Ch. 3: Vitamins, Cofactors, Coenzymes, Enzymes * Readings

Ø P. J. Bruice, Organic Chemistry, 2nd ed., Prentice-Hall, Upper Saddle River, New Jersey, 1998, Chapter 22. Ø H. Dugas, Bioorganic Chemistry - A Chemical Approach to Enzyme Action, 3rd ed., Springer Verlag, New York, 1996, chapter 7. Ø O. Isler, G. Brubacher, Vitamins I: Fat-Soluble Vitamins, Thieme Verlag, Stuttgart, 1982. Ø O. Isler, G. Brubacher, S. Ghisla, B. Kraeutler, Vitamins II: Water- Soluble Vitamins, Georg Thieme Verlag, Stuttgart, 1988. Ø http://www.indstate.edu/thcme/mwking/vitamins.html

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 1 Glossary • vitamin (water soluble vitamin, fat soluble vitamin): a substance needed in small amounts for normal body functions that the body cannot synthesize in adequat amounts • enzyme: a protein that is a catalyst • metalloenzyme: an enzyme that has a tightly bound metal ion cofactor: an organic molecule or a metal ion that certain enzymes need in order to catalyze a reaction or a process • coenzyme: an organic molecule (vitamin) as cofactor • prosthetic group: a cofoctor permanently associated with the protein, often covalently bound • holoenzyme: catalytically active enzyme-cofactor complex. • apoenzyme: an enzyme without its cofactor (enzymatically inactive protein)

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 2 Glossary

• metabolism: reactions that living organism carry out • catabolism: energy and simple molecules are formed from complex molecules • anabolism: synthesis of complex biomolecules using energy

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 3 Problems - 1

@ Vitamin C and Vitamin E are both natural radical inhibitors. Explain why this is so. @ Explain why vitamin C is a „strong“ acid @ Ascorbinsäure can be methylated by diazomethane. Which functional group is most sensitive towards methylation? @ (L)-Sorbose which is an intermediate in the technical synthesis of vitamin C, forms a furanosid by reaction with aceton/H+. Discuss the mechanism of formation. Are there diastereotopic methyl groups?

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 4 Problems - 2

@ Apply Woodward-Hoffmann-Rules for the synthesis of Vitamin D3. Sum up the WH-rules for the electrocyclic and sigmatropic reactions under thermal respectively photochemical conditions. a) Which stereoisomer is formed by irradiating (2E,4Z,6Z)- octatriene? @ Stereoselective oxidoreduction of ketones and alcohols using horse liver alcohol dehydrogenase (HLADH) needs NADH as cofactor. NADH is rather expensive and has to be recycled. Recyclization can be achieved by natrium dithionite. Formulate the reaction sequence for the keton reduction by HLADH/NADH and the subsequent recyclization of NAD+ by dithionite. @ Assign the pro-R hydrogen and the pro-S hydrogen of NADH. @ Compare typical applications of NAD+/NADH und FAD as oxidoreductases with regard to their involvements in the biochemical processes.

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 5 Problems - 3 @ Nature exploits the reactivity of C-8 methyl group of flavins in order to link to the apoenzyme via a SH group of a cystein. The overall process is as given below. The last step is the oxidation of the cystein bound

FADH2 to enzyme-FAD.

R R - CyS CySCH N N O Me N N O 2 N N Me N H Me N H O O

@ Discuus the similarity of decarboxylation of a b-keto acid and enzymatic pyruvate decarboxylation. @ The pyrimidine-amino-group in thiamin pyrophosphate contributes in the enzymatic TPP-dependent processes. How can this be explained?

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 6 Enzymatic reactions and catalysis

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 7 Vitamins, coenzymes, cofactors, enzymes

protein + coenzyme (vitamin)

enzyme

protein + cofactor (metal ion)

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 8 Vitamins, coenzymes, enzymes cofactors

Vitamins that are coenzyme precursors

Ø D. Voet, J. G. Voet, Biochemistry, second edition ed., John Wiley & Sons, New York, 1995.

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 9 Structure of water soluble vitamins BO/BBM

CH OH NH 2 2 H CH2OH OH H OH + HOCH OH N N 2 O S OH O HO H OH H3C N H C N CH 3 3 HO OH Me N N O OH thiamine N pyridoxine vitamin C vitamin B Me N H vitamin B 1 O 6 ascorbic acid

O riboflavin O vitamin B2 OH HN NH

H H N CO2H n CO2H S S S niacin n = 3 H nicotinic acid lipoate biotin vitamin H

Me Me O O O HO CO2H H N CO H N N N 2 H N vitamin B12 OH H H H2N N N pantothenate CO2H folate subunit of CoA-SH

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 10 BO/BBM/SKB Vitamin B12

Ø Discovering Nature's Diverse Pathways to Vitamin B12: A 35-Year Odyssey: A. I. Scott, J. Org. Chem. 2003, 68, 2529-2539..

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 11 BO/BBM Fat soluble vitamins

CH3 O HO CH3

CH3 H3C O n

CH3 O vitamin E n = 3 a-tocopherol vitamin K1

Me Me CH2OH Me Me H vitamin A (all-trans) HO

vitamin D3

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 12 Vitamin C

• mp 190-192°C

• pK1 = 4.17; pK2 = 11.57 • anti-scorbutically factor (essential for the synthesis of collagen) • reducing agent in hydroxylation reactions • radical scavenger (inhibitor)

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 13 Industrial synthesis of vitamin C (Reichstein, 1934)

Ø H. Beyer, W. Walter, Lehrbuch der Organischen Chemie, 1998.

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 14 Enzymatic synthesis of vitamin C

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 15 Vitamin D

• Metabolism of calcium and phosphorous • Deficiency impairs bone formation (rickets) • Excess causes calcification of soft tissues

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 16 Photochemical formation of vitamin D3

C-1 C-25

* two step reaction - first step: conrotatory photochemical ring opening - second step: [1,7] sigmatropic antarafacial hydrogen shift (thermal) * Woodward-Hoffmann rules

* Active form 1,25-dihydroxyvitamin D3

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 17 Oxidoreduction: Pyridine nucleotide coenzymes + + (NAD /NADP ) (Vitamin B3, Niacin)

* NAD+/NADH; NADP+/NADPH - Structure - Oxidation reactions - Reduction reactions (Pyruvate ® lactate; lactate dehydrogenase)

- Comparison with LiAlH4 - Mechanism and stereochemistry (Prochirality)

H(R) H(R) and H(S) enantiotopic hydrogens H C H(S) 3 OH

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 18 Bioactive flavins

H * vitamin B2 – riboflavin O

OH * flavin mononucleotide HO OH (FMN) Me N N O * flavin adenine dinucleotide N Me N H (FAD) O

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 19 Flavin Biochemistry

* Dehydrogenation * Activation of oxygen, hydroxylation, oxygenation * Electron transfer, electron transport, respiration chain, etc. * Light emission, bacterial luciferase (bioluminescence) * Photobiology: Photolyase, phototropism, photomorphogenesis, circadian processes (Cryptochrome, Phototropin, blue-light photo receptors)

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 20 Flavin structure

Ø F. Müller, Chemistry and Biochemistry of Flavoenzymes, Vol. I, CRC, Boca Raton, 1991.

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 21 Isoalloxazine synthesis

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 22 Redox processes of flavins (electron transfer/protonation)

R2 H R2 R2 + H R1 N N O R1 N N O red R1 N N O N N N R1 N H R1 N H R1 N H O O O Fl - FlH ox Fl red rad

red

R2 H + R2 H R1 N N O H R1 N N O

N N R1 N H R1 N H H O O FlH - 2 FlH red

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 23 Some enzymatic processes of flavin nucleotides

• dihydrolipoyl dehydrogenase • L-amino acid oxidase • succinate dehydrogenase • NADH dehydrogenase • luciferase catalyzed bioluminescence

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 24 Enzymatic processes of flavin nucleotides

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 25 8a-S-Cysteinylflavin: Binding of flavins at the protein

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 26 FAD-dehydrogenase – oxidation of a dithiol

O O dihydrolipoyl dehydrogenase O FADH O + FAD + 2 SH SH S S

H+ R Me N N O S S N Me N H O R

H+ S S R

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 27 Bioluminescence

• Photoluminescence Population of excited state by • Chemoluminescence some process XYZ • Bioluminescence • Triboluminescence Electronically • Electroluminescence excited state • Lyoluminescence • Aquoluminescence hn • Sonoluminescence • Galvanoluminescence • … Ground state • … • XYZ-Luminescence hn = Ee - Eg

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 28 Bioluminescence

light

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 29 Bacterial bioluminescence

AA

• enzyme catalyzed reaction: luciferase • reaction cascade ultimately leads to the formation of the

excited state (S1) of 4a- hydroxyflavin

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 30 LOV domain of Phototropin

· Genetic information translated into protein structure · Enzymatic synthesis of FMN · Molecular self-assembly · Input light, output morphogenesis D S. Crosson, K. Moffat, 2001. D See also: http://www.uni- regensburg.de/Fakultaeten/nat_Fak_III/GK/SP/download/Kottke _Phototropins.pdf 19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 31 Pyruvate decarboxylase – cofactor thiamine pyrophosphate (TPP)

O O O H C + CO2 3 H C H O 3

NH2 H + N N S

H3C N H C 3 O O O P O P O O O thiamine pyrophosphate TPP

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 32 Pyruvate decarboxylase

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 33 Benzoin-type condensation – compare its mechanism to TPP catalyzed processes (transketolase)

- O H H CN O 2 R R O H R

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 34 Enzyme bound biotin

O

HN NH H N E

S O

E: enzyme

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 35 Biotin – catalyzes carboxylation as a CO2 carrier

* carboxylation of activated carbon

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 36 Pyridoxal phosphate (PLP)

Enzyme CH OH H O H N 2 O O O O H HOCH OH 2 P OH P O O O O O N CH3 N CH 3 N CH3 H

pyridoxine pyridoxal phosphate enzyme covalently

vitamin B6 PLP bound to the cofactor

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 37 PLP-catalyzed processes of amino acids in Nature

* transimination Enzyme H N O * O H decarboxylation P O O O * transamination N CH + 3 * racemization H enzyme covalently * C -C bond cleavage bound to the a b PLP cofactor („b-elimination“) * a,b-elimination

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 38 Decarboxylation of an amino acid

* First step: Synthesis of amino acid-bound PLP from enzyme- bound PLP * Second step: Decarboxylation * Third step: Transimination leading to enzyme-bound PLP and „free“ decarboxylated amino acid

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 39 Vitamin B12

D Discovering Nature's Diverse Pathways to Vitamin B12: A 35-Year Odyssey, A. I. Scott, J. Org. Chem. 2003, 68, 2529-2539. D Total synthesis of cobyric acid: historical development and recent synthetic innovations, D. Riether, J. Mulzer, European Journal of Organic Chemistry 2003, 30-45. D W. Kaim, B. Schwederski, Bioanorganische Chemie, Teubner, Stuttgart, 1991.

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 40 Coenzym B12

• vitamin B12 coenzyme (adenosylcobalamin)

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 41 General scheme for the structure interconversion cataylyzed by B12

Y C1 C2 C1 C2 Y

X H H X

1,2 shift process

• radical mechanism

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 42 Processes requiring coenzym B12 - 1

glutamate mutase

19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 43 Processes requiring coenzym B12 - 2

Ø H. Dugas, Bioorganic Chemistry - A Chemical Approach to Enzyme Action, 3rd ed., Springer Verlag, New York, 1996, chapter 7. 19.07.04 lcns_03k03aw.ppt - SBO/SS 2003 44