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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5987_Библиотеки_им_академика_М_И_Перельмана.pdf
132
Pharmaceutical Chemistry of Natural Products
 chain
␣ chain
Heme
Fe
2⫹
Proteins of Pharmaceutical Importance
Glutathione (GSH)
O
–
O
NH
2
O
O
N
H
O
SH
O
–
H
N
H—
H—
Glutathione (GSH)
Gamma-glutamyl-cysteinyl-glycine
It is a tripeptide hormone, γ-L-glutamyl-L-cysteinyl glycine, occurring nearly in all living cells.
Functions
1. It protects haemoglobin against oxidation by hydrogen peroxide.
2. It protects the liver by detoxicating the foreign substances.
3. It functions as a coenzyme of various enzymes.
4. It reduces the ascorbic acid into the more stable form and hence it protects the body store of
ascorbic acid.
5. It helps in maintaining the structure of red cell and globin part of the haemoglobin
molecule.
6. The effect of alloxan which causes permanent diabetes is prevented by the use of glutathione.
Structural elucidation
1. Hydrolysis: Upon hydrolysis glutathione yields 1 mole of glutamic acid, cysteine and
glycine which indicates that it is a tripeptide.
2. Presence of sulphydryl group (–SH): The presence of a free sulphydryl group (–SH) of
the cysteine molecule is confi rmed by the following reactions:
(a) It undergoes oxidation readily to form oxidized glutathione (GSSG).
(b) It undergoes alkylation to form S-alkyl derivatives.
(c) It undergoes acylation to form acyl derivatives.
Amino acids, Peptides and Proteins
133
3. Titration studies: Using titration studies the sequence of the three amino acids in
glutathione is established as follows:
(a) From the titration curves of the reduced glutathione molecule, it is found to possess
four titratable groups having pK
a
values of 2.12, 3.53, 8.66 and 9.62; the fi rst two values
correspond to carboxyl group, while the last two correspond to the amino and thiol
groups, respectively.
(b) The pK
a
values of 2.12 and 8.66 are typical for a carboxyl group having an adjacent
amino group and an amino group having an adjacent carboxyl group, respectively.
(c) This suggests that glutamic acid is present on the one end and it is linked to other amino
acid through its γ-carboxyl group. This is further confi rmed by the chemical method as
discussed herein.
COOH C
H
CH
2
CONH C
H
CH SH
2
pK 2.12
pK 9.62
CONH
CH
2
COOH
pK 3.53
NH
2
Glutathione (reduced
glutathione or GSH)
CbOCl(C H OCOCl)
65
COOH C
H
CH
2
CONH
H
CH SH
2
C CH
2
COOH
NH
Cbo
CH
2
CONH
(I)
1.2N HCl
heat for
1 hour
94°C
COOH CH C
CH
2
CH
2
N
Cbo
NH CH
CH SH
2
CONH CH
2
COOH
H
2
HC
2
CH
2
COOH
CH
CbO
NH
COOH
CbO derivative of
glutamic acid, II
HC
2
CH
2
CO
CH
N
H
COOH
III
H NCHCONHCH COOH
22
CH SH
2
⫹
1.2N HCl
94°C, 1 hour
Cysteinylglycine, IV
⌬
HC CO
OC
CH
2
H
N
H
N
HSH C
2
Diketopiperazine, V
CH
2
pK 8.66
OH
134
Pharmaceutical Chemistry of Natural Products
(d) The carbobenzoxy (Cbo) derivative (I) of glutathione is heated with water at 62°C for
5 days or with 1.2 N HCl at 94°C for 1 hour when pyrrolidone carboxylic acid (III)
and cysteinylglycine (IV) are obtained. The compound (III) may also be obtained by
treating the Cbo derivative of glutamic acid under the same conditions. The formation
of the compound (III) indicates that the γ-carboxyl group of the glutamic acid residue
is involved in glutathione. When the amino-end degradation method is applied to
dipeptide (IV), the amino group of cysteine and carboxyl group of glycine are found to be
free. Further, compound (IV) on heating or simply on standing at ordinary temperature
gives diketopiperazine (V).
4. Oxidation of glutathione
[O]
HOOCCHCH CH CONHCHCONHCH COOH
22 2
NH
2
CH
2
SH
Glutathione
HOOCCHCH CH CONHCHCONHCH COOH
222
NH
2
CH
2
S
S
CH
2
HOOCCHCH CH CONHCHCONH CH COOH
22 2
×
Oxidized glutathione (GSSG)
NH
2
In the structure of glutathione, the linkage between glutamic acid and cysteine residues is
not typical of the peptides of the protein type, because the amino group of the glutamic acid
residue is not coupled. Glutathione was the fi rst compound with abnormal peptide linkage to be
discovered. The above proposed structure of glutathione is proved by its synthesis.
SCH CHCOOH
2
NH
2
SCH CHCOOH
2
NH
2
2C H CH OCOCl
65 2
SCH CH COOH
2
×
NHCbO
SCH CHCOOH
2
NHCbO
(i) PCl
5
(ii) CH
2
(NH )COOC H
225
SCH CHCONHCH COOC H
2225
NHCbO
SCH CHCONHCH COOC H
2225
NHCbO
SHCH CHCONHCH COOC H
2
25
2
NH HI
2
PH I
4
(–2C H
65 2 2
CH I, –2CO )
(acid chloride of -methyl
carbobenzenyloxy glutamate
a
ClCOCH CH CHCOOCH
22 3
NHCbO
NHCOCH CH CHCOOCH
22 3
NHCbO
HSCH CHCONHCH COOC H
2225
(i) Hydrolysis of ester group
(ii)PH l or Na/NH
43
COOHCHCH CH CONHCHCONHCH COOH
22 2
NH
2
CH
2
SH
Glutathione
Amino acids, Peptides and Proteins
135
Oxytocin
Oxytocin is the hormone secreted in the posterior pituitary gland. It exerts the biological activity
of contraction of the uterus (oxytocic effect) coupled with milk ejection. It is a nine–amino acid
peptide synthesized in hypothalamic neurons and transported down the axons of the posterior
pituitary gland for secretion into blood. Oxytocin is also secreted within the brain and from a
few other tissues, including the ovaries and testes. Oxytocin differs from antidiuretic hormone in
two of the nine amino acids. Both hormones are packaged into granules and secreted along with
carrier proteins called neurophysins. It differs from vasopressin (the other hormone secreted in the
posterior pituitary gland) by only two amino acids. Biochemical functions of oxytocin includes
contraction of pregnant uterus (smooth muscles) and inducing labour, contraction of myoepithelial
cells of breast which stimulates the squeezing effect and causes milk ejection from the breast. In
situations where continued pregnancy is a risk to the mother or foetus, oxytocin is the drug of
choice for labour induction. Atosiban, a competitive inhibitor for the interaction of oxytocin with
its membrane receptor, is used for the treatment of preterm labour.
Mode of action: Oxytocin (in human myometrium) acts via specifi c GPCRs, coupled to Gq
and G11, to activate the PLC–IP3–Ca
2+
pathway and improve the activation of voltage-sensitive
Ca
2+
channels. Further, it stimulates uterine contractions through the increased production of
prostaglandin locally.
Oxytocin
Tyr
Cys
Gly
Leu
Pro
Asp
Ile
Gln
Cys
Isolation: The powdered form of posterior pituitary gland is extracted with ethanol or acetic
acid and the resultant solution is treated with NaCl. The coagulated proteins are then treated with
trichloroacetic acid to dissociate the various proteins such as oxytocin, noradrenaline, vasopressin
and other impurities. Using paper chromatography technique, proteins are separated and fi nally
purifi ed using electrophoresis.
Structural elucidation
1. Presence of free –NH
2
and absence of –COOH group: Presence of free amino group
and absence of carboxyl group is indicated by the isoelectric point of 7.7.
2. Presence of eight amino acids: It yields eight different amino acids (glycine, leucine,
isoleucine, proline, cystine, tyrosine, aspartic acid and glutamic acid) in equimolecular
amount upon complete hydrolysis with acid along with 3 moles of ammonia. The hydrolysis
products indicate that oxytocin is built up of eight amino acids which have the amide–CONH
2
136
Pharmaceutical Chemistry of Natural Products
(which gives ammonia molecule) groups. The molecular weight of oxytocin is found
to be 1000 Da which corresponds to octapeptide, and therefore, oxytocin must be an
octapeptide.
3. Presence of disulphide unit: Oxytocin gives a disulphonic acid having a molecular
weight in the range of oxytocin disulphonic acid upon oxidation with performic acid. This
suggests that oxytocin is a cyclic compound and has a disulphide unit which is oxidized by
performic acid to sulphonic acid in the following manner:
Oxytocin
HCOOOH
Oxytocin disulphonic acid
R—S—S—R
HCOOOH
2 RSO H
3
4. Acid hydrolysis: Upon controlled hydrolysis with hydrochloric acid, the oxidized form of
oxytocin yields 2 moles of cysteic acid, four dipeptides (I–IV) and two tripeptides (V and
VI). The structures of these peptides were established by treatment with FDNB followed by
hydrolysis and identifi cation of the DNP derivative using chromatography.
(a) Asp–CysSO
3
H (d) ILeu–Glu
(b) CysSO
3
H–Tyr (e) Tyr–Glu, ILeu
(c) Leu–Gly (f) CysSO
3
H–(Leu,Pro)
The amino acid units when separated by comma and written within the bracket indicate
that their sequence is not known.
On the basis of the structure of the dipeptides and amino ends of the tripeptides, the
partial structure of oxytocin can be written according to following points.
(a) From the sequence of dipeptide (IV), the structure of tripeptide (V) may be written as
Va: Tyr–lleu–Glu.
(b) On the basis of structure of the dipeptide (II), the structure of Va may be extended as
Vb: CySO
3
H–Tyr–lleu–Glu which also accounts for Va.
(c) On the basis of structure of the dipeptide (III), the structure of compound (VI) may be
written as CySO
3
H–Pro–Leu–Gly.
5. Enzymatic hydrolysis: Using usual methods with proteinase (isolated from Bacillus subtilis)
partial hydrolysis of oxidized oxytocin yields glycine amide and two acidic tetrapeptides
(VII) and (VIII), whose amino acid components and amino-end groups are established.
CysSO
3
H–(Glu, Tyr, ILeu) Asp–(CysSO
3
H, Leu, Pro)
VII VIII
6. Structure of oxidized oxytocin: The structure of the tetrapeptide (VII) has already been
established in point 4 (b) as CySO
3
H–Tyr–lleu–Glu. In addition, from the structure of (VI)
point 4 (c), the structure of (VIII) may be extended as Asp–CysO
3
H–Pro–Leu–Gly. The
formation of glycine amide indicates that glycine is present as an end group and hence
the structure of (VIII) may be written as below.
Asp—CysSO
3
H—Pro—Leu—Gly (NH
2
)
Based on the structure of the two tetrapeptides and the fact that glycine is present as
an end group, the structure of oxidized oxytocin may be written as follows:
CysSO H—Tyr—ILeu—Glu— —Asp—CysSO H—Pro—Leu—Gly (NH )
332
NH
2
NH
2
Amino acids, Peptides and Proteins
137
The dotted lines indicate the points of cleavage (achieved using bacterial proteinase) of
the oxidized oxytocin to the tetrapeptides (VII) and (VIII), and the glycine amide.
7. Position of the amide groups: The position of the three amide groups as pointed out by
the formation of 3 moles of ammonia is established by the following facts.
(a) As glycine is isolated as glycine amide in point 5, one of the amide groups must be
present in the glycine molecule.
(b) Among the eight amino acids isolated from oxytocin, only glutamic and aspartic acids
are monoamino dicarboxylic acids. So one of the carboxylic groups of each of these
acids which is not involved in peptide linkage may only be present as amide groups.
8. Structure of oxytocin: The structure of oxidized oxytocin and its formation from oxytocin
by using performic acid without chain fi ssion indicates the presence of S–S ring in the
compound. Therefore, oxytocin must possess the structure as shown below (assuming that
α-carboxyl group of glutamic acid and aspartic acid are involved in the peptide linkages).
Cy—Tyr—ILe—Glu—Asp—Cy—Pro—Leu—Gly(NH ) or
2
NH
2
NH
2
S S
Cy—Tyr—ILeu
S
S
Cy—Asp—Glu (NH )
2
NH
2
Pro—Leu—Gly (NH )
2
Cystine
CC
H
HC
2
S
S
CH
2
C
H
NH
CO
p_HOC H
64
CH
2
NH
CH C
O
Tyrosine
Isoleucine
CH
25
CH
3
CH
CH
NH
CO
O
CCH
NH
CH
2
CONH
2
CH CONH
22
CH
2
C
H
C
O
NH
Asparagine
Glutamine
HC
2
CH
2
N
HC
Proline
NH
H
C
CO
CH
2
CHCH
3
CH
3
Leucine
NHCH CONH
22
Glycinamide
or
NH
2
O
O
C
CH
2
Abbreviated and detailed formula for oxytocin
138
Pharmaceutical Chemistry of Natural Products
Finally, the above structure of oxytocin is proved by a number of syntheses. The four
important steps in the synthesis are as follows:
(i) By treating the amino acid with carbobenzoxy chloride (C
6
H
5
CH
2
OCOCl), the amino group
is protected by carbobenzoxy group.
(ii) The amino acid having protected amino group Cbo is reacted with the ester of other amino
acid molecule. The various esters used are ethyl, benzyl and p-nitrophenyl.
(iii) The protective group is removed by hydrogen bromide–acetic acid and the resulting product
is treated with the ester of other amino acid whose amino group is protected by Cbo. Steps
(ii) and (iii) are repeated till the amino acids are reacted.
(iv) By catalytic reduction or by using sodium in liquid ammonia, the protective group is fi nally
removed as toluene and carbon dioxide molecule to form the product.
In the abbreviated form the overall synthesis is depicted as below:
HGlyOEt
Cbo.LeuNP
Cbo Leu Gly OEt
HBr—CH COOH
3
H Leu GlyOEt
Cbo ProNP
Cbo Pro Leu Gly OEt
NH /CH OH
33
Cbo Pro Leu Gly NH
2
(i) HBr—CH COOH
3
(ii) CboCys(Bzl)NP
Cbo Cys Pro Leu GlyNH
2
(i) HBr—CH COOH
3
Bzl
(ii) CboAsp(NH ) NP
2
×
Cbo Asp Cys Pro Leu GlyNH
2
NH
2
Bzl
(i) HBr—CH COOH
3
(ii) CboGlu(NH )NP
2
CboGlu Asp Cys Pro Leu GlyNH
2
NH
2
NH
2
Bzl
(i) HBr—CH COOH
3
(ii) Cbo Ileu NP××
Cbo Ileu Glu Asp Cys Pro Leu GlyNH
2
NH
2
NH
2
Bzl
(i) HBr—CH COOH
3
(ii) Cbo Tyr(Bzl) NP×
Cbo Tyr Ileu Glu Asp Cys Pro Leu GlyNH
2
Bzl
NH
2
NH
2
Bzl
(i) HBr—CH COOH
3
(ii) Cbo Cys(Bzl) NP
Cbo Cys Tyr Ileu Glu Asp Cys Pro Leu GlyNH
2
Bzl
NH
2
NH
2
Bzl
(i) Na—NH
3
(ii) O (air)
2
Bzl
Cy Tyr Ileu Glu AspCy Pro Leu GlyNH
2
NH
2
NH
2
S S
Oxytocin
Thyroxine
It is a hormone secreted by thyroid gland in the form of a protein called thyroglobulin. The latter
on hydrolysis yields mainly thyroxine (tetraiodothyronine) with the formation of other products
such as mono- and di-iodotyrosine, and di- and tri-iodothyronine. The synthesis of the thyroid
hormones [thyroxine (tetraiodothyronine) (T
4
) and tri-iodothyronine (T
3
)] is regulated by
thyrotropin [thyroid-stimulating hormone (TSH)] by stimulating the synthesis of thyroglobulin
Amino acids, Peptides and Proteins
139
thyroperoxidase and hydrogen peroxide. Exogenous supply of iodide is important in the formation
of the thyroid hormones (daily requirement of iodine ranges from 40 to 120 µg for children, 150
µg for adults, 220 µg for pregnant women and 270 µg for lactation). In the thyroid gland, about
25% of the body’s supply of iodide is located. Thyroid gland is the only tissue of the body which
can accumulate iodine in large quantities and incorporate it into hormones.
By the following sequence of events, biosynthesis of thyroid hormones happens in the body:
(i) active uptake of iodide by the follicular cells; (ii) oxidation of iodide and formation of iodotyrosyl
residues of thyroglobulin; (iii) formation of iodothyronines from iodotyrosines; (iv) proteolysis of
thyroglobulin and release of (tetraiodothyronine) (T
4
) and triiodothyronine (T
3
) into blood; (v)
conversion of T
4
to T
3
.
Defi ciency of thyroxine disturbs the metabolism of carbohydrates, lipids, proteins, electrolytes
and water which leads to cessation of growth. It plays a critical role in brain development. The
defi ciency of thyroid hormone during active neurogenesis (up to 6 months postpartum) leads to
irreversible mental retardation (cretinism). In hypothyroidism, cardiovascular effects observed are
bradycardia and elevation of mean arterial pressure. Therapeutic uses of thyroid hormones include
hormone replacement therapy in patients with hypothyroidism or cretinism and TSH suppression
therapy in patients with thyroid cancer.
Liver is the major site of nondeiodinative degradation of thyroid hormones; T
4
and T
3
are
conjugated with glucuronic and sulphuric acids through the phenolic hydroxyl group and excreted
in the bile. Some thyroid hormone is liberated by hydrolysis of the conjugates in the intestine and
reabsorbed.
In hyperthyroidism, cardiovascular effects observed are tachycardia, decreased peripheral
vascular resistance and increased pulse pressure. Antithyroid drugs such as propylthiouracil,
methimazole and carbimazole are used to treat this condition.
1’
2’
3’
4’
5’
6’
HO
I
I
O
1
2
3
4
5
6
I
I
CH CHCOOH
2
NH
2
Thyroxine
Isolation
The dried thyroid gland is treated with Ba(OH)
2
solution—fi rst with 10% solution and then with
40%. In 1% hot NaOH solution, barium salt of thyroxine is suspended and treated with sodium
sulphate solution to remove barium as BaSO
4
. By the acidic hydrolysis of the sodium salt thyroxine
is obtained. Further purifi cation may be carried out with alcohol and CH
3
COOH. The hormone so
obtained is a racemic mixture which can be resolved into L-isomer by trypsin.
Structural elucidation
1. Molecular formula: The molecular formula of thyroxine is found to be C
15
H
11
NI
4
O
4
.
2. Presence of acidic group: By the usual reactions, it was found to contain one acidic group.
3. Presence of –NH
2
group: Upon acetylation, thyroxine forms monoester indicating the
presence of –OH or –NH
2
group. Upon treatment with HONO, thyroxine gives a hydroxyl
acid with evolution of nitrogen, which indicates the presence of –NH
2
group.
140
Pharmaceutical Chemistry of Natural Products
4. Catalytic reduction: Upon catalytic reduction (H
2
–Pd) by replacing iodine atoms with
hydrogen, it gives thyronine (C
15
H
15
NO
4
).
5. Constitution of thyronine ( thyronin):
(a) The molecular formula of thyronine is found to be C
15
H
15
NO
4
.
(b) With Millon’s reagent (Hg in HNO
2
) thyronine gives a brick red colour indicating the
presence of hydroxyphenyl group.
(c) With ninhydrin it gives blue colour, which indicates the presence of α-amino acid.
(d) When fused with KOH at 300°C it yields quinol, p-hydroxybenzoic acid, diphenyl
derivative (OH–C
6
H
4
–O–C
6
H
4
–CH
3
), oxalic acid and ammonia. Formation of these
products reveals that the thyronine molecule contains two benzene nuclei linked by an
ether linkage (–C
6
H
4
–O–C
6
H
4
–) with one of the nuclei carrying a hydroxyl group in the
para position to the ether linkage. So the rest of the carbon atoms (C
15
– C
12
= C
3
) must
be present in the side chain. Moreover, thyronine is also found to be an α-amino acid.
Hence, the part structure of thyronine may be either I or II.
HO
O
C C COOH HO O C COOH
C
NH
2
NH
2
I, Straight side chain
II, Branched side chain
HO C COOH
NH
2
H
CH
2
Tyrosine
HO O
I a
C COOH
NH
2
H
CH
2
Thyronine
The next step in elucidating the structure of thyroxine is to specify the position of
the iodine atoms in thyronine molecule. This can be demonstrated with the following
experiment.
When fused with alkali, thyroxine gives two pyrogallol compounds (replacement of
iodine by –OH groups), which reveals that the two iodine atoms are in o-positions to
the original hydroxyl group in each benzene nuclei. Hence, the structure of thyroxine
is assigned to be II.
Amino acids, Peptides and Proteins
141
HO O
C COOH
NH
2
H
CH
2
I
I
I
I
KOH
HO OH
C COOH
NH
2
H
CH
2
OH
OH
OH
OH
HO⫹
6. The above proposed structure can further be proved by the following specifi c reaction of
iodinated phenols in both the ortho positions.
7. Thyroxine with HONO shows yellow colour upon boiling. The colour deepens on boiling
and changes to red on cooling followed by addition of ammonia.
8. The structure of thyroxine can be fi nally proved by its synthesis.
HN
2
NO
2
p-Nitroaniline
I
2
HN
2
NO
2
I
I
(Sandmeyer's reaction)
(i) NaNO HCl
(ii) KI
2
⫹
I
NO
2
p-Methoxyphenol
I
I
HCO
3
O
HCO
3
OH
NO
2
I
I
(i) Reduction
(ii) C H ONO–HCl
Diazotization
511
HCO
3
O CN
I
I
SnCl –HCl
(Partial reduction)
2
CuCN
HCO
3
O NCl
2
I
I
K CO –Butanone
23