TETRODOTOXIN
O
H2 N
HO
H
N
OH
O
O
H N HO
OH
OH
Presentation by Neil Vasan
Baran Lab Group Seminar
August 13, 2003
TTX: Background
• Toxic venom component of puffer fish or fugu (Spheroides
rubripes), a Japanese delicacy (1 fish = ~$400)
• First isolated in 1909 and named after puffer fish order
Tetraodontidae
• Structure first elucidated in 1964 by Woodward (confirmed by
Kishi in 1965)
• First synthesis by Kishi, et. al. in 1972
• Toxicity attributed to selective blockage of Na+ channels of
skeletal muscles
• Lethal dose for adult human = .001 mg
• Upon ingestion, one feels tingling and lightheadedness but is
lucid; paralysis and death ensue within 6-24 hours
• 70-100 deaths each year, mostly in rural Japan
• No known antidote exists
1
TTX: Structure
O
H2N
HO
H
N
OH
O
O
H N HO
OH
OH
O
O
O
O
H
N
H2N
H2N
H N HO
OH
O
HO
H
N
OH
OH
O
H N HO
OH
OH
OH
Equilibrium mixture among ortho ester, anhydride, and lactone forms
TTX: Kishi Synthesis
Synthesis of Cyclohexane chiral core
O
H
, SnCl4
Me
O
N
O
OH
o
Me
H
O
H2O, 100 C
Beckmann
(61%)
NH
O
Ac
Me
O
Me
Et3N
(quant.)
MeCN, rt
(83%)
Me
H
MsCl
Me
O
N OH
H
NaBH4
MeOH
(96%)
Me
O
N Ms
H
OH
NH
O
Ac
Me
O
H
m-CPBA
camphorsulfonic acid HO
(75%)
H
H
Me
NH
O
Ac
2
TTX: Kishi Synthesis
Towards Tetrodamine
H
O
H
H
H
O
Me
H
H
O
(90% in 2 steps) O
HO
H
NH
O
Ac
O
H
O
H
(76% in 6 steps) EtO
NH H
OAc
Ac
EtO
H
H
O
CH2OAc 1. o-DCB, reflux
H
2. m-CPBA, K2CO3
O
CH2Cl2
EtO
Me
O
MPV-reduction
2) O(COCH3)2, pyr.
(95% in 2 steps)
NH
O
Ac
O
NH
OAc
Ac
H
CH2OAc
O
O
H
H
Me 1) Al OCHMe2 3
H
NH H
OAc
Ac
AcOH, rt
(70% from
diethyl ketal)
TTX: Kishi Synthesis
Towards Tetrodamine
H
H
H
O
H
CH2OAc
O
O
AcO
AcO
NH H
OAc
Ac
mCPBA
CH2Cl2, rt
(quant.)
H
O
H
CH2OAc
O
O
O
AcO
KOAc, AcOH
90 oC, 2h
(quant.)
NH H
OAc
Ac
H
O
1) O(COCH3)2, pyr.
H
H
o
OAc
OH
2) pyrolysis, 300 C, vacuum
CH
OAc
2
OAc
OAc CH2OAc
(80% in 2 steps)
AcHN
H
AcHN
H
O
O
(acetylated
AcO
AcO
tetrodamine)
H
H
O
O
H
O
H
3
TTX: Kishi Synthesis
Tetrodamine to Tetrodotoxin
H
AcHN
AcO
O
H
OAc
OAc CH2OAc
H
O
H
O
(acetylated
tetrodamine)
H
BF3 Et2O
CH2Cl2
(92%)
H2N
AcO
H
O
SEt
H
H
OAc
OAc CH2OAc
H
O
AcN
SEt
120 oC, 12h
SEt
C
AcHN
N
AcO
O
H
O
H
OAc
OAc CH2OAc
H
O
O
(20% in 3
steps from
tetrodamine)
HO
HO
TTX
O
H
H
OAc
NH2
OAc CH2OAc
o
C
1) NaIO4, THF, 0 C
OsO4
AcHN
N
H
O
THF,
-20 oC
2) NH4OH, MeOH
AcO
H
(25% from monoO
acetylguanidine)
H
NH2
C
AcHN
N
AcO
H
O
H
OAc
OAc CH2OAc
H
O
O
TTX: Isobe Synthesis
Retrosynthesis
4
TTX: Isobe Synthesis
Sonogashira Coupling and Claisen Rearrangement
OH
O
OTBS
O
Oi-Pr
Oi-Pr
TMS
(61% in 4 steps)
OH
I
O
(available in 2 steps from
)
AcO
OAc
OH
TMS
OAc
OTBS
O
OMe
Oi-Pr
PPTS/THF
Oi-Pr
Pd(OAc)2,
Ph3P, CuI,
Et3N, PhH
(99%)
OH
OAc
OTBS
O
OTBS
O
K2CO3
o-DCB
O
(50% in 6 steps)
150 oC
(89%)
OTBS
O
Oi-Pr
Me
(94%)
BzO
5
O
OTBDPS
O
TMS
TMS
Oi-Pr
TTX: Isobe Synthesis
Cyclohexenone and Exoolefin Synthesis
H2SO4
MeOH
and then
HgO (cat.)
(82%)
H
O
OMe
O
2) TBS-Cl
imid., DMF
5
TBDPSO OHOBz
OTBS
O
1) TBS-OTf
Et3N, lutidine
1) TBAF
THF H2O
OMe
BzO
O
O
2) Cl3CCOCl
DMAP, pyr.
(74% in 4 steps)
OTBS
OTBDPS
OH
OTBS
O
O
OMe
BzO
BzO
O
OTBDPS
OH
(67% in 6 steps)
OBOM
OTBDPS
1) NaBH4,
O
MeOH, rt
2) Me2C(OMe)2,
CSA, acetone BzO
3) PPTS, MeOH
(93% in 3 steps)
O
OH
OBOM
OTBDPS
5
TTX: Isobe Synthesis
Previous Overman Rearrangement
Problem with Overman Rearrangement
TTX: Isobe Synthesis
Installation of Nitrogen Functionality: Conjugate Carbamate Addition
O
O
OH
BzO
OBOM
OTBDPS
1) DIBAL-H
O
2) TEMPO, NCS
1) Cl3CCONCO
O
COOMe
3) NaClO2, NaH2PO4,
CH2Cl2
Me2C=CHMe-H2O HO
2) Et3N, MeOH
4) TMS-CHN2
(99% in 2 steps)
OBOM
(78% in 4 steps)
OTBDPS
O
1) t-BuOK, THF
2) LiBH4, THF
3) MMTrCl, pyr.
(88% in 3 steps)
O
O
O
O
O
H2N
COOMe
O
OBOM
OTBDPS
O
O
NH OMMTr
OBOM
OTBDPS
1) Boc2O, Et3N
DMAP, THF
2) LiOH, MeOH
(CH2Cl)2-H2O
(84% in 2 steps)
O
HO
Boc
NH OMMTr
OBOM
OTBDPS
6
TTX: Isobe Synthesis
Construction of Lactone Bicycle
O
O
HO
Boc
NH OMMTr
(70% in 7 steps)
OBOM
OTBDPS
TTX: Isobe Synthesis
Introduction of Guanidine
Me
O
HO
H
Me
Me
O
O
BocHN
O
BOM
AcO
BocHN
BocN
OAc
O
OH
AcO
Me
O
BocHN
O
(96% in 4 steps)
OBz
Ac
OBz
OBz
O
HO
O
HO
N
O
H
OBz
Ac
OAc
AcO
H
N
N O
H
1) NaIO4
MeOH-H2O Boc
2) TFA, MeOH
N
(90% in 2 steps)
AcO
Ac
N
Ac
N
N O
Ac
1) TFA, MeOH
2) CAN
MeCN-H2O
3) HgCl2
NHBoc
OBz
Et3N-DMF
OAc
SMe
(53% in 3 steps) BocN
O
O
O
OBz
OH
Ac
O
O
OBz
(50% in 3 steps)
OAc
OBz
Me O
O
O
Ac
OBz
OAc
7
TTX: Isobe Synthesis
Deprotection and Final Conversion
OBz
AcO
Ac
N
Me O
O
O
Ac
N
N O
Ac
O
HO
OBz
Ac
Et3N
MeOH-H2O H2N
(85%)
Me O
O
O
H
N
N
H
O
O
H
OAc
+
O
O
O
H
N
H2N
N
H
OH
O
H
OH
OH
OH
(TTX-anhydride)
TFA-d/D2O
H2N
HO
H
N
O
OH
O
O
(-) TTX
H N HO
OH
OH
(TTX-ortho ester)
TTX: Du Bois Synthesis
Retrosynthesis
OH
HO
HN
O
OH
OH
N OH
H
NH2
HO
OH
O
HO
O
O
OH
OH
HO
O
OH
OH
OH
H
CO2H
OHC
H OH
HO
stereospecific C-H
amination
Me
HO
HO
OHC
HO
O
OH
OH
OH
H
CO2H
Me
O
Me
O
OR2
Me
O
N2
OR2
H
O
O
1
R O
O
Rh-catalyzed
C-H insertion
O
O
R1O
O
8
TTX: Du Bois Synthesis
Rh-carbene C-H insertion
O
O
HO
O
O
OH
Me
Me
O
CO2Bn
(64% in 5 steps)
OH
BnO2C
Me
Me
OH
H
OTBS
OH
(53% in 4 steps)
O
O
O
COOt-Bu
TBSO
Me
O
Me
1) Rh2(tpacam)4, CCl4
2) NH3 BH3, CH2Cl2-MeOH
(75% in 2 steps)
O
O
1) H2 (1200 psi), Rh-C,
CF3CO2H-MeOH
2) p-TsOH, 2,2-DMP;
then t-BuCOCl, pyr., THF
(77% in 2 steps)
OTBS
O
t-BuO2C
O
O
O
N2
Me
Me
O
O
O
Me
O
Me
O
H
CO2t-Bu
O
TTX: Du Bois Synthesis
Construction of Lactone Bicycle
Me
Me
Me
O
O
O
O
Me
O
O
O
O
Me
O
Me (56% in 3 steps)
H
Me2NOC
CO2t-Bu
O
Me
O
o
Me 100 C, pyr., PhCl
(70%)
H
CO2t-Bu
O
O
Ph2Se2, PhIO2
Me2NOC
O
(77% in 2 steps)
O
Me
CO2t-Bu
Me
O
O
1) t-BuCO2H
Me
O Me
H
OCOt-Bu
Me2N
Me
Me
Me
O
HO
O
H
Me
O
Me
Me
PhCl, 200 oC
2) NaOMe, THF-MeOH
(78% in 2 steps)
O
Me
O
Me
O
O
H
OH
O
9
TTX: Du Bois Synthesis
Rh-nitrene C-H insertion
O
Me
Me
Me
O
O
Me
O
Me
Me O
OH
O O
O
Me
Me O
H
O
(66% in 3 steps)
O
H
Me
MgO, DCE, 50 oC
Me
Me
OH
Rh2(tfacam)4, PhI(OAc)2,
(55% with 38% reactant)
O
Me
O
(59% in 4 steps)
NH2
Cl
Me
Me
Me O
OH
NHBoc
O
O
H
Cl
O O
O
O
NH
O
O
Me
Me O
O O
O
Me
O
O
Me
O
O O
O
O
Me
TTX: Du Bois Synthesis
Guanidine Insertion and Final Steps
Me
Me O
O O
O
Me
OH
NHBoc
O
Me
O O
O
H2O, 110 oC
O
Me
Me
BocHN
Me
O
HO
OH
O3, Me2S,
OH
CH2Cl2-MeOH; HO
N OH
then H2O-CF3CO2H HN
H
(1:1 mixture)
H2N
([-] TTX-ortho ester)
Me
Me O
O O
O
SMe
HgCl2, Et3N,
MeCN-CH2Cl2
(80%)
O
OH
N
O
Me
NHBoc
NHBoc
O
Me
OH
OH
O
NBoc
OH
NH2
(95%)
O
Me O
O
HN
H2N
O
O
OH
OH
N OH
H
H2O-CF3CO2H
5 days
(4:1 mixture)
(65% from
after deprotection)
(-) TTX
([-] TTX-anhydride)
10
Comparisons and Contrasts
Chemist
KISHI
ISOBE
DU BOIS
Date
Sept. 1972
Jan. 2003
June 2003
Type
Racemic
Highlights
Diels-Alder synthesis
of skeleton; 6 chiral
centers established
on cyclohexane core
ring; Only ketal and
acetyl protecting
groups; Common
reagents employed
to carry out elegant
chemistry
Claisen
rearrangement;
Sonogashira
coupling;
Intramolecular
carbamate-ester
conjugate addition;
All OH groups
differently protected
for future analog
synthesis
Rhodium-catalyzed
carbene and nitrene
C-H insertions;
Ph2Se2-promoted
allylic oxidation;
Shows power of
organometallic
chemistry in total
synthesis
# of Steps
29
67
32
Yield
0.66%
1.22%
0.49%
Asymmetric Asymmetric
TTX: References
Structure:
Woodward, R. B. Pure. Appl. Chem. 1964, 9, 49-74.
Tsuda, K., et. al. Chem. Pharm. Bull. 1964, 12, 1357-1374.
Goto, T.; Kishi, Y.; Takahashi, S.; Hirata, Y. Tetrahedron 1965, 21, 2059-2088.
Absolute stereochemistry:
Furusaki, A., et. al. Bull. Chem. Soc. Jpn. 1970, 43, 3332-3341.
Bioorganic studies:
Narahashi, T.; Moore, J. W.; Scott, W. R. J. Gen. Physiol. 1964, 47, 965-974.
Hucho, F. Angew. Chem, Int. Ed. Engl. 1995, 34, 39-50.
Numa, S.; Noda, M. Ann. N.Y. Acad. Sci. 1986, 479, 338-355.
Other analog syntheses:
Nishikawa, T., et. al. Angew. Chem., Int. Ed. 1999, 38, 3081-3084.
Asai, M., et. al. Tetrahedron 2001, 57, 4543-4558.
Nishikawa, T., et. al. J. Am. Chem. Soc. 2002, 124, 7847-7852.
11
TTX: References
Kishi synthesis:
Kishi, Y., et. al. Tetrahedron Lett. 1970, 59, 5127-5128.
Kishi, Y., et. al. Tetrahedron Lett. 1970, 59, 5129-5132.
Kishi, Y., et. al. J. Am. Chem. Soc. 1972, 94, 9217-9219.
Kishi, Y., et. al. J. Am. Chem. Soc. 1972, 94, 9219-9221.
Isobe synthesis:
Isobe, M., et. al. J. Am. Chem. Soc. 2003, 125, 8798-8805.
Overman Rearrangement:
Sonogashira Coupling:
α-hydroxyl Lactone:
Overman, L. E. Acc. Chem. Res. 1980, 13, 218-224.
Sonogashira, K., et. al. Tetrahedron Lett. 1975, 16, 4467-4470.
Corey, E. J.; Ghosh, A. K. Tetrahedron Lett. 1988, 26, 3205-3206.
Du Bois Synthesis:
Hinman, A.; Du Bois, J., personal communication. (to be published in JACS)
Rh-carbene C-H insertion:
Rh-nitrene C-H insertion:
Ph2Se2 allylic oxidation:
Espino, C. G.; Du Bois, J. Angew. Chem., Int. Rd. Engl. 2001, 40, 598-600.
Guthikonda, K.; Du Bois, J., J. Am. Chem. Soc. 2002, 124, 13672-13673.
Barton, D. H. R.; Crich, D. Tetrahedron 1985, 41, 4359-4364.
TTX: Acknowledgements
• Prof. Justin Du Bois, Stanford University
• The Baran Laboratory, TSRI
12