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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5467_Библиотеки_им_академика_М_И_Перельмана.pdf
AMINO ACIDS
Peptides and proteins are polyamides containing amino acids bonded through the carboxyl
function of one amino acid and the amino function of other amino acid in a head to tail manner.
The amide linkage is called peptide bond. In other words, amino acids are building blocks of peptides
and proteins. Simple proteins contain only amino acids whereas the conjugated proteins have
additional components. Proteins upon hydrolysis cleaved (hydrolysed) to their constituent amino
acids by a variety of methods. Twenty different amino acids are commonly found in proteins.
Asparagine is the fi rst amino acid to be isolated from its natural source (it was purifi ed from
asparagus juice in 1806) and threonine (isolated from the protein fi brin in the year 1938) is
the last one identifi ed. Amino acids were also isolated from antibiotics, bacterial cell wall and
biochemical metabolites.
Amino acids are compounds having one or more amino groups and one or more carboxyl
groups in the same molecule. They are usually classifi ed into α-amino acid, β-amino acid, γ-amino
acid, etc., according to the relative positions of the amino (NH
2
) and carboxyl (COOH) functional
groups. The term ‘amino acids’ often refers to α-amino acids (those in which amino group is
present on the α-carbon atom) that are isolated from natural sources.
CH CH CHCH COOH
32 2
NH
2


-Aminopentanoic acid
( -Amino acid)
CH CH CH CHCOOH
322
NH
2
␣
␣
-Aminopentanoic acid
( -Amino acid)
CH CHCH CH COOH
32
2
NH
2
␥
␥
-Aminopentanoic acid
( -Amino acid)
More than 200 amino acids have been isolated and identifi ed but only 25 are obtained from
typical proteins of which only 20 amino acids occur generally and are found in all proteins.
Among the 20 amino acids, 10 are called essential amino acids. These amino acids cannot be
produced by the body and must be supplied by food. They are also called indispensable amino acids.
Defi ciency in any one of the essential amino acids prevents growth and may even cause death.
Although there are 10 essential amino acids to humans, the following 8 are more important:
Amino acids, Peptides
and Proteins
4
CHAPTER
94
Pharmaceutical Chemistry of Natural Products
methionine, tryptophan, threonine, valine, isoleucine, leucine, phenylalanine and lysine. Two
amino acids are less important: arginine and histidine, as they are partly made by the body (for
the students to remember these 10 essential amino acids, it can be mentioned as MATT VIL PHLY
which corresponds to Methionine, Arginine, Tryptophan, Threonine, Valine, Isoleucine, Leucine,
Phenylalanine, Histidine and LY sine). The other amino acids are called nonessential amino acids or
dispensable amino acids and are synthesized by the body. The nonessential amino acids are alanine,
asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, proline, serine and tyrosine.
The amino acid components of peptides and proteins generally have the L-confi guration, but many
peptides contain one or more D-amino acids in their structures. Hence, abbreviations assume the
L-confi guration applies and D-amino acids must be specifi cally mentioned.
Leucine, isoleucine and valine are sometimes called branched chain amino acids, human beings
cannot survive unless these amino acids are present in the diet. The combination of these three
amino acids makes up approximately one-third of skeletal muscle in the human body.
Metabolic disorder of amino acids in humans may lead to certain diseases, which passes from
generation to generation. For example, a body that is unable to metabolize phenylalanine leads
to feebleness of mind. Some of the amino acids also help in the removal of undesirable acids from
the body by a detoxifi cation process. For example, glycine combines with benzoic acid to form
hippuric acid which is excreted in the urine.
In addition to their role in protein and enzyme synthesis, amino acids are extremely crucial for
good health. Amino acids contribute signifi cantly to the health of the nervous system, muscular
structure, hormone production, vital organs and cellular structure.
Nomenclature of Amino Acids
All the amino acids have trivial or common names and in some cases the names are given on the
basis of the source from which they were fi rst isolated. For example, asparagine was fi rst isolated
from asparagus and glutamate from wheat gluten; tyrosine was fi rst isolated from cheese (its name
is derived from the Greek word tyros which means ‘cheese’) and glycine (Greek: glykos means
‘sweet’) was so named because of its sweet taste.
In the IUPAC system, the amino acids are named as amino derivatives of the corresponding
acid, with the position of the amino group defi ned by an appropriate number. The carboxyl carbon
of an amino acid would be C-1 and the α-carbon would be C-2, the β-, γ-, δ- and ε-carbon would
be C-3, C-4, C-5 and C-6, respectively. In amino acids with heterocyclic R groups, the Greek
lettering system is confusing and hence the numbering system is used.
NH — CH —
22
CH — CH — CH — CH — COOH
222
NH
2
123456
⑀
␦
␥␣
2,6-Diamino heptanoic acid
Qualitative Chemical Tests for Amino Acids
1. Ninhydrin test (general test): Heat 3 mL test solution and three drops of 5% ninhydrin solution
in boiling water bath for 10 min. The resulting solution shows purple or bluish colour.
Amino acids, Peptides and Proteins
95
2. Test for tyrosine: Heat 3 mL of test solution and three drops of Millon’s reagent solution. The
resulting solution shows dark red colour.
3. Test for tryptophan: To 3 mL of test solution add few drops of glyoxalic acid and concentrated
sulphuric acid, reddish violet ring appears at junction of two layers.
4. Test for cysteine: To 5 mL of test solution add few drops of 40% NaOH solution and 10% lead
acetate solution. When boiled, black-coloured precipitate of lead sulphide is formed.
Classification of Amino Acids
Only proline and hydroxyproline are imino acids. The rest are all amino acids obtained by hydrolysis
of typical proteins. The amino acids are classifi ed into different ways as follows:
1. Based on essentiality of amino acids, they are classifi ed as follows:
(a) Essential amino acids: Not synthesized by the body, supplied in the diet (e.g. methionine,
tryptophan, threonine, valine, isoleucine, leucine, lysine, phenylalanine, arginine and
histidine).
(b) Nonessential amino acids: Synthesized by the body (e.g. alanine, asparagine, aspartic acid,
cysteine, glutamine, glutamic acid, glycine, proline, serine and tyrosine).
2. Based on the affi nity of amino acid’s side chain for water, they are classifi ed as follows:
(a) Hydrophilic amino acids: Having affi nity for water (e.g. aspartic acid, glutamic acid and
histidine).
(b) Hydrophobic amino acids: Not having affi nity for water (e.g. glycine, serine and threonine).
3. Based on the charge of amino acids, they are classifi ed as follows:
(a) Neutral amino acids: Contain one amino and one carboxyl group (e.g. glycine and
alanine).
(b) Acidic amino acids: Contain one amino and two carboxyl groups (e.g. aspartic acid and
glutamic acid).
(c) Basic amino acids: Contain two amino and one carboxyl group (e.g. arginine and lysine).
4. Based on the structure of the side chain, they are classifi ed as follows:
(a) Aliphatic amino acids: Have aliphatic side chain (e.g. glycine and alanine).
(b) Aromatic amino acids: Have aromatic side chain (e.g. phenylalanine and tyrosine).
(c) Heterocyclic amino acids: Have heterocyclic ring side chain (e.g. proline and histidine).
Aliphatic amino acids
1. Neutral (monoamino monocarboxylic acid)
Glycine NH
2
CH
2
COOH
Alanine CH
3
CH(NH
2
)COOH
Serine OHCH
2
CH(NH
2
)COOH
Threonine CH
3
CH(OH)CH(NH
2
)COOH
Valine (CH
3
)
2
CHCH(NH
2
)COOH
Leucine (CH
3
)
2
CHCH
2
CH(NH
2
)COOH
Isoleucine CH
3
CH
2
CH(CH
3
)CH(NH
2
)COOH
2. Acidic (monoamino dicarboxylic acid)
Aspartic acid COOHCH
2
CH(NH
2
)COOH
Asparagine NH
2
COCH
2
CH(NH
2
)COOH
96
Pharmaceutical Chemistry of Natural Products
Glutamic acid COOHCH
2
CH
2
CH(NH
2
)COOH
Glutamine NH
2
COCH
2
CH
2
CH(NH
2
)COOH
3. Basic amino acids
Arginine NH
2
C
||
NH
NH(CH
2
)
3
CH(NH
2
)COOH
Lysine NH
2
(CH
2
)
4
CH(NH
2
)COOH
Hydroxylysine NH
2
CH
2
CH(OH)(CH
2
)
3
CH(NH
2
)COOH
4. Sulphur containing amino acids
Cysteine SHCH
2
CH(NH
2
)COOH
Cystine (dicysteine) –[SCH
2
CH(NH
2
)COOH]
2
Methionine CH
3
SCH
2
CH
2
CH(NH
2
)COOH
Aromatic amino acids
HO O
CH CH(NH )COOH
22
CH CH(NH )COOH
22
CH CH(NH )COOH
22
HO
Phenylalanine
Tyrosine
Thyroxine
l
l
l
l
Heterocyclic amino acids
N
H
CH
2
CH
COOH
NH
2
N
H
COOH
HO
N
H
COOH
CH
2
CH
COOH
NH
2
HN
N
Proline Hydroxyproline Tryptophan Histidine
Note: Bolded amino acids are essential amino acids.
Isolation and Separation of Amino Acids from Proteins
Peptide bonds: A covalent bond formed between two molecules when the carboxyl group of one
molecule reacts with the amino group of the other with the releasing of a water molecule. This is
a condensation reaction and usually occurs between amino acids. The resulting CO–NH bond is
called as peptide bond and the resulting molecule is an amide (peptide).
Amino acids, Peptides and Proteins
97
These peptide bonds present in proteins are hydrolysed to amino acids by proteolytic (protein
splitting) enzymes or water in the presence of acid or base. Proteins can be obtained in pure
crystalline form by breaking the cell membrane to leak all the protoplasm.
H
N
H
H
H
O
OH
N
H
H
H
H
O
H
N
H
H
O
OH
Peptide bondAmino acid
N
H
H
H
H
OH
O
Amino acid
+
–H O
2
Acidic hydrolysis is performed by refl uxing the protein using hydrochloric or sulphuric acid.
This process destroys tryptophan. Alkaline hydrolysis of a protein is performed by heating the
protein with either 6 N NaOH or Ba(OH)
2
. This method destroys arginine, cystine, lysine and leads
to racemization.
Proteins are also hydrolysed by proteolytic enzymes (enzymatic hydrolysis). These enzymes
are obtained from digestive system of mammals, higher plants, bacteria and fungi (e.g. trypsin from
the pancreas, erepsin from the intestine, papain from papaya tree and pepsin from the stomach).
This method has the disadvantage of diffi culty in removing the enzymes after hydrolysis and
the hydrolysis is also slow and incomplete. But this method will not lead to decomposition and
racemization.
Synthesis of ␣-Amino Acids
Nowadays most commercially available amino acids are prepared synthetically because of the
development in methods for synthesizing specifi c amino acids. Generally, synthesis of amino
acid is more convenient than preparing it from natural sources. Several important methods for
synthesizing amino acids are discussed herein.
l. Gabriel phthalimide synthesis: From α-halogenated acid ester and potassium phthalimide.
C
C
O
O
NK ⫹
R
C
C
O
O
N
R
NaOH
C
C
O
O
⫹
RR
AgOH
OH
OH
HCl
Potassium phthalimide
Amino acid
BrCHCOOC H
25
␣-Halogenated acid ester
CHCOOC H
25
R
NH CHCOONa
2
HClNH CHCOOH
2
NH CHCOOH
2
98
Pharmaceutical Chemistry of Natural Products
2. Amination of a-halogen acids: From α-halogenated acid and excess of ammonia.
ClCH COOH
2
2 NH
3
⫹
Chloroacetic acid
NH CH COONH
22 4
⫹
H
⫹
-NH Cl
4
NH CH COOH
22
Glycine
Cl
⫺
α-Halo acids (or esters), the starting material for this reaction, are prepared by Hell–Volhard–
Zelinsky reaction as follows:
CH CH COOH
32
CH CH
3
Br P
2,
Br
COOH
R—CH
Br
COOH
(i) Hydrolysis (–2 C H OH)
(ii) Heat (–CO )
25
2
Br
R—C
COOC H
25
COOC H
25
Br
2
HC
2
R
HC
(i)C H ONa
(ii) RX
25
COOC H
25
COOC H
25
COOC H
25
COOC H
25
(a)
(b)
Applications: Amino acids prepared by this method are glycine, alanine serine, threonine,
valine and leucine.
Drawbacks: Because of the following drawbacks, Gabriel phthalimide synthesis is preferred
over this method.
(a) This method requires a large amount of ammonia.
(b) By-products of the type NH–(CH
2
COOH)
2
and N–(CH
2
COOH)
3
are formed in this
reaction.
3. Malonic ester synthesis: This method is an extension of amination of α-halogen acids method,
as it proceeds via the preparation of α-halogen acids.
CH (COOC H )
2522
(i)C H ONa
25
(ii)CH Br
3
CH CH(COOC H )
3252
CH CH(COOH)
32
⫺HBr
Br
2
Br
CH C(COOH)
32
CH CHCOOH
3
Br
Heat
⫺CO
2
NH
3
⫺HBr
CH CHCOOH
3
NH
2
Amino acid (Alanine)
(i) KOH
(ii) HCl
Malonic ester
Applications: Amino acids prepared by this method are phenylalanine, proline, leucine,
isoleucine and methionine.
4. Phthalimidomalonic ester synthesis: It is a combination of malonic ester synthesis and Gabriel
phthalimide synthesis. Amino acid synthesis starts from phthalimidomalonic ester (obtained
by the reaction of potassium phthalimide and monobromomalonic ester).
Amino acids, Peptides and Proteins
99
Synthesis of phenylalanine
C
O
C
O
NK
C
O
C
O
NCH(COOC H )
252
⫹
BrCH(COOC H )
252
⫺HBr
Phthalimido malonic ester
C
O
C
O
NC(COOC H )
252
CHCH
265
COOH
COOH
NH CHCH C H
2265
.
COOH
⫹
Phenylalanine
(i) KOH
(ii) HCl
(iii) Heat
(i) C H ONa
25
(ii) C H CH Cl
65 2
Synthesis of proline
C
O
C
O
NCH(COOC H )
252
(i) C H ONa
25
(ii) Br(CH ) Br
23
C
O
C
O
NC(COOC H )
252
CH CH CH Br
222
CH COOK
KBr
3
⫺
C
O
C
O
NC(COOC H )
252
CH CH CH OCOCH
222 3
(i)NaOH
(ii)HCl
(iii)Heat
Phthalimido malonic ester
NH .CHCOOH
2
CH CH CH OH
222
HC
2
HC
2
OH
HN
2
Ch
2
CH
COOH
HCl
⫺HO
2
COOH
Proline
N
H
100
Pharmaceutical Chemistry of Natural Products
Synthesis of cysteine
C
O
C
O
NCH(COOC H )
252
(i) C H ONa
25
(ii) C H CH —SCH Cl
65 2 2
C
O
C
O
NC(COOC H )
252
CH —S—CH —C H
2265
(i) NaOH
(ii) HCl
Phthalimido malonic ester
⫺Phthalic acid
COOH
NH CHCH SCH C H
22265
NH —CH—CH SH
22
Na–liq. NH
3
[H]
⫺CHCH
65 3
COOH
Oxidation by air
COOH
C
H
HN
2
CH S
2
CH S
2
H
C
HN
2
COOH
( )-Cystine⫾
( )-Cystine⫾
Applications: Amino acids prepared by this method are phenylalanine, cysteine, cystine, tyrosine,
proline, serine, aspartic acid, lysine and methionine.
5. Hofmann degradation: Hofmann degradation of malonic ester amide affords the method for
preparing amino acids.
RCH
R
CH
COOC H
25
COOC H
25
NH
2
CONH
2
Br –KOH
2
(Hofmann reaction)
Hydrolysis
RCH(NH )COOH
2
6. Curtius reaction: From malonic and cyanoacetic ester.
(a)
CH
2
COOC H
25
COOC H
25
(i) C H ONa
(ii) RX
25
KOH
RCH
COOC H
25
COOC H
25
RCH
COOK
COOC H
25
NH NH
22
RCH
COOK
CONHNH
2
HONO
C H ONa
25
RCH
COOK
CON
3
RCH
COOH
NHCOOC H
25
Pot. salt of malonic
acid azide
HCl
Heat
RCH(NH ).COOH
2
Amino acid
Amino acids, Peptides and Proteins
101
(b)
HC
2
CN
COOC H
25
(i) C H ONa
25
NH NH
22
RCH
CN
COOC H
25
RCH
CN
CONHNH
2
HONO
RCH
CN
CON
3
CHOH
25
HCl
RCH
CN
NH.COOC H
25
RCH(NH ).COOH
2
Amino acid
(ii) RX
Curtius reaction
Cyano acetic ester
Applications: Amino acids prepared by this method are phenylalanine and tyrosine.
7. Darapsky synthesis: From aldehyde and cyanoacetic ester.
RCHO
CN
COOC H
25
H —Ni
2
i) NH NH
ii) HONO
iii) Curtius reaction
iv) HCl
22
RCH CH
2
CN
COOC H
25
RCH CH(NH ).COOH
22
⫹
HC
2
8. Acylamidomalonic ester synthesis: From malonic ester.
(a)
CH (COOC H )
2252
HONO
C(COOC H )
252
NOH
H –Ni
2
NH CH(COOC H )
2252
CH COCl
3
CH COOC H
225
CH CONHC(COOC H )
3252
(i) C H ONa
25
(ii) CH Br.COOC H
225
CH CONHCH(COOC H )
3252
Hydrolysis
CH COOH
2
H —CHCOOH
2
N
Aspartic acid
(b)
CH NMe SO CH
2343
⫹⫺
N
H
C H CONHCH(COOC H )
65 252
⫹
C H ONa
25
Benzamidomalonic esterGramine methosulphate
N
H
(ii) HCl
N
H
NH
2
Tryptophan
CH C(COOC H )
2252
NHCOC H
65
(i) NaOH
CH CHCOOH
2
Applications: Amino acids prepared by this method are serine, leucine, valine, methionine,
lysine, glutamic acid and ornithine.