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142
Pharmaceutical Chemistry of Natural Products
HCO
3
O CH
I
I
NH
Hydrolysis
HCO
3
O CHO
I
I
HC
2
COOH
NH COC H⭈
65
HCO
3
O CH
I
I
C
CO
N
CCH
65
⭈
O
Heat
–HO
2
HCO
3
O
I
I
C
NH COC H
65
⭈
CH
COOH
HI + P
Azalactone
HO
O
I
I
CH CH COOH
2
⭈⭈
NH
2
I /NH
23
HO
O
I
CH CH COOH
2
⭈⭈
NH
2
( )-Thyroxine±
II
I
Insulin
Insulin is a polypeptide hormone secreted by the β-cells of islets of Langerhans of the pancreas
(islets of Langerhans contain four major cell types, each of which synthesizes and secretes a distinct
polypeptide hormones mentioned here: insulin in the β-cell, glucagon in the α-cell, somatostatin
in the δ-cell and pancreatic polypeptide in the PP- or F-cell). The β-cells make up 60–80% of the
islet. It regulates the metabolism of carbohydrate, fat and protein, and control the sugar level
in the blood. The β-cells synthesize insulin from a single-chain precursor of 110 amino acids
termed as preproinsulin. Insulin promotes the synthesis of glycogen, triacylglycerols and proteins
and hence it is considered as anabolic hormone. The defi ciency of this hormone leads to diabetes
mellitus, characterized by increased blood glucose and excretion of sugar in the urine.
Human pancreas secretes about 40–50 units of insulin daily. The normal insulin concentration
in plasma is 20–30 µU/mL. The factors that infl uence the release of insulin from the β-cells of
pancreas are glucose, amino acid and GIT hormones such as secretin and gastrin.
Insulin initiates its actions by binding to its cell surface receptor. Insulin receptors are present
in virtually all cells, not only in the classic targets for insulin action (i.e. liver, muscle and fat) but
also in nonclassic targets such as circulating blood cells, neurons and gonadal cells.
Insulin has a normal half-life of 4–5 min. This short half-life permits rapid metabolic changes
in accordance to the alterations in the circulating levels of insulin. Insulinase (found in liver and
kidney) degrades insulin.
Mode of action: Insulin binds to specifi c plasma membrane receptors present on the target
tissues, such as muscle and adipose tissues. This leads to a series of reactions ultimately leading to
the biological action. Three distinct mechanisms of insulin action are known. The fi rst mechanism
Amino acids, Peptides and Proteins
143
is concerned with the induction of transmembrane signals (signal transduction), the second
mechanism is concerned with glucose transport across the membrane and the third mechanism is
concerned with induction of enzyme synthesis.
Gly
Ile
Val
Glu
Gln
Cys Cys
Thr
Ser
Ile
Cys Ser
Leu Tyr
Gln
Leu
Glu
Asn Asn
Tyr
His
Leu Cys
Gly
Ser
His
Leu
Val
Glu Ala
Leu
Try
Leu
Val
Thr
L
y
s Pro
Thr
T
y
r
Phe
Phe
Cys
Cys
Gln
Asn
Val
Phe
Gly
Glu
Arg
Gl
y
Chain B
Chain A
Human insulin
Functions of insulin
In addition to its role of regulating glucose metabolism, insulin also helps in the following:
Stimulating lipogenesis
Diminishing lipolysis
Increasing amino acid transport into cells
Modulating transcription
Altering the cell content of numerous messenger RNAs
Stimulating growth
DNA synthesis
Cell replication
Isolation
The slaughtered animal’s pancreas is immediately frozen and then minced and extracted with
80% ethanol (adjusted to a pH of 3). The pH of the extract is then increased to about 8 after the
fats and proteins are separated. The solution is fi ltered; the fi ltrate is acidifi ed and then evaporated
in vacuum to remove fatty materials. The solution in ethanol is free from fatty material and is
treated with picric acid. The insulin picrate is dissolved in acetone and insulin is reprecipitated as
its hydrochloride. Further purifi cation may be done by means of chromatography.
Structural elucidation
Insulin is one of the most complex proteins with an established structure. Its structure was
established in 1955 by tremendous contribution of Sanger for which he was awarded the Nobel
Prize in 1958.
1. Hydrolysis: Upon complete hydrolysis, insulin gives the below mentioned 17 different
types of amino acids of the total 51 amino acids indicating the fact that it has 51 amino acid
molecules.
144
Pharmaceutical Chemistry of Natural Products
(a) Glycine
(b) Alanine
(c) Valine
(d) Leucine
(e) Isoleucine
(f) Proline
(g) Phenylalanine
(h) Tyrosine
(i) Serine
(j) Threonine
(k) Lysine
(l) Glutamic acid
(m) Glutamine
(n) Asparagine
(o) Arginine
(p) Histidine
(q) Cysteine or cystine
2. Presence of two polypeptide chains: Sanger, using FDNB technique confi rmed that
in a molecule of insulin, glycine and phenylalanine are present as N-terminal residues
indicating that insulin has two chains of polypeptides: one as glycine and the other having
phenylalanine as the terminal amino acid. As insulin is found to have cystine, the two
chains may be linked with each other by disulphide bonds.
3. Separation of polypeptide chain by oxidation: Performic acid oxidation of insulin
yields two peptide chains, i.e. chain A and chain B. By electrophoresis or chromatography,
the two fractions are separated and degraded individually.
NH CH CO
CH
2
S
S
CH
2
HN CH CO
HCOOOH
NH CH CO
CH
2
SO H
3
SO H
3
CH
2
HN CH CO
Cystine residue 2 Cysteic acid residue
Structure of fraction B
The chain B of insulin is found to contain 30 amino acid residues and is basic in nature. By
means of different reagents, the fraction B was partially hydrolysed into various fragments of
polypeptides. The fragments were separated by ion-exchange resins, by adsorption on charcoal
and by ionophoresis. Further fractionation was achieved by paper chromatography.
Amino acids, Peptides and Proteins
145
Agent Polypeptides
Acid NH
2
NH
2
| |
(i) Phe—Val—Asp—Glu—His—Leu—Cys—Gly
(ii) Ser—His—Leu—Val—Glu—Ala
(iii) Ala—Glu
(iv) Ala—Leu—Tyr
(v) Tyr—Leu—Val—Cys—Gly
(vi) Gly—Glu—Arg—Gly
(vii) Gly—Phe
(viii) Thr—Pro—Lys—Ala
Pepsin NH
2
NH
2
| |
(i) Phe—Val—Asp—Glu—His—Leu—Cys—Gly—Ser—His—Leu
(ii) Val—Glu—Ala—Leu
(iii) Leu—Val—Cys—Gly—Glu—Arg—Gly—Phe
(iv) Tyr—Thr—Pro—Lys—Ala
Chymotrypsin (i) His—Leu—Cys—Gly—Ser—His—Leu
(ii) Val—Glu—Ala—Leu—Tyr
(iii) Leu—Val—Cys—Gly—Glu—Arg—Gly—Phe—Phe
Trypsin (i) Gly—Phe—Phe—Tyr—Thr—Pro—Lys
(ii) Ala
By DNP method, the different polypeptide fragments are degraded and thus the sequences
of amino acids in various fragments are established by DNP method. Adopting the structure
of different polypeptides, the chain B is established by tracing the overlapping patterns of the
different peptides isolated.
NH
2
NH
2
SO
H
3
Phe-Val-Asp-Glu-His-Leu-Cy-Gly-Ser-His-Leu-Val-Glu-ala-Leu-
Tyr-Leu-Val-Cy-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala
SO H
3
Structure of fraction A
The chain A of insulin is found to contain 21 amino acids and is acidic. In chain A, the sequence
of amino acids was elucidated in a similar manner as shown below:
NH
2
SO
H
3
Gly-Ile-Val-Glu-Glu-Cy-Cy-Ala-Ser-Val-Cy-Ser-Leu-Tyr-Glu-Leu-Glu-Asp-Tyr-Tyr-Cy-Asp(NH )
2
SO
H
3
SO H
3
NH
2
NH
2
NH
2
146
Pharmaceutical Chemistry of Natural Products
This step is carried out to establish the linkage between two chains in insulin molecule.
Performic acid oxidation of insulin yields cysteic acid residues, which suggests that the two chains
must be linked through disulphide bridges. The position of the disulphide bridges is established by
getting peptides containing the cysteine residues intact from unoxidized insulin. Such fragments
(i.e. the fragments containing disulphide bonds) are obtained in the following ways:
In the presence of thiol inhibitor such as N-ethylmaleimide (the inhibitor is used to check the
cleavage of disulphide bonds), insulin is hydrolysed by enzyme and the peptides containing the
cystine residues having intact disulphide linkages are separated, which is then oxidized to cysteic
acid peptides which are separated and then identifi ed by the usual DNP method. Insulin is found
to have the following three types of disulphide linkages based on the above type of reactions:
Leu-Val-Cy-Gly
S
S
Tyr-Cy-Asp-NH
2
His-Leu-Cy-Gly
S
S
Cy-Ala
Ser-Val-Cy
S
S
Gly-Ileu-Val-Glu-Glu-Cy
I II III
Chain A was found to contain four cysteic acid residues and chain B contains only two.
Hence, chain A contains a disulphide ring and is linked to chain B by two disulphide bonds.
Thus, the complete structure of insulin can be depicted below.
Tyr: TyrosineAla: alanine
Ser: SerineVal: Valine
The: ThreonineLeu: Leucine
Lys: LysineIleu: Isoleucine
Glu: Glutamic acidPro: Proline
CyS-SCy: Cysteine
NH
2
—
Glu
: Glutamine His : Histidine
NH
2
—
Asp
: Asparagine
Gly-Ile-Val-Glu-Glu-Cy-Cy-Ala-Ser-Val-Cy-Ser-Leu-Tyr-Glu-Leu-Glu-Asp-Tyr- Cy-Asp
NH
2
S S NH
2
NH
2
NH
2
123 4 5 6 78 910111213141516171819
20
21
S
S
S
S
Chain A
Chain B
123456 789101112131415161718192021222324
Phe-Val-Asp-Glu-His-Leu-Cy-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cy-Gly-Glu-Arg-Gly-Phe-
Phe-Tyr-Thr-Pro-Lys-Ala
26 27 28 29 3025
NH
2
NH
2
Synthesis of insulin
The above structure of insulin was confi rmed by the synthesis by Katsoyannis and his co-
workers in 1960. It was the synthesized by Eli-Lilly and co-workers. It was commercially called
humulin.
Amino acids, Peptides and Proteins
147
PROBABLE QUESTIONS
1. Defi ne and classify amino acids. Write the nomenclature of amino acids.
2. Write any three methods for the preparation of amino acids.
3. Write the importance of following reactions in the synthesis of amino acids.
a. Curtius reaction
b. Gabriel phthalimide reaction
4. What are α-amino acids? How are they prepared?
5. Write a note on the following:
a. Zwitterion
b. Isoelectric point
6. Write the general structure of α-amino acid. How are α-amino acids related to polypeptides and proteins?
7. What are peptides? What is the difference between polypeptides and proteins? How are polypeptides synthesized?
Describe in detail how the study of polypeptides has thrown light on the structure of proteins.
8. Write a note on the following:
a. Sorensen formol titration
b. Sydnones
9. Write any two methods for the separation of α-amino acids produced by hydrolysis of a protein.
10. Enumerate the chemical reactions of amino acids and explain any three of them.
11. Explain the steps involved in polypeptide synthesis.
12. Describe the methods employed for determining the sequence of amino acids in proteins and polypeptides.
13. Write the various ways by which a protein molecule can be hydrolysed, and discuss their merits and demerits.
14. Describe the methods adopted to determine the N-terminal and C-terminal amino acid residues used to determine the
molecular structure of proteins. How are they helpful in determining the sequence of amino acids in insulin?
15. Write in detail about the primary, secondary, tertiary and quaternary structures of proteins.
16. Write a note on the following:
a. Amino-end degradation
b. Carboxyl-end degradation
17. What are the various types of bonds found in protein?
18. Write a note on the isoelectric point of an amino acid.
19. Write a note on denaturation of proteins.
20. Explain Sanger’s method which is used to determine the sequence of amino acids in a polypeptide or a protein chain.
21. What are the advantages of carbobenzoxy chloride over benzoyl chloride as a protecting group in the synthesis of a peptide?
22. Defi ne and classify polypeptides. Write any two methods for the synthesis of polypeptides.
23. Defi ne and classify proteins with suitable examples.
24. Describe in detail about the structural elucidation of the following compounds:
a. Insulin
b. Thyroxine
25. Write a note on essential amino acids.
148
Pharmaceutical Chemistry of Natural Products
SUGGESTED READINGS
Anson, ML (1944). Advances in Protein Chemistry, Academic Press.
Bailey, JL (1967). Techniques in Protein Chemistry, 2nd Ed. Elsevier, Amsterdam.
Bodanszky, M and Ondetti, M (1966). Peptide Synthesis. Interscience, New York.
Branden, C and Tooze, J (1991). Introduction to Protein Structure. Garland Publishing, Inc., New York.
Budzikiewicz, H, Djerassi, C and Williams, DH (1964). Structure Elucidation of Natural Products by Mass Spectrometry,
Vol 2. Holden-Day, Inc., San Francisco.
Cook, JW (1953). Progress in Organic Chemistry, Vol 2. Academic Press, 212.
Creighton, TE (1992). Proteins: Structures and Molecular Properties, 2nd Ed. W.H. Freeman and Company, New York.
Dell, A and Morris, HR (2001). Glycoprotein structure determination by mass spectrometry. Science 291: 2351–2356.
Eliel, EL and Allinger, NL (eds) (1970). Topics in Stereochemistry, Vol 5. Wiley-Interscience, 69.
Elmore, DT (1968). Peptides and Proteins. Cambridge University Press.
Florkin, M and Stotz, EH (eds) (1963). Comprehensive Biochemistry, Vols 7 and 8. Elsevier.
Fox, SW and Foster, JF (1957). Introduction to Protein Chemistry. Wiley.
Freedman, RB (1971). Applications of the chemical reactions of proteins in studies of their structure and functions. Quart Rev
25: 431.
Geveart, K and Vandekerckhove, J (2000). Protein identifi cation methods in proteomics. Electrophoresis 21: 1145.
Gillard, RD and Laurie, SH (1969). Amino Acids, Peptides and Proteins. The Chemical Society Specialist Periodical Reports,
Vol 1.
Gilman, H (1943). Organic Chemistry: An Advanced Treatise, Vols 1 and 2. Wiley, New York.
Greenstein, JP and Winitz, M (1961). Chemistry of the Amino Acids, 3 Vols, John Wiley & Sons, New York.
Haurowitz, F (1963). The Chemistry and Function of Proteins, 2nd Ed. Academic Press.
Jones, JH (1968). The mass spectra of amino-acid and peptide derivatives. Quart Rev 22: 302.
Kendrew, JC (1961). The three-dimensional structure of a protein molecule. Sci Am 205 (Dec): 96–111.
Kreil, G (1997). D-Amino acids in animal peptides. Annu Rev Biochem 66: 337.
Lederer, E (1968). Mass spectrometry of natural and synthetic peptide derivatives. Pure Appl Chem 17: 489.
Li, WH and Graur, D (2000). Fundamentals of Molecular Evolution, 2nd Ed. Sinauer Associates, Inc., Sunderland, MA.
McLafferty, FW et al. (1999). Biomolecule mass spectrometry. Science 284: 1289.
Meister, A (1965). Biochemistry of the Amino Acids, 2nd Ed, Vols 1 and 2. Academic Press, Inc., New York.
Scopes, RK (1994). Protein Purifi cation: Principles and Practice, 3rd Ed. Springer-Verlag, New York.
Sheldrick, B (1970). Application of computers in chemical analysis; Amino-acid analysis and sequence determination. Quart
Rev 24: 454.
Shemyakin, MM (1968). Primary structure determination of peptides and proteins by mass spectrometry. Pure Appl Chem 17:
313.
Thomas, PJ, Qu, B-H and Pederson, PL (1995). Defective protein folding as a basis of human disease. Trends Biochem Sci 20:
456–459.
West, ES and Todd, WR (1961). Textbook of Biochemistry, 3rd Ed. Macmilla.
INTRODUCTI0N
Alkaloids are pharmacologically active, complex organic compounds containing one or more
nitrogen atoms, characteristically as primary, secondary or tertiary amines, which provide basicity
to the alkaloid. The term alkaloid (alkaloid means alkali-like) cannot be defi ned exactly, as there
is no clear-cut boundary between alkaloids and naturally occurring complex amines. In practice,
those substances obtained from plant sources and answer for the standard qualitative tests are
called alkaloids. The name protoalkaloid is applied to compounds such as ephedrine and colchicine
which are not having certain properties of typical alkaloids. They are found mainly in plants, but
to lesser extent in animals and microorganisms.
By keeping the knowledge of alkaloids in view, alkaloids may be defi ned as basic nitrogen
containing (usually in a heterocyclic system) plant products, possessing marked pharmacological
action at very low dose level.
Generally, from an amino acid, the nitrogen atoms in alkaloids are instigated and the carbon
skeleton of the particular amino acid precursor is also mostly retained in the structure of alkaloid.
However, through the transamination reactions a major group of alkaloids are found to obtain
their nitrogen atoms, incorporating only the nitrogen from an amino acid, while the remaining
part of the molecule may be derived from acetate or shikimate, or may be terpenoid or steroid in
origin.
Alkaloids are generally bitter in taste and optically active (except papaverine), usually
levorotatory in nature (exception is coniine, which is dextrorotatory), colourless (except berberine
which is yellow; harmaline and betanidine which are reddish), crystalline solids (except nicotine
and coniine which are liquids) and soluble in organic solvents like chloroform and ethanol but
insoluble in water. As they are basic in nature and form salts with the acids, some of the alkaloids
exist as salts called quaternary amines (e.g. cinchona alkaloids with quininic acid) while some of
them exist as free bases (e.g. nicotine). Some of the alkaloids also occur as glycosides (e.g. solanum
alkaloids) and esters (e.g. atropine). Biological activity of the alkaloids frequently depends on the
amine function being transferred into a quaternary amine at physiological pH by protonation.
Alkaloids
5
CHAPTER
150
Pharmaceutical Chemistry of Natural Products
Alkaloids were fi rst isolated successfully in the nineteenth century and the alkaloid-containing
drugs were marketed. The structure of the fi rst alkaloid, namely coniine was established in the
year 1870 by Schiff. In search of plant drugs with anticancer activity, catharanthus alkaloids and
paclitaxel came into the market.
NOMENCLATURE OF ALKALOIDS
There is no systematic nomenclature of alkaloids because of their complex molecular structure
and some historical reasons. They are named by adopting different methods as described below:
1. According to their physiological action: Examples include emetine (Greek word: emetikos means
to vomit), morphine (German word: morphine means God of Dreams) and narcotine (Greek
word: narkoo means benumb).
2. According to the plants from which they are obtained: Examples include papaverine from Papaver
somniferum and berberine from Berberis vulgaris.
3. Prefi xes like epi, iso, neo, pseudo or Greek letters are used to name isomeric or slightly modifi ed structures:
The prefi x nor indicates the structure which does not have a methyl group attached to the
nitrogen atom (e.g. norephedrine).
4. According to the name of the discoverer: For example, pelletierine (discovered by P.J. Pelletier).
5. The minor alkaloids are named by adding one prefi x or suffi x to the name of principal alkaloids: For
example, cinchonidine derived from cinchonine.
CLASSIFICATION OF ALKALOIDS
Alkaloids are classifi ed in different methods. Some of these methods are described briefl y and the
chemical classifi cation is described in detail:
1. Pharmacological classifi cation: It is based on the clinical use or pharmacological activity. Examples
include analgesic alkaloids and cardioactive alkaloids.
2. Taxonomic classifi cation: It is based on the family or genus, without reference to the chemical
type of alkaloid present (e.g. solanaceous alkaloids). However, the most common classifi cation
is according to the genus in which they occur (e.g. rauwolfi a alkaloids and cinchona
alkaloids).
3. Biosynthetic classifi cation: It is based on the type of precursors or building block compounds
used by plants to synthesize alkaloids. This is a more fundamental method than chemical
classifi cation. For example, morphine, papaverine, narcotine and colchicine may be listed as
phenylalanine- and tyrosine-derived bases.
4. Chemical classifi cation: It is based on the chemical structure of the alkaloid. The chemical
classifi cation of alkaloids is universally adopted and depends on the basic ring structure
present. For example, atropine is a tropane alkaloid; quinine is considered as a quinoline-
type alkaloid; papaverine is an isoquinoline and reserpine, strychnine and ergometrine are
indole alkaloids.
Alkaloids
151
Alkaloids
Derived from amino acids
冧
True alkaloids Proto/amino alkaloids Pseudo alkaloids
Example: Steroidal, terpenoidal alkaloids
Contain heterocyclic nitrogen Simple amines Not derived from amino acids
but from acyl CoA units
Example: Conestine, caffeine
Based on the chemical nature, alkaloids are further classifi ed into two major groups as
mentioned below:
1. Heterocyclic or typical alkaloids
2. Nonheterocyclic or atypical alkaloids [protoalkaloids (or) biological amines]
They are further subdivided as follows:
Heterocyclic Alkaloids
1. Pyridines and piperidines
O
O
CCCCC
HHO
N
HH
Piperine
Nicotine
N
N
CH
3
Arecoline
COOCH
3
N
CH
3
N
H
O
Pelletierine
OCH
3
CN
O
N
CH
3
Ricinine
Lobeline
N
CH
3
O
OH
Me
N
H
Conine