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Bio-Organic Mechanism Game – Simplistic Biochemical Structures and Simplistic Organic Reaction Mechanisms Are Used to Explain Common Biochemical Transformations

Bio-Organic Mechanism Game – Simplistic Biochemical Structures and Simplistic Organic Reaction Mechanisms Are Used to Explain Common Biochemical Transformations

1 Bio-Organic Mechanism Game – Simplistic biochemical structures and simplistic mechanisms are used to explain common biochemical transformations. Simplified biochemical are presented first.

Many have a somewhat complex structure that makes it difficult to write out step by step mechanisms. However, if we simplify those structures to the essential parts necessary to explain the mechanistic of each step, it becomes much easier to consider each step through an important cycle. I have proposed possible simplified structures that are used in the later examples of biochem cycles and problems. The usual strategy in biochem cycles is to just write names, or perhaps, names and a structure. Occasionally a few mechanistic steps are suggested, but almost never is a detailed sequence of mechanistic steps provided. Since it is hard to find such detailed mechanistic steps anywhere (sometimes they are not known) our proposed steps are, of necessity, somewhat speculative. In this book we are not looking for perfection, which is not possible, but for sound organic logic that is consistent with the biochemical examples presented below. There is great satisfaction in blending organic knowledge with real life reactions that help explain how life works. In working through some of the problems, you may develop an alternative mechanism that is just as good, or even better than the one I have proposed. If you do, I hope you will share it with me and if an improved version of this book ever gets written I can include it the next edition (and give you credit). It is almost certain that I have made some errors and I would appreciate it if you would let me know about them.

Biomolecules and our simplified representation.

1. ATP – – phosphorylation, energy source NH2

N N O O O O

O P O ATP O P O P O P O CH2 N = O N O O O O H H

simplified structure H H actual structure OH OH

2. NAD+ and NADP+ - nicotinamide adenine dinucleotide (hydride acceptor)

CONH2 NH2

N N = O O N N H2C O P O P O CH2 N O N OH HO R O O H H O simplified H H actual OH OH NADP+ has a structure structure phosphate here.

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 2 3. NADH and NADHP - nicotinamide adenine dinucleotide (hydride donor)

H CONH2 NH2 H H H N N O O =

N H2C O P O P O CH2 N O N N OH HO O O H H R O H H simplified OH OH NADPH has a structure phosphate here.

4. Vitamin B-6 – pyridoxal phosphate (amino , transamination with -ketoacids, , removal of some amino acid side chains, epimerizations)

1o version of Vit B-6 version of Vit B-6

NH2 NH2 H O H O H interconvert H via , -2 O , -2 O O PO 3 O3PO = N N N = N H H H H simplified actual simplified actual structure structure structure structure

5. TPP – Thiamine diphosphate (decarboxylation and chemistry with or )

N NH2

R R N

N N B N H = H S S O O S ylid form simplified O P O P O CH2 structure of TPP actual O O structure

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 3 6. Coenzyme A (acyl transfer)

All of this is "Co-A" O O O O

S O P O P O N H N N O NH H H 2 O O N OH form here. N O HO N H CoA OH S CoA This is an S acetyl group actual simplified simplified structure structure structure of CoA of acetyl Co-A

7. FAD / FADH2 – Flavin adenine dinucleotide (oxidation – reduction) – used to deliver hydride to C=C or take hydride from CH-CH (fatty acid metabolism, etc.)

O O

O P O P O N O NH2 O O N HO OH N HO N actual structure OH OH

N N O FAD - flavin dinucleotide (a hydride acceptor) NH N H O N N

N N FAD FADH H 2

simplified structures for FAD and FADH2

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 4

H B H H B

N C C C C H H

N FAD N N H flavin dinucleotide (a hydride acceptor) C C N N H FADH2 B B H flavin dinucleotide (a hydride donor)

Hydride transfer reduces FAD to FADH2 which can be oxidized to FAD.

8. THF – tetrahdrofolate (transfer of one units) –recycles cysteine to methionine and other 1C metabolic functions, many variations

Tetrahydrofolate (THF) one carbon transfers as R "CH3", "CH2". 2 H2N N N V2 -p762 R H N N actual H N structure One glutamate is H shown, but several 5 = O HN can be attached. N H 10 HN One carbon groups H N CO2 Ar can bond here in various ways (see simplified below structures. structure O CO2

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 5 different forms R R R R N N N N NAD+ NADP+ +H2O 5

5 N NADH 5 NADPH H -H2O N N N 10 O N 10 10 Ar CH3 HN N HC N H2C 10 Ar Ar Ar N -formyl THF 5 5 10 H N -methyl THF 5 10 N ,N -methenyl THF N ,N -methylene THF HCO2 ATP glycine -H2O +H2O or +NH3 R THF serine R THF -NH3 N N

5 5 histidine N N THF 10 10 HN HN H NH Ar H O Ar 5 = N5-formyl THF N -formimino THF

9. SAM = S-adenosylmethionine (methyl transfer agent), The methyl group (CH3) attached to the methionine sulfur in SAM is chemically reactive. This allows donation of this group to an acceptor substrate in transmethylation reactions. More than 40 metabolic reactions involve the transfer of a methyl group from SAM to various substrates, such as nucleic , proteins, lipids and secondary metabolites. SAM can be made from methionine and N5-methyl THF (just above).

was triphosphate N SAM = S-adenosylmethionine methionine NH2 NH2 Rmet Rad O O N S S N CH = 3 OH CH3 N simplified structure actual HO OH adenosine structure SAM = S-adenosylmethionine Rmet = methionine Rad = adenonine N O O O O O O NH2 NH2 P P P O N O O O O O S N N OH methionine CH3 HO OH ATP

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 6 10. Cytochrom P-450 are oxidizing agents in the body. They can convert inert sp3 C-H bonds into C-OH bonds and they can make groups at and aromatic pi bonds.

Oxidations in the body often use cytochrom P-450 enzymes.

N N N +3 N N N +3 Fe simplified simplified Fe N N structure Fe structure N N N N S S Enz Enz HO2C

CO2H O heme, protoporphyrin IX, found in This is the structure cytochrom P-450 oxidative enzymes that we will use. +4 +3 Fe Fe simplified structure

11. Halogenase Enzymes (related to cytochrom P-450 enzymes, can have imidazole ligands from histadine amino acids

Halogenations in the body often halogen bonds. His

N O N N O N N +3 This is the structure Fe N that we will use. His Fe+4 Cl H Cl N O O N +4 +3 N Cl Fe Fe simplified structure His

Biochemical Examples

Mechanism arrows used in the “Bio-Org Game” are meant to suggest how the move over a single transformation, and are not necessarily meant to imply that all of the electrons and transfer in one huge “domino” cascade. Organic mechanisms are often multistep transformations, but it’s harder to pin down biochemical transformations. The symbolism used in these examples represents a concise way to show movement involving making and breaking bonds. Lone pairs are rarely drawn (or used). They are included on the generic (B:) used to show proton transfers. A generic acid (H-B+) is used to provide a proton. Very occasionally a pair of electrons is used when it provides some special effect (enamine reaction,

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 7 stabilization in formation or breakdown). Multiple resonance structures are not drawn. Only very occasionally is an intermediate drawn, when confusion arises from too many arrows going in too many different directions. Do not confuse these examples for real mechanisms! They are designed to show the essential how changes might occur in complex biochemical reactions. Also, at physiological pH (7) a few organic groups are ionized (RCO2H is -- + anionic as RCO2 , and RNH2 is cationic as RNH3 ). They are drawn in their neutral forms in this game. The initial examples of biochemical transformations can serve as foundational reactions in endless biochemical sequences or cycles. Knowing how these reactions work can provide insight into many biochemical aspects of anabolism and catabolism and can help improve your organic “mechanistic” logic. First, bare-bones examples are provided to show the essence of each type of reaction. The problems that follow use several “typical” types of biochemical transformations in made-up sequences and many real biochemical cycles in which to practice. With such practice using these simple model reactions you can learn to recognize where (when) and how similar transformations might be occurring in real biochemical reactions that are presented without any mechanistic detail in a book or article. Nature uses simple strategies applied to limited classes of molecules (carbohydrates, lipids, fats, steroids, amino acids, nucleic acids, neurotransmitters, alkaloids, terpenoids and more) having enormous variation of patterns. It’s amazing what you can speculate upon using these few reactions.

An example of mechanistic simplification.

An “organic” mechanism, showing keto  tautomerization in acid, is shown without simplification, having all of the normal mechanistic details (lone pairs, formal charge, resonance, etc.). We won’t do it this way in the Bio-Org game.

H B H H H O O O O B C H C H C H C C C C resonance C

A complete organic mechanism shows lone pairs, each individual step and resonance structures.

The same mechanism in the Bio-Organic Mechanism Game is shown in the first “biochem” example, using the simplified mechanistic conventions of this game.

1. Keto / enol tautomerization (two proton transfers and a shift of pi electrons).

H B B H B = general base, O O possibly on B H B C C H H B = general acid, C C possibly on enzyme

keto enol tautomer

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 8

B H B H O H B O

C C H B C C

enol tautomer keto tautomer

Quite often in the acid and base functions are a cooperative action in the active site of an enzyme, much in the manner used in this Game. This avoids the necessity of very strong acid or very strong base, often used by in their reactions. Such conditions are not tolerated by living organisms. We arbitrarily use neutral base, B: and cationic acid H-B+.

2. Carbonyl hydration – a regioselective of H2O to a . This also requires some proton tranfers. A carbonyl hydrate can be dehydrated via an which also requires some proton transfers. These steps are very similar to hemiacetal/hemiketal reactions (Example 6), but use H-O-H instead of R-O-H. The carbonyl hydrate can be used to oxidize an aldehyde (example 8) or allow a reverse (example 3).

Forward Direction H B B BH H H B O OH hydration H O O C (addition C reaction) aldehyde or carbonyl hydrate Reverse Direction BH B H B H H B O H dehydration OH O O C C (elimination reaction) carbonyl hydrate aldehyde or ketone

3. Aldol reactions make a new carbon-carbon bond, forming a -hydroxycarbonyl compound. A carbonyl C position becomes the (as enol or enolate) and reacts with a separate electrophilic carbonyl carbon. Reverse aldol reactions cleave the C-C bond, leaving the electrons on a C position and forming a C=O at the C-OH position. The aldol product can proceed on an additional step as shown in Example 5 (reverse Michael  ,-unsaturated carbonyl compounds)

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 9 Forward Aldol

B B H BH O O H B O aldol (addition O reaction) C C H C C C C This product also looks like a . See Example 5. carbonyl nucleophile -hydroxycarbonyl carbonyl at C position Reverse Aldol H B BH B B H reverse O aldol O O O (elimination reaction) H C C C C C C

-hydroxycarbonyl

4. Claisen reactions make a new carbon-carbon bond, forming -ketocarbonyl compounds. A carbonyl C position becomes the nucleophile (as enol or enolate) and reacts with a separate electrophilic carbonyl carbon (similar to Example 3, except carbonyl substitution occurs instead of carbonyl addition). groups are common in and thiol ester groups (acetyl Co-A) are common in biochemistry. Reverse Claisen reactions cleave the C-C bond leaving the electrons on the C position and forming a carboxyl at the C=O position. The tetrahedral intermediate is omitted in the Bio-Organic Game.

Forward Claisen B H B B O O H B O O Claisen C C (acyl substitution) H C C OR C C OR OR RO H -ketocarbonyl carbonyl electrophile with carbonyl a leaving group, (often and or thiol ester or nucleophile ester or thiol ester or a thiol ester mixed anhydride) Reverse Claisen O B -ketocarbonyl O H B H C O O reverse Claisen (acyl substitution) C C OR OR C C ester or carbonyl electrophile thiol ester C OR with a leaving group, (often and ester or H B RO thiol ester) alcohol H B or thiol

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 10 5. Reverse Michael reaction (elimination = dehydration) eliminates H2O between the C-C bonds (E1cB mechanism). Dehydration carries the aldol (Example 3) one step farther along, forming ,-unsaturated carbonyl compounds. Michael reaction (addition / hydration) is the reverse reaction and adds the elements of water across the C=C, a resonance extension of the C=O. We have taken liberties with the name “Michael”. It is probably better to describe these reactions as conjugate addition and reverse conjugate addition. A close variation of this reaction eliminates instead of water. Other possibilities also exist.

Reverse Michael Reaction B BH H H O reverse H O O O Michael reaction C C C C C C (elimination)

H -unsaturated carbonyl H B B -hydroxycarbonyl (Michael acceptor) Reverse Michael Reaction H B H O BH This product looks like H O Michael O O an aldol product. See C C reaction Example 3. It is now C C able to do a reverse aldol, C or be oxidized to a (addition) C 1,3-, maybe followed by -unsaturated carbonyl H decarboxylation, etc. H B (Michael acceptor) -hydroxycarbonyl B

6. Hemiacetal (or hemiketal) formation is an addition reaction to a carbonyl by alcohol, similar to carbonyl hydration and dehydration, Example 2. The reverse reaction reforms the carbonyl group and alcohol in an elimination reaction., A second alcohol can react with the hemiacetal/ketal and undergo an SN1 reaction with the OH to form an acetal or ketal (Example 7, just below). The example shown here is an and typically forms rings of 5 or 6 atoms.

Forward Direction

H B H H B BH O O O addition O B reaction C C

alcohol aldehyde or ketone hemiacetal / hemiketal

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 11 Reverse Direction

BH B B H H B O H O elimination O O reaction C C

hemiacetal / hemiketal alcohol aldehyde or ketone

7. Acetal (or ketal) formation from a hemiacetal (or hemiketal). The “OH” becomes a water leaving group that is replaced by an “OR” in an SN1 reaction, producing an linkage. In the reverse reaction (acetal or ketal forming a hemiacetal or hemiketal) an alcohol leaving group is replaced by a water molecule in an SN1 reaction. These are reversible reactions that require acid . Because arrows are used in both directions on the same bonds, we show the intermediate in this example. These reactions often occur when one sugar molecule “OH” connects to another sugar molecule at its hemiacetal site (such as galactose + = lactose). Such linkages can go on for hundreds of sugar molecules (glycogen in animals and cellulose in plants).

Forward Direction B BH H H H R R O OH O O O O O eliminate add H2O ROH

hemiacetal / hemiketal intermediate acetal / ketal Reverse Direction H B BH BH R OH H O O O O O

eliminate add ROH H2O acetal / ketal intermediate hemiacetal / hemiketal

8. a. Oxidation of CH(OH) to C=O (1o ROH  aldehyde, 2o ROH  ketone, hydrated aldehyde  ) with an equivalent of NAD+. NAD+ accepts a hydride via conjugate addition, quenching the positive charge on the nitrogen and forms NADH. A base removes a proton from the adjacent oxygen atom allowing an elimination reaction to produce the C=O (or in Example 9, a C=N).

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 12 H B H H BH H O oxidation of C an alcohol C O H N elimination N reaction R R NAD+ NADH o o aldehyde or ketone 1 or 2 alcohol equivalent equivalent

b. Reduction of C=O to CH(OH) with an NADH equivalent is the opposite of the above reaction. NADH is a hydride donor that becomes aromatic (forms NAD+) with the transfer of the nucleophilic hydride to the electrophilic C=O. A nearby acid protonates the oxygen completing the addition reaction. H H H BH B H O reduction of a carbonyl O H C C N addition N reaction R R NADH 1o or 2o alcohol NAD+ aldehyde or ketone equivalent equivalent

9. a. Oxidation of an amine, CH(NHR), to imine (C=N-R) with an NAD+ equivalent that is reduced to NADH, followed by hydrolysis to a C=O. This is the opposite of 9b, below. The first step is similar to reaction 8a above with an alcohol. Overall, this is a transformation of an amine into a carbonyl group and a primary amine.

Oxidation of an amine H B H H R H B H N elimination R reaction C C N H N oxidation of N an amine R R NAD+ NADH amine equivalent imine equivalent

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 13 Hydrolysis of an imine BH BH B B H B H H H R H B R N N OH R N O O H C C addition elimination C reaction reaction imine aminal aldehyde or ketone

b. Formation of an imine, C=N-R, from a C=O, followed by reduction to CH(NHR) (an amine) with an NADH equivalent that is oxidized to NAD+. This is the opposite of 9a, above. The second step is similar to reaction 8b above with an alcohol. Overall, this is a transformation of a carbonyl group into an amine.

Formation of an imine B B H H B B H H H B H B R N R O R N O N H H C OH C addition elimination C reaction reaction aldehyde or ketone aminal imine Reduction of an imine H H BH B H R reduction of H N an imine R N H

C N addition C reaction N R NAD+ imine NADH amine equivalent equivalent R

10. Decarboxylation of a -ketocarboxylic acid, forming an enol, which tautomerizes to a keto group.

H B O B B H B H C H O O H B O O O decarboxylation tautomers C H B C C C C C carbon C O dioxide

keto tautomer -ketocarboxylic acid enol tautomer

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 14

11. Decarboxylation of an -ketocarboxylic acid with TPP (thiamine diphosphate). Also includes both “TPP ylid” and “TPP enamine” chemistry. The enamine can be protonated to form an aldehyde or it can react with another carbonyl compound to make a new carbon-carbon bond (a larger in this game) The TPP ylid is also regenerated, which can react again or protonate to make TPP. See another reaction of -ketocarboxylic acids with Vit B-6 in Example 13.

TPP reaction with an a-ketoacid, decarboxylation and formation of enamine. OH R R H B H B B O C R O N N O CO C H N R S S C OH TPP -ketoacid TPP ylid TPP ylid S pK  18 addition to R a a carbonyl

decarboxylation (-CO2) R

N OH C

S R TPP enamine Reaction of enamine nucleophile with a carbonyl electrophile followed by an E2-like reaction to from a new (larger) carbohydrate. B R R R H B H O O OH N OH N N C C C C R H R S elimination S S R R H reaction forms TPP enamine a carbonyl group, TPP ylid TPP enamine addition to similar to a reaction with C=O a carbonyl reverse aldol O OH

C CH R CH R

OH a new (larger) carbohydrate

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 15 Reaction of enamine nucleophile with an acid to from a new (smaller) carbohydrate. B R R R H O O N N N OH C H B C C H R H S S R S R elimination a new (smaller) reaction forms TPP ylid carbohydrate TPP enamine reaction acid/base a carbonyl group, with a proton protonation of similar to a TPP enamine reverse aldol

12. a. Phosphorylation of an OH with an ATP equivalent (making an inorganic phosphate ester).

O O O O O OPO P O ADP O P O P O P O ATP O O O O O CO acyl-like Mg+2 Mg+2 substitution H reaction ADP = leaving group B O

+2 Complexing with Mg can make one phosphorous atom COP O more electrophilic and the other one a better leaving group. The Mg+2 is not required to show this reaction. Mg+2 O is not used in the other reactions below, but it could be. H B inorganic phosphate ester

b. Dephosphorylation of an inorganic phosphate ester to an alcohol and phosphate. B H H O O inorganic BH O COP O ester H hydrolysis CO O P O O H acyl-like O BH B

c. An elimination reaction of a phosphate leaving group to make an (pi bond). Could actually be E1 or E2. O H O H O O P P O O E1 or E2 (anti) mechanisms C O C O are possible C C

B H BH

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 16

d. acyl phosphate (inorganic anhydride) and formation of a thiol ester (like acetyl Co-A) ADP leaving group O O phosphate leaving group OPO P O ADP O O O O OPO Mg+2 organic-inorganic O P O ATP anhydride O Mg+2 O O O O CO acyl-like O COP O H C H substitution 3 reaction B H C Co-A 3 O Mg+2 acyl-like substitution S reaction very reactive H thiol ester B (acetyl Co-A) Co-A S H B

13. Vit B-6 reactions – 1. imine formation with -keto acid and the amino version of Vit B-6 (similar to Example 9b), 2. tautomerization (Example 1) and 3. imine hydrolysis to amino acid and the aldehyde version of Vit B-6 (similar to Example 9a).

BH OH H B O H OH OH N O N H N R O O -keto acid O B R H R

imine dehydration N synthesis H B (-H2O) N N H vitamin B-6 H H O (1o amine version) Similar to Example 9b. H H

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 17

BH OH OH H H N N O O B H Similar to Example 1. R R

keto / enol tautomerization, N makes imine on N the other side H H

B OH H B H B H BH H2N H H OH H H OH O O H H O H N O N R O O hydrolysis -amino acid of imine R addition R of water

N N N H H H vitamin B-6 Similar to Example 9a. (aldehyde version)

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 18 Three additional vit B6 reactions from aromatic imine

1. Decarboxylation B H B O C O H H O O H BH H B H N N H O N H

R H R R

decarboxylation N N N H H H

H B B

O H H H H

N O N H

R R

N

H N

H

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 19 2. Elimination of side R group, like serine or threonine.

B serine aa H B H2CO H H O O H BH H B N N N

CO2H CO2H CO2H

decarboxylation N N N H H H vitamin B-6 (imine version) H B B H also threonine aa O O O H H H H

N N O N H H

H CO2H CO2H CO2H glycine aa N N H

N H N CO2H glycine aa vitamin B-6 (aldehyde version) H H

3. Epimerize a proton at the C position of an amino acid.

S amino acid proton adds on H B the opposite face H B R amino acid N R R N R N H

CO2H CO2H CO2H

N N N H H H vitamin B-6 vitamin B-6 (imine version) (imine version)

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 20 14. FAD / FADH2 reduction of C=C to CH-CH or the reverse reaction oxidation of CH-CH to a C=C, FAD can be recharged with NADH.

Simplified mechanism of action for reduction of C=C by FADH2 FAD and mechanism for reforming FADH2 from NADH. O H H O

CoA CoA R S R S H H B H H N N B

BH N N B FADH2 H FAD

H H H

N N

N N NAD+ R N N BH NADH R FADH2 FAD H

H H B N N FADH2 - flavin dinucleotide FAD - flavin dinucleotide (a hydride donor) (a hydride acceptor) used N N to reoxidize fats for energy. FADH2 H

H H O H O C C Enz R C S C C Enz R C S H H B H A saturated fatty acid chain. An ,-unsaturated fatty acid chain.

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 21 15. Cytochrom P-450 oxidation of sp3 C-H bonds to make sp3 C-OH groups

sp3 C-H bonds alcohols

H C H O C O H C O Fe +3 9 Fe +4 8 Fe +4 The carbon free abstracts hydroxyl (OH) from The free radical-like oxygen atom abstracts iron, making an C-OH bond where a C-H bond had a hydrogen atom from a C-H bond in the +3 enzyme cavity, forming an O-H bond and a been. The iron is reduced back at Fe to begin the carbon free radical. process all over again.

16. Cytochrom P-450 oxidation of C=C pi bonds (alkenes and aromatics) to make , which can be opened to .

R R R R R R R C C R C C CC R R O O O R R epoxides Fe +3 Fe +4 Fe +4 The carbon free radical abstracts the oxygen atom from The free radical-like oxygen atom adds to a the iron, making an epoxide ring. The iron is reduced C=C bond (alkene or aromatic) in the enzyme back at Fe+3 to begin the process all over again. Reactive cavity, forming a O-C bond and a carbon free epoxides can be opened up to diols (more water soluble). radical.

17. Cytochrom P-450 oxidation of sulfur and nitrogen lone pairs.

S S R R O R O sulfur O R Fe +3 S substrate sulfur R (1e-) substrate R Fe +4 Fe +4 sulfoxides, further oxidation is -2 possible, all the way to sulfate, SO4

R/H R/H N N O R R R R O O Fe +3 N nitrogen nitrogen R/H substrate substrate R (1e-) R Fe Fe +4 +4 N-oxides, further oxidation is -1 possible, all the way to nitrate, NO3

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 22

18. of sp3 C-H bonds to make C-X groups (X = Cl, Br, I) using halogenase enzymes.

sp3 C-H bonds

C H H O O H C CCl O

Cl Cl Fe +5 Fe +5 Fe +4

The free radical-like oxygen atom abstracts The carbon free radical abstracts a halogen (Cl or Br) a hydrogen atom from a C-H bond in the forming an unusal halogenated bioorganic molecule. enzyme cavity, forming an O-H bond and a carbon free radical.

19. of aromatic rings using X-OH to make sp2 C-X bonds (like thyroxine).

Iodide is stored in the thyroid gland. Iodoperoxidase enzyme makes it electrophilic (instead of nucleophilic iodide). It could react as hypoiodous acid, or the oxygen could be made into an even better leaving group if was a phosphate (using ATP).

H B O O O H H H O O P O ATP O H O P O I O I I H O O B possibly an even a good leaving group on iodine better leaving group Speculative mechanism for iodinating tyrosine and formation of thyroxine from two tyrosines. O I H B I CO H 2 CO2H H

H NH 2 H NH2 O O tyrosine

I CO2H I CO2H

H NH2 repeat O H NH2 O I diiodotyrosine - makes thryoxine

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 23 20. S-adenosyl methionine (SAM-e) to methylate biomolecules.

NH NH2 2 NH B 2 6 H C H 3 H3C 1 N Rmet 5 N H N S CH3 2 4 N O N O Rad 3 N O H H H cytosine 5-methylcytosine

21. Anti-oxidation reactions using vit E (fat soluble), vit C (water soluble), (also possible are glutathione, resveratrol and other bio-antioxidants).

possible dangerous free reduced radical neutralized free radical protection by vitamin E radical reduced by vit E by body's buffer system (possibly in cell membrane) R R RH

HB

O R O R O R H H H

R R R O O O

O vitamin E located resonance and inductive in cell membranes effects stabilize radical so quenches radicals it does not do damage O H O reduces R RH vitamin E back to normal and ultimately H O R HB washes out of the body

R O O O O R H

O O O H O H O H O R O B O R H O R H O R vitamin E is recharged B and still in cell membrane protects a second time O

O oxidized vitamin C O form washes out of the body

O R

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 24 other possible anti-oxidants OH glutathione SH NH2 O H O

O N N OH OH OH H O resveratrol glutaric acid HO ( linkage) cysteine glycine

3 types of problems are possible

1. Fill in the missing mechanistic details. 2. State what transformation occurred (and provide the missing mechanistic details). 3. Given the term, draw the step (and provide the missing mechanistic details).

Summary of Biochemical Topics having examples provided above:

1. Keto/enol tautomerization (proton transfer, resonance, proton transfer).

2. Carbonyl hydration (addition reaction of H2O to a C=O) / carbonyl hydrate dehydration (elimination reaction forms a C=O and H2O).

3. Aldol (makes a -hydroxy carbonyl compound). Reverse aldol, (makes 2 C=O from a -hydroxy carbonyl compound).

4. Claisen (makes a -keto ester). Reverse Claisen (makes two esters). Often occurs using thiol esters in biochemistry (such as acetyl Co-A).

5. Reverse Michael reaction (elimination / dehydration) of -hydroxy carbonyl compounds, an elimination reaction forms ,-unsaturated carbonyl compounds and carries an aldol reaction one step farther. Michael reaction (addition / hydration) adds a nucleophile at the beta carbon of an ,-unsaturated carbonyl compound (usually OH in this game) and adds a proton at the alpha carbon.

6. Hemiacetal (or hemiketal) formation (an addition reaction) of an alcohol to a C=O, forms an ether and an alcohol group on the same carbon. The reverse reaction reforms the carbonyl and alcohol from an elimination reaction. Typical ring sizes in the intramolecular reaction are 5-6 atoms. These transformations are very similar to carbonyl hydration / dehydration, presented in example 2 above.

7. Acetal (or Ketal) formation from a hemiacetal (or hemiketal) makes a water molecule leaving group that is replaced by an alcohol in an SN1 reaction, producing a second ether linkage. In the reverse reaction (acetal or ketal forming a hemiacetal or hemiketal) an alcohol leaving group is replaced by a water molecule in an SN1 reaction. Both are reversible reactions. Because arrows are used in both directions on the same bonds, we show the intermediate in this reaction.

8. a. Oxidation of CH(OH) to C=O with an NAD+ equivalent, which is reduced to NADH (opposite of 8b, below).

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 25 b. Reduction of C=O to CH(OH) with an NADH equivalent, which is oxidized to NAD+ (opposite of 8a, above).

9. a. Oxidation of an amine, CH(NHR), to C=N-R (an imine) with an NAD+ equivalent which forms NADH, followed by imine hydrolysis to a C=O (opposite of 9b, below). b. Formation of an imine, C=N-R, from a C=O, followed by reduction to CH(NHR) (an amine) with an NADH equivalent which forms NAD+ (opposite of 9a, above).

10. Decarboxylation of a -ketocarboxylic acid, liberates CO2 and forms an enol which tautomerizes to a keto group.

11. Decarboxylation of an -ketocarboxylic acid with TPP (thiamine pyrophosphate = diphosphate). The “TPP ylid” adds to an -keto group, liberates CO2 and becomes a “TPP enamine”, which can protonate or react with a C=O of another carbohydrate.

12. a. Phosphorylation of an OH with an ATP equivalent (making a phosphate ester) – common in enzyme signaling b. Dephosphorylation of a phosphate ester to an alcohol and phosphate – common in enzyme signaling c. Making an alcohol OH into a phosphate ester makes it a better leaving group. An elimination (E1 or E2) or substitution (SN2) reaction with a phosphate leaving group is possible. d. Formation of acyl phosphates (mixed anhydrides) also allows for exothermic carbonyl substitution reactions (can make thiol esters).

13. Vit B-6 reactions – Many reactions are possible. The only example shown is 1. imine formation with an -keto acid and the primary amine version of Vit B-6, 2. tautomerization to a different imine, and 3. imine hydrolysis to an amino acid and the aldehyde version of Vit B-6. The imine complex also allows for the loss of various groups on amino acids (the acid part, CO2H, an alpha C-H proton, and certain amino acid side groups, -CH2OH in serine, and -CHCH3OH in threonine). are also seen in Example 9 and -keto acids in Example 11.

14. FAD / FADH2 reduction of C=C to CH-CH or the reverse reaction oxidation of CH-CH to a C=C, FAD can be recharged with NADH.

15. Cytochrom P-450 oxidation of sp3 C-H bonds to make sp3 C-OH groups.

16. Cytochrom P-450 oxidation of C=C pi bonds (alkenes and aromatics) to make epoxides, which can be opened to diols.

17. Cytochrom P-450 oxidation of sulfur and nitrogen lone pairs.

18. Halogenation of sp3 C-H bonds to make C-X groups (X = Cl, Br, I) using Fe halogenase enzymes.

19. Halogenations of aromatic rings using X-OH to make sp2 C-X bonds (X = Cl, Br, I) (thyroxine).

20. S-adenosyl methionine (SAM-e) to methylate biomolecules.

21. Anti-oxidation reactions using vit C, vit E, resveratrol, glutathione, and other bio-antioxidants.

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 26 Many of the steps of biochemical cycles can be explained with the above reactions.

Problem – Use B-H+ / B: and any necessary cofactors to accomplish the following transformations using simplistic mechanisms (you do not need to show lone pairs of electrons and you can combine multiple steps using several arrows). 1. H H H H O O O O reverse reverse those steps, aldol do a forward aldol

(acyl (acyl O O O substitution substitution H H reaction) reaction) 2. H H H H O O O O hemiacetal reverse that reaction formation back go a carbonyl 6 atom ring and an alcohol

(elimination (addition reaction) O O O reaction) H H 3. H H H H O O O O reverse forward Michael Michael (dehydration) (hydration)

O O O (elimination (addition reaction) reaction) H H 4. H H H H O O O O keto/enol tautomerization carbonyl to form an (hydration) aldehyde

(twice) (addition O O O reaction) H H 5. H H H H O O O O keto/enol tautomerization hemiacetal to form a new formation ketone 5 atom ring

(twice) (addition O O O reaction) H H

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 27 6. OH OH O keto/enol tautomerization to form a beta decarboxylation OH keto acid

OH OH keto/enol tautomerization to form an aldehyde

+ NAD carbonyl oxidation (hydration)

(elimination (addition reaction) reaction)

7. OH OH O keto/enol tautomerization reaction with to form an alpha TPP ylid OH keto acid (addition reaction) OH OH decarboxylation to TPP enamine elimination reaction to TPP enamine reaction form new 6C with 2C carbohydrate carbohdrate O and TPP ylid

H (addition OH reaction) 8. OH O O reverse forward Claisen R Claisen O (acyl substitution (acyl substitution OH reaction) reaction)

9. OH OH O NAD+ NADH oxidation reduction to an aldehyde H (addition (elimination OH OH reaction) reaction)

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 28 10. O H acetal H R formation O HO O (2 steps)

(elimination (addition reaction) reaction) HO OH intermediate overall = SN1 11. O R acetal hydrolysis H H to hemiacetal HO O (2 steps) O

(elimination (addition reaction) reaction) HO OH intermediate overall = SN1 12. Vit B-6 O imine formation (2 steps) OH (addition (elimination reaction) reaction) O 13. O

OH hydrolysis of N imine to amino keto/enol acid and the tautomerization aldehyde version to new imine of Vit B-6 (2 steps)

(addition and elimination N reactions)

H 14. H

NR OH O H imine formation NADH (2 steps) reduction H to an amine (addition and OH elimination (addition reactions) reaction)

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 29 15. R H + imine OH N NAD hydrolysis oxidation to an aldehyde to an imine (2 steps) H (addition and (elimination elimination OH reaction) reactions) 16. H O O O P O PP ATP phosphorylation of 3o alcohol withATP O OH (acyl-like substitution reaction) 17. O O elimination P reaction to O form an alkene O alcohol (show as an E2 reaction)

OH

H 18. O O P O O hydrolysis of phosphate ester to di-alcohol

OH (acyl-like substitution H reaction) 19. O O formation of mixed anhydride H O P O PP ATP O O (acyl-like substitution reaction) 20. O O O Claisen O condensation P H Co-A O OH S (acyl-like substitution reaction)

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 30 21. O O NADH reduction of keto group Co-A S (addition reaction)

22. H O O reverse Michael Co-A reaction S (elimination reaction) 23. NADH hydride O reduction of the conjugated C=C Co-A by a Michael S reaction (addition reaction)

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 31 Methylates choline, norepinephrine, DNA (epigenetics)

Biotin (carboxylations)

Lipoic acid (acyl transfer)

Possible reactions

Aldol reverse Aldol Hemi-acetal formation reverse hemi-acetal reaction (includes ketal reactions) Acetal formation reverse acetal reaction (includes ketal reactions) Michael reaction reverse Michael reaction Carbonyl carbonyl Tautomeric changes (keto  enol and/or enol  keto) (enamine chem.?) DNA / RNA base synthesis and degradation Additional possibilities??? Enamine chemistry Imine chemistry Amine oxidation to carbonyl Phosphate ester / anhydride synthesis and hydrolysis (tyrosine, serine, ATP, throxine, etc.) xxxxxxxxxx Lipid chemistry – glycerol esters, , phosphates, carbohydrates

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 32

Forward Direction B BH H H H R R B H H B O OH B O O O O O O O C C lose add ROH H2O

hemiacetal / hemiketal alcohol aldehyde or ketone intermediate acetal / ketal Reverse Direction H B B B BH BH H H B R OH O O H O O O O O C C lose add ROH H2O acetal / ketal intermediate hemiacetal / hemiketal alcohol aldehyde or ketone

Forward Direction B BH H H H R H H B B O B OH O O O O O C C lose H2O

hemiacetal / hemiketal alcohol aldehyde or ketone intermediate add ROH R O O

acetal / ketal

B Reverse Direction H B B BH BH H R OH O O O O O

lose add ROH H2O acetal / ketal intermediate hemiacetal / hemiketal

H B H O O

C C

alcohol aldehyde or ketone y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 33

Problem - What kind of reaction occurred in each part? Use a simplistic mechanism to show how the reaction could have proceeded. The following problems are more limited questions from the original “carbohydrate game” and do not include many of the biochemical “co-factors”.

OH O O O

1 ? H

OH OH OH OH OH OH

O O OH O OH OH

2 ? H

OH OH OH OH OH OH

OH OH O OH OH O OH OH O O HO OH 3 ? 4 ? OH HO OH OH OH HO OH OH OH OH HO OH

O OH O OH HO 5 ?

HO OH OH OH OH O OH O OH OH O OH O OH

H 6 ? 7 ? 8 ?

OH OH OH OH OH OH OH O OH OH O O

9 ? O OH

H 11 ? H 12 ? 10 ?

(2 steps) O OH O O

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 34 H O OH OH OH OH OH OH OH OH O

13 ? 14 ? 2 rotations (2 steps) OH OH OH OH O OH HO OH O OH O

15 ? H H H O O O O OH OH 18 ? 17 ? OH 16 ?

HO OH HO O HO O HO OH O O O O

1. reverse aldol 7. keto/enol tautomerization 13. 2 x keto/enol tautomerization

2. forward aldol 8. reverse aldol 14. reverse Michael reaction 3. hemi-ketal formation to 6 atom ring 9. keto/enol tautomerization 15. intramolecular Michael reaction using the OH of an alcohol group 4. reverse of hemi-ketal to 5 atom ring 10. structure shown below 16. reverse Michael on the other side of the ring 5. reverse of hemi-ketal to form open chain 11. keto/enol tautomerization 17. keto/enol tautomerization

6. reverse Michael reaction 12. 2 x keto/enol tautomerization 18. reverse Michael reaction OH enol at both ends of the , can form a carbonyl on either side, one as a ketone and one OH O as an aldehyde

Problem – Use B-H+ / B: to accomplish the following transformation using simplistic mechanisms (you do not need to show lone pairs of electrons and you can combine multiple steps using several arrows).

OH OH OH reverse aldol to 3C dehydration keto/enol to form aldehyde and 4 carbon (reverse Michael) 1,2-diketo structure 2,3-diketo structure

OH OH O OH

OH OH OH reverse aldol to 1C dehydration keto/enol to form aldehyde and 6 carbon (reverse Michael) 3,4-diketo structure 2,3-diketo structure

OH OH O OH

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 35 OH OH OH

hemi-acetal formation acetal formation to 5 atom ring with ROH

OH OH O OH

OH OH OH

hemi-acetal formation acetal formation to 6 atom ring with ROH

OH OH O OH

OH OH dehydration carbonyl O keto/enol to form to form carbonyl 2-keto structure hydration H

OH OH O H

H O

HO O hemi-acetal ring opening OH HO

OH

O

HO Michael addition cyclic ester ring opening O of water (hydration) addition of water (hydration)

OH OH OH OH OH reverse forward aldol aldol (reverse step)

OH O O H

OH OH OH OH reverse reaction back to hemi-ketal formation carbonyl and alcohol to 6 atom ring

O OH O H

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 36 OH OH HO H OH

dehydration hydration (reverse Michael) (Michael)

O OH O H

OH OH OH OH keto/enol tautomerization reverse H (form an aldehyde) aldol H (2 steps) OH OH O

OH OH HO H OH keto/enol tautomerization reverse aldol to form (form a ketone) a 3 carbon ketone and (2 steps) 4 carbon aldehyde

OH OH O OH OH OH OH

hydration of dehydration of carbonyl carbonyl hydrate

OH OH O

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 37 A few possible answers OH OH OH OH OH OH OH OH H reverse H forward aldol OH OH OH OH aldol H (reverse steps) OH O O OH O O H B H B OH O O BH B H

OH OH OH OH OH OH hemiacetal reverse reaction formation OH OH OH OH OH (6 atom ring) back to carbonyl & alcohol O OH O O OH B H H B BH O OH O O OH H H B

B H B OH OH HO H OH OH OH OH OH OH OH HO H OH dehydration hydration (reverse Michael) (Michael) OH OH O OH O OH OH O H B B HOH

B OH OH OH OH H H OH OH OH O OH OH OH O H keto/enol H OH OH O B tautomerization (form an aldehyde) OH OH O OH OH O H BH H BH H

B B OH OH HO H OH H OH OH O OH keto/enol OH OH O OH tautomerization (form a new ketone) OH OH O OH OH OH BH H B OH OH OH

OH OH OH OH OH OH OH OH hydration of reverse reaction carbonyl dehydration of OH OH OH OH OH OH OH O OH OH O HOH H OH OH O B B BH H B

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 38 Problem - State what type of transformation occurred and show a simplistic arrow pushing mechanism for how it occurred, adding in any B: and/or B-H+ that is necessary. (an older problem set)

a. OH OH OH OH OOH O O H H H H H O OH OH O OH O OH O OH

b. OH OH OH OH OH OH OH HO OH HO O OH O OH OH O OH c. OH OH O OH OH OH OH OH OH H

OH OH OH OH OH OH OH OH O

d. OH OH OH OH HO HO HO HO O O O O HO H O O OH O O OH OH OH O e. OH OH OH OH HO OH OH OH H H HO OH OH O OH O OOOH O f. HO H R R OH OH O OH H H O OH O OH O HO HO O HO O O

OH OH OH OH OH HO HO OH HO intermediate g. HO OH R O OH OH O OH H HO R OH HO H O H HO O O O O HO OH OH OH HO HO OH OH intermediate OH h. R OH H O R H H HO OH HO O OH HO O O HO OH OH O O O HO HO HO OH OH OH OH OH intermediate OH i. OH OH OH OH OH OH OH H H HO H O H H

O HO OH OH OO OO O y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 39

Partial Answers

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc 40 a. OH OH OH OH OOH O O H H H H H reverse reverse O OH OH O OH tautomerism O OH O Michael aldol OH

b. OH OH OH OH OH OH OH HO OH HO Michael hemi-acetal O OH O OH OH formation O OH c. OH OH O OH OH OH OH OH OH H tautomerism tautomerism OH OH OH OH OH OH OH OH O

d. OH OH OH OH HO HO HO HO O O O O H O HO reverse O OH dehydration O tautomerism O OH Michael OH OH O e. OH OH OH OH HO OH OH OH H H HO OH aldol OH reverse O OH O aldol OOOH O f. HO H R R OH OH O OH H H O OH O OH O HO HO O HO O O hemi-acetal acetal acetal OH OH OH OH formation formation OH formation HO HO OH HO (2 steps) intermediate (2 steps) g. HO OH R O OH OH O OH H HO R OH HO H O H O HO O O O hemi-acetal acetal HO acetal OH OH OH formation HO HO formation OH formation OH (2 steps) intermediate (2 steps) OH h. R OH H HO O R H H OH HO O OH HO O O HO OH OH O O O reverse acetal reverse acetal HO formation reverse HO formation HO OH (2 steps) hemi-acetal OH OH OH (2 steps) OH intermediate OH formation

i. OH OH OH OH OH H OH OH HO H H O H H reverse carbonyl carbonyl O HO OH OH aldol OOhydration dehydration OO O

y:\files\classes\Organic Hunger Games\Bio-Org Game newer.doc

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