H at 7.21ppm and
ds (30°C). Chemical exchange between hydroxyl protons ofCTX-1 and C at 123,5ppm).
H2O was detected as direct and relayed cross-peaks from each hydroxyl
protontothewaterresonanceat4.94ppm(mixingtime55msec). Datawere RESULTS
zero filled togiveamatrixconsistingof4 K x 1 K points.
HMQC
The spectrum of
coherences are converted to observable proton CTX-1 (Fig.2) detected all 'Jch except those
signals, ratherthan using magnetisation from the associated with the proton resonances at 4.86
lesssensitive heteronucleus, ill the so-called"in- ppm which were obscuredby the H2O resonance
verse"orprotondetectedcorrelationexperiment. which was not suppressed in this experiment.
The one-bond correlation variant ofthis experi- This spectrum allowed us to inde-
ment is known as the inverse-detected HMQC pendentlydetermine the l3C chemical shifts for
and the somewhat less sensitive long-range eachcarbononCTX-1 andthe 'Hchemicalshifts
mvualrtiainptleabsontdhecoirnrveelrastei-onde(teHcMteBdC)heetxepreorniumcelneta.r orfestphoenadtttoacthheedaspsroitgonnme(nTtabolfeMu1r).aTthaeesteatd.a(t1a99c2o)r,-
Theseprocedureswereappliedtotheciguatoxins widi the minorexception ofcarbon 36 to which
as described below. we assign a 13C chemical shift of Sl.Oppm, as
(HThMeQCsheoxrpte-rriamnengte) (txJwcon-)diimnevnesrisoen-adletNecMteRd moeptphoisneedstion 8C3T.X6-9p1pma.ndThteheseasdsaitganmceonntfiorfmtahlel
spectrum ofCTX-1 was obtained at 400MRz on double bond in the flexible portion of CTX-1
a BrukerAMX-400 at 30°C (206 ti values, each (ring F). In addition, all carbons assigned as at-
with 2048 points (512 scans averaged) over a tached tooxygen had ,3Cchemical shiftscharac-
spectral width of 4000Hz}. The long-range teristic ofsuch a chemical environment. The 13C
(**Vch)NiMnvRerse-detected (HMBC) two-dimen- assignments were confirmed by careful com-
sional experiment on CTX-1 was per- parison of the coupling patterns of protons ob-
formed at 500MHz on a Bruker AMX-500 at served in the HMQC, HOHAHA and reference
30°C (495 ti values, each with 2048 points (96
MEMOIRSOFTHEQUEENSLANDMUSEUM
mM
ona 1 solution). However,
we were able to confirm the
location offour ether linkages
by this experiment(Figs 1.4).
DISCUSSION
C&ttUn*IC5MHMI
4U We HMQC
obtained and
«Iftflfffl o' (411H57fi HMBC spectraofa ImM solu-
tionofCTX-1 inpyridine-ds at
HMQC
30°C. The spectnimin-
dependently confirmed the nC
assignments of CTX-1 given
- by Murata et al. (1992). The
HMBC
spectra confirmed the
position ofcarbons 33 and 52,
I ,,111 wC<W - tphreesetnwtoinqCuTaXte-r1na(rFyig.cl)a.rbTohniss
tti/Utt* spectrum also confirmed the
location offourofthe 13 ether
linked rings (Fig. 1). The loca-
- tion of these quaternary car-
bons and 12 of the 13 ether
—i—i—r T i—T T T—«—iiH—/IcMJt I lfeirnrkeadgefsromwaUsCpcrheevmiiocuasllsyhifitns-
4.0 V5 3.0 2.5 2.0 15 1-0 and one-dimensional nOe ex-
Protnitrhnnii:il shift (ppm> perimentsH(MMuBrCataetal.t1990).
The experiment
nG.4.HMBCsr^ctmmofciguatoxin-l(CTX^lUt5(K)MHzinpyndine-d3 detected all carbons two or
(30°C). 2iJcu arc labelled according lo the proposed structure ofCTX-1 three bonds from methyl
(Fig. 1). Data were zero Filled to give a matrix consisting of 4 K x 1 K protons but owing to the rela-
points. tively poor signal to noise of
this experiment (compared to
1-D JH NMR spectra. All 'H chemical shifts the HMQC experiment) it was not sufficiently
HMBC
assignedfromthe experimentoverlapped sensitive to detect many ofthe 2Vch couplings.
HOHAHA
thechemical shifts assigned from the These couplings may also be either larger or
experiment also obtained at 30°C (Fig.3). In ad- smaller than 8.3Hz, the coupling size for which
dition to scalar coupled connectivities, the theexperimentwasoptimised. Inconclusion,our
HOHAHA
(55msec mixing time) also detected datasupportthe structureproposedoriginally for
thechemicalexchangebetweenhydroxy1protons CTX-1 by Murata et al. (1989).
ofCTX-1 andresidualH2Ointhesolvent.Similar
exchange was detected previously for CTX-2 ACKNOWLEDGEMENTS
(Lewisetal.,1993).Suchsignalsprovideauseful
means ofidentifying the exchangeable hydroxyl We thank Peter Barron (Bruker, Australia) for
HMQC
protons in molecules. running the experimentand Ray Norton
The HMBCspectraofCTX-1 (Fig.4) includes and David Doddrell forencouragement and sup-
^
2"Vchforallmethyl protons. Fortunately, thetwo port.
quaternarycarbonsinCTX-1 (carbons33and52)
were within three bonds ofamethyl, allowingus LITERATURECITED
toconfirm unambiguously the l3Cchemical shift
(Table 1) and location (Fig.l) of these carbons. BAX A. & DAVIS, D.G. 1985. MLEV-17-basedtwo-
Mustotherlong-rangecouplingsexpectedforthe dimensional homonuclear magnetisation transfer
proposed structure ofCTX-1 were not detected, spectroscopy.JournalofMagneticResonance65:
at least in part owing to the small quantity of 355-360.
CTX-1 available (experiments were performed LEWISR.,S.R,.JM.,ACSELLELOIDN,, MJ..K,.P&OUS,HEMI.LA,.,M.NOM.RT1O9N91,.
NMROFCIGUATOXIN- 559
TABLE 1. 13Cand 'HNMRchemicalshiftsforCTX-I Purification and characterization of ciguatoxins
HMQC
at30°C(400MHz, pyridine-^) from the ex- from moray eel (Lxcodomis javaniats, Murae-
periment. LEWInSi,dae)R..ToJ.xi&conS2E9L:L1I1N1,5-1M1.27.1992. Multiple
Poa 13C 'H Pos 13C 'H ciguatoxins inthe fleshoffish.Toxicon30: 915-
ition (ppm) (ppm) iuon (ppm) (ppm) 919
1 67.1 3.96 31 40.0 2.52,2.52 LEWISE.CCR.LJ.E.SN,ORCTDO.N,19R.9S3..T BCRiEguRaEtToOxNin.-2I.Mi.s&a
2 72.9 4.68 32 74.5 4.16
diastereomer ofciguatoxm-3. Toxicon 31: 637-
3 132.0 6.36 33 78.6* - 643
J 131.0 6.36 34 80.8 3.31 MARTIN, G.E. & CROUCH, R.C. 1991. Inverse-
NMR
5 78.0- 4.86 35 36.4 1.92,2.26 detected two-dimensional methods: ap-
plications in natural products chemistry. Journal
6 136.0 5.91 36 81.0 3.34 ofNatural Products54: 1-70,
7 126.8 5.78 37 73! 350 MURATA, M.. LEGRAND, A.M., ISHIBASHI, Y.&
8 34.6 2.54,2.73 38 46.8 1.54. 1 84 YASUMOTO, T. 1989. Structures ofciguatoxin
9 76.6 3.50 39 27.8 1.91 and its congener. Journal American Chemical
Society 111:8929-8931.
10 hi a 3 75 40 46.1 1.71,2.03 MURATA> M LEGRAND. A.M. ISHIBASHI, Y..
11 74.1 4.10 41 81.5 3.21 FUKUI, M,.& YASUMOTO,T. 1990.Structures
12 82.0 3.43 42 84 ."' ':; and configurations ofciguatoxin from the moray
eel Gymnothoraxjavanirus and its likelyprecur-
!3 73.6 3.34 43 41.4 1.78,2.59 sor from the dinoflagellate Gambierdiscus
14 37.5 1.85,2.56 44 74.8 4.47 toxtcus.Journal AmericanChemical Society 112:
15 7V.7 3.55 45 87.6 3.20 438CM386.
1176 18303.56 45..0734 i 4476 4747..00 24..2519 MURAYcTiAgAuS.aUtoMMx.iO.nTLbOEy,GiRTn.Ave1Nr9sD9e2.-.dAe1.t3CeMc.tN.eMdSRC2HDaEssUspiEegRcnt,mreoPns.tcJs.opo&yf
NMR
18 131.1 5.89 48 72.6 4.06 and an explanation of signal broadening.
19 83.3 4.07 : 7W7.M0 ', 96 SUZUTKetIr,ahedTr.o.n LeStAteTrsO.33: 5O2.5.-52H6.IRAMA, M.,
20 83.6 4.21 50 2.0] YAMAMOTO, YM MURATA, M.,
21 132.3 5.67 51 42.: 1.68 YASUMOTO, T. & HARADA, N. 1991. Enan-
22 135.9 6.04 52 109.7* - tioselective synthesisofthe AB ring fragment of
23 85.7 4.02 53 15.9 2.34,2.40 gambiertoxin 4B. Implication for the absolute
configurationofgambiertoxin4Bandciguatoxin.
24 85.0 3.64 54 70.7a -'. t Tetrahedron Letters35: 4505^1508.
25 32.5 -2.2,2.96 55 75.1 4.18, i is
26 128.2 5.97 56 9.7 1.37
27 128.2 5 96 57 28.3 0.94
28 32.7 -2.3,2.93 58 20.2 132
29 83.9 3.76 59 16.2 1.31
30 85.4 3.63 60 13.9 1.23
1H chemical shifts at 4.86ppm (2 protons) were
obscured by the water resonance 13C values from
(
Murataeta!.,1992).
b 13Cchemicalshift valuesforquaternarycarbonsfrom
theHMBCexperimentat30°C (500MHz, pyridine-
ds).
