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Vitamin B12
Vitamin B12, is an essential co-factor for carrying out numerous rearrangements in biosynthetic pathways. The common form of Vitamin B12 is cyanocobalamin which is co-enzymatically inactive. The cyanide ligand is an adventitious, high-affinity ligand picked up during isolation and purification. The active form of co-enzyme B12 has the top axial ligand modified depending on the specific function required. Two main types of reactions are mediated by co-enzyme B12: 1) rearrangements involving 1,2-shifts, and 2) methyl transfer in methionine biosynthesis and in the biomethylation of trace metals. For the rearrangements, cyanocobalamin is reduced by FADH2 to the Co(I) oxidation state and then condenses with ATP forming a Co-C bond with 5’-deoxyadenosyl as the top axial ligand. Vitamin B12 is the only natural product known that contains a carbon-metal bond.
N
NN
N
Me
Me
H2NOCMe
MeH2NOC
H2NOC
Me
CONH2Me
CONH2
Me
Me
CONH2
Co
CN
OHN
OMeN
NMe
MeO
OH
PO
-O OHO
cyanocobalamin
FADH2
cyanocobalamin Co(IiI)
..
Co-enzyme B12
N
NN
N
CH3
H3C
H2NOC
H3CH3C
H2NOC
H2NOC
H3C
CONH2CH3
CONH2
CH3
CH3
CONH2
Co
CN
OHN
OH3CN
NCH3
CH3O
OH
PO
-O OHO
N
NN
N
CH3
H3C
H2NOC
H3CH3C
H2NOC
CONH2
H3C
CONH2CH3
CONH2
CH3
CH3
CONH2
Co+
OHN
OH3C
N
NCH3
CH3O
OH
PO
-O OHO
O
OHHO
N
N
N
N
H2NO P
O
O_O P
O
O_
OPO
O_
O_
N
NN
N
CH3
H3C
H2NOC
H3CH3C
H2NOC
CONH2
H3C
CONH2CH3
CONH2
CH3
CH3
CONH2
Co
OHN
OH3CN
NCH3
CH3O
OH
PO
-O OHO
O OH
OH
NN
NN
H2N
Co(I)Co(III)
R.B. Woodward, Harvard University
Albert Eschenmoser Robert Burns Woodward
The achievement of the total synthesis of vitamin B12 is perhaps, one of the most impressive and technically complex synthesis endeavors ever accomplished. The completion of this mammoth undertaking, spanned over a decade and was the result of a unique collaborative effort by the laboratories of the late Professor Robert Burns Woodward of Harvard University and Professor Albert Eschenmoser of the ETH, Zurich. The formal total synthesis of vitamin B12 was accomplished with the synthesis of cobyric acid, the simplest B12 derivative and has been recounted as a series of lectures given by Woodward. A complete paper with full experimental details was never published. The conversion of cobyric acid into vitamin B12 itself, was previously reported by Bernhauer in 1960. Notwithstanding, Woodward took the synthetic cobyric acid and had this converted into synthetic vitamin B12 to complete the “total” synthesis effort. (Bindra & Bindra, Creativity in Organic Synthesis, Vol. 1,1975)
Woodward, R.B., Pure Appl. Chem. 1968, 17, 519Woodward, R.B., Pure Appl. Chem. 1971, 25, 283Woodward, R.B., Pure Appl. Chem. 1973, 33, 145
O
Me MeMe BF3, Ac2O
OAcMe
HMe
MeCO2H
1. (COCl)2
2. NH3
OAcMe
HMe
MeCONH2
1. O3
2. Zno, MeOH
HN
O
O
MeMeMeO
HCl
MeOH HNCO2Me
O
MeO Me MeMe
!HN
CO2MeO
MeMe
A
O
Synthesis of the Eschenmoser Fragment
O
Me MeMeO
O
Me MeMeO
BF3, Ac2O
O
Me Me
Me
AcO
AcO
AcF3B
OAcMe
HMe
Me
O O
O Me
H2O (work-up)OAc
Me
HMe
MeCO2H
Wagner-Meerwein Rearrangement
Me MeO
CO2H
PhH, SnCl4 O
Me CO2H
Me CrO3O
O
O
O
MeMe
CO2H
1. SOCl2
2. CH2N23. MeOH4. NH3, MeOH
O
HN
O
O
MeMe
CO2Me
P2S5 O
HN
S
O
MeMe
CO2MeB
(resolution)
Synthesis of the Eschenmoser Fragment
Eschenmoser Sulfide Contraction
O
HN
S
O
MeMe
CO2Me
HNCO2Me
O
MeMe
+ (PhCO2)2
CH2Cl2, HCl
ON
S
O
Me
MeCO2Me
HN
CO2Me
O MeMe
P(OEt)3
xylenes
ON
O
Me
MeCO2Me
HN
MeO2C
O MeMe
MeHgOi-Pr
Me3BF4, H2S(i-Pr)2NEt
ON
O
Me
MeCO2Me
HN
MeO2C
S MeMe
C
A
B
Mechanism of the Eschenmoser Sulfide Contraction
O
HN
O
SCO2Me
MeMe
OOO
O
!O
OO
N
O
SCO2Me
MeMe
HN
OCO2MeO
N
O
SCO2Me
MeMe
HN
OCO2Me
O
OH
O
O
O
N
O
SCO2Me
MeMe
N
OCO2Me
H
O
N
O
SCO2Me
MeMe
HN
OCO2Me
H
P(OEt)3
O
HN
O
SCO2Me
MeMe
N
O
CO2Me
HP(OEt)3
O
HN
O
SCO2Me
MeMe
N
O
CO2Me
H P(OEt)3
O
N
O
CO2Me
MeMe
HN
O
CO2Me
H
+ S POEtOEt
OEt
Synthesis of the Woodward Segment
NH
Me
Me
MeO
1. MeMgI
2. CH2Br NMe
MeO
MeMeOH, HgO
BF3HN
Me
OMeMe
HN
OMe
O
Me
Me
1. resolution
2.
MeMe
COClMe
N OMe
O Me
Me
MeMe
MeO
KOt-Bu
N
OMe
O
Me
MeO
H
Me
Me
Me
N
OMe
MeH
Me
Me
Me
H
1. NH2OH
2. NaNO2 HOAc
OO
N
OMe
MeH
Me
Me
Me
H
HONO
1. O3, MeOH
2. HIO43. CH2N2
CO2MeO
N
Me
MeH
Me
Me
Me
H
HONO
OO
H-HOAcN
1.
2. MsCl
CO2MeO
N
Me
MeHMe
H
MsONO
Me
O
pentacyclic enone
N
OMe
O
Me
MeO
H
Me
Me
Me
1. (CH2OH)2, H+
2. Et3OBF43. NaOMe, MeOH
N
OMeMe
MeH
Me
Me
Me
O
O OMeOMe
1. Lio, NH3
t-BuOH, THF2. H+
Synthesis of the Woodward Segment
Discovery of the Woodward-Hoffman RulesThe Total Synthesis of Vitamin B12
NO
MeO2C
Me
O
O
Me
Me
CN
OH
Δ
R
Me
MeO2C
Δ HR
MeCO2Me
NO
MeO2C
Me
O
O
Me
Me
CN
OH
H
Hdisrotatory
disrotatory
6 π-e-
Synthesis of “Cornersterone”
CO2MeO
N
Me
MeHMe
H
MsONO
Me
O
1. O3, H2O, MeOAc
2. HIO43. CH2N2
CO2MeO
N
Me
MeHMe
H
MsONO
MeMeO2C
O
H+, MeOH
170 oC
H2ON N
HO
OO
Me
Me
Me
H
CO2Me
CO2Me!-cornersterone
H
MeO2CMe
HN NH
OO
Me
Me
Me
H
CO2Me
CO2Me
HO HN N
H
OO
Me
Me
Me
H
CO2Me
CO2Me
H
O
O
Beckmann rearrangement
N NH
O
OO
Me
Me
Me
H
CO2Me
CO2Me
HHO
-H2O
Dieckmann
Synthesis of the Woodward Segment
N NH
O
OO
Me
Me
Me
H
CO2Me
CO2Me!-cornersterone
H1. strong base
2. H3O+
3. CH2N2
N NH
O
OO
Me
Me
Me
CO2Me
CO2Me"-cornersterone
H H+, PhSH
MeOHN
NH
SPh
O
Me
Me
Me
MeO2C
MeO2C
MeO2C
O3
MeOHNH
OMe
Me
MeO2C
MeO2C
N
CHO
PhSOC
MeO2C
NH
OMe
Me
MeO2C
MeO2C
N
CHO
H2NOC
MeO2C
NH3(l) 1. NaBH4
2. Mes2O, py.3. LiBr, DMF
NH
OMe
Me
MeO2C
MeO2C
N
NC
MeO2C
Br
cyanobromide
Me MeMe
RBW AE
NH
OMe
Me
MeO2C
MeO2C
N
NC
MeO2C
Br
cyanobromide
ON
O
Me
MeCO2Me
HN
MeO2C
S MeMe
C
+KOt-Bu NH
OMe
Me
MeO2C
MeO2C
N
NC
MeO2C
S
O
N
O
Me
Me
CO2MeN
MeO2C
MeMe
thioether type I thioether type II
(NCCH2CH2)3P
TFA, MeNO2
NHOMe
Me
MeO2C
MeO2C
N
NC
MeO2C
O
N
O
MeMe
CO2MeHN
MeO2C
MeMe
cyanocorrigenolide
NHOMe
Me
MeO2C
MeO2C
N
NC
MeO2C
S
O
N
O
Me
Me
CO2MeHN
MeO2C
MeMe
P2S5, tol.
!-picolinecat
NHSMe
Me
MeO2C
MeO2C
N
NC
MeO2C
S
HN
O
MeMe
CO2MeN
MeO2C
MeMe
dithiocyanocorrigenolide
Me3OBF4
MeMe
Me
MeMe
Joining of the Woodward and Eschenmoser Fragments(performed at Harvard)
In the 1971 Pure and Applied Chemistry paper, Woodward makes a special citation to Kishi:
“This first preparation of corrigenolide afforded striking testimony of the experimental skill of its discoverer, Dr. Yoshito Kishi. All of the operations had to be conducted with every conceivable precaution in respect to purity of reagents, exclusion of oxygen and moisture, and with the greatest possible speed. It will be easily imagined that it would be a difficult, if not impossible task to develop so intricate and complicated procedure into a reproducible method for a relatively large-scale preparation of the desired key intermediate.”
Yoshito Kishi & R.B. Woodward, 1972
Yoshito Kishi & Tohru Fukuyama 2004
NSMeMe
Me
MeO2C
MeO2C
N
NC
MeO2C
S
HN
O
MeMe
CO2MeN
MeO2C
MeMe
Me2NH
MeOHN
SMeMe
Me
MeO2C
MeO2C
N
NC
MeO2C HN
Me
CO2MeN
MeO2C
MeMe
CONMe2 N
NN
S
N
MeO2C
MeMe
MeO2C
MeO2C
Me
CONMe2Me
CO2Me
Me
Me
CO2Me
Co
CN
CN
CoCl2, THF
charcoal, KCN
Me
CN
DBN
DMF N
NN
N
MeO2C
MeMe
MeO2C
MeO2C
Me
CONMe2Me
CO2Me
Me
Me
CO2Me
Co
CN
CN
CN
N
NN
N
MeO2C
MeMe
MeO2C
MeO2C
Me
Me
CO2Me
Me
Me
CO2Me
Co
CN
CN
CN
I2
HOAc
O
O1. ClCH2OBz sulfolane
2. PhSH
N
NN
N
MeO2C
MeMe
MeO2C
MeO2C
Me
Me
CO2Me
Me
Me
CO2Me
Co
CN
CN
CN
O
OSPh
SPh
N
NN
N
MeO2C
MeMe
MeO2C
MeO2C
Me
Me
CO2Me
Me
Me
CO2Me
Co
CN
CN
CN
O
OPhS
SPh
1. Ra-Ni
2. CH2N23. HPLC separation
N
NN
N
MeO2C
MeMe
MeO2C
MeO2C
Me
CO2MeMe
CO2Me
Me
Me
CO2Me
Co
CN
CN
CN
Me
Me
1. conc. H2SO4
2. HPLC separation of C13-epimers
N
NN
N
MeO2C
MeMe
MeO2C
MeO2C
Me
CO2MeMe
CO2Me
Me
Me
CO2Me
Co
CN
CONH2
CN
Me
Me
NO
1.
2. H3O+
3. Me2NH, PrOH
N
NN
N
MeO2C
MeMe
MeO2C
MeO2C
Me
CO2MeMe
CO2Me
Me
Me
CO2Me
Co
CN
CO2H
CN
Me
Me
cobyrinic acid abcdeg hexamethyl ester
NH3(l)
(CH2OH)2NH4Cl, 75oC
N
NN
N
H2NOC
MeMe
H2NOC
H2NOC
Me
CONH2Me
CONH2
Me
Me
CONH2
Co
CN
CO2H
CN
Me
Me
cobyric acid
Completion of Cobyric Acid
N
NN
N
H2NOC
MeMe
H2NOC
H2NOC
Me
CONH2Me
CONH2
Me
Me
CONH2
Co
CN
CO2H
CN
Me
Me
totally synthetic cobyric acid
N
NN
N
Me
Me
H2NOC MeMe
H2NOC
H2NOC
Me
CONH2Me
CONH2
Me
Me
CONH2
Co
CN
OHN
OMeN
NMe
MeO
OH
PO
-O OHO
totally synthetic cyanocobalaminFriedrich, W.; Gross, G.; Bernhauer, K.; Zeller, P., Helv. Chim. Acta 1960, 43, 704
Conversion of Totally Synthetic Cobyric Acid to Totally Synthetic Vitamin B12
Dr. Mark Wuonola
Mark Wuonola & R.B. Woodward March 17, 1976
Origins of Vitamin B-12There is no active vitamin B-12 in anything that grows out of the ground; storage vitamin B-l2 is found only in animal products where it is ubiquitous and where it isultimately derived from bacteria. All the vitamin B-12 in plants is there fortuitously in bacteria contaminating the food. That contamination is usually on the outside of the plant but occasionally is internal. (Am. J. Clin. Nutr. 1988, 48, 852)
Reactions Mediated by Co-enzyme B12
Vitamin B12, is an essential co-factor for carrying out numerous rearrangements in biosynthetic pathways. The common form of Vitamin B12 is cyanocobalamin which is co-enzymatically inactive. The cyanide ligand is an adventitious, high-affinity ligand picked up during isolation and purification. The active form of co-enzyme B12 has the top axial ligand modified depending on the specific function required. Two main types of reactions are important:
1) rearrangements involving 1,2-shifts, and 2) methyl transfer in methionine biosynthesis and in the biomethylation of trace metals.
For the rearrangements, cyanocobalamin is reduced by FADH2 to the Co(I) oxidation state and then condenses with ATP forming a Co-C bond with 5’-deoxyadenosyl as the top axial ligand.
N
NN
N
Me
Me
H2NOC MeMe
H2NOC
H2NOC
Me
CONH2Me
CONH2
Me
Me
CONH2
Co
CN
OHN
OMeN
NMe
MeO
OH
PO
-O OHO
cyanocobalamin
FADH2
ATP
N
NN
N
Me
Me
H2NOC MeMe
H2NOC
H2NOC
Me
CONH2Me
CONH2
Me
Me
CONH2
Co
OHN
OMeN
NMe
MeO
OH
PO
-O OHO
co-enzyme B12
O OH
OHN
N
NN
H2N
The classes of reactions involving dehydration with rearrangements include three enzymes:
1) propanediol dehydrase. This enzyme converts 1,2-propanediol to propionaldehyde.
H3C OHOH
H3C O
H
2) glycerol dehydrase. This enzyme acts on glycerol to from β-hydroxypropionaldehyde which spontaneously dehydrates to acrolein.
OHOH
HOH3C O
OH
H
H2C O
H
3) ethanolamine deaminase. This enzyme transforms ethanolamine into acetaldehyde and ammonia.
OHNH2
H3C O
H+ NH3
The rearrangements involving 1,2-shifts follows the general equation shown below:
C CH
34
1
2R
C CR
34
1
2H
co-enzyme B12
Based on a large body of labeling studies, Abeles has proposed the following mechanism for the 1,2-shifts mediated by co-enzyme B12.
O OH
OHN
N
NN
H2N
N
N
N
NCo
O OH
OHN
N
NN
H2N
N
N
N
NCo
CH3
HOHO H
O OH
OHN
N
NN
H2N
N
N
N
NCo
HHH
CH3
HOHO
N
N
N
NCo
CH3HO
HOO OH
OHN
N
NN
H2N
HHH
N
N
N
NCo
CH3HO
O OH
OHN
N
NN
H2N
HHH
HO
N
N
N
NCo
CH3HO
HOO OH
OHN
N
NN
H2N
HHH
N
N
N
NCo
O OH
OHN
N
NN
H2N
O OH
OHN
N
NN
H2N
N
N
N
NCo
CH3H
O+
1) glutamate mutase
CO2H
NH2
HO2CH
L-glutamate
1
2
3
4
5CO2H
NH2
HO2CHH3C
lyaseCO2H
HO2C
H3C
threo-β-methyl-L-aspartate
formerly C3
2) methylmalonyl-CoA mutaseCOSCoA
CO2HH
HH
R-methylmalonyl CoA
HO2C COSCoA
succinyl-CoA3) methyleneglutarate mutase
CO2H
CH2
HO2CH
α-methyleneglutarate
CO2HH2C
HO2C CH3
Enzymes 1 and 3 are apparently restricted to the anaerobic bacterium Clostridia sp.; enzyme 2 is present in both microbial cells and animal cells and constitutes the only known B12-dependent rearrangement enzyme in higher organisms. In bacterial and animal cells, methylmalonyl-CoA mutase is important in the catabolism of valine and isoleucine. Both of these amino acids are catabolically degraded to propionyl-CoA which in turn, undergoes carboxylation to the S-isomer of methylmalonyl-CoA.
Co-enzyme B12 also catalyzes the migration of amino groups in Clostridia sp.. The three known examples are:
1) β-lysine mutase
NH2
HCO2HH2N
β-lysineNH2
H3CH
CO2HNH2
3,5-diaminohexanoate
2) D-α-lysine mutase
CO2H
NH2
HH2N CO2H
NH2
HH2N
CH3
D-lysine 2,5-diaminohexanoate
3) D-ornithine mutase
CO2H
NH2
HH2N
D-ornithine
CO2H
NH2
H3CH
NH2
2,4-diaminopentanoate
In all three cases, these enzymes are involved in fermentative pathways in Clostridium sp. The D-lysine and D-ornithine are formed by the PLP-dependent racemase action on the respective L-isomers. However, the L-b-lysine is formed from L-lysine by lysine-2,3-amino mutase. This enzyme contains PLP and has no corrinoid dependency. This enzyme appears to be similar mechanistically to the amino mutases phenylalanine amino mutase (important in the biosynthesis of the taxol side chain) and tyrosine amino mutase.
CO2H
NH2
HH2NNH2
HCO2HH2N
lysine-2,3-amino mutase
Methyl-Co-enzyme B12 N
NN
N
CH3
H3C
H2NOC
H3CH3C
H2NOC
H2NOC
H3C
CONH2CH3
CONH2
CH3
CH3
CONH2
Co
CH3
OHN
OH3CN
NCH3
CH3O
OH
PO
-O OHO
Methyl Co-enzyme B12
Methyl co-enzyme B12 is involved in two important methyl transfer reaction sub-types:
1) The methylation of homocysteine to form methionine (E. coli). There are two types of methylation enzyme systems in E. coli: one requires methyl co-enzyme B12 and the other does not. The ultimate source of the methyl group is N5-methyl-THF (tetrahydrofolate).
N
N
N
NCoI
"CH3 + " S
CO2H
NH2
H
homocysteine N
N
N
NCoI
H3CS
CO2H
NH2
H
methionine
2) Trace heavy metal methylations. Methylation of metals such as mercury, palladium, thallium, lead, platinum, gold, tin, arsenic, selenium and chromium are known to be mediated by methyl co-enzyme B12. Depending on the nature of the methyl acceptor, it has been proposed that the methyl co-enzyme B12 utilizes the Co(I), Co(II) or Co(III) oxidation states. The movement of trace metals in the environment by enzyme-mediated processes is an area of increased study.
CH3N
N
N
NCoIII
N
N
N
NCoIII
"CH3 _ "H3C Hg
N
N
N
NCoI
"CH3 + "
H3C SR
N
N
N
NCoII
"CH3 "
H3C SnX3
Hg+2
SR
SnX3