Spinosyn G: Proof of Structure by Semisynthesis

Graupner, P. R.; Martynow, J.; Anzeveno, P. B.J. Org. Chem. 2005, 70, 2154-2160.

Tom Graham @ Wipf Group 1 6/6/2005

Natural products that contain the tricyclic core

H

HO H

H

O

MeO

OO Et

R

(-)-spinosyn A, R = Ha.k.a. (-)-A83543A, (-)-lepicidin A

(-)-spinosyn D, R = Me

OMe

OMeMeOMeO

OMeN(Me)2

H

H H

HMeO

O NH

O

O

NH

OH

O

H

capsimycin

H

H H

HNH

O

O

NH

OH

O

H

ikarugamycin

• •H H

•Me

Me

HO

HO

OH

Me

FR182877 hexacyclinic acid

OMe

O

•••H

•Me

CO2H

HO

AcO

OH Me

OMe

O

H H

OH

H

Tom Graham @ Wipf Group 2 6/6/2005

• Spinosyn A isolated in 1990 from the fermentation broth of the soil microbe Saccharopolyspora spinosa.

• Spinosad (Tracer® Naturalyte®, SpinTor® and Conserve®) is an environmentally benign insecticide marketed by Dow-Elanco for crop protection. Spinosad is marketed to the retail market for the control of fire ants.

• Manufactured by fermentation and consists of a 90:10 mixture of spinosyn A and spinosyn D.

• 24 spinosyns have been characterized.

• Based on tentative assignments, Spinosyn G is the only compound with a sugar of the L-configuration (L-ossamine).

• Is the assignment of spinosyn G correct?

H

HO H

H

O

MeO

OO Et

R

(-)-spinosyn A, R = H(-)-spinosyn D, R = Me

OMe

OMeMeOMeO

OMe

N(Me)2

Tom Graham @ Wipf Group 3 6/6/2005

Proposed Mechanism for the formation of Spinosyn from the PKS Product

Martin, C. J.; Timoney, M. C.; Sheridan, R. M.; Kendrew, S. G.; Wilkinson, B.; Staunton, J.; Leadley, P. F. Org. Biomol. Chem. 2003, 1, 4144-4147.

O

OO

MeOH

Et

HO

H H

O

OOH

MeOH

Et

HO OH-2H

O

OOH

MeOH

Et

HO OH

H+ O

OO

MeOH

Et

HO OH H

Enz - B:

O

OO

MeOH

Et

H

H

- H2O

HO

Tom Graham @ Wipf Group 4 6/6/2005

Biosynthesis of Forosamine

Zhao, Z.; Hong, L.; Liu, H,-w. J. Am. Chem. Soc. 2005, 127, 7692-7693.

O

OTDPHOHO

HOOH - H2OO

OTDPHOHO

MeOO

OTDP

MeO

O

O

OTDP

MeOHO

NADP-HO

OTDP

MeN

HO

HN O-Me

-O3PO

H

HO

OTDP

NHN O-

Me

-O3PO

H

NADP-H

O

OTDP

MeNHN O-

Me

-O3PO

HO

OTDP

MeO aminotransferase O

OTDP

MeH2N

O

OO

MeO

Et

RhamO

H H

O MeNMe2

(-)-spinosyn A

1" 5"

4"

- H2O

+ H2O

Tom Graham @ Wipf Group 5 6/6/2005

Roush’s Synthesis of (-)-Spinosyn A

Mergott, D. J.; Frank, S. A.; Roush, W. R. PNAS, 101, 11955-11959.

CHO

RhamOBr

O

Me

OPMB

OEt

OPO

OEtOEt

O

OO

MeOPMB

EtBr

RhamO

H Hi-Pr2NEt, LiCl

CH3CN (1 mM)23 °C, 19 h, 75%E/Z = >95:5ds = 73:12:9:6

O

OO

MeOPMB

EtBr

RhamO

H H

Me3P (8 eq)t-amyl alcohol (0.005M)

23 °C, 6 h, quantitative26:27:C(3)-epi-26 = 88:7:5

O

OO

MeOPMB

EtBr

RhamO

H3

26 27

1) tris(trimethylsilyl)3SiH,AIBN, dioxane, 80 °C, 1.5 h2) DDQ, CH2Cl2/pH 7 buffer0 °C, 3 h, 73% (2-steps)

O

OO

MeOH

Et

RhamO

H H3

(-)-spinosyn A pseudoaglycon

Tom Graham @ Wipf Group 6 6/6/2005

Completion of the Synthesis

O

OO

MeOH

Et

RhamO

H H

OMe

AcON3 O CCl3

NH

BrO

OO

MeO

Et

RhamO

H HBr

O MeN3

AcO

TMSOTf, CH2Cl2, 90-97%

O

OO

MeO

Et

RhamO

H HBr

O MeNMe2

HO

1) guanidinium nitrate,NaOMe, MeOH, CH2Cl2, 95%

2) SnCl2, PhSH, Et3N, THF, 92%3) CH2O, NaBH3CN,MeOH, HOAc, NaOAc, 87%

1) thiocarbonyldiimidazole,DMAP, toluene

2) Bu3SnH (25 eq), AIBN,dioxane, 35%

O

OO

MeO

Et

RhamO

H H

O MeNMe2

23 steps (longest linear)31 steps total3% overall yield

(-)-spinosyn A

Mergott, D. J.; Frank, S. A.; Roush, W. R. PNAS, 101, 11955-11959.

Tom Graham @ Wipf Group 7 6/6/2005

Evans’ Synthesis of (+)-Spinosyn A

Evans, D. A. and Black, W. C. J. Am. Chem. Soc. 1993, 115, 4497-4513

O

O

N

O

OR1

TBSO

Me

O

Et

OTIPS

ORON

O O

R1

TBSO

H

H

R2

ON

O O

R1

TBSO

H

H

R2

1, R = TES, R1 = H2, R = TES, R1 = Bn

71%, 6:1 74%, 1:10

Et2AlCl

1a2a

1b2b

Tom Graham @ Wipf Group 8 6/6/2005

Evans’ Synthesis of (+)-Spinosyn A

ON

O O

R1

TBSO

H

O

Me

O

Et

OTIPS

OR OEtS

TBSO

H

O

Me

O

Et

OTIPS

OR

OH

TBSO

H

O

Me

O

Et

OTIPS

O

TBSO

H

O

Me

O

Et

OTIPS

OH

HHO

H

TBSO

H

O

Me

O

Et

OTIPS

OH

H

R = TES

LiSEt, THF, rt

1) AcOH, aq THF, rt2) TBS-Cl, imidazole,CH2Cl2, rt

3) (ClCO)2, DMSO,Et3N, CH2Cl2,90% (3 steps)4) Et3SiH, 5% Pd/CaCO3/PbOacetone, rt

NaHMDS,THF, -78 °C

diastereoselection12:1

Martin Sulfurane

CH2Cl2, 0 °C81% (3 steps)

Evans, D. A. and Black, W. C. J. Am. Chem. Soc. 1993, 115, 4497-4513

Tom Graham @ Wipf Group 9 6/6/2005

Paquette’s Synthesis of Spinosyn A

Paquette et. al. J. Am. Chem. Soc. 1998, 120, 2543-2552Paquette et. al. J. Am. Chem. Soc. 1998, 120, 2553-2562

H

HO H

H

O

MeO

OO Et

R

OMe

OMeMeOMeO

OMe

N(Me)2

Br

OTBSO

OMeMeO

t-BuLi, CeCl3

86%

OH

OMeMeO

OTBS

NaH, THF;CH3OH, H2O

77%

TBSO H

HO

H

H

OMeOMe

1) DiBAl-H;2) p-TsOH, aq acetone, 92%

3) MOM-Cl, i-Pr2NEt, 71%

TBSO H

HMOMO

H

H

O

TBSO

MOMOH

O

K2CO3,MeOH

reflux78%

H

(-)-Spinosyn A

TBSO

H

H

O

H

H

OTBDPS

OPMB

10 steps

degradation(12 steps)

PivO

H

H H

H OPMB

O

6 steps

11 steps

Tom Graham @ Wipf Group 10 6/6/2005

Which of the Structures is Spinosyn G?

O

OO

MeO

Et

RhamO

H H

O MeNMe2

(-)-spinosyn A O

OO

MeO

Et

RhamO

H H

O NMe23 (5" epi-spinosyn A)

Me

O

OO

MeO

Et

RhamO

H H

O Me

NMe2

4 (4" epi-spinosyn A)

1"

4"

5"

1"

4"

5"

1" 5"

4"

α-L-ossamine

O

OO

MeO

Et

RhamO

H H

19 (5" epi-spinosyn A)OMe

NMe2

β-D-ossamine

β-L-ossamine

α-D-forosamine

1"

4"

5"

Tom Graham @ Wipf Group 11 6/6/2005

Synthesis of β-D-ossamine Spinosyn

O

OO

MeO

Et

RhamO

H H

O MeNMe2

(-)-spinosyn A

F-TEDA, pyridineMeCN-H2O, 0°C O O Me

R

OLi(t-BuO)3AlH

Et2O, 0° CO O MeR

OH

1) MeSO2Cl, (i-Pr)2NEt, CH2Cl2, 0°C

2) NaN3, DMF, 120 °CO OR

Me

N3 1) SnCl2, MeOH, rt

2) MeI (excess), MeCN, reflux3) m-xylene, reflux O

OO

MeO

Et

RhamO

H H

O Me

NMe2

4

N+N+

Cl

F2 BF4

-

F-TEDA(SelectfluorTM)

Tom Graham @ Wipf Group 12 6/6/2005

Synthesis of the Ossamines

O OAcOAc

O

MeO

N3O

MeO

NMe2

1) H2, 45 psi,Pd(OH)2/C,EtOH, rt

2) formalin,MeOH, reflux,1 h, then NaBH4

TROC-Cl,K2CO3

toluenereflux

O

MeO

N

O

O CCl3Me

ON

O

O CCl3Me+ O N

Me

TrOC

Me

ROHROH

O N

Me

TrOC

MeO N

Me

TrOC

MeOR

RO

Major Minor

Tom Graham @ Wipf Group 13 6/6/2005

α-L-Ossamine and β-L-Ossamine

O

OO

MeO

Et

RhamO

H H

O NMe23Me

1"

4"

5" α-L-ossamine

O

OO

MeO

Et

RhamO

H H

19 OMe

NMe2β-L-ossamine

O

OO

MeOH

Et

RhamO

H H

O NMe

Me

TrOC

1) PPTS, BF3•OEt2,1,2-dichloroethane

2) Zn, 1 M NH4OAcTHF-MeOH, rt80%, 2:1 eq/ax3) MeI (2.5 equiv),DIPEA, THF, rt49% combined

Tom Graham @ Wipf Group 14 6/6/2005

O

OO

MeO

Et

RhamO

H H

O MeNMe2

(-)-spinosyn A O

OO

MeO

Et

RhamO

H H

O NMe23 (5" epi-spinosyn A)

Me

O

OO

MeO

Et

RhamO

H H

O Me

NMe2

4 (4" epi-spinosyn A)

1"

4"

5"

1"

4"

5"

1" 5"

4"

α-L-ossamine

O

OO

MeO

Et

RhamO

H H

19 (5" epi-spinosyn A)OMe

NMe2

β-D-ossamine

β-L-ossamine

α-D-forosamine

1"

4"

5"

• The structure of Spinosyn G has been unambiguously assigned and contains the L-ossamine residue.

• The result has important implications for understanding the biosynthesis of the spinosyn class of molecules.

Conclusions:

Tom Graham @ Wipf Group 15 6/6/2005