1

TETRODOTOXIN

Presentation by Neil Vasan

Baran Lab Group SeminarAugust 13, 2003

OHOH

HO

OO

NH2N N

H

H

O

OH

HO

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

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TTX: Structure

OHOH

HO

OO

NH2N N

H

H

O

OH

HO

OHOH

HO

OO

NH2N N

H

H

OO

OHOH

HO

OOH

NH2N N

H

H

O

OH

HO

Equilibrium mixture among ortho ester, anhydride, and lactone forms

TTX: Kishi SynthesisSynthesis of Cyclohexane chiral core

, SnCl4

MeCN, rt(83%)

OMe

Me

NOH

OMe

NH

H

OAc

NaBH4MeOH

OMe

MeN OH

H

O

Me

NH

HOH

OAc

MsClO

Me

MeN Ms

H

O

Me

NH

HO

HOH

H

OAc

H

Et3N(quant.)

H2O, 100 oCBeckmann

(61%) (96%)

m-CPBAcamphorsulfonic acid

(75%)

O

3

TTX: Kishi Synthesis

(90% in 2 steps)

(76% in 6 steps) (70% from diethyl ketal)

Towards Tetrodamine

Me

NH

HO

HOH

H

OAc

H Me

NH

HO

O

O

H

OAc

H Al OCHMe2

NH

HO

EtO

EtO

H

OAcAc

HOCH2OAc

H NH

HO H

OAcAc

HOCH2OAc

HEtOO

Me

NH

HO

O

O

H

OAcAc

H3

MPV-reduction

1)

2) O(COCH3)2, pyr.(95% in 2 steps)

1. o-DCB, reflux2. m-CPBA, K2CO3

CH2Cl2

AcOH, rt

TTX: Kishi SynthesisTowards Tetrodamine

O

O

O

AcO

HOHCH2OAc

HOAc

AcHNAcO

H

H

H

NH

HO H

OAcAc

HOCH2OAc

HOAcO

mCPBACH2Cl2, rt

NH

HO H

OAcAc

H

OCH2OAc

HOAcO

O

O

O

O

HOAcCH2OAc

HOAc

AcHNAcO

H

H

KOAc, AcOH90 oC, 2h

(quant.) (quant.)

1) O(COCH3)2, pyr.2) pyrolysis, 300 oC, vacuum (80% in 2 steps)

(acetylatedtetrodamine)

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TTX: Kishi SynthesisTetrodamine to Tetrodotoxin

O

O

O

HOAcCH2OAc

HOAc

AcHNAcO

H

HBF3 Et2OCH2Cl2

O

O

O

HOAcCH2OAc

HOAc

H2NAcO

H

HAcN

SEt

SEt

O

O

O

HOAcCH2OAc

HOAc

NAcO

H

H

CNH2

AcHN

HOHO

TTX O

O

O

HOAcCH2OAc

HOAc

NAcO

H

H

CNH2

AcHN

O

O

O

HOAcCH2OAc

HOAc

NAcO

H

H

CSEt

AcHN120 oC, 12h

OsO4

THF, -20 oC1) NaIO4, THF, 0 oC2) NH4OH, MeOH

(92%)

(acetylated tetrodamine)

(25% from mono-acetylguanidine)

(20% in 3 steps from tetrodamine)

TTX: Isobe SynthesisRetrosynthesis

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TTX: Isobe SynthesisSonogashira Coupling and Claisen Rearrangement

OMe

OHO

OH

Oi-Pr

OOTBS

O

Oi-Pr

TMS

O

OAcOAc

AcO

OAc

OOTBS

OH

Oi-Pr

I

OOTBS

Oi-Pr

TMS

OMe

TMS OOTBS

OH

Oi-Pr

TMS

OOTBS

Oi-Pr

OOTBDPS

BzO

Pd(OAc)2, Ph3P, CuI,Et3N, PhH

(99%)

PPTS/THF

(89%)

K2CO3

o-DCB150 oC(94%)

5(50% in 6 steps)

(61% in 4 steps)

(available in 2 steps from )

TTX: Isobe Synthesis

H2SO4MeOH

OOTBS

OMe

OTBDPS

BzO

O

OO

OOBzOHTBDPSO

HOMe

OOH

OTBDPS

BzO

OBOM

OH

OOTBS

OMe

OTBDPS

BzOO

OTBS

OTBDPS

BzO

OBOM

OHOO

and thenHgO (cat.)

(82%)

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1) TBS-OTf Et3N, lutidine

2) TBS-Cl imid., DMF

1) TBAF THF H2O

2) Cl3CCOCl DMAP, pyr.(74% in 4 steps)

(67% in 6 steps)

1) NaBH4, MeOH, rt2) Me2C(OMe)2, CSA, acetone3) PPTS, MeOH(93% in 3 steps)

Cyclohexenone and Exoolefin Synthesis

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TTX: Isobe SynthesisPrevious Overman Rearrangement Problem with Overman Rearrangement

TTX: Isobe SynthesisInstallation of Nitrogen Functionality: Conjugate Carbamate Addition

OTBDPS

BzO

OBOM

OHOO

OMMTr

OTBDPS

O

OBOM

OO

O

NH

COOMe

OTBDPS

HO

OBOM

OO

OTBDPS

HO

OBOM

OO

NHBoc

OMMTr

COOMe

OTBDPS

O

OBOM

OO

O

H2N

1) DIBAL-H2) TEMPO, NCS3) NaClO2, NaH2PO4, Me2C=CHMe-H2O4) TMS-CHN2 (78% in 4 steps)

1) Cl3CCONCO CH2Cl22) Et3N, MeOH(99% in 2 steps)

(88% in 3 steps)

1) t-BuOK, THF2) LiBH4, THF3) MMTrCl, pyr.

(84% in 2 steps)

1) Boc2O, Et3N DMAP, THF2) LiOH, MeOH (CH2Cl)2-H2O

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TTX: Isobe Synthesis

OTBDPS

HO

OBOM

OO

NHBoc

OMMTr

(70% in 7 steps)

Construction of Lactone Bicycle

TTX: Isobe Synthesis

OAcOBz

O

OOAc

NN N

Ac

OBz

O

AcO

Ac

Ac

Me

Introduction of Guanidine

OHOBz

O

OOAc

O

BOM

OO

Me

Me

HOH

BocHN

OAcOBz

O

OO

OBz

Ac

OO

Me

Me

AcO

BocHN

OAcOBz

O

OO

OBz

Ac

HOHO

AcO

NH

BocHN

BocN

OAcOBz

O

OO

NN N

H

H

OBz

OH

AcO

Boc

Ac

BocN

NHBoc

SMe

(96% in 4 steps)

1) TFA, MeOH2) CAN MeCN-H2O3) HgCl2 Et3N-DMF(53% in 3 steps)

1) NaIO4

MeOH-H2O2) TFA, MeOH(90% in 2 steps) (50% in 3 steps)

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TTX: Isobe SynthesisDeprotection and Final Conversion

OAcOBz

O

OOAc

NN N

Ac

OBz

O

AcO

Ac

Ac

Me

OHOHO

OOH

NH2N N

H

O

O

HOMe

H

H

OHOH

HO

OO

NH2N N

H

O

OH

HO

OHOHO

OOH

NH2N N

H

O

H

O

Et3NMeOH-H2O

(85%)+

TFA-d/D2O

(TTX-anhydride)

(TTX-ortho ester)

(-) TTX

TTX: Du Bois Synthesis

OHOH

OH

OO

OH

HO

NHNH

NH2

OHOH

OH

OO

OH

HO

HO

OH

OH

HOOH

HO

OHCHCO2HHO

O

OO

Me

Me

OR1O

O

OR2

O

OO

Me

Me

OR1O

O

OR2

HN2

OH

OH

HOOH

HO

OHCHCO2HHO

stereospecific C-Hamination

Rh-catalyzedC-H insertion

Retrosynthesis

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TTX: Du Bois SynthesisRh-carbene C-H insertion

O OH

OH

O

HOOH

O

OO

Me

Me

Ot-BuO2C

O

OTBS

H

OOO

MeMe

OTBS

CO2Bn

OHBnO2CO

O

MeMe

TBSO

OO

COOt-Bu

ON2

O

OOMe

Me

O Me

MeHO

O CO2t-Bu

(53% in 4 steps)(64% in 5 steps)

1) Rh2(tpacam)4, CCl42) NH3 BH3, CH2Cl2-MeOH

(75% in 2 steps)

1) H2 (1200 psi), Rh-C, CF3CO2H-MeOH2) p-TsOH, 2,2-DMP; then t-BuCOCl, pyr., THF (77% in 2 steps)

TTX: Du Bois SynthesisConstruction of Lactone Bicycle

O

OO Me

O Me

MeHO

O CO2t-Bu

Me

O

OO Me

O Me

MeH

Me2NOCCO2t-Bu

Me

O

OO Me

O Me

MeH

Me

HO

Me2NOCOt-Bu

O

O

OO Me

O Me

MeH

Me2NOCCO2t-Bu

Me

O

OO Me

O Me

Me

Me

HOH

O

(56% in 3 steps)

Ph2Se2, PhIO2

100 oC, pyr., PhCl(70%)

(77% in 2 steps)

1) t-BuCO2H PhCl, 200 oC2) NaOMe, THF-MeOH (78% in 2 steps)

O

O

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TTX: Du Bois Synthesis

O

OO Me

O Me

Me

Me

HOH

O

OO

O

OO

MeMe

HO

MeMe

OH

OO

O

OO

MeMe

OMe

Me

Cl

NH

OO

OO

O

OO

MeMe

HO

MeMe

O

Cl

O

NH2

OO

O

OO

MeMe

OMe

Me

NHBoc

OH

(66% in 3 steps)

Rh2(tfacam)4, PhI(OAc)2, MgO, DCE, 50 oC

(55% with 38% reactant) (59% in 4 steps)

Rh-nitrene C-H insertion

TTX: Du Bois SynthesisGuanidine Insertion and Final Steps

OO

O

OO

MeMe

OMe

Me

NHBoc

OH

OHOH

OH

OO

OH

HO

NHNHH2N

HO

OO

O

OO

MeMe

OMe

Me

NH2

OH

OHOH

OH

OO

OH

NHNHH2N

O

NBoc

BocHN SMe

H2O-CF3CO2H5 days

(4:1 mixture)(65% from

after deprotection)

OO

O

OO

MeMe

OMe

Me

N

OHNHBoc

NHBocH2O, 110 oC

(95%) HgCl2, Et3N, MeCN-CH2Cl2

(80%)

O3, Me2S, CH2Cl2-MeOH;

then H2O-CF3CO2H(1:1 mixture)

([-] TTX-ortho ester) ([-] TTX-anhydride)

(-) TTX

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Comparisons and Contrasts

0.49%1.22%0.66%Yield326729# of Steps

Rhodium-catalyzed carbene and nitrene

C-H insertions; Ph2Se2-promoted allylic oxidation; Shows power of organometallic

chemistry in total synthesis

Claisenrearrangement;

Sonogashiracoupling;

Intramolecularcarbamate-ester

conjugate addition; All OH groups

differently protected for future analog

synthesis

Diels-Alder synthesis of skeleton; 6 chiralcenters established on cyclohexane core ring; Only ketal and

acetyl protecting groups; Common

reagents employed to carry out elegant

chemistry

HighlightsAsymmetricAsymmetricRacemicTypeJune 2003Jan. 2003Sept. 1972DateDU BOISISOBEKISHIChemist

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.

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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: Overman, L. E. Acc. Chem. Res. 1980, 13, 218-224.Sonogashira Coupling: Sonogashira, K., et. al. Tetrahedron Lett. 1975, 16, 4467-4470. α-hydroxyl Lactone: 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: Espino, C. G.; Du Bois, J. Angew. Chem., Int. Rd. Engl. 2001, 40, 598-600.Rh-nitrene C-H insertion: Guthikonda, K.; Du Bois, J., J. Am. Chem. Soc. 2002, 124, 13672-13673.Ph2Se2 allylic oxidation: Barton, D. H. R.; Crich, D. Tetrahedron 1985, 41, 4359-4364.

TTX: Acknowledgements

• Prof. Justin Du Bois, Stanford University

• The Baran Laboratory, TSRI