Download Chapter 16. Biological Reagents

CHAPTER 16
BIOLOGICAL REAGENTS
Reactions that occur in living systems require a very defined set of reaction
conditions (aqueous, dilute, pH 7) and reagents. The reagents, also called cofactors
or coenzymes, combine with the enzyme and substrate (and sometimes metal ions)
to form an enzyme substrate complex that converts the substrate to products. Both
the enzyme and the cofactor can be reused for further reactions, perhaps after some
modification. Some biological reactions synthesize new larger molecules in a
process called anabolism. Metabolism is the breakdown of large molecules and the
process is called catabolism.
16.1 Anhydride Reactions
A common reaction found in organic systems and discussed the chapter on
carboxylic acids is the reaction of an alcohol with an anhydride to form an ester.
Acetic anhydride, which is a dehydrated form of acetic acid, is considered a high
energy molecule because it is ready to react with hydroxyl functions and return to
acetic acid. Thus the reaction occurs readily in the organic laboratory.
O
O
CH3-C
+ CH3COOH
CH3-C-O
O + HO
CH3-C
O
Living systems perform a similar task but usually instead of using
anhydrides of carboxylic acid, anhydrides of phosphoric acid are used. Phosphoric
acid may be dehydrated to polyphosphoric acids, of which triphosphoric acid is
shown below. These are used in organic chemistry as dehydrating and
phosphorolating agents.
OH
OH
OH
O
O
O
O
- 2 H2O
3 H3PO4
H
P
P
P
H
O
O
O
Most living systems cannot use such a highly reactive substance as
triphosphoric acid, but living systems use a reagent called adenosine triphosphate
(ATP). ATP is used to make phosphates of molecules in the body so that the
314 Ch 16 Biological Reagents
phosphate can be used as a substrate for an enzyme reaction. Below is a sketch of
the first reaction in the metabolism of glucose, which ultimately yields carbon
dioxide and water. Glucose is not a substrate for the metabolism and it must be
converted to the higher energy phosphate in order for enzymes to use it. Thus
glucose-6-phosphate is generated and used further in the metabolism scheme (not
shown here). The biological reagent ATP functions as an anhydride of phosphoric
acid and delivers a phosphate to the glucose and gives up the energy in the process.
The ATP is converted into ADP. ADP can phosphorolate other molecules and
produces adenosine monophosphate (AMP), or it can be reconverted to ATP.
Nearly all biological reagents can be recycled in living systems.
NH2
N
N
HOH H
O
O
O
N N
O
HO
H
H
O P O P O P O
OH
O
enzyme
HO
O
O
O
H OH
H
H
H
H
Mg+2
HO OH
β-D-glucose
Adenosine triphosphate
ATP
NH2
N
N
O
O
O
N N
HO P-O
H
H
H
O
O P O P O
O
O
HO
O
+
O
OH
H
HO
H
Mg+2 HO OH
H OH
H
H
Adenosine diphosphate
β-D-glucose-6-phosphate
ADP
A model of ATP without hydrogen atoms is shown below.
16.1 Anhydride Reactions 315
A sketch of the reaction proceeding through an enzyme-substrate complex is
shown below.
OO
ATP
O
O
-2 Mg+2
O
O
O
OPO3
O
O
OO O
O O
OH
O O O
O
O
glucose
O O O
O
NH2
O O
O O
O O
O
O
O
O
O O
O O O
O
O
O
O
COOH
O
ATP Mg+2
H OH
HO
HO
H
O
HO P-O
O HO
O
H OH
H
H
β-D-glucose
OH
HO
HO
+ ADP
OH
H
H OH H
β-D-glucose-6-phosphate
16.2 Oxidation-Reduction
The reductions of the carbonyl group to an alcohol or the oxidation of an
alcohol to a carbonyl group represent very important transformations in organic
chemistry. The reagents used for these transformations have been discussed in
previous chapters. Common reducing agents include NaBH4, LiAlH4 and H2 with
a catalyst. Common oxidizing agents include compounds of chromium and
manganese. Although these procedures work well in preparative organic chemistry,
none of them would work in a biological setting because of their toxicity and lack of
specificity.
Living systems have developed several reagents for oxidation and reduction.
A commonly used oxidation agent is nicotinamide adenine dinucleotide (NAD+ ).
The positively charged pyridinium ring attracts a hydride from the α-carbon in
alcohols in an enzyme catalyzed reaction that produces the carbonyl compound. A
316 Ch 16 Biological Reagents
common reaction in living systems is the conversion of ethanol to acetaldehyde.
Acetaldehyde is responsible for the "hangover" and other long term effects in
alcohol intoxication.
OH
H
CH3
H
O
ethanol
H2N
NH2
N
+N
HO H
O
O
N
O P O P O H OH
O
O
H
H
H
H
OH OH
OH OH
N
N
dehydrogenase
- H+
Nicotinamide adenine dinucleotide oxidized form (NAD+)
O H
NH2
H
N
H2N
N
N
O
O
N N
O
H O H O P O P O H OH
+
CH3
H
O
O
H
H
H
H
acetaldehyde
OH OH
OH OH
Nicotinamide adenine dinucleotide reduced form (NADH)
A ball and stick model of NAD+ is shown below.
Reduced nicotinamide adenine dinucleotide (NADH) is also a product of the
reaction. Living systems use NADH as a reducing agent in the conversion of
carbonyl groups to alcohols. NADH is used in an enzyme catalyzed reaction to
reduce pyruvate to lactate. NAD+ is produced in this reaction and is now available
for oxidation reactions. Both the oxidation and reduction reactions are stereospecific
16.3 Claisen Condensations 317
in that only the hydrogen atom indicated by the arrows are transferred in the
reactions.
OH
H
O
OH
enzyme
H
H2N
+ NAD+
COO
+
CH3
+ H+ CH
N
COO
3
pyruvate
lactate
R NADH
16.3 Claisen Condensations
The synthesis of organic compounds in the laboratory requires methods that
form new carbon-carbon bonds. Many of these reactions involve the formation of
an enolate, a carbanion stabilized by an adjacent carbonyl group, and subsequent
addition of the enolate nucleophile to the carbonyl function of an aldehyde or
ketone. The Claisen condensation is normally a reaction in which the enolate from
an ester reacts with another ester. But a modification of the reaction involves a
reaction of an ester enolate with an aldehyde or ketone, and this modification is
known as the Claisen reaction. As shown below it is a very useful method for the
formation of a new carbon-carbon bond.
O
1) Li+ -CH2CO2Et
OH
2) H3O+
CH2CO2Et,
Living systems must also synthesize new carbon-carbon bonds for many
different anabolic processes. One of these processes is the tricarboxylic acid or
Kreb's cycle, shown in the chapter on carboxylic acids. The biological reagent used
in this process is known as acetyl coenzyme A (acetyl CoA). Acetyl CoA is a thio
ester instead of an oxo ester. The sulfur in acetyl CoA does not efficiently donate
electrons to the carbonyl group thus making the acetyl group more reactive than an
oxo ester. Thus in the tricarboxylic acid cycle the acetyl group condenses with the
carbonyl group of oxaloacetate to produce citrate. Acetyl CoA will also react with
alcohols to produce esters.
318 Ch 16 Biological Reagents
NH2
N
O
O
CH3
HN
O P
OH CH3
O
O
O
O P O
O
NH
S
CH3
H
O
N
N
H
N
H
H
HO
OH
Acetyl-CoA
O
O
O HO
O
O
O
CH2COO
enzyme
O
O
O
oxaloacetate
+ HS-CoA
O
citrate
A model of coenzyme A is shown below.
16.4 Carboxylation-Decarboxylation
Carbon dioxide is a useful reagent in organic synthesis and it is easily
available from cylinders or dry ice. In living systems carbon dioxide is an essential
compound in respiration and in the building (anabolism) or degradation
(catabolism) of biological materials.
A common organic procedure is the reaction between organometallics
(Grignard) with carbon dioxide to produce a carboxylic acid. The procedure is very
simple and can be used to make a wide variety of carboxylic acids.
16.4 Carboxylation-Decarboxylation 319
CH3O
1) CO2
MgBr
CH3O
2) H3O+
COOH
Living systems use enzyme catalyzed reactions of course and mild
conditions. The coenzyme used is biotin that reacts with bicarbonate to form a
carboxylated biotin that then reacts with pyruvate and transfers the carbon dioxide.
The product is oxaloacetate required in the Kreb's cycle.
O
HN
NH
OH
S
Biotin
Vitamin H
O
O
O C N NH
S
carboxylase
enzyme
Mn2+ HCO3
O
O
O
O
C
H2
pyruvate
R
H
O
O C N
O
NH
S
O
O
O
CH2
R
+ biotin
C O
O oxaloacetate
Removal of carbon dioxide from a substance in organic chemistry is often a
thermal process. β-Ketoacids decarboxylate readily to yield acetic acid derivatives,
a process used in the Malonic ester synthesis of organic compounds. The
decarboxylation occurs in a cyclic six-membered ring process.
320 Ch 16 Biological Reagents
H
O
H
O
HO
heat
-CO2
O
R H
O
O
R
HO
H
CO2H
CO2Et
1) NaOEt
CO2Et
2) CH3CH2Br
3) H3O+
heat
- CO2
CH3CH2-CH2COOH
CH2
R
HO
CH3CH2-CH
CO2H
Thiamin (Vitamin B1) is the reagent used in living systems to catalyze
decarboxylation reaction. Thiamin forms a ylid at biological pH. The ylid adds to
the carbonyl group of pyruvate to give an intermediate that resembles a β-ketoacid, a
β-iminoacid in this case, that easily decarboxylates. The thiamin intermediate is
hydrolyzed to acetaldehyde which eventually becomes acetyl CoA.
NH2
N
CH3
+
N
S
OH OH
N
P
P
CH3
OH
O
O
O
O
Thiamine diphosphate
Vitamin B1
+
N
pH 7
CH3
H3C
+
N
CH3
S
CH3-C-C-O
+
N
OO
O
S
CH3
O
OH O
C
C
O
S
O
16.4 Carboxylation-Decarboxylation 321
H3C
- CO2
N
CH3
OH
C
S
thiamine + CH3CHO
O
Thiamin Diphosphate
Many other biological reagents are used in biological systems, and the
reactions are too numerous to elaborate now. All of the vitamins can be considered
as a biological reagent as they combine with an enzyme to complete a very specific
reaction. The specificity of biological reactions as a result of enzyme catalyzed and
enzyme-cofactor catalyzed reactions is one of the most remarkable phenomenon that
occurs in nature.
16.5 Summary
Biological systems must conduct reactions under mild conditions. The
reactions are similar to those observed in organic chemistry, but special biological
reagents are needed.
Enzymes recognize phosphate derivatives of many substrates. By analogy
with organic chemistry that uses high energy anhydrides to make acetyl derivatives,
biological systems use adenosine triphosphate (ATP), a high energy reagent, to
make the phosphate derivatives.
Biological oxidations are conducted with the coenzyme nicotinamide
adenine dinucleotide (NAD+ ) which accepts a hydride to produce reduced
nicotinamide adenine dinucleotide (NADH). NADH is used as a hydride reducing
agent, delivering its hydride stereospecifically.
322 Ch 16 Biological Reagents
Claisen-like addition of an acetic acid unit is accomplished by acetyl
coenzyme A (Acetyl Co-A).
Addition of carbon dioxide to organometallic reagents is a very useful
organic reaction. Biological systems cannot tolerate an organometallic reagent but
instead use biotin to activate carbon dioxide for incorporation into biological
systems. Decarboxylation occurs readily in β-keto acids (malonic acid) systems in
organic reactions. Biological systems form activated carboxylic acids that undergo
decarboxylation by using thiamin. Biotin and thiamin are both vitamins and
coenzymes.
16.6 Problem Set
16.1 The first step in the metabolism of glucose (glycolysis) involves the formation
of the glucose 6-phosphate shown in this chapter. The next step is the conversion
of glucose-6-phosphate by an enzyme and ATP to fructose 1,6-diphosphate. Show
the structure of fructose 1,6-diphosphate.
16.2 The oxidation of ethanol by NAD+ is stereospecific. Only the pro-R
hydrogen atom is removed. Which hydrogen atom is the pro-R hydrogen?
16.3 Show a crossed Claisen reaction between benzaldehyde and acetyl-CoA. Do
you expect this to be a significant biological reaction?
16.4 The decarboxylation of acetoacetic acid produces acetone and carbon dioxide
is lost. The decarboxylation of biotin does not release carbon dioxide but instead
uses the carbon dioxide in a synthetic step to produce oxaloacetate. Have you seen
any organic reactions that use the carbon dioxide lost from acetoacetic acid (or
malonic acid) use in a coupled synthetic step?
16.5 Show the analogous mechanism between decarboxylation of a β-ketoacid and
the thiamin decarboxylation of pyruvate.
16.6 Another biological reagent is vitamin B6 which displays activity through
pyridoxal phosphate as shown in chapter 15. Show the structure of pyridoxal
phosphate and explain its function.