Bio-Organic Mechanism Game - Cal Poly Pomonapsbeauchamp/pdf/bio_org_game.pdf · Bio-Organic Mechanism Game – Simplistic biochemical structures and simplistic organic reaction mechanisms

1

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

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

Many biomolecules 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 chemistry 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 – adenosine triphosphate – phosphorylation, energy source

N

NN

N

NH2

O

OHOH

HH

HH

P

O

O

OP

O

OP

O

O

O O

CH2O=

ATPOP

O

O

O

simplified structure actual structure

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

N

NN

N

NH2

O

OHOH

HH

HH

P

O

O

OP

O

OH2C

O

CH2O

=

O

OH HO

N

CONH2

N

R

NADP+ has a phosphate here.

actual structure

simplified structure

2


3. NADH and NADHP - nicotinamide adenine dinucleotide (hydride donor)

N

NN

N

NH2

O

OHOH

HH

HH

P

O

O

OP

O

OH2C

O

CH2O

O

OH HO

N

CONH2

N

R

NADPH has a phosphate here.

HHHH

=


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

NH2

N

H

O

N

H

H


actual structure

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

NH2

N

H

O3POO

H


actual structure

O

N

H

H

O3POO

H

-2

interconvert via imine, tautomers, hydrolysis

-2

==

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

N

N

NH2

P

O

O

OP

O

O

O

CH2OS

N

HS

N

H

R

actual structure


B

S

N

R

ylid formof TPP

=

3


6. Coenzyme A (acyl transfer)

O P

O

O

O P

O

O

OO

HOOH

N

N

NN

NH2

OH

O

NH

O

NH

S

Thiol esters form here.O

S

simplified structure of acetyl Co-A

All of this is "Co-A"

actual structuresimplified

structure of CoA

This is an acetyl group

H

CoAH

SCoA

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.)

N

N

NH

N

O

O

OH

HOOH

O P

O

O

O P

O

O

O

O

HOOH

N

N

NN

NH2

FAD - flavin dinucleotide (a hydride acceptor)

actual structure

simplified structures for FAD and FADH2

N

N

FAD FADH2

N

N

H

H

4


N

N

FADflavin dinucleotide

(a hydride acceptor)

BH

FADH2flavin dinucleotide(a hydride donor)

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

BH

C CC

H

C

H

N

N

H

H

B

C

H

C

H

B

N

N

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

NH

N

N

N

O

H2N

R

H

HN

O

HN

CO2

CO2

Tetrahydrofolate (THF)one carbon transfers as "CH3", "CH2".

H

One carbon groupscan bond here in various ways (see below structures.simplified

structure

actual structure

NH

HNAr

N

RV22-p762

5

10

One glutamate is shown, but severalcan be attached.=

5


=

N

HNAr

N

Rdifferent forms

CH3

N5-methyl THF

N

NAr

N

R

5

10

N5,N10-methylene THF

H2C

N

NAr

N

R

5

10

N5,N10-methenyl THF

HC

NH

NAr

N

R

5

10

N10-formyl THF

O

H

N

HNAr

N

R

5

10

N5-formyl THFH O

N

HNAr

N

R

5

10

N5-formimino THF

H NH

THF

glycineor

serine

NAD+

NADH

NADP+

NADPH

+H2O

-H2O

+H2O-H2O+NH3

-NH3

histidine

THF

THF

HCO2ATP

9. SAM = S-adenosylmethionine (methyl transfer agent), The methyl group (CH3) attached to the methionine sulfur atom 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 acids, proteins, lipids and secondary metabolites. SAM can be made from methionine and N5-methyl THF (just above).

O

OH

NH2

S

CH3

O N

OHHO

N

NN

NH2

SAM = S-adenosylmethionine

RmetS

Rad

CH3

methionine

adenosinesimplified structure actual structure

SAM = S-adenosylmethionine

=

leaving group was triphosphate

Rmet = methionineRad = adenonine

O

OH

NH2

S

CH3

O N

OHHO

N

NN

NH2

OP

OP

OP

O

O O O O O O

methionineATP

6


10. Cytochrom P-450 enzymes are oxidizing agents in the body. They can convert inert alkane sp3 C-H bonds into C-OH bonds and they can make epoxide groups at alkenes and aromatic pi bonds.

Fe

N

N

N

N

HO2C

CO2H

N

N

N

N

Fe+3


+3

Enz

S S

Enz

N

N

N

N

Fe

heme, protoporphyrin IX, found in cytochrom P-450 oxidative enzymes


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


Fe

O

+4 Fe+3

This is the structure that we will use.

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

Cl

Fe+3

NCl

N

N

N

Fe+4

Halogenations in the body often iron halogen bonds.


Fe

O

+4 +3

This is the structure that we will use.

N

N

N

His

His

His

ON

N

O

Cl Fe

OH

Biochemical Reaction Mechanism Examples

Mechanism arrows used in the “Bio-Org Game” are meant to suggest how the electrons move over a single transformation, and are not necessarily meant to imply that all of the electrons and atoms 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 electron movement involving making and breaking bonds. Lone pairs are rarely drawn (or used). They are included on the generic base (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,

7


resonance stabilization in acetal 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” arrow pushing mechanism, showing keto enol 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.

C

O

CH C

O

CH

H

C

O

CH

H

resonance

C

O

C

H

B

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

BH

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).

C

O

CH

B

= general base,possibly on enzyme

= general acid, possibly on enzyme

B

C

O

C

H

keto tautomer enol tautomer

BBH

BHBH

8


C

O

C

H

enol tautomer

C

O

CH

keto tautomer

B

B

BH

BH

Quite often in biochemistry 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 chemists 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 addition reaction of H2O to a carbonyl group. This also requires some proton tranfers. A carbonyl hydrate can be dehydrated via an elimination reaction 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 aldol reaction (example 3).

C

O

aldehyde or ketone carbonyl hydrate

B

Forward Direction

O H

H

hydration

C

O

H

OH

B

(addition reaction)

B H BH

Reverse Direction

carbonyl hydrate

C

O

H

OH

BH

B

dehydration

C

O

aldehyde or ketone

B

O H

H

(elimination reaction)

B H

3. Aldol reactions make a new carbon-carbon bond, forming a -hydroxycarbonyl compound. A

carbonyl C position becomes the nucleophile (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)

9


C

O

B

carbonyl electrophile

C

O

CH

carbonyl nucleophile at C position

C

OH

CC

O

B

-hydroxycarbonyl

This product also looks like a Michael reaction. See Example 5.

aldol(addition reaction)

B H BH

Forward Aldol

C

OH

CC

O

B

-hydroxycarbonyl

C

O

C

O

CH

B reverse aldol(elimination reaction)

BH B HReverse Aldol

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). Ester groups are common in organic chemistry 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.

C

O

OR

B

carbonyl electrophile with a leaving group, (often and ester or thiol ester or a mixed anhydride)

ORC

O

CH

carbonyl nucleophile

C

O

CC

O

OR

B

-ketocarbonyl

R OH

Claisen (acyl substitution)

alcohol or thiol ester or

thiol ester

BHBH

Forward Claisen

C

O

CC

O

OR

B

R OH B

ORC

O

CH

C

O

OR

-ketocarbonyl

carbonyl electrophile with a leaving group,(often and ester or thiol ester)

reverse Claisen (acyl substitution)

alcohol or thiol

ester or thiol ester

BH

BH

Reverse Claisen

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 alcohols instead of water. Other possibilities also exist.

C

OH

CC

O

B

-hydroxycarbonyl

C

O

CC

BH -unsaturated carbonyl

(Michael acceptor)

reverse Michael reaction

OH

H

(elimination)

B H

BH

Reverse Michael Reaction

Reverse Michael Reaction

C

O

CC

-unsaturated carbonyl (Michael acceptor)

OH

HB

C

O

CC

O

H

H

-hydroxycarbonyl B

This product looks like an aldol product. See Example 3. It is now able to do a reverse aldol, or be oxidized to a 1,3-dicarbonyl, maybe followed by decarboxylation, etc.

Michael reaction

(addition)

BH

B H

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 intramolecular reaction and typically forms rings of 5 or 6 atoms.

C

OH

C

O

aldehyde or ketone

B

OO

H

hemiacetal / hemiketalalcohol

Forward Direction

addition reaction

B

BHB H

11


OO

H

hemiacetal / hemiketal

B

C

OH

C

O

alcohol aldehyde or ketone

Reverse Direction

elimination reaction

BB HBH

7. Acetal (or ketal) formation from a hemiacetal (or hemiketal). The “OH” becomes a water

molecule leaving group that is replaced by an “OR” in an SN1 reaction, producing an 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. These are reversible reactions that require acid catalysis. 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 + glucose = lactose). Such linkages can go on for hundreds of sugar molecules (glycogen in animals and cellulose in plants).

OO

H


O H

HB

O

OH R

intermediate

Forward Direction

OO

R

acetal / ketal

addROH

eliminate H2O

B H

OO

R

acetal / ketal

O

intermediate

O H

H B

OO

H


Reverse Direction

eliminate ROH

addH2O

B H B H

8. a. Oxidation of CH(OH) to C=O (1o ROH aldehyde, 2o ROH ketone, hydrated aldehyde

carboxylic acid) 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).

12


1o or 2o alcohol aldehyde or ketone

oxidation of an alcohol

B

C

OH

H N

H

R

NAD+

equivalent

C O

N

H H

R

NADHequivalent


B H

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.

aldehyde or ketone

C

O

N

H H

R

NADHequivalent

reduction of a carbonyl

N

H

R NAD+

equivalent

C

O H

H

B

1o or 2o alcohol

additionreaction

B H

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.

amine imine

oxidation of an amine

C

NH

H N

H

R NAD+

equivalent

C NN

H H

R NADHequivalent

R

R

Oxidation of an amine


B

BH

13


Hydrolysis of an imine

C

NR

imine

O H

HB

C

N O

HH

R

aminal

B

C

ONR

H

H

B

aldehyde or ketone

additionreaction


B HB H

BH

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

C

N O

HH

R

aminal

C

ONR

H

H

aldehyde or ketone

BB

C

NR

O H

H

B

imine

additionreaction


BH BHBH

amineimine

reduction of an imine

B

C

N

H

H

N

H

R

NAD+

equivalent

C

N

N

H H

R NADHequivalent

R

R

Reduction of an imine

additionreaction

B H

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

C

O

C

H

enol tautomer

C

O

CH

keto tautomer

B

BC

O

CC

-ketocarboxylic acid

O

O

C

O

O

carbon dioxide

decarboxylation tautomers

HB BH

BH

BH

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 carbohydrate 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.

S

N

H

R

B

pKa 18

S

N

R

TPP ylid-ketoacid

C O

CO

OH

R

S

N

C

R

OH

R

C

O O

S

N

C

R

OH

R

TPP enamine

TPP reaction with an a-ketoacid, decarboxylation and formation of enamine.

decarboxylation(-CO2)

H B

TPP TPP ylid addition to a carbonyl

BH

Reaction of enamine nucleophile with a carbonyl electrophile followed by an E2-like reaction to from a new (larger) carbohydrate.

S

N

C

R

OH

R

C

O

H RS

N

C

R

OH

R

C

OH

R

H

B

S

N

R

CR

O

CH

OH

CH

OH

R

TPP ylid

a new (larger) carbohydrate

TPP enamine reaction with C=O

TPP enamine addition to a carbonyl

elimination reaction forms a carbonyl group, similar to a reverse aldol

BH

15


Reaction of enamine nucleophile with an acid to from a new (smaller) carbohydrate.

S

N

C

R

OH

R S

N

C

R

OH

R

B

S

N

R

CR

O

H

TPP ylida new (smaller) carbohydrate

H

TPP enamine reaction with a proton

elimination reaction forms a carbonyl group, similar to a reverse aldol

acid/baseprotonation of TPP enamine

BH

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

C O

H

P

O

O

O

O P

O

O

O P

O

O

O ATP

B

BH

O P

O

O

O P

O

O

O ADP

C O P

O

O

O

Mg+2

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

Mg+2 acyl-like substitution reaction

inorganic phosphate ester

ADP = leaving group

b. Dephosphorylation of an inorganic phosphate ester to an alcohol and phosphate.

C O P

O

O

O

B

OH

H

B H

C O P

O

O

OH

O

H

B

acyl-like substitution reaction

inorganic ester hydrolysis

B H

c. An elimination reaction of a phosphate leaving group to make an alkene (pi bond). Could actually be E1 or E2.

CC

H

OP

O

O

O

B

H

CC

OP

O

O

O

H

E1 or E2 (anti) mechanismsare possible

B H

16


d. acyl phosphate (inorganic anhydride) and formation of a thiol ester (like acetyl Co-A)

C O

H

P

O

O

O

O ATP

B

O P

O

O

O P

O

O

O ADP

C O P

O

O

O

Mg+2


O

H3C

O

H3C

organic-inorganic anhydride

Co-A S

HB


O

SCo-A

very reactive thiol ester(acetyl Co-A)

O P

O

O

O

ADP leaving group

phosphate leaving group

Mg+2

Mg+2

BH

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).

N

N

H vitamin B-6(1o amine version)

H

B

BHdehydration (-H2O)

iminesynthesis

H

B

OH

OO

R

-keto acid

N

N

H

H

R

OH

O

OH

N

N

H

R

OH

O

HO

HSimilar to Example 9b.

B H

17


N

N

H

R

OH

O

keto / enol tautomerization, makes imine on the other side

H

HB

N

N

H

R

OH

O

H

Similar to Example 1.

B H

addition of water

HO

H

B

hydrolysis of imine

N

N

H

R

OH

O

H

N

N

H

R

OH

O

HH

O

H

B

N

H2N

H

R

OH

O

H

O H

vitamin B-6(aldehyde version)

-amino acid

Similar to Example 9a.

BHBHH

18


Three additional vit B6 reactions from aromatic imine 1. Decarboxylation

decarboxylation

N

N

H

O

O

H B

R H

N

N

H

R

H

CO O

BH

N

N

H

R

HH

HO

H

B

BHH

N

N

H

R

O

H H

BBH

N

N

H

R

O HH

H

19


2. Elimination of side R group, like serine or threonine.

decarboxylation

N

N

H

O

vitamin B-6(aldehyde version)

H B

N

N

H

CO2H

H2C O

BH

N

N

H

CO2H

HO

H

B

BHH

N

N

H

CO2H

O

H H

BBH

N

N

H

CO2H

O HH

H

CO2H

serine aa

glycine aa

vitamin B-6(imine version)

also threonine aa

N

N

H

OH

CO2H

N

CO2H

H

H

glycine aa

H

O

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

N

N

H

B

N

N

H

CO2H

BH

CO2H

S amino acid


R

H

R

N

N

H

CO2H

H

RR amino acid


proton adds on the opposite face

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.

N

N

H

H

FADH2

BH

B

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

N

N

FAD

R

O

SCoA

R

O

SCoA

HH

H H

B H

B

R

N

HH

R

N

N

N

FADB H

N

N

H

H

FADH2

NAD+NADH

FAD - flavin dinucleotide (a hydride acceptor) used

to reoxidize fats for energy.

CC

O

SEnzC

R

H H

HH

FADH2 - flavin dinucleotide

(a hydride donor)

B

A saturated fatty acid chain.

CC

O

SEnzC

R

H

H

An ,-unsaturated fatty acid chain.

BH

N

N

N

N

H

H

FADH2

21


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

The free radical-like oxygen atom abstracts a hydrogen atom from a C-H bond in the enzyme cavity, forming an O-H bond and a carbon free radical.

CCO

H

Fe +4

O CH

Fe +4

OH

The carbon free radical abstracts hydroxyl (OH) from iron, making an C-OH bond where a C-H bond had been. The iron is reduced back at Fe+3 to begin the process all over again.

Fe +3

sp3 C-H bonds alcohols

89

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

be opened to diols.

The free radical-like oxygen atom adds to a C=C bond (alkene or aromatic) in the enzyme cavity, forming a O-C bond and a carbon free radical.

Fe +4

O

Fe +4

O

The carbon free radical abstracts the oxygen atom from the iron, making an epoxide ring. The iron is reduced back at Fe+3 to begin the process all over again. Reactiveepoxides can be opened up to diols (more water soluble).

Fe +3

CC

O

R

RRR

epoxides

C C

R

R

R

R

CR

R

C

R

R

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

R

SR

sulfursubstrate

(1e-)

R

SR

sulfursubstrate

S

RR

O

sulfoxides, further oxidation is possible, all the way to sulfate, SO4

-2

R

NR

N

RR/H

O

N-oxides, further oxidation is possible, all the way to nitrate, NO3

-1

R

NR

nitrogensubstrate

(1e-)

R/H R/H

R

Fe +4

O

Fe +4

OFe +3

Fe +4

O

Fe +4

O Fe +3nitrogensubstrate

22


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

The free radical-like oxygen atom abstracts a hydrogen atom from a C-H bond in the enzyme cavity, forming an O-H bond and a carbon free radical.

C

C Cl

Fe +5

O CH

Fe +5

OH

The carbon free radical abstracts a halogen (Cl or Br) forming an unusal halogenated bioorganic molecule.

sp3 C-H bonds

Cl ClFe

OH

+4

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

OOH

HI

BH

IO

H

HO

H

B

P

O

O

O

O ATP P

O

O

O

O

I

possibly an even better leaving group

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).

a good leaving group on iodine

CO2H

OH

tyrosine

NH2

IO

H

CO2H

OH NH2

I

H

B

CO2H

OH NH2

ICO2H

OH NH2

I

repeat

I

Speculative mechanism for iodinating tyrosine and formation of thyroxine from two tyrosines.

diiodotyrosine - makes thryoxine

23


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

N

N

O

NH2

H1

2

34

5

6

cytosine

Rmet

S

Rad

CH3

H

N

N

O

NH2

H

H

H3CB

N

N

O

NH2

H3C

H

5-methylcytosine

21. Anti-oxidation reactions using vit E (fat soluble), vit C (water soluble), (also possible are

glutathione, resveratrol and other bio-antioxidants).

free radical protection by vitamin E (possibly in cell membrane)

R

O

ROH

vitamin E located in cell membranes quenches radicals

OR

ROH

OR

ROH

O

O

O

O

H

H R

OR

ROH

vitamin E is recharged and still in cell membrane

O

O

O

O

H

H R

O

O

O

O

H

H

O

O

O

O

H

RR

R

Bprotects a second time

O

O

O

O R

oxidized vitamin C form washes out of the body

R R H

H B

reduced radical neutralized by body's buffer system

resonance and inductive effects stabilize radical so it does not do damage

vitamin C reduces vitamin E back to normal and ultimately washes out of the body

B

R

possible dangerous free radical reduced by vit E

R R H

H B

24


other possible anti-oxidants OH

OH

HO

N

O

SH

N

O

O

OH

NH2 O

OH

glycinecysteineglutaric acid ( linkage)

glutathione

resveratrol

H

H

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).

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). Imines 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.

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.

O O

O O

O

O

O

reverse aldol

reverse those steps, do a forward aldol

(acyl substitution reaction)


HHHH

H H

2.

O O

O O

O

O

OHHHH

H H

hemiacetal formation6 atom ring

reverse that reactionback go a carbonyl and an alcohol

(addition reaction)


3.

O O

O O

O

O

OHHHH

H H

reverse Michael(dehydration)

forward Michael(hydration)


(addition reaction)

4.

O O

O O

O

O

OHHHH

H H

keto/enol tautomerizationto form an aldehyde

carbonyl (hydration)

(addition reaction)

(twice)

5.

O O

O O

O

O

OHHHH

H H

keto/enol tautomerizationto form a new ketone

hemiacetal formation5 atom ring

(twice) (addition reaction)

27


6. OH

OH

OH

OH

O keto/enol tautomerizationto form a beta keto acid

decarboxylationOH

NAD+

oxidation

keto/enol tautomerizationto form an aldehyde

carbonyl (hydration)

(addition reaction)


7. OH

OH

OH

OH

O keto/enol tautomerizationto form an alpha keto acid

reaction with TPP ylid

OH

elimination reaction to form new 6C carbohdrate and TPP ylid

decarboxylationto TPP enamine

TPP enamine reaction with 2C carbohydrate

O

H

OH

(addition reaction)

(addition reaction)

8. OH

OH

O Oreverse Claisen

forward Claisen

OR



9. OH

OH

OH ONADH reduction

H

OH

NAD+

oxidation to an aldehyde

(addition reaction) (elimination

reaction)

28


10. acetal formation (2 steps)

O

OHO

HO OH intermediate overall = SN1


(addition reaction)

H

OH R

11. O

OHO

HO OH intermediate overall = SN1


(addition reaction)

R

OH H

acetal hydrolysis to hemiacetal (2 steps)

12. Vit B-6 imine formation(2 steps)

O

OH

O

(addition reaction)


13.

keto/enoltautomerization to new imine

O

OH

N

N

H

hydrolysis of imine to amino acid and the aldehyde version of Vit B-6 (2 steps)

(addition and elimination reactions)

14.

O

H

OH

imine formation (2 steps)

OHN R

H

H NADH reduction to an amine


(addition reaction)

29


15.

N

H

OH

imine hydrolysisto an aldehyde (2 steps)

OH NAD+

oxidation to an imine

R H



16.

P

O

O

O

O PP ATPO

OH

phosphorylation of 3o alcohol withATP

H

(acyl-like substitution reaction)

17.

O

OH

P

O O

O

H

elimination reaction to form an alkenealcohol (show as an E2 reaction)

18.

O

OH

P

O O

O

H

hydrolysis of phosphate ester to di-alcohol


19.

P

O

O

O

O PP ATP

formation of mixed anhydride

O

OH


20. O

SCo-AH

Claisen condensation

O

OP

OO

OH (acyl-like substitution reaction)

30


21. O

SCo-A

O NADH reduction of keto group

(addition reaction)

22.

O

SCo-A

Oreverse Michaelreaction

H


23.

O

SCo-A

NADH hydride reduction of the conjugated C=C by a Michael reaction

(addition reaction)

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 hydration reaction carbonyl dehydration reaction

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, ethers, phosphates, carbohydrates

32


C

OH

C

OB

aldehyde or ketone

B BH

OO

H


B H

O H

HB

O

OH R

intermediate

OO

R

acetal / ketal

B H B

O

intermediate

O H

H B

OO

H


BB H

C

OH

C

O


Forward Direction

Reverse Direction

loseROH

addH2O

BH

OO

R

acetal / ketal

addROH

loseH2O

C

OH

C

OB

aldehyde or ketone

B BH

OO

H


B H

O H

HB

O

OH R

intermediate

OO

R

acetal / ketal

B H B

O

intermediate

O H

H B

OO

H


B

B H

C

OH

C

O


Forward Direction

Reverse Direction

loseROH

addH2O

BH

OO

R

acetal / ketal

addROH

loseH2O

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”.

O

OH OHOH

OH

1 ?

O

OH OHOH

O

H

O

OH OH OH

O

H

OH

2 ?

O

OHOH OH

OH OH

O

OHOH OH

OH OH

3 ?

OOH

OH

OHHO

HO

O

OHOH OH

OH OH

4 ?

O

OH

OH

OHHO

HO

O

OH

OHHO

HO 5 ?

O

OH OHOH

OH

O

OHOH OH

OH OH O

OHOH OH

OH

6 ? 7 ?

O

OOH OH

OH O

OOH O

OH

H8 ?

9 ?

10 ?11 ?

OH

OO

H12 ?

O

OHO

H

(2 steps)

34


O

OHOH OH

OH OH

13 ?

OH

OOH OH

OH OH

(2 steps)

14 ?

OH

O OH

OH OH

2rotations

O

H

OH

O

HO

15 ?

OOH

OH

O

HO

O

H

HO

O

OH

OH

O

H

HO

O

O

OHO

H

HO

O

O

16 ?17 ?18 ?

1. reverse aldol

2. forward aldol

3. hemi-ketal formation to 6 atom ring

4. reverse of hemi-ketal to 5 atom ring

5. reverse of hemi-ketal to form open chain

6. reverse Michael reaction

7. keto/enol tautomerization

8. reverse aldol


10. structure shown below


12. 2 x keto/enol tautomerization

13. 2 x keto/enol tautomerization


15. intramolecular Michael reaction using the OH of an alcohol group16. reverse Michael on the other side of the ring17. keto/enol tautomerization


OH

OH O

enol at both ends of the double bond, can form a carbonyl on either side, one as a ketone and one 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

OHOH O

OH OH

OH

dehydration (reverse Michael)

keto/enol to form1,2-diketo structure

reverse aldol to 3C aldehyde and 4 carbon

2,3-diketo structure

OH

OHOH O

OH OH

OH


keto/enol to form3,4-diketo structure

reverse aldol to 1C aldehyde and 6 carbon

2,3-diketo structure

35


OH

OHOH O

OH OH

OH

hemi-acetal formation to 5 atom ring

acetal formationwith ROH

OH

OHOH O

OH OH

OH

hemi-acetal formation to 6 atom ring

acetal formationwith ROH

OH

OHOH O

OH

OH

H

dehydration to form carbonyl

keto/enol to form2-keto structure

carbonylhydration

O

O

HO

HO

OH

OH

H

hemi-acetalring opening

O

O

HO

OH

Michael addition of water (hydration)

cyclic ester ring opening addition of water (hydration)

OH OH

OH OH

OH

O

OH

reversealdol

forward aldol(reverse step)

OH OH

O OH

OH

O

OH

hemi-ketal formation to 6 atom ring

reverse reaction back tocarbonyl and alcohol

H

36


OH OH

O OH O

OH


hydration(Michael)

H

HO H

OH OH

OH OH O

OHketo/enol

tautomerization(form an aldehyde)

(2 steps)

reversealdol

OH

H

H

OH OH

OH OH O

OHreverse aldol to form a 3 carbon ketone and

4 carbon aldehyde

HO H keto/enoltautomerization(form a ketone)

(2 steps)

OH OH

OH OH O

OH

dehydration of carbonyl hydrate

hydration ofcarbonyl

OH

37


A few possible answers OH

OH

OH

O

OH

O

OH

H

reverse aldol

forward aldol(reverse steps)

B BH

OH

OH

OH

O

H

OH

O

OH

H

H

BB H

OH

OH

OH

O

OH

O

OH

H

hemiacetal formation(6 atom ring)

OH

O

OH

OH

OH

O

OH

HO

OH OH

OH

OH

O OHH

B BH

reverse reactionback to carbonyl & alcohol

B

B H

OH

O

OH

OH

OH

O

OH

H

OH

OH

OH

OH O

OHHO H dehydration(reverse Michael)

B

BH

hydration(Michael)

OH

OH

OH

O

OHOH

H OHB

BHOH

OH

OH

OH O

OHHO H

keto/enol tautomerization(form an aldehyde)

OH

OH

OH

OH O

OHOHH

HB

B H

OH

OH

OH

OH O

OOH

H

HB

B H

OH

OH

OH

OH O

OOH

H

HH

hydration of carbonyl

reverse reactiondehydration ofdiol

OH

OH

OH

OH O

OHOH

H OHBB H

OH

OH

OH

OH

OHOH

O

H

OH

BBH

OH

OH

OH

OH O

OHOH

keto/enol tautomerization(form a new ketone)

OH

OH

OH

OH O

OHHO H

B H

B

OH

OH

OH

OH

OHO

H

OH

B

BH

OH

OH

OH

OH

OHO

OH

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.

H

O

OH

OH

OH

OH

H

O

OH OH

OH

H

O

O OH

OH

H

O

O O

OH

H

b.

O

OH OH

OH

OH

O

OH OH

OH

OH

OH OOH

OH

OHHO

HO

c.O

OHOH

OH

OH

OH OH

OHOH

OH

OH

OH

OHOH

OH

O

OH OH

H

d.

e.

f.

g.

h.

OOH

O

OHHO

HOOH

OH

O

OHHO

OH

OH

O

OHHO

OO

O

OHHO

O

O

OH OH

O

OH

H

OHO

HO

OH

OHHO

OH

H

O OOH

OH

OHOH

O

OHOH

OH

OH

OH

O

HOOH

HO OH

HO

O

OHOH

OH

OH

OH

O

HOOH

OH

HO

HO

O

OH

HO OH

HO

OR

OH H

O

OH

HO OH

HOO

R H

intermediate

OH H

OR HO

OH

OH

HO

HO

O

OOH

OH

HO

HO

R

intermediate

O

HOOH

OH

HO

HOO

OOH

OH

HO

HO

R

OR H

O

OH

OH

HO

HO

intermediate

OH H

O

OHOH

OH

OH

OH

O

HO

OH

OH

i.

O O

H

OHOH

OH H

O

H

OHOH

OHHOO O

H

OHOH

39


Partial Answers

40


a.

H

O

OH

OH

OH

OH

H

O

OH OH

OH

H

O

O OH

OH

H

O

O O

OH

H reverseMichael tautomerism reverse

aldol

b.

O

OH OH

OH

OH

Michael O

OH OH

OH

OH

OH OOH

OH

OHHO

HOhemi-acetal formation

c.O

OHOH

OH

OH

OH

tautomerism

OH

OHOH

OH

OH

OH

tautomerismOHOH

OH

O

OH OH

H

d.

e.

f.

g.

h.

OOH

O

OHHO

HOOH

OH

O

OHHO

OH

OH

O

OHHO

OO

O

OHHO

O reverseMichael

dehydration tautomerism

O

OH OH

O

OH

H

OHO

HO

OH

OHHO

OH

aldol reverse aldol

H

O OOH

OH

OHOH

O

OHOH

OH

OH

OH

hemi-acetal formation

O

HOOH

HO OH

HO

O

OHOH

OH

OH

OH

hemi-acetal formation

O

HOOH

OH

HO

HO

O

OH

HO OH

HO

OR

OH H

O

OH

HO OH

HOO

R H

intermediate

OH H

OR HO

OH

OH

HO

HO

O

OOH

OH

HO

HO

R

intermediate

acetal formation (2 steps)




O

HOOH

OH

HO

HO

reverse acetal formation (2 steps)

O

OOH

OH

HO

HO

R

OR H

O

OH

OH

HO

HO

intermediate

reverse acetal formation (2 steps)

OH H

reverse hemi-acetal formation

O

OHOH

OH

OH

OH

O

HO

OH

OH

reverse aldol

i.

O O

H

OHOH

OH H

O

H

OHOH

OHHOcarbonylhydration

carbonyldehydration O O

H

OHOH

Documents

Bio-Organic Mechanism Game - Cal Poly Pomonapsbeauchamp/pdf/bio_org_game.pdf · Bio-Organic Mechanism Game – Simplistic biochemical structures and simplistic organic reaction mechanisms