Cerulenin©

1. Discovery1,2), producing organism1,2,55) and structure3,4,24)
Cerulenin was originally isolated from the culture broth of Sarocladium oryzae KF-140 as an
antifungal antibiotic with a structure of (2R,3S)-2,3-epoxy-4-oxo-7,10-trans, trans-dodecadienoylamide. The first paper on the total synthesis of cerulenin was reported by Boeckman et al.5) (SeeAppendix I).
OH3CO

CONH2

Cerulenin

Cephalosporium caerulens KF-140T(Sarocladium oryzae KF-140)
Bar: 5 µM

2. Physical data1)
White powder. C12H17NO3; MW 223.12. Sol. in EtOH, acetone, benzene. Slightly sol. in H2O.
Practically insol. in petroleum ether.
3. Biological Activity2,6,8-10)
1) Inhibition of fatty acid synthesis6-8)
Cerulenin is a potent inhibitor of fatty acid synthase (FAS). It inhibits all known types of FASs;
both multifunctional enzyme complexes (Type I) (from yeast, rat liver, mammalian cells, andcertain bacteria) and unassociated enzymes (Type II) (from most bacteria, and higher plants).
NADP+SpHEnz

ScCOCH2CH2CH3

Enoyl Reduction
NADPH + H+(FMN)
Enz

Enz

ScCOCH3SpHHS CoA

ScCOCH=CHCH3Acetyl Transfer

Dehydration

Enz

SpH

CH3CO S CoA

H2OEnzSpH

HS CoACerulenin
Enz

NADPH + H++CoA
HO2CCH2CO S
Malonyl Transfer

ScCOCH2CHCH3β-Ketoacyl Reduction

ScH

ScH

ScH

NADP+SpH

SpCOCH3SpH

OH

Enz

Enz

Final Reaction:

ScCOCH2CO2H

CO2SpHScCOCH2COCH3CH3(CH2CH2)nCO S CoAEnz

SpCOCH3Enz

Condensation

SpH
ScCO(CH2CH2)nCH3+HS CoA

Palmitoyl Transfer

ScH and SpH are abbreviations for central and peripheral SH groups
indicating different functions in the enzymatic reaction.

2) Inhibition of polyketide synthesis9,10)
Cerulenin blocks the synthesis of polyketides in a wide variety of organisms, including actinomycetes, fungi and higher plants. In addition, cerulenin is suggested to inhibit the condensation
step in polyketide synthesis as well as fatty acid synthesis.
CH3

CH3COOH
3X

O CO S Enzyme

COOH
O

COOH

CH3
COOH
OHO

6-MSA

Cerulenin

Biosynthesis of 6-methylsalicylic acid (6-MSA)

3) Antimicrobial activity
Cerulenin has a wide range of antimicrobial activity as shown below. Most noteably, the drug
significantly inhibits growth of yeast like fungi, such as Candida, Saccharomyces and Cryptococcus.
2)

Test Organism

MIC (µg/ml)

Staphylococcus aureus FDA 209p
Micrococcus luteus PCI 1001
Streptococcus haemolyticus
Escherichia coli NIHJ
Salmonella typhosa
Shigella dysenteriae Shiga
Shigella flexineri E-20
Bacillus subtilis PCI 219
Corynebacterium diphtheriae
Haemophilus influenzae
Mycobacterium avium F
Mycobacterium smegmatis
Mycobacterium tuberculosis
Nocardia asteroides
Streptomyces griseus SN-15100
25100
12.5502550
12.550100
1.5
3.71001.53.1

Test Organism		 MIC (µg/ml)

Alternaria kikuchiana
Aspergillus flavus
Aspergillus fumigatus
Botrytis cryptoneriae
Candida albicans
Cephalosporium caerulen
Cryptococcus neoformans
Penicillium crysogenum
Penicillium notatum
Pyricularia oryzae
Pullularia pullulans
Rhizopus nigricans
Saccharomyces cerevisiae
Trichophyton asteroides
Trichophyton interdigitale

50100
50
12.5
1.5
100
1.5
12.5
12.5
6.25
25
25
0.8
6.2
12.5

4. Mode of Action7,8,11-19,25-29)
1) Inhibition of the condensing enzyme of FAS7,8,11-13)
The inhibition of FAS activity by cerulenin is based on its covalent binding to the cysteine residue (SpH) in the condensation reaction domain. Cerulenin was initially thought to take a lactam
form in order to react with the SpH,12) however X-ray crystallographic analysis of the cerulenin-condensing enzyme complex revealed that cerulenin itself is covalently attached in a hydrophobic cavity of the active site as illustrated below18).
O 4'-Phosphopantetheine
HN
ScH CH HO Ser Acyl transferase
3

Malonyltransferase Ser–OHOO
H2NOHSpCysCondensing enzyme

Proposed model for cerulenin binding to the active center of the cysteine residue in the condensation reaction

2) Mechanism of cerulenin-resistance in a cerulenin producer14-13) and mutant yeast17)
The cerulenin-producing fungus Acremonium (Cephalosporium) caerulens KF-140 was resistantto cerulenin. The mechanism of self-resistance revealed that cerulenin cannot bind to the significantly altered condensation reaction domain of the producer FAS13-15).
In a cerulenin resistant mutant (KNCR-1) (a 30-fold increase) of Saccharomyces cerevisiae, theGGT codon encoding Gly-1257 of FAS 2, (the gene encoding the α subunit of the fatty acid synthase) was changed to AGT, resulting in the codon for Ser16). As predicted, Gly-1257 is incorporated in the hydrophobic cavity of the active site to fit cerulenin18). Therefore, the change causes
steric hindrance to cerulenin binding, leading to cerulenin resistance in mutant yeast.
1257
Ser Gly Ser Gly Met Gly Gly
TCT GGT TCT GGT ATG GGT GGT
TCT GGT TCT AGT ATG GGT GGTSer Gly Ser Ser Met Gly Gly
1257

Wild type
KNCR-1

FAS 21307 1312
β-KS1038 1040
β-KRComparison of the DNA sequence of the FAS 2 gene.

5. Total synthesis20)
The total synthesis of cerulenin has been reported by many groups (See Appendix-I). Below is S.
Ōmura’s approach.
1) EtMgBr
2) CuCl

CO2H

3)Li/NH3

CO2H

Cl

LiAlH4

CO2H

92%

98%

72% (3 steps)

CO2Me1) pyridine2•CrO32) Li
CO2MeOH

H2, Pd-BaSO4

67% (2 steps)

H

NaOClOO

H

NH4OH
83%

67%

H

O
OH

66%

OH

H

H
CONH2O
OHO

H

H
CO2H

pyridine2•CrO390%

H

H

OO

H

H
O

CONH2O(±)-Cerulenin
Overall yield 14 % in 11 steps

6. Research reagent as a chemical probe30-47,50-54)
Cerulenin is commercially available as a biochemical reagent for widely use in the field of obesity, cancer biology, posttranslational protein modification system, drug discovery research andso on. Until now, Cerulenin is a typical chemical probe to research fatty acid synthesis in many
biological mechanisms.
7. Synthetic derivative CM-55
1) Synthesis and structure21)
CM-55 was synthesized as an analog of antilipogenic antibiotic cerulenin (See also “Cerulenin”).
The general procedure for the synthesis is shown below.
OH3CO
Ac2OOH

Me2NH

H3C

Et3NOOOOO
H3CNO

CH3CH3CM-552) Biological activity21-23,48)i) Antimicrobial activity21,22,48)
CM-55 shows antifungal and antimicrobial activity with rather low MIC values (6.25-12.5 µg/ml, see below)21). The mode of action appears to be attributed to inhibition of acetyl-CoA by incorporation into the non-saponifiable fraction22).
Antimicrobial activity
Test organism
Bacillus subtilis PCI 219
Staphylococcus aureus FDA 209PMycobacterium smegmatis ATCC607
Escherichia coli NIHJ
Pseudomonas aeruginosa P-3
Candida albicans
Pyricularia oryzae
Trichophyton rubrum

MIC (µg/ml)
6.25
3.126.25
3.12>100
12.5
6.25
6.25ii) CM-55 was found to inhibit epithelial transport of sodium ions across the toad urinary bladderand to inhibit hydro-osmotic response of the bladder to vasopressin after 2 hours of pretreatment
with CM-5523).
8. Biosynthesis49)
Biosynthesis of cerulenin is closely related with fatty acid synthesis.

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