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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5987_Библиотеки_им_академика_М_И_Перельмана.pdf
112
Pharmaceutical Chemistry of Natural Products
2.
Alanine
Glycine
Alanylglycine (a dipeptide)
⫹
NH CH COOH
22
NH CHCOOH
2
CH
3
NH CHCONHCH COOH
22
CH
3
3.
GlycineGlycylalanine (a dipeptide)
NH CH CONHCHCOOH
22
CH
3
NH CH COOH
22
⫹
NH CH CONHCHCONHCH COOH
22 2
Glycylalanylglycine (a tripeptide)
CH
3
4.
GlycylalanineGlycine
NH CH COOH
22
NH CH CONHCHCOOH
22
⫹
NH CH CONHCH CONHCHCOOH
22 2
Glycylglycylalanine (a tripeptide)
CH
3
CH
3
General Principles of Peptide Synthesis
The steps involved in the peptide synthesis are as follows:
1. Protection of the amino group
2. Activation of carboxyl group
3. The coupling step
4. Removal of the protecting group
Let us discuss each of these steps in detail.
1. Protection of the amino group: The fi rst step is the protection of the amino group. This step is
essential to avoid the interaction of the two moles of the same amino acid.
2NH CH COCl
22
⫺HCl
NH CH CONHCH COCl
22 2
Expected reaction when the amino groups are not protected
This process might continue until all of the amino acid is converted into peptide. Protective
group is introduced on one of the amino acid molecules which will thus act as an acylating
agent.
A good protecting group must possess the following characteristics:
1. It must be inert during the reactions when peptide bonds are formed.
2. It should be attachable to the amino group easily without causing racemization of an optically
active amino acid.
3. It should be easily removable under mild condition without disturbing the peptide linkage or
sensitive functional groups on the amino acid side chains.
4. Special blocking groups are used for blocking amino acids such as Cys, Tyr, Asp and Glu.
These blocking groups should be stable to conditions which cleave the protecting groups.
Amino acids, Peptides and Proteins
113
The two important protecting groups that comply with all the above conditions are as
follows:
Phthalyl (–CO–C
6
H
4
–CO–) group
By the direct reaction of the phthalic anhydride with an amino acid or by the reaction of an
amino acid with N-carboxyethylphthalimide, the phthalyl group can be introduced into an amino
acid molecule.
NH CH COOH
22
O
CO
CO
CO
CO
NCH COOH
2
CO
CO
NCOOC H
25
⫹ NH CH COOH
22
CO
CO
NCH COOH
2
⫹ NH COOC H
225
⫹
HO
2
Carbobenzyloxy (C
6
H
5
CH
2
OOC–) group
By using benzyloxycarbonyl chloride, also known as carbobenzoxy chloride or carbobenzyloxy
chloride, the carbobenzyloxy (CbO) protecting group is introduced into the amino acid. The
carbobenzoxy chloride is prepared by the reaction of phosgene (COCl
2
) with benzyl alcohol in
toluene.
CH OH
2
⫹
COCl
2
⫺20°C
CH OCOCl
2
⫹
HCl
Benzyloxy carbonyl chloride or
carbobenzoxy chloride or
carbobenzyloxy chloride (95%)
CH OCOCl
2
⫹
NH CH COOH
22
(i) Dil. NaOH
(ii) HCl
CH OCONHCH COOH (75%)
22
Other protecting groups are as follows:
Carbethoxy (–COOC
2
H
5
) group: This is introduced by using ethyl chloroformate (Cl–COO–C
2
H
5
).
p-Toluenesulphonyl or p-tosyl (–SO
2
–C
6
H
4
–CH
3
–) group: This is introduced by using p-toluenesul-
phonyl chloride.
Triphenylmethyl (CPh
3
) group: This is introduced by using trityl chloride (ClCPh
3
).
Benzyl group (–CH
2
C
6
H
5
) group: This is introduced by using benzyl chloride.
Me
3
COCO group: This is introduced by using Me
3
COCON
3
.
2. Activation of carboxyl group: By converting the carboxylic group into acid chloride, ester or mixed
anhydride, the amino group–protected amino acid is activated. Conversion into an appropriate
mixed anhydride using ethyl or isobutyl chloroformate is the most adopted method. The chemical
reactions are as follows:
114
Pharmaceutical Chemistry of Natural Products
RHNCH COOH
2
(i) Et N
3
(ii) ClCOOC H
25
RHNCH COOCOOC H
252
+ HCl
(R = Protecting group)
3. The coupling step: Peptide bond formation takes place in this step. Nucleophilic attack of the
free amino group of an amino acid ester (or peptide ester) on the activated carboxyl end of the
N-protected amino acid leads to the formation of a peptide bond. If the leaving group is more
electron withdrawing, then peptide bond formation takes place faster.
RHNCH COOCOOC H
225
Activated carboxyl
compound
⫹ NH CHCOOCH
23
Nucleophilic amino
component
RHNCH CONHCHCOOCH
23
⫹
CO
2
CHOH
25
⫹
Dipeptide, where R= protecting group
R¢
R¢
The dipeptide ester is hydrolysed to peptide which is further activated and treated with another
molecule of an amino acid to form tripeptide and so on.
4. Removal of the protecting group: The protecting group is removed after forming a polypeptide
chain of desired length to yield the fi nal product. The carbobenzyloxy group is removed by
catalytic reduction of the peptides (if they do not have sulphur). If sulphur is present, then the
carbobenzyloxy group is removed either by sodium metal in liquid ammonia at 40°C or by using
triethylsilane and palladium chloride. The N-phthalyl group is removed by means of hydrazine.
CO
CO
NCH C⭈⭈
2
O
NH NH
22
NH
NH
NH CH C
22
⭈⭈
⫹
O
O
O
CHCHO
65 2
C
NHR
H /Pd
2
HBr
CHCH
65 3
CHCH
65 2
Br
⫹
⫹
H
HO
O C NHR
O
C
NHR
O
O
⫺CO
2
H N—R
2
⫺CO
2
H N—R
2
Amino acids, Peptides and Proteins
115
5. Other methods: Other protecting groups are removed by the following ways:
p-CH C H SO NHCHCO
364 2
⭈
R
p-Tosylsulphonyl protecting
group
HI
50–60°C
p-CHCH SH
364
⭈
⫹
NH CHCO
2
⫺
R
ClCH CONHCHCO
2
⫺
R
Chloroacetyl protecting
group
NH
3
NH CH CONHCH CO
22
⭈⫺
R
Ph CNHCHCO
3
⫺
R
Trityl group
Boil
CH COOH
3
Ph COCOCH
33
⫹
NH CHCO
2
⫺
R
C H CH NHCHCO
65 2
⫺
R
CHCH
65 3
⫹
NH CHCO
2
⫺
R
Na–NH
3
Me C
3
O
OC
NH
CHCO
R
In HCl
CH COOH
3
CH
3
C CH
2
CH
3
⫹
CO
2
⫹
NH
2
CHCO
R
(a)
(b)
(c)
(d)
(e)
General Methods of Peptide Synthesis
Some of the important general methods of polypeptide synthesis are the following:
1. Methods involving the protection of amino group of one of the amino acids: The above four basic
principles of polypeptide synthesis are utilized in these methods.
(a) Fischer’s second method: A polypeptide prepared by the condensation of an amino acid
whose amino group is protected by using ethyl chloroformate with an amino acid ester as
depicted below:
ClCOOC H
25
⫹
Ethyl chloroformate
NH CH COOC H
22 25
Ethyl ester of glycine
NHCH COOC H
225
COOC H
25
Ethyl ester of
alanine
NH CHCOOC H
225
CH
3
NHCH CONHCHCOOC H
225
COOC H
25
CH
3
Dipeptide derivative
Hydrolysis
NH CH CONHCHCOOH
22
Glycylalanine
CH
3
116
Pharmaceutical Chemistry of Natural Products
By condensing in further with another amino acid ester, the dipeptide derivative can
be converted into tripeptide and hence a polypeptide may be prepared. Without affecting
the peptide bond, the protecting N-carbethoxyl group cannot be eliminated by hydrolysis,
and therefore, the yield of the product is very low.
(b) Fischer’s third method: In this method, p-toluenesulphonyl chloride is used as a protective
agent (instead of ethyl chloroformate used in the above method). The p-toluenesulphonyl
(p-tosyl) group can be easily cleaved by using HI without affecting the peptide bond.
p-CHCHSOCl
364 2
p-Toluenesulphonyl
chloride
p-CH C H SO
364 2
⫹
NH CH COOC H
2225
Ethyl ester of glycine
NHCH COOC H
225
Ethyl ester of
alanine
CH
3
NH CHCOOC H
225
NHCH CONHCHCOOC H
225
p-CHCH SO
364 2
CH
3
50–60°
HI
NH CH CONHCHCOOC H
2252
Glycylalanine ester
CH
3
p-CHCHSH
364
⫹
Thiophenol
(c) Bergmann’s method: By the use of benzyl chloroformate, the amino group is protected
whereby carbobenzyloxy (–OCOCH
2
C
6
H
5
) group is introduced. The protecting group is
removed by catalytic hydrogenation. If the peptide has sulphur, it poisons the catalyst
palladium, and hence, the protecting group should be removed using sodium in liquid
ammonia or by using triethylsilane and palladium chloride.
C H CH OCOCl
65 2
Benzyl chloroformate
CHCHOCO
65 2
⫹
NH CH COOH
22
Glycine
NHCH COOH
2
(i) PCl
5
NHCH CONHCHCOOH
2
CHCHOCO
65 2
CH
3
H –Pd
2
NH CH CONHCHCOOH
22
Glycylalanine
CH
3
⫹
CH
3
(ii) NH CHCOOH
2
(Alanine)
CHCH
65 3
CO
2
⫹
2. Mixed anhydride method: Amino acids are converted into anhydrides, which when dissolved in
organic solvent and treated with water (catalyst) undergo polymerization and form peptide.
The anhydrides may be obtained as follows:
(a)
COCl
2
⫹
NH CH COOH
22
CONHCH CO
2
O
⫹
2HCl
Anhydride
Amino acids, Peptides and Proteins
117
(b)
CH COOCl
3
⫹
NH CH COOH
22
CH OOCNHCH COOH
32
SOCl
2
CH OOCNHCH COCl
32
Heat in
vacuum
50-60°
CONHCH CO
2
O
CH Cl
3
⫹
Anhydride
Methyl chloroformate
(c)
n
CONHCH CO
2
O
Anhydride
Water
(COCH NH)
2n
nCO
2
⫹
Polymer (polypeptide)
3. From diketopiperazine: E. Fischer in 1901 prepared glycylglycine (dipeptide) by the partial
hydrolysis of a diketopiperazine using hydrochloric acid.
HC
2
HN
C
C
CH
2
HN
O
O
HCl
NH CH CONHCH COOH
22 2
Glycylglycine
2,5-Diketopiperazine
HC
2
HN
C
C
NH
CHCH
3
O
O
a
b
HCl
HCl
(a)
(b)
NH CHCONHCH COOH
22
Alanylglycine
*
NH CH CONHCHCOOH
22
CH
3
CH
3
Glycylalanine
Structure of Peptides
The peptide to be analysed is fi rst hydrolysed to its constituent amino acids, which are separated,
identifi ed and quantifi ed.
The next diffi cult task is to determine the sequence of the various amino acids present in the
peptide. Even in a dipeptide, having glycine and alanine, the two amino acids may be present in
either of the two ways.
NH CH CONHCHCOOH
22
Glycylalanine
NH CHCONHCH COOH
22
Alanylglycine
CH
3
CH
3
118
Pharmaceutical Chemistry of Natural Products
The two structures differ in the respect that in the fi rst structure, N-terminal amino acid is
glycine (i.e. the amino group of glycine is free) and C-terminal amino acid is alanine, while in the
second, the N-terminal amino acid is alanine and C-terminal amino acid is glycine.
The arrangement of the various amino acids in a polypeptide can be established by a variety of
chemical methods, which remove either of the two terminal amino acid residues of a polypeptide
in a stepwise manner.
‘ N’-Terminal (amino end) degradation
1. Sanger’s method or dinitrophenyl method: The amino acid present in the amino end of the
polypeptide is determined by this method using the reagent, 1-fl uoro-2,4-dinitro benzene
(FDNB). The polypeptide is treated with FDNB at room temperature in presence of sodium
hydrogen carbonate solution to form a N-2,4-dinitrophenyl (DNP) derivative of the
polypeptide. The product obtained is then hydrolysed to cleave the peptide bond to yield
DNP derivative of the N-terminal amino acid along with the rest of peptide or amino acid
residues.
NO
2
F
ON
2
NH CHCONHCHCONHCHCOOH
2
⫹
R
1
R
2
R
3
NO
2
ON
2
NHCHCONHCHCONHCHCOOH
⫺HF
H
⫹
Hydrolysis
NO
2
ON
2
NHCHCOOH
⫹
NH CHCOOH
2
⫹
NH CHCOOH
2
2,4-DNP derivative of amino acid
identified via TLC
Amino acids mixture
R
1
R
2
R
3
R
1
R
2
R
3
The reagent FDNB reacts with the groups such as –NH
2
, –OH, –SH and imidazole of
the amino acid even though these groups do not present in the free end of the peptide.
Hence, the amino acids such as lysine (diamino derivative), tyrosine (hydroxyl derivative),
cysteine (thiol derivative) and histidine (imidazole derivative) will also react with FDNB
even though these amino acids are not present in the free end of the peptide. As a result of
this, it may form a number of DNP derivatives which can be readily isolated and identifi ed
by chromatographic techniques.
Amino acids, Peptides and Proteins
119
It cannot be used repetitively, as the DNP derivative is completely hydrolysed (a difference
from Edman’s method), and hence, only the N-terminal amino acid can be identifi ed.
2. Dansyl method: The amino acid present in the amino end of the polypeptide is determined by
this method using the reagent 1-dimethylaminonaphthalene-5-sulphonyl chloride ( Dansyl
chloride, DNS–Cl).
N(CH )
32
SO Cl
2
Dansyl chloride or DNS-Cl
NH CHCONHCHCONHCHCOOH
2
⫹
⫺HCl
N(CH )
32
SO NHCHCONHCHCONHCHCOOH
2
6M HCl, 100°C
N(CH )
32
SO NHCHCOOH
2
⫹
NH CHCOOH
3
⫹
NH CHCOOH
3
Amino acids mixture
Dansyl derivative of amino acid
(DNS-derivative) identified via
TLC method
Polypeptide
R
1
R
2
R
3
R
1
R
2
R
3
R
2
R
3
R
1
The polypeptide is treated with dansyl chloride to form a dansyl derivative of the
polypeptide. The product obtained is then hydrolysed to cleave the peptide bond to yield
dansyl derivative of the N-terminal amino acid along with the rest of peptide or amino
acid residues. This method has the advantage that the TLC identifi cation of the amino acid
derivatives is 100 times more sensitive than in the Sanger’s method, as even the presence of
10
–9
mole of a dansyl amino acid can be detected.
In both the above methods, the amino end of the amino acid is converted into its
derivatives (DNP or dansyl derivative), hence the component amino acids and therefore the
120
Pharmaceutical Chemistry of Natural Products
amino acid sequence in a peptide cannot be easily determined. Selective enzymatic or partial
chemical hydrolysis to smaller fragments followed by N-terminal degradation leads to better
applicability of these methods.
3. Edman’s method or phenylthiohydantoin method: The amino acid present in the amino end of the
polypeptide is determined by this method using Edman’s reagent (phenyl isothiocyanate).
The polypeptide is treated with phenyl isothiocyanate in the presence of dilute alkali to form
a phenylthiohydantoin (PTH). The product obtained is then hydrolysed to cleave the PTH,
which is separated and identifi ed by paper chromatography by leaving the remainder of the
polypeptide or protein intact at the end of the reaction.
CH
65
NCS⫹ NH
2
C
H
R
C
O
NH C
H
R’
C
O
NH
⫺⫹HH
+
+
,
CH
NC
CS
R
HN
CH
65
O
⫺
H
N
C
H
C
O
NH
⫹
R’
⫺⫹H; H
⫹⫹
HN
2
C
H
C
O
N
H
R’
CH
NC
CS
R
O
CHHN
65
Thiazolone
H
⫹
⫹
HN
CHR
CC
OS
N
CH
65
The newly obtained polypeptide is treated with a fresh molecule of phenyl isothiocyanate
to yield the second-end amino acid and so on. Thus, this method can be used to determine
the amino acid sequence in polypeptides in a stepwise manner.
4. Enzymatic method: The enzyme used in this method is leucine aminopeptidase, which attacks
the peptides that have free amino group. After the terminal amino acid is eliminated, the
enzyme attacks the new terminal that has free amino group.
5. IR spectroscopy: Using IR spectroscopy, the peptide can be characterized. From the characteristic
band IR spectra of the peptide, the crystalline part of silk fi broin was found to have glycine
and alanine residues arranged alternatively.
‘ C’-Terminal (Carboxyl end) degradation
1. Schlack and Kumpf method: By benzoylation, the amino group is fi rst protected and then the
C-terminal amino acid is converted into thiohydantoin which is hydrolysed to the amino
acid. The amino acid liberated is identifi ed and the procedure can be repeated to determine
the amino acid sequence in polypeptides.
Amino acids, Peptides and Proteins
121
NH CHCONHCHCONHCHCOOH
2
R
1
R
2
R
3
C H COCl
65
CHOC
65
NHCHCONHCHCONHCHCOOH
R
1
R
2
R
3
NH NCS
Ac O
4
2
⌬
CHOC
65
NHCHCONHCHCO
R
1
R
2
N
C
CHR
3
C
O
N
H
S
NaOH
CHOC
65
NHCHCONHCHCOOH
R
1
R
2
⫹
HN
C
3
C
O
N
H
S
Thiohydantoin of C-termina
l
amino acid
Ba(OH)
2
NH CHCOOH
2
R
3
C
-Terminal amino acid
2. Hydrazinolysis: Heating of the peptide with anhydrous hydrazine at 100°C converts all of the
amino acids (except the carboxyl terminal one) of the chain into amino acid hydrazides. The
product is then subjected to chromatographic separation on a column of a strong cation-
exchange resin. The free amino acid is eluted, upon the elution, retaining the basic hydrazides.
This is then identifi ed.
NH CHCONHCHCONHCHCOOH
2
R
1
R
2
R
3
Heat with NH NH
22
NH CHCONHNH
22
R
1
⫹
NH CHCONHNH
22
R
2
⫹
NH CHCOOH
2
R
3
3. Reductive method: Using lithium aluminium hydride or lithium borohydride, the free terminal
carboxyl group of the peptide is reduced to primary alcohol group and the product obtained
on hydrolysis yields amino alcohol (from the C-terminal amino acid) along with degraded
protein or a mixture of amino acids. Using paper chromatography, the aminoalcohol
is separated and identifi ed. The procedure can be repeated to determine the amino acid
sequence in polypeptides.