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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5709_Библиотеки_им_академика_М_И_Перельмана.pdf
102
Pharmaceutical Chemistry of Natural Products
9. Schmidt reaction: From alkylacetoacetic ester.
RCHCOOC H
25
COCH
3
NH
3
⫹
RCHCOOC H
25
NHCOCH
3
Hydrolysis
Schmidt reaction
⫺N
2
R.CHCOOH
NH
2
10. Strecker synthesis: From aldehyde or ketone and ammonium chloride and potassium cyanide
(ammonium cyanide).
(a)
NH Cl
4
⫹
KCN
NH
3
⫹
HCN
KCl
⫹
(b)
R
CO
⫹
NH
3
R
COH
NH
2
HH
⫺HO
2
R
CH
NH
CN
⫺
R
CH CN
NH
2
⫹
H
⫹
R
CH CN
NH
2
HO
3
⫹
R
CH COOH
NH
2
Applications: Amino acids prepared by this method are glycine, alanine, serine, valine,
methionine, glutamic acid, leucine, isoleucine, norleucine and phenylalanine.
General Physical and Chemical Properties of Amino Acids
Physical properties of amino acids
As the amino acids have both basic and acidic groups in the same molecule, they may be regarded
as salts. Amino acids are colourless, crystalline compounds generally soluble in water, alkalies and
acids, but sparingly soluble in organic solvents. All the amino acids are optically active as they
contain at least one chiral carbon atom (except glycine). All of the amino acids obtained from
proteins are confi gurationally related to L(–)-glyceraldehyde (except D-phenylalanine which occurs
in the polypeptide antibiotic gramicidin S) and are thus assigned L-series. Among the synthetic
amino acids only those belonging to L-series are found to possess biological activity. They have
characteristically high melting points varying from 200 to 300°C or even more.
NH
2
C H
COOH
R
OH
C H
CHO
CH OH
2
L( ) or ( ) Amino acid⫹⫺ L( ) or ( ) Glyceraldehyde⫹⫺
As the acidic and basic groups are present in the same molecule, most amino acids exist largely
as dipolar or zwitterion (compounds containing double charges, i.e. both a positive and a negative
charge in a same molecule and electrically neutral) due to the transfer of proton from carboxyl
group to the amino group.
R
C COOH
H
NH
2
Amino acid
Zwitterion
R
C COO
⫺
H
⫹
NH
3
Amino acids, Peptides and Proteins
103
The following observations confi rm the presence of dipolar ion structure of amino acids:
1. Absence of the characteristic vibration frequencies for the free carboxyl and amino groups in
the spectroscopic studies (IR and NMR).
2. Solution of amino acids has very high dielectric constant.
3. Solubility in water and insolubility in organic solvents and the high melting point.
4. Titration of an amino acid with base leads to neutralization of the –
+
NH
3
group and not the
–COOH group. In the same way, titration of amino acid with acid involves the reaction of
–COO
–
group of the amino acid and not its –NH
2
group.
R
C COOH
H
Cation, III
Zwitterion, I
R
C COO
⫺
H
⫹
NH
3
HCl
⫹
NH
3
NaOH
R
C COO
⫺
H
NH
2
Anion, II
5. Amino acids exhibit different electrolytic behaviour in different media. In an acidic solution,
an amino acid acts as a protonated derivative and hence migrates to the cathode, while in an
alkaline medium the same amino acid acts as an anion derivative and hence migrates to the
anode.
R
CH.COOH
⫹⫺
NH Cl
3
HCl
NaOH
R
CH.COO
⫺
ZwitterionCation
(Migrates to cathode)
⫹
NH
3
NaOH
HCl
R.CHCOO Na
⫺⫹
NH
2
Anion
(Migrates to anode)
6. Isoelectric point: The addition of a proton to an amino acid converts it into a cation and the
addition of a base converts it into an anion. Therefore, at a particular pH value, the acidic
and basic properties of the amino acid must be balanced, and hence, the amino acid at this
particular pH can exist as a neutral dipolar ion (i.e. electrically neutral zwitterions). This pH
at which there is no net charge on the amino acid molecule and does not migrate to any
electrode under the infl uence of electric current is known as isoelectric point. The isoelectric
point of each amino acid is constant. An amino acid with pH values lower than its isoelectric
point mainly exists as an anion II, while at pH values higher than that of its isoelectric point
it mainly exists as a cation III.
Chemical properties of amino acids
The chemical properties of amino acids are due to the amino group and the carboxyl group present
in it.
Reactions involving both the carboxyl and the amino groups:
1. Reaction with isocyanates (formation of hydantoins): When isocyanates are treated with α-amino
acids, they yield the corresponding carbamides, which upon warming with hydrochloric acid
afford hydantoins. This reaction is used for the synthesis of amino acids.
104
Pharmaceutical Chemistry of Natural Products
R
O
CH
NH
2
C
OH
␣−Amino acid Phenylisocyanate
O
CH
65
C
N
R
O
CH
NH
C
OH
C
HN
O
CH
65
H
⫹
⫺HO
2
R
O
CH
NH
C C
N
O
CH
65
Hydantoin
The mechanism of this reaction is shown below:
R CH
NH
2
CH
65
C
O
OH
O
C
N
R CH
NH
C
O
OH
CH
65
O
⫺
C
N
R CH
NH
2
C
O
OH
CH
65
O
C
N
⫺
R CH
NH
C
O
OH
CH
65
O
C
HN
H
⫹
(HCl)
R CH
NH
C
HO
OH
CH
65
O
C
HN
⫹
R CH
NH
C
CH
65
OC
N
⫹
H
HO
HO
R CH
NH
C
O C
R CH
NH
C
CH
65
OC
N
⫹
HO
2
O
+H
⫹
H
H
⫹
⫺HO
2
N
CH
65
Hydantoin
⫹
␣
-Amino acid
Phenylisocyanate
O
+
⫹
–H
⫹
2. Action of heat: The action of heat on amino acids is a useful reaction for distinguishing the
different types of amino acids (α and β), because different amino acids yield different products
upon heating.
(a) α-Amino acids: 2,5-Diketopiperazine is the resultant product upon reaction of two
molecules of amino acid, which react in such a way that the amino group of one forms
amide with the carboxylic group of the other.
Amino acids, Peptides and Proteins
105
CH C
R
HN
2
O
OH
Heat
⫺2H O
2
Diketopiperazine
(a cyclic diamide)
CH
C
O
R
HN NH
C
CH
OR
2
(b) β-Amino acids: α,β-Unsaturated acids are the resultant product upon heating β-amino
acids, which undergo β-elimination of ammonia.
RCHCH COOH
2
NH
2
Heat
RCH CHCOOH
⫹
NH
3
-Amino acid ␣, -Unsaturated acid
(c) γ-Amino acids:
R C
H
NH
2
CH
2
CH
2
COOH
Heat
R CH
NH
CH
2
CH
2
CO
␥-Amino acid ␥-Lactam
(d) δ-Amino acids:
HC
2
C
O
OH
HC
2
CH NH
22
HC
2
Heat
HC
2
C
O
NH
HC
2
CH
2
HC
2
␦-Aminovaleric acid ␦-Valerolactam
3. Formation of sydnones: With nitrous acid, N-alkyl or aryl amino acids yield N-nitroso derivatives,
which on dehydration with acetic anhydride give sydnones.
RN
CH .COOH
2
H
HONO
RN
CH .COOH
2
NO
Ac O
2
RN
CH
N
⫹
CO
O
⫺
Sydnone
4. Reaction with ninhydrin: A complex reaction in a sequential way occurs with the formation of
brilliant blue colour when amino acids are heated with an alcoholic solution of ninhydrin.
This reaction is useful for the qualitative and quantitative estimation of amino acids. Blue or
violet coloured compound is believed to be formed by the following sequence of steps.
106
Pharmaceutical Chemistry of Natural Products
(a) Formation of Schiff’s base (I)
⫺H,
⫹
⫹H
⫹
N
C COOH
R
H
OH
H
O
O
C COOH
R
H
⫺HO
2
O
O
N
O
O
O
⫹
HN
2
C COOH
R
H
HN
2
C COOH
R
H
⫹
O
⫺
O
O
(b) Decarboxylation of (I) to aldimine (II)
C C
R
H
O
O H
O
O
N
⫺CO
2
O
O
N
H
CHR
III
(c) Hydrolysis of aldimine (II) to (III)
II
+H O
2
O
O
N
H
CH
R
O
O
N
H
OCH H
R
H
⫺RCHO
O
O
NH
2
H
III
Amino acids, Peptides and Proteins
107
(d) Condensation of (III) with a second molecule of ninhydrin to form the coloured product
(IV)
O
O
NH
2
H
O
+
⫺HO
2
O
O
N
H
O
O
O
O
⫺H, H
⫹
⫹⫹
O
O
N
HO
O
IV
. Coloured compound
Other reagents used are sodium 2,4,6-trinitrobenzene-1-sulphonate. Specifi c reagents may be
used to detect particular amino acids (e.g. diazotized sulphanilic acid couples with tyrosine and
histidine in alkaline solution to give red colour).
Reactions of amino group
1. Alkylation: Upon reaction with alkyl halides (in alkaline solution), amino acids form N-alkyl–
substituted amino acids. Internal quaternary alkyl ammonium salts are formed when a large
excess of alkyl halides is used. These salts possess zwitterionic character and are known as
betaines.
(a)
⫹
NH
3
R CHCOO
⫺
⫹
R’X
R CH.COO
⫺
⫹
X
⫺
⫹
NH R'
2
(b)
⫺
OOC
CH
2
NH
3
⫹
3CH I
3
CH
2
COO
⫺
(CH)N
33
⫹
Betaine
(c)
F
ON
2
2,4-Dinitrofluorobenzene
or Sanger's reagent or 2,4-DNF
⫹
NO
2
NH CHCOONa
2
R
OH
⫺
N
H
ON
2
NO
2
CHCOON
a
R
2,4-Dinitrophenyl derivative
This reaction (using DNF) is useful in determining N-terminal amino acid in a polypeptide
which has a free amino group.
108
Pharmaceutical Chemistry of Natural Products
2. Deamination: Upon heating with HI at 200°C, amino acids yield the corresponding fatty acids
with the elimination of ammonia.
RCHCOOH
NH
2
HI
200°
C
RCH COOH
2
NH
3
⫹
Upon reaction with hydrogen peroxide, amino acids may also be converted into a keto acid
which is similar to biological oxidation, catalysed by enzymes called amino acid oxidases. This is
the mechanism behind the conversion of proteins into carbohydrates in living organisms.
2RCHCOOH
NH
2
HO
22
2RCOCOONH
4
H
2
⫹
3. Reaction with nitrous acids: α-Amino acids react with nitrous acid and yield α-hydroxy acids
and nitrogen.
CH COOH⭈
NH
2
⫹
R
HONO
CHCOOH
OH
RN+ HO
22
⫹
By measuring the liberated nitrogen (Van Slyke’s amino nitrogen determination), amino
acids are determined quantitatively.
4. Acylation: Upon reaction with acid chlorides or anhydrides, the amino group may be acylated.
The acetylated derivatives are useful in peptide synthesis.
CHCOOH
NH
2
⫹
R
(CH CO) O
32
CHCOOH
NHCOCH
3
R CH COOH
3
⫹
5. Reaction with mineral acids: Amino acids on treatment with mineral acids form salts. Upon
adding strong organic base (e.g. pyridine), the free acids may be liberated.
⫹⫺
NH CHCOO
3
R
HCl
⫹
Cl H NCHCOOH
3
R
⫺⫹
Salts
6. Reaction with nitrosyl chloride or bromide: Upon reaction with nitrosyl chloride or bromide,
amino acids yield the related halogen acids.
NH
2
NOCl
RCHCOOH
CI
RCHCOOH
⫹⫹
N
2
⫹
H
2
O
Reactions involving carboxyl group
1. Esterifi cation: Amino acids do not undergo esterifi cation in the usual condition, but when
they are dissolved in anhydrous alcohol, which is fi rst saturated with hydrogen chloride,
and then heated, esterifi cation takes place smoothly. Thus, alcohol does not react with the
zwitterions itself but react with acidic form of the zwitterions to form ester.
AgOH
NH CH COOC H
2
225
⫹
AgCl
HO
2
⫹
⫹⫺
NH CH COO
32
HCl
⫹
⫺⫹
ClH NCH COOH
32
CHOH
25
⫺HO
2
Cl H NCH COOC H
⫺
32 25
Ester salt
⫹
Amino acids, Peptides and Proteins
109
2. Formation of acid chlorides: Direct treatment of amino acids with PCl
5
or SOCl
2
to form
corresponding acid chlorides is a diffi cult reaction, because of the presence of amino group.
Hence the amino group is fi rst protected by acetylation and then the product is treated with
PCl
5
or SOCl
2
to form corresponding acid chlorides.
⫹⫺
NH CH COO
32
CH COHNCH COOH
32
CH COHNCH COCl
32
HCl
HCl H NCH COCl
22
(CH CO) O
32
PCl
3
3. Decarboxylation: By heating or using acids, bases or specifi c enzymes, amino acids can be
decarboxylated to the primary amines. Decarboxylation of histidine to histamine is one such
decarboxylation reaction having great importance in the body.
NH
2
RCHCOOH
Ba(OH)
2
Heat
RCH NH
22
⫹
BaCO
3
H
2
O
⫹
N
CH
HC C
N
H
CH CHCOOH
2
NH
2
⫺CO
2
N
CH
HC C
N
H
CH CH NH
222
Histamine
Histidine
4. Sorensen formol titration: Amino acids cannot be titrated with bases directly, as they largely
exist as zwitterions. By protecting the amino group using formaldehyde (–NHCH
2
OH), the
carboxyl group becomes normal and thus the acid can be titrated using suitable indicators.
RCHCOOH
NH
2
2HCHO
RCHCOOH
N(CH OH)
22
Glycine Dimethylolglycine
(major product)
NaOH
RCHCOONa
N(CH OH)
22
5. Reaction with acetic anhydride in pyridine (Dakin–West reaction): Amino acids upon heating with
acetic anhydride in pyridine solution form methyl α-acetamido ketones.
RCHCOOH
NH
2
(CH CO) O
32
RCHCOCH
3
NHCOCH
3
Pyridine
6. Amino acids form salts with alkalies: Amino acids form salts with alkalies.
⫹
NH
3
CH COO
2
⫺
NaOH
NH CH COONa
22
HO
2
⫹
The salts of heavy metals are chelate compounds.
7. Reaction with ammonia: The esters of amino acid react with ammonia and yield amides.
RCHCOOC H
25
NH
2
⫹
NH
3
RCHCONH
2
NH
2
⫹
CHOH
25
8. Reduction: When reduced with lithium aluminium hydride, they are converted into amino
alcohols without any loss of optical activity.
⫹
NH
3
H
C
LiAlH
4
NH
2
R
COO
⫺
R
CH CH OH
2
110
Pharmaceutical Chemistry of Natural Products
PEPTIDES
A peptide is formed when two or more amino acids are joined together by peptide linkage (–CO–
NH–); a polypeptide is a chain of many amino acids. Proteins are made up of one or more polypeptide
molecules. The amino acids are linked covalently by peptide bonds. The image shown below explains
how three amino acids linked by peptide bonds into a tripeptide. One end of every polypeptide,
called the amino terminal or N-terminal, has a free amino group. The other end, called the carboxyl
terminal or C-terminal, has a free carboxyl group. Conventionally, the left-hand amino acid in this
sequence is the one with the free amino group or the N-terminus, whereas the right-hand amino
acid has the free carboxyl group or the C-terminus. Occasionally, the terminals are indicated by
showing H− and −OH. This convention can have no use in cyclic peptides, and hence, arrows are
incorporated into the sequence to indicate peptide bonds in the direction CO→NH. When this
becomes longer, one-letter abbreviations are used instead of the three-letter abbreviations. Thus,
the following peptide Gly–Ala–Cys becomes GAC.
H
N
⫹
C C O
⫺
H
H
H
H
O
H
N
⫹
C C O
⫺
H
CH
3
H
HO
H
N
⫹
C C O
⫺
H
CH
2
SH
H
HO
H
N
⫹
C C N
⫹
H
H
H
H
O
H
C C
H
CH
3
O
N
⫹
H
C C O
⫺
H
CH
2
SH
O
HO
2
HO
2
Glycine Alanine Cysteine
Peptide bonds
Amino acid molecules contain both basic and acidic groups, and therefore, an intermolecular
reaction takes place between the carboxyl group of one amino acid and the amino group of another
amino acid. The product obtained still has a free amino and a carboxyl group, and hence it may
react with other amino acid and yield condensation product with higher molecular weight. By this
mechanism polypeptides, and therefore, proteins are synthesized in nature.
Peptides are classifi ed into various types depending upon the number of the amino acid residues
present in them. Example, a peptide derived from two amino acids is known as dipeptide. A peptide
with three, four and indefi nite moles of amino acids is called tripeptide, tetrapeptide and polypeptide,
respectively. Adrenocorticotropic hormone, glucagon and vasopressin are the examples for the
naturally occurring polypeptides.
Amino acids, Peptides and Proteins
111
Generally, a chain length of more than 40 amino acid residues refers to protein, whereas
the term polypeptide can be used to cover all chain lengths. Proteins and polypeptides can also
be distinguished according to the molecular weight: substances with molecular weight less than
10,000 are called polypeptides, while those above 10,000 are called proteins.
Naming a peptide
By indicating the sequence of amino acids beginning with N-terminal, residue peptides are named.
NH CH CONHCHCONHCH CONH CH CONH CH CONH CH COOH
22 2
⭈⭈⭈⭈⭈
CH
3
CH
2
OH CHCH
265
CH CONH
22
Gl
y
cine
Alanine
Serine Phen
y
lalanine As
p
ara
g
ine Gl
y
cine
This hexapeptide is called Glycyl–alanyl–seryl–phenylalanyl–asparaginyl–glycine or it can be
mentioned using the common abbreviations as H–Gly–Ala–Ser–Phe–Asn–Gly–OH. However, all
naturally occurring important peptides possess a shorter individual name.
Classification of Peptides
They may be classifi ed in various ways as explained below:
Based on the hydrolytic products, peptides are classifi ed into two types:
1. Homomeric peptides: Those peptides that on hydrolysis give only one type of amino acids are
called homomeric peptides.
2. Heteromeric peptides: Those peptides that form other products in addition to amino acids are
called heteromeric peptides.
Based on their structure, peptides are classifi ed into two types:
1. Linear peptides: They possess an open chain structure.
2. Cyclic peptides: They possess a cyclic structure. Cyclic peptides are further divided into two
types, namely homodetic [whose structures contain only peptide linkages (e.g. glutathione)]
and heterodetic [whose structures contain both peptide and other type (e.g. disulphide) of
linkage (e.g. oxytocin, insulin)].
Synthesis of Peptides
Peptide synthesis is a chemical process in which coupling of the carboxyl group of one amino
acid with the amino group of another amino acid occurs. Usually chemical techniques are used to
synthesize peptides of up to 30–40 amino acids length. The fi rst peptide synthesis was carried out
by T. Curtius and the fi rst pure dipeptide (Gly–Gly) was synthesized by E. Fischer in 1901. Some
of the simple peptide synthesis are depicted below.
1.
Glycine
Alanine
Glycylalanine (a dipeptide)
NH CH COOH
22
CH
3
NH CH CONHCHCOOH
22
CH
3
⫹
NH CHCOOH
2