17.07.2014, Darius Schwazrer

Synthesis of Vitamins & CofactorsGaich Group Seminar

Definition

Cofactor/Coenzyme: A nonprotein component of enzymes is called the cofactor. If the cofactor is organic, then it is called a coenzyme. Coenzymes are relatively small molecules compared to the protein part of the enzyme. Many of the coenzymes are derived from vitamins

Protein + Cofactor/Coenzyme = Holoenzyme Proteinpart of Enzyme = Apoenzyme Vitamin:

1912 Kazimierz Funk defined that the essential substances contain an amine function are called Vita = life; amine

!Essential, organic compounds which are not synthesized in the human or animal organism or formed only in insufficient amounts . Must be regularly consumed with diet

Vitamin A & ß-Carotene

Retinol acetate (BASF)

3

ß-Carotoene (BASF)

Me

MeMe

O

Me

MeMe Me Me

OAc

Retinoacetate (4)

ClMg

Me

MeMe OH

Me

Me

MeMe Me

PPh3

HSO4

1. THF

98 %

2. PPh3, H2SO4, i-PrOH, Heptane

95 %

Me

AcOO

3. BuLi, THF, - 50 °C

80 %1 2 3

c. KOAc, MeOH, rflx. then KOH, H2O, (90 %)d. Acetone, MnO2 (85 %)e. EtOAc, Lindlar, H2 (95 %)f. Benzene, Iodine, hexane, hv (?? %)

BrMgC CMgBrO

a. Et2Othen 10% H2SO4

52 %

b. PBr3, (CH2OH)2

75 %

OHHO

CH2Br

BrH2C

85 6 7

Me

MeMe Me

PPh3

OO

2x

DMF, NH3, thenH2O2 isomerization

Me

MeMe Me Me

Me Me

Me

Me Meβ-Carotin (9)

80 %

8

3

Mildner; Weedon Journal of the Chemical Society, 1953 , 3294, Inhoffen; v.der Bey Justus Liebigs Annalen der Chemie, 1953 , 583.

Ascorbic Acid

4

Vitamin C synthesis by Reichstein process

step 3+4 can be replaced by oxidation with Air & PtO

Reichstein synthesis was optimized so that each step gives over 90 % yield !Overall yield from D-glucose is now 60 % !Direct synthesis via microalgae possible, but yield and concentration are too low

OOH

OH

OH

OHHO

D-glucose (10)

O

OHOO

Me

Me

O O

Me Me

OHO

OH

OH

OHHOOH

OH

OH

OH

OHHO 37 %

3. Acetone,H2SO4

AcetobactersuboxydanspH 4-6, 30-35 °CH2, RaNi

quant. >90 %

11 12 13

OH

OHOHO

O

HO

OHO

OH

OH

OHHO

O

4.NaOCl, NiCl, > 93 %5. H2O, H+, 82 %

14

6. HCl, PhMe, Δ

> 60 %

Ascorbic acid (15)

T. Reichstein, A. Güssler Helv. Chim. Acta 1934, 17, 311; Bullmanns Enzykl. Tech. Chem., 3. Ed., Vol 18, 233 ff.

Ascorbic Acid

Metabolism of Vitamin C

5

Industrial use • Inhibition of nitrosamine in

cured meat• Improvement of flour quality• Increasing of clarity of wine &

beer• protecting fruits from browning

Economic aspects • production >

600 kt/a• cost ~3000 €/t

Biological activity • anti oxidant• radical scavenger• electron donator for 8

enzymes(hydroxylases)

• prevents from scurvy

HO

OHHO O

O

OH O

OHHO O

O

OHO

OHHO O

O

OHO

OHHO O

O

O

OH

HOO

OOHO

OH

-H+

+H+

-e- -H+

+H+

pka = -0.86pka = 4.1

-e-

O

OHHO O

O

O

+H2O

-H2O

+H2O

-H2O HO

OHHO O

O

OHOHOH

15

16 17 18

Thiamine

Synthesis of Vitamin B1 Step 1. Synthesis of 22

6

22 and 23 commercially available

Step 2. Condensation & formation of thiazolium ring

Me

N

NH2

CN CN

Me

N

NH

CN

CN

a. i-PrOH, max. 40 °C

87 %

b.Na(powdered), PhEt, Styrol & PhNHMe in THF over 2 h, rt, then rflx, 20, then H2O

77 %

N

NH

Me

NH2

CNc. MeOH, NH3, RaCo,

110 °C 60 atm, 13 h

50 %

N

NMe

NH2

NH2

19 20 21 22

N

NMe

NH2

NH2

O

Cl

AcO

O

SH

AcO1. KSH, MeOH,

0 °C

74 %

N

NMe

NHN2. 80 °CMe2N

OMe

OMe

89 %

3. HCO2H, < 40 °C

89 % OHS

N+

Me

NH2NMe

NCl-

Thiamine (26)

2324

22 25

A. Edenhofer, H. Spielberg, W. E. Oberhänsli Helv. Chim. Acta 1975, 58, 1230; P. Contant, L. Forzy, U. Hengartner, G. Moine Helv. Chim. Acta 1990, 73, 1300.

Thiamine

Biological activity • Thiaminediphosphate (TDP) is the active form• Essential for carbohydrate & energy metabolism

• Present in pyruvat dehydrogenase, oxoglutarate dehydrogenase,transketolase, pyruvate decarboxylase, 2-hydroxyphytanoyl-CoA lyase.

7

Deficiency • Beriberi —> effects peripheral nervous system; cardiovascular system,• Polyneuropathy• Anemia• many more

N

S

RMe

PPO

HN

S

RMe

PPO

N

S

RMe

PPO

MeHO

OOH

N

S

RMe

PPO

OH

Me

CoAS Ph

O

N

S

RMe

PPO

OH

Me

O

Ph

MeO

PhO

Me

OHO

O

27 29

Biotin

Vitamin B7 Synthesis by Hoffmann-La Roche

8

US 2 489 232 (Roche; 1949; appl. 1946); US 2 489 233 (Roche; 1949; appl. 1946), US 2 489 234 (Roche; 1949; appl. 1946), US 2 489 235 (Roche; 1949; appl. 1946), US 2 489 236 (Roche; 1949; appl. 1946), US 2 489 237 (Roche; 1949; appl. 1946), US 2 489 238 (Roche; 1949; appl. 1946), US 2 519 720 (Roche; 1950; appl. 1948), US 3 740 416 (Roche; 1973; CH-prior. 29.11.1969).

HO2C

CO2H BnN NBn

O

HO2C CO2H

BnN NBn

O

HO2C CO2R

BnN NBn

O

RO2C CO2H

BnN NBn

S O

O1. Br2, H2O, 90 %2. BnNH2, MeOH,

90 %3. COCl2, KOH

5. LiBH4, THF6. AcSK, DMF,

150 °C

> 90 %

4. PhH, C6H12O, Δ, then(+)-Ephidrine, iPrOH,80 °C

2 steps

30 3132

33

34

OH

NHO

HHN

H S

OBnN NBn

S

O

OMe

BnN NBn

S

O

Br

7. then AcOH, Δ

8. Ra/Ni, H2 40 atm.35 °C, MeOH

9. 48% HBr,95 °C, 2 h

82 % 90 %

10. Malonic acid diethyl ester,NaOMe, PhMe, 80 °Cthen, 48% HBr, rflx.

(25% over all)

35 36

Biotin (37)

ClMg OMe

Biotin

Biological activity • present in carboxylases (pyruvat-, acetyl-CoA-, propionyl-CoA-, methylcrotonyl-

CoA-carboxylase)• catalyzes carboxylation as a CO2 carrier• Formation of malonyl-CoA from acetyl-CoA

9

Biotin deficiency causes Neurological symptoms (depression, lethargy, etc.)

ATP HCO3- HN NH

S

O

CO2H4

+ ADP + OP

HO

O

OHOH

O

N NH

S

O

CO2H4

HO

O

CoAS

OO

SCoA

O

HO

Biotin+

37

3839

Cobalamine

Vitamin B12 • Synthesis by fermentation• No industrial total synthesis available

• Total synthesis with 113 steps, 12 PhD, 91 Postdocs,17 years

• optimized to 70 steps• cobalt can be complexed by CN-, SO32-, OH-, halides • 10t/a; market volume 77*106 EUR!Biological activity • catalyzes 1,2 H shift• Coenzyme for Methionin-synthase & Methylmalonyl-CoA-mutase• Hydroxy-cobalamine —> treatment of cyanide poisoning• deficiency causes anaemia

10

Crowfoot-Hodgkin, D. et al. Nature, 1956, 178, 64; Woodward, R.B. Pure & Appl. Chem. 1968, 17, 519; Woodward, R.B. Pure & Appl. Chem. 1971, 25, 283; Woodward, R.B. Pure & Appl. Chem. 1973, 33, 145; Eschenmoser, A.; Wintner, C.E. Science 1977, 196, 1410; Woodward, R.B. In Vitamin B12, Proceed. 3rd European Symposium on Vitamin B12 and Intrinsic Factor, Zagalak, B.; Friedrich, W., Eds., Walter de Gruyter: Berlin, 1979, P.37; Benfey, O. T.; Morris, P. Robert Burns Woodward: Architect and Artist in the World of Molecules. Philadelphia: Chemical Heritage Foundation, 2001.Nicolaou, K.C.; Sorenson, E.J.; Classics in Total Synthesis: Targets, Strategies, Methods. Weinheim, Germany: VCH, 1996.

N N

N N

Me

Me

Me

Me

H2NOC

H2NOC

H

H2NOCMe

MeMe

CONH2

Me CO2NH2

NHO

OMe PO

OO O

HO N

NMe

Me

CONH2

HO

CoCN

40

Calcitriol

Synthesis of Vitamin D3

11

MeMe

Me

HOH

H

H

OH1. Ac2O, Pyridine,

100 °C, 17 h

96 %

N

NO

O

MeMe

Br

Br

2. NaHCO3,Hexane,20 min.

0.5 Eq.

N

Me

MeMe

3. Xylene, Δ2 eq.

then pTSA, Dioxane, 70 °C, 30 min., 50 %

MeMe

OH

3

MeMe

Me

AcOH

H

H

OAc

MeMe

OAc

3 MeMe

Me

AcOH H

OAc

MeMe

OAc

3

41 4243

5. -15 °C KOH, MeOH, THF78 % Diacetate, 16 % Monoacetate

6. 450 W Hg, -5 °C, THF, hexane, 20 %25 % unconverted

7. KOH, MeOH, 30 h, quant.

MeMe

Me

HOH

OH

MeMe

OH

3

MeMe

H

MeMe

OH

8. Δ, Dioxane, 30 min

50 %

3

OHHO

Calcitriol (45)

44

US 3 993 675 (Hoffmann La Roche Inc.; 23.11.1976; prior 12.11.1973; 24.02.1975.

Calcitriol

Biological activity of Vitamin D3

• hormonally active vitamin• increases the Ca2+ concentration in the blood, by

increasing the uptake in the gastrointestinal tract• decreases the probability to get some of up to 17

different cancer diseases Hypervitaminose causes calcification of tissues and arteries !!

Production & prize • No t/a given• cost 50 $/kg

12

MeMe

H

MeMe

OH

3

OHHO

Calcitriol (45)

Folic Acid

Vitamin B9 synthesis

13

E. L. Bennett J. Am. Chem. Soc., 1952, 74 (9), pp 2420; S. Nishigaki, K. Ogiwara, F. Yoneda Chemical & Pharmaceutical Bulletin 1971, 19, 418, Wailer, C.W. et al.: J. Am. Chem. Soc. (JACSAT) 70, 19 (1948). Hultquist, M.E. et al.: J. Am. Chem. Soc. (JACSAT) 70, 23 (1948); Angier, R.B. et al.: J. Am. Chem. Soc. (JACSAT) 70, 25 (1948).; Boothe, J.H. et al.: J. Am. Chem. Soc. (JACSAT) 70, 27 (1948). US 2 436 073 (American Cyanamid; 1948; appl. 1945)., US 2 442 836 (American Cyanamid; 1948; appl. 1945).; US 2 443 078 (American Cyanamid; 1948; appl. 1945).; US 2 443 165 (American Cyanamid; 1948; appl. 1946).; US 2 444 002 (American Cyanamid; 1948; appl. 1946).; US 2 472 482 (American Cyanamid; -1949; appl. 1947).; US 2 477 426 (American Cyanamid; 1949; appl. 1948). US 2 547 501 (American Cyanamid; 1951; appl. 1946). US 2 599 526 (American Cyanamid; 1952; appl. 195 1).; US 2 719 157 (Shionogi; 1955; appl. 1951). US 2 956 057 (Kongo Kagaku Kabushiki Kaisha; 1960; J-prior. 1955).

Folic acid is metabolized to „THF“ what is the active compound

NH

H2N NH2

CN

CO2EtN

N NH2H2N

OH

N

N NH2H2N

OHH2N

2. H2SO4/H2O, NaNO2 5 °C,then room temperature

82%

3. MeOH, Pd/C, H21. MeOH, Δ, quant.

46 47 4849

NH

O

HO

OOH

OH2NCH2ClO

CHCl2

O OH

NH

O

NH

HN

O

N

H2N NH

NH

O

OH4. Dioxane, H2O,

NaHSO3, pH 4-55. AcOH, 2-mercapto- ethanol NaCNBH3,

85 %N

N NH2H2N

OHH2N

49 50 51 Tetrahydrofolic acid (52)

RNH

HN

O

N

H2N NH

NH

PLP, Serine

PLP, Glycine

Michael-Additionthen Retro-Mannich N

O

N

H2N NH

NH

HNR

NO

N

H2N NH

NH

NR

"THF" 5253 54

Folic Acid

Biological activity of „THF“

14

Economic aspects • 400 t/a available as pure

crystals• cost 36 - 77 €/kg• used for feed enrichment

Formyl transfer in the Histidine biosynthesis

Methyl transfer in the SAM biosynthesis

Deficiency • anaemia• neural tube defects• depression

Folic acid & derivatives (antifolates) are used in anti cancer therapy Acts as an antidepressant

NO

N

H2N NH

NH

NR

NADH + H+ NO

N

H2N NH

NH

NHRMe

NAD+

Homocysteine,Methioninesynthase "THF"

NH2

SO

OH

Methionine (56)5455

NADH

NAD+ NO

N

H2N NH

NH

NR

H2OHN

O

N

H2N NH

NH

NROHC

Histidine &

THF

NO

N

H2N NH

NH

NR

54 57 58

Nicotinamide

Vitamin B3 synthesis (Lonza)

15

active coenzyme

redox reactions using NAD+ & NADPH

CONH2

NO

OHOH

OPO

OO

PO

OO

ONN

OH

OX

H2N X=H NAD+ (63)X= PO3

2- NADP+ (64)

O

O

O

Me

Me Me

1. (NH4)3PO4, H2O,200 °C, 15 bar

68 %

NMe

Me

N

CO2H

2. HNO3, Δ6-8 Mpa

N

CONH2

3. NH3, > 200 °Cneat

96 % 85 %

Nicotinamide (62)Niacine (61)59 60

R

H2NOC

N-O

O

O-

OO

Malate (65)NAD+ (63)

H B-

H

Hmalate

dehydrogenase

R

H2NOC

N

NADH (66)

H H

-OO

O-

OO

Oxaloacetate (67)

R

H2NOC

N-O2C

O

ß-Aspartat-semialdehyde (68)

NADP+ (64)

HB

Hhomoserine

dehydrogenase

R

H2NOC

N

NADPH (64)

H H

NH3+

-O2C

OH

NH3+

Homoserine (69)

DOS 2 046 556 (Lonza; appl. 22.09.1970; CH-prior. 24.09.1969); US 2 905 688 (Abbott; 1959; appl. 1954); US 2 280 040 (SMA Corp.; 1942; appl. 1939); US 2 314 843 (American Cyanamid; 1943; appl. 1941); US 2 993 051 (Cowles Chem. Comp.; 18.07.1961; appl. 26.02.1958).

Nicotinamide

16

Economic aspects • world capacity >>22000

t/a• cost 10-100 $/kg• mainly produced in China

Vitamin B3 deficiency • Pellagra

diarrhea, dermatitis, dementia, death

• Mental confusion• Ataxia• Dilated

cardiomyopathy• peripheral neuritis• etc.

R

H2NOC

N

NADP+

H

R

H2NOC

N

NADPH

HR H

H3C O

HSH3C O

HR HS

H BBH

• re- (pro R) & si- (pro S) specific enzymes• stereospecific dehydrogenation also possible

Pyridoxine

Vitamin B6 synthesis

17

O

O-

NH3+

RO

O-

NH3+

RO

O-

NH3+

R

L-amino acid L-amino acid D-amino acid

PLP

Transamination

Decarboxylation Isomerization Retro-Aldol

1. EtOH, HCl, 35 °C,red. pressure

NH2

MeO

OH

80 %2. formamide,

slow to 105 °C,45 min

HN

MeO

OEt

CHO

3. P2O5, CHCl3,then KOH,MeOH, H2O

40 - 60 % N

O

Me

OEt80 %

O OMe Me

4.then HCl(aq)

180 °C

Me

NHOHO

OHPyridoxine (73)70 71 72

OH

OHOH

N Me NH

Me

OH

OOP

O

O

O

PyridoxalphosphatePLP (74)

Pyridoxine (73)

O

O-

NH3+

RO

O-

O

-OO

ketoglutarate (76)

O

O-

O

RO

O-

O

-ONH3

+

glutamate (78)

PLP

75 77

O

O-

NH3+

R

NH3+

RCO2

PLP

75 79

O

O-

NH3+

OH

R R

O O

O-

NH3+

PLP

80 81 82

Review: H. König, W. Böll Chem. -Ztg 1976, 100, 105; E. E. Harris et. al. J. Org. Chem. 1962, 27, 1962; DAS 1 470 022 (Merck & Co; appl. 10.05.1962; USA pripr. 15.05.1961, 16.01.1962); US 3 227 721 (Merck & Co; 04.01.1966; prior. 15.05.1961, 16.01.1962, 24.01.1965); US 3 227 724 (Merck & Co 04.01.1966; prior. 15.05.1962; 16.01.1962, 16.06.1964).

Pyridoxine

Mechanism for the decarboxylation reaction

18

NH

Me

O

NOiP H

(CH2)4EnzCO2-R2

NH2

NH

Me

O

NHOiP H

(CH2)4EnzHN

R2 CO2-

NH

Me

O

NOiP H

CO2-R2

Enz(CH2)4NH2

NH

Me

O

NOiP H

CO2-R2

NH

Me

O

NOiP H

R2

NH

Me

O

NOiP H

R2

NH

Me

O

NOiP H

(CH2)4Enz

R2 NH2

regeneration by transimination

H B B-

Economic aspects • world capacity

>>4100t/a• cost 50$/kg• mainly produced in

China

Pyridoxine deficiency causes • anaemia• nerve damage• skin problems

Used for treatment of Wilson’s & rheumatic diseases carpal tunnel syndrome, autism, schizophrenia, mental retardation, etc.

K. H. Blässler, Int. J. Vitam. Nutr. Res. 1988, 58, 105; A. H. Merill, J. M. Henderson Annu. Rev. Nutr. 1987, 7, 137.

Riboflavin

Vitamin B2 synthesis

19

An efficient & cheap synthesis of D-ribose (90) is essential for the industrial riboflavin production

Me

Me

NH2 OHOH

OH

OHO 1. MeOH, rflx., then

RaNi/H2 25 atm. Me

Me

HN OH

OH

OH

OH

70 %

83 8485

NO2

NN

Cl Me

Me

HN

Rib.

NN

PhNO2

2. EtOH, 10 °C

92 %

3. Barbituric acid, AcOH, rflx. 5 h

67 %

OH

Me

NNH

O

NOH

HOOH

N O

Me

Riboflavin (87)

86

OOH

OH

OH

OHHO

D-Glucose (10)

a. KOH, O2

OK

O

OH

OH

OHHO

b. CaCl2c. H2O, 140 °C

d. H2SO4f. Na/HgOCa0.5

O

OH

OH

OHHO

O

OH

OH

OHHO

8889 90

IJS 2 384 105 (Roche; 1945; appl. 1943).; Tishler, M. et al.: J. Am. Chem. Soc. (JACSAT) 69, 1487 (1947).; US 2 350 376 (Merck & Co.; 1944; appl. 1941).; US 2 370 093 (Merck & Co.; 1945; appl. 1941). similar processes: Tishler, M. et al.: J. Am. Chem. Soc. (JACSAT) 67, 2165 (1945).; US 2 807 61 1 (Merck & Co.; 1957; appl. 1955).

Riboflavin

Biological activity

20

Me

NNH

O

NR

N O

Me

Me

NH

NH

O

NR

HN O

MeSred Sox

Economic aspects • world capacity 2400t/a• cost 45-54 €/kg• produced by HLR,

BASF, Takeda, ADM

Deficiency • atrophic glossitis• seborrheic dermatitis• eye problems

(all in all nothingserious)

• Most reactions followa radical mechanism

• Is able to form Cl+species with oxygen &chloride

Me

NNH

O

NR

N O

Me

B HS-SH

R2

Me

NH

NH

O

NR

N O

Me

SSH R2B-

HB

Me

NH

NH

O

NR

HN O

Me

S SR2

Methoxatin (non vitamin)

Synthesis of Methoxatin • 1st. Synthesis by Corey in 50 mg scale

21P. Martin, E. Steiner, K. Auer, T, Winkler Helv. Chim. Acta 1993, 76, 1667.

NH2

OMe

NO2

NHCHOOMe

NH2

NHCHOOMe

N2BF4

NHCHOOMe

NN

Me CO2EtCOMe

1. Ac2O, HCO2H, 35 °C (92 %)2. Pd/C, H2, DMF, 35 °C, 2 bar (80 %)

3. EtOH, HCl, NaNO2,then HBF4, EtOH

4. Ethyl-2-methyl acetoacetate, NaOAc, EtOH

91 92 93 94

NHCHOOMe

HNN

Me CO2Et

NHCHOOMe

NHEtO2C

OMe

NHEtO2C

NH

CO2MeHO

CO2Me5. Ph 6.5-5.2 6. HCO2H, 80 °C

7. HCl, Acetone 80 °C (85 %)8. 2-oxoglutaconic acid-dimethylester, CH2Cl2 (73 %)

70 % 62 %

95 96 97

N

HN

HO2C

CO2H

OO

CO2H

9. Cu(OAc)2, CH2Cl2, HCl/Air (95 %)10. CAN, H2O, MeCN (69 %)11. LiOH, H2O, THF; then H2SO4 (73 %)

(13 % over all)

98

Synthesis of Vitamins & Cofactors

22

Thank you for your attention

Synthesis of Vitamins & Cofactors

23

General Literatur Ullmann's Encyclopedia of Industrial Chemistry, 2005 Wiley VCH Verlag GmbH & Co. KGaA, Weinheim (10.1002/14356007.a27_443) Pharmaceutical Substances: Syntheses, Patents, Applications, 4th Ed. 2001 Stuttgart, Thieme, ISBN-13: 978-3135584058. !Naturstoffe der chemischen Industrie, 1. Auflage 2007, Elsevier GmbH, München; ISBN-13: 978-3-8274-1614-8. !Handbook of Vitamins, 4th Ed. 2007, Taylor & Francis Group, LLC; ISBN-13: 978-0-8493-4022-2.