Synthesis of Vitamins & Cofactors Gaich Group Seminar 17.07.2014, Darius Schwazrer 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 insufcient amounts . Must be regularly consumed with diet Vitamin A & ß-Carotene Retinol acetate (BASF) 2. PPh3, 3. BuLi, THF, - 50 °C Me Me Me Me OH H2SO4, Me Me Me AcO Me Me 1. THF Me Me i-PrOH, O ClMg Heptane Me OAc O Me PPh3 95 % 80 % 98 % Me Me 1 Me 2 Me 3 HSO4 Retinoacetate (4) ß-Carotoene (BASF) c. KOAc, MeOH, rflx. then KOH, H2O, (90 %) CH2Br d. Acetone, MnO2 (85 %) a. Et O BrMgC CMgBr b. PBr e. EtOAc, Lindlar, H2 (95 %) 2 OH 3, f. Benzene, Iodine, O then 10% H SO (CH OH) 2 4 HO 2 2 hexane, hv (?? %) 8 5 6 75 % 52 % 7 BrH2C O O Me 8 Me Me DMF, NH3, then Me Me H2O2 isomerization Me Me Me 80 % Me Me Me Me PPh3 2x Me β-Carotin (9) Me 3 Mildner; Weedon Journal of the Chemical Society, 1953 , 3294, Inhoffen; v.der Bey Justus Liebigs Annalen der Chemie, 1953 , 583. 3 Ascorbic Acid Vitamin C synthesis by Reichstein process Me Me Acetobacter OH O O O OH OH OH OH suboxydans 3. Acetone, O H RaNi pH 4-6, 30-35 °C OH H SO O 2, OH 2 4 Me HO HO quant. HO O OH >90 % OH OH 37 % OH OH OH OH Me O D-glucose (10) 11 12 13 OH O HO 4.NaOCl, NiCl, > 93 % OH + OH 5. H2O, H , 82 % 6. HCl, PhMe, Δ HO O step 3+4 can be replaced OH OH O > 60 % O OH 14 OH by oxidation with Air & Ascorbic acid (15) 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 T. Reichstein, A. Güssler Helv. Chim. Acta 1934, 17, 311; Bullmanns Enzykl. Tech. Chem., 3. Ed., Vol 18, 233 ff. 4 Ascorbic Acid Metabolism of Vitamin C OH OH OH OH HO O HO HO HO O O O -H+ O O -H+ O -e- O +H+ O HO +H+ O O O OH OH OH 15 pka = 4.1 pka = -0.86 OH HO OH HO O O HO - +H2O +H O O -e O O 2 O O OH OH O -H2O OH -H2O HO O OH OH OH 16 17 18 Biological activity Economic aspects Industrial use • anti oxidant • production > • Inhibition of nitrosamine in • radical scavenger 600 kt/a cured meat • electron donator for 8 • cost ~3000 €/t • Improvement of flour quality enzymes • Increasing of clarity of wine & (hydroxylases) beer • protecting fruits from browning• prevents from scurvy 5 Thiamine Synthesis of Vitamin B1 Step 1. Synthesis of 22 NH b.Na(powdered), PhEt, 2 NH2 Styrol & PhNHMe in CN CN CN a. i-PrOH, CN THF over 2 h, rt, then N c. MeOH, NH RaCo, N NH N N 3, 2 max. 40 °C rflx, 20, then H2O 110 °C 60 atm, 13 h CN Me N Me N Me NH2 87 % Me N 77 % H 50 % H 22 19 20 21 Step 2. Condensation & formation of thiazolium ring O O 1. KSH, MeOH, 0 °C AcO AcO 74 % 24 SH Me 23 Cl Cl- + 3. HCO2H, < 40 °C N N 89 % S OH NH2 Me N NH2 2. OMe 80 °C N NH Me2N OMe Thiamine (26) N NH 2 N 89 % Me N Me N 22 25 22 and 23 commercially available 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. 6 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. PPO PPO PPO PPO PPO O Me HO O S S HO S Me OH O Me H S OH S H O CoAS Ph O O N N Me N N Ph O Me Me Me N Me Me Me R R R O R Ph 27 R 29 Deficiency • Beriberi —> efects peripheral nervous system; cardiovascular system, • Polyneuropathy • Anemia • many more 7 Biotin Vitamin B7 Synthesis by Hofmann-La Roche 1. Br H O, 90 % O 2, 2 O O 2. BnNH2, MeOH, 5. LiBH THF 4. PhH, C H O, Δ, then 4, 90 % BnN NBn 6 12 6. AcSK, DMF, CO2H (+)-Ephidrine, iPrOH, BnN NBn 3. COCl2, KOH BnN NBn 80 °C 150 °C > 90 % HO2C HO2C CO2H 30 31 RO2C CO2H O 32 S O 34 2 steps BnN NBn HO2C CO2R 33 ClMg OMe O O 7. 10. Malonic acid diethyl ester, then AcOH, O Δ 9. 48% HBr, NaOMe, PhMe, 80 °C NH O 8. Ra/Ni, H 40 atm. BnN NBn H 2 95 °C, 2 h BnN NBn then, 48% HBr, rflx. 35 °C, MeOH HN OH 82 % 90 % (25% over all) H S Biotin (37) S OMe Br S 35 36 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). 8 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 O O O O O O O O - P ATP + HCO3 ADP + HO O OH HN NH N NH HO SCoA OH CoAS HO 39 38 + Biotin S 4 S 4 37 CO2H CO2H Biotin deficiency causes Neurological symptoms (depression, lethargy, etc.) 9 Cobalamine Vitamin B12 H2NOC • Synthesis by fermentation CO2NH2 Me Me H2NOC • No industrial total synthesis available CONH Me 2 • Total synthesis with 113 steps, 12 PhD, 91 Postdocs, N CN N Me Co H 17 years N N Me Me H2NOC • optimized to 70 steps Me Me CONH2 - 2- - • cobalt can be complexed by CN , SO3 , OH , halidesO N NH 6 Me • 10t/a; market volume 77*10 EUR HO N O ! Me Me O P O O Biological activity O HO • catalyzes 1,2 H shift 40 • Coenzyme for Methionin-synthase & Methylmalonyl-CoA-mutase • Hydroxy-cobalamine —> treatment of cyanide poisoning • deficiency causes anaemia 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. 10 Calcitriol Synthesis of Vitamin D3 Me OH Me OAc Me Me Me OAc Me Me Me 3 Br Me Me 3 Me 2. NaHCO3, O N OAc Hexane, Me O 0.5 Eq. Me 3 OH 20 min. Me N Me H 1. Ac2O, Pyridine, Me H Br OAc 100 °C, 17 h Me Me H H 96 % H H 3. Xylene, Δ 2 eq. N H H HO AcO Me Me AcO 41 42 then pTSA, Dioxane, 70 °C, 43 30 min., 50 % Me OH Me OH Me Me Me 5. -15 °C KOH, MeOH, THF Me Me 3 78 % Diacetate, 16 % Monoacetate Me 3 6. 450 W Hg, -5 °C, THF, hexane, 20 % 25 % unconverted OH Me 8. Δ, Dioxane, 30 min 7. KOH, MeOH, 30 h, quant. H H 50 % HO 44 HO OH Calcitriol (45) US 3 993 675 (Hoffmann La Roche Inc.; 23.11.1976; prior 12.11.1973; 24.02.1975. 11 Calcitriol Biological activity of Vitamin D3 Me OH Me Me • hormonally active vitamin Me 3 • increases the Ca2+ concentration in the blood, by increasing the uptake in the gastrointestinal tract H • decreases the probability to get some of up to 17 diferent cancer diseases HO OH Hypervitaminose causes calcification of tissues and Calcitriol (45) arteries ! ! Production & prize • No t/a given • cost 50 $/kg 12 Folic Acid Vitamin B9 synthesis 2. H SO /H O, NaNO 5 °C, H N N NH 2 4 2 2 H2N N NH2 2 2 then room temperature H2N NH2 CO2Et 1. MeOH, , quant. Δ 3. MeOH, Pd/C, H 2 N N H N NH CN 82% 2 46 49 47 48 OH OH O OH 4. Dioxane, H O, O O O 2 NaHSO3, pH 4-5 OH OH 5. AcOH, 2-mercapto- O N H2N N NH2 H H CHCl HO ethanol NaCNBH3, 2 85 % N O NH N N N H H2N O CH2Cl H2N H2N N N OH O H H 49 Tetrahydrofolic acid (52) 50 51 Folic acid is metabolized to „THF“ what is the active compound O R H PLP, Serine Michael-Addition O HN O NR N R N N then Retro-Mannich N N H N N H2N N N H H H N N N H2N N N PLP, Glycine 2 H H "THF" 52 H H 53 54 E.