DESIGN, SYNTHESIS AND STUDIES OF SOME LINEAR AND CYCLIC PEPTIDES AND THEIR IRON CHELATES AS ANTIOXIDANT NUTRACEUTICALS IN ANAEMIA Department of Pharmaceutical Sciences Birla Institute of Technology Mesra, Ranchi – 835 215. 2012 A Thesis Submitted in partial fulfillment of the requirements for the award of the Degree of MASTER OF PHARMACY IN PHARMACEUTICAL CHEMISTRY By Shroff Prashantkumar Balubhai (MPH/1014/2010) Under the guidance of Dr. S. SAMANTA (Professor)
The objective of our work is to design and synthesize small synthetic nutraceutical peptides, using more economical liquid phase method (CPE method) and their iron chelates, which will be helpful in treatment of anemia by lowering red blood cell oxidative stress and by increasing iron absorption in body.
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DESIGN, SYNTHESIS AND STUDIES OF SOME LINEAR AND CYCLIC PEPTIDES AND THEIR IRON CHELATES AS ANTIOXIDANT
NUTRACEUTICALS IN ANAEMIA
Department of Pharmaceutical SciencesBirla Institute of Technology
Mesra, Ranchi – 835 215.2012
A Thesis
Submitted in partial fulfillment of the requirements for
the award of the Degree of MASTER OF PHARMACY
IN
PHARMACEUTICAL CHEMISTRY
By
Shroff Prashantkumar Balubhai
(MPH/1014/2010)
Under the guidance of
Dr. S. SAMANTA
(Professor)
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INTRODUCTION LITERATURE REVIEW OBJECTIVE OF THE WORK PLAN OF WORK EXPERIMENTAL WORK RESULT AND DISCUSSION SUMMARY AND CONCLUSION FUTURE SCOPE REFERENCES
CONTENTS
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1. INTRODUCTION
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NUTRACEUTICAL1,2,
3
• The term “Nutraceutical” was coined from “Nutrition” & “Pharmaceutical” in 1989 by Stephen DeFelice, MD Founder and Chairman of the Foundation for Innovation in Medicine (FIM).
1
•Nutraceuticals may range from isolated nutrients, herbal products, dietary supplements and diets to genetically engineered ''designer'' foods and processed products such as cereals, soups and beverages. Doubtlessly, many of these products possess pertinent physiological functions and valuable biological activities.
DEFINITION :•Food, or parts of food, that provide medical or health benefits, including the prevention and treatment of disease.
•The term also has been defined as a product isolated from food and generally sold in medicinal forms not usually associated with food and demonstrated to have physiological benefit or provide protection against chronic disease.
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• It is important to note that this definition pertains to all categories of foods. It ranges from dietary supplements such as folic acid used for the prevention of spine bifida, to chicken soup taken to lessen the discomfort of the common cold. It includes a bioengineered designer vegetable food rich in antioxidant ingredients to a stimulant functional food or pharmafood.
• With the passage of the Dietary Supplement Health and Education Act of 1994 in US, the definition of nutraceuticals has been expanded to include vitamins, minerals, herbs and other botanicals, amino acids and any dietary substance for use by humans to supplement the diet by increasing total dietary intake and subsequently increased the use of nutraceuticals dramatically.
Nutraceuticals can be grouped into the following three broad categories:• Nutrients- The most commonly known nutrients are antioxidant, water and fat-soluble
vitamins.• Herbals- Numerous nutraceuticals are present in medicinal herbs as key components.
E.g. Aloe Vera, Ephedra, Garlic etc.• Dietary supplements- For e.g. Ketogenic diets, comprised of foods high in fat and low
in protein and carbohydrate content, have been reported to improve seizure control.
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• Iron, as a therapeutic agent, was first documented about 2735, B.C., when it was declared by the Chinese Emperor, Shen Nung, as a cure for “Anemia”.4
Iron is an essential in human body for5:• the formation of hemoglobin and certain enzymes, many proteins and
enzymes that maintain good health• transporting oxygen in the blood to all parts of the body• many metabolic reactions and the regulation of cell growth and differentiation• immune activity• proper functioning of the liver• protection against the actions of free radicals.
IRON Iron(Fe) is an absolute requirement for most forms of life, including humans and most bacterial species, because plants and animals all use iron; hence, iron can be found in a wide variety of food sources.
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Iron absorption in human body6:
Fig 1.1: Iron transport through the enterocytes6
Nonetheless, intestinal iron absorption and cellular iron transport are poorly understood. Iron deficiency anemia is, in part, a result of diet.
The availability of iron from many foods is very low. No more than 5% of vegetable iron is absorbed and while the absorption of iron from meat, poultry and fish may be somewhat higher, significant worldwide consumption of animal proteins is limited to the more affluent.
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•Consumption of certain foods, such as coffee or tea, phosphates, phytates and bran will generally reduce iron absorption. Besides a diet, an individual’s iron status is also related to age, sex, lifestyle, lactations etc.
•The use of oral contraceptives, aspirin, antacids, antiinflammatories, anticoagulants and steroids may all increase the risk of iron deficiency.
•Diseases of the gastrointestinal tract, including cancer, hemorrhoids will result in a greater risk of iron deficiency due to increased iron requirements and/or an increased inability to efficiently absorb iron.
•Illness associated with a fever may also reduce iron utilization, even if the iron is absorbed.
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• Iron deficiency is the most common micronutrient deficiency in the world and has far-reaching and serious adverse effects on health. Nearly one fourth of the world’s population is currently anemic.
• As per WHO, Anaemia(Anemia) is a condition in which the number of red blood cells or their oxygen-carrying capacity is insufficient to meet physiologic needs, which vary by age, sex, altitude, smoking, and pregnancy status.7
• Anemia detection is often used as a screening test for iron deficiency. Anemia is a late sign of deficient iron stores.
ANAEMIA
Figure 1.2: Anemic blood cells60
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Anemia has four basic causes. One or more of these causes must be operating to produce anemia8:•Hemorrhage -- bleeding•Hemolysis -- excessive destruction of red blood cells•Underproduction of red blood cells•Not enough normal hemoglobin
There are many forms of anemia, some of them are common. They include, for example:8
Iron deficiency anemia: ( iron deficiency results in anemia)Aplastic anemia: (due to failure of the bone marrow to produce blood cells)Fanconi anemia: (A genetic disease of the bone marrow elements, mainly in children)Pernicious anemia: (A blood disorder caused by inadequate vitamin B12 in the blood.)Sickle cell disease: (A genetic blood disorder caused by the presence of an abnormal form of hemoglobin.)
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Figure 1.3: Symptoms of Anemia61
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IRON DEFICIENCY ANEMIA7
The World Health Organization considers Iron Deficiency is the number one nutritional disorder in the world. As many as 80% of the world's population may be iron deficient, while 30% may have iron deficiency anemia.
In iron deficiency anemia, the red cells appear abnormal and are unusually small (microcytic) and pale (hypochromic). The pallor of the red cells reflects their low hemoglobin content.
The prevalence of iron deficiency anemia is the highest in children and women of childbearing age (particularly pregnant women).
The treatment of iron deficiency anemia, whether it be in children or adults, is with iron and iron-containing foods.
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How anemia is detected?7
Anemia is most commonly detected by measuring Hemoglobin or by determining Hematocrit (the volume of red blood cell in the specific amount of blood).
WHO proposes the following cut-off hemoglobin(Hb) values:
WHO lists the following ranges for normal hematocrit(Hct) values:
Children under 5 year of age Hb < 110g/L
Non-pregnant women Hb < 120g/L
Pregnant women Hb < 110g/L
Men Hb < 130g/L
Children under 5 year of age Hct 38-44%
Women Hct 37-43%
Men Hct 40-50%
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Oxidative stress in Anemia9
Figure 1.4: A hypothetical mechanism of anemia and autoantibody production against erythrocytes due to reduced activity of the antioxidant system in blood.9
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Fenton reaction in Erythrocytes10
The erythrocytes represent an important component of the antioxidant capacity of blood, comprising in particular intracellular enzymes, e.g. superoxide dismutase and catalase, but also the glutathione system.
Iron deficiency anemia enhances red blood cell oxidative stress and reduces the erythrocyte levels of catalase, superoxide dismutase and glutathione.10
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GLUTATHIONE REDUCTASE11
Glutathione reductase, also known as GSR or GR, is an enzyme that reduces glutathione disulfide (GSSG) to the sulfhydryl form GSH, which is an important cellular antioxidant. GSR is used as an indicator of oxidative stress in red blood cell in anemia.
Reaction mechanism of human glutathione reductase:
NADPH reduces FAD present in GSR to produce a transient FADH- anion. This anion then quickly breaks a disulfide bond (Cys58 - Cys63) and leads to Cys63's nucleophilically attacking the nearest sulfide unit in the GSSG molecule, which creates a mixed disulfide bond (GS-Cys58) and a GS- anion. His467 of GSR then protonates the GS- anion to form the first GSH. Next, Cys63 nucleophilically attacks the sulfide of Cys58, releasing a GS- anion, which, in turn, picks up a solvent proton and is released from the enzyme, thereby creating the second GSH. So, for every GSSG and NADPH, two reduced GSH molecules are gained, which can again act as antioxidants scavenging reactive oxygen species in the cell. 9,10
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Iron - Amino acid chelates12:
In order to enhance iron bioavailability and still avoid interaction with food ingredients, chelating iron with amino acids has been employed. A nutritionally viable iron amino acid chelate must have a stability constant which would result if the iron were chelated or complexed to the food ligands found in the stomach and intestines.
If the chelate dissociates in the gut, it has no more value than ionized iron from a soluble salt. The stability constant should also be high enough to allow chelate to cross the intestinal cell membrane into the cytoplasmic ligands are capable of removing the iron from the absorbed amino acid chelate by complexing with the absorbed iron.17
For an iron amino acid chelate to be absorbed into the mucosal tissue, it must be a low molecular weight chelate less than 1500 daltons if it is to be absorbed in humans. Or we can prepare iron chelated peptides with amino acids which can be absorbed from gastrointestinal track into the mucosal cell as a chelate than a soluble salt of iron in treatment of iron deficiency anemia.4
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Bioactive Antioxidant Peptides13,14:
• Opportunities have arisen to formulate food products which deliver specific health benefits, in addition to their basic nutritional value. In this respect, bovine milk and colostrum are considered the most important source of natural bioactive components.
• All amino acids are susceptible to oxidation, although their susceptibilities vary greatly. Organisms have evolved complex antioxidant defenses to minimize oxidative damage to proteins and other macromolecules.
• The antioxidative activities of peptides generated from the digestion of various proteins have been reported. Several amino acids, such as Tyr, Met, Asp, Pro, His, Lys, and Trp are generally accepted to be antioxidative and exhibit higher antioxidative activities when incorporated into peptides. However, neither the structure-activity relationship nor the antioxidant mechanism of peptides is fully understood.
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2. Literature review
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IRON AND ANEMIA
•William Dameshek in 1950Has shown that Ferrous ion has become established as the material of choice and ferrous sulphate is highly acceptable; however oral preparations are good, but injectable iron is better due to severe reactions in GIT with oral iron.15
•Dainel et al. in 1955Have shown that iron stores are exhausted before anemia occurs and patients shows response to oral iron but if they are unable to take iron because of gastrointestinal symptoms, parenteral administration of iron has advantages.16
•Hugh in 1958Has shown that local factors ( reducing agents, GI acidity, presence of phosphate) and General factors (Diet etc…) are influencing absorption of iron, has also shown supplementation with inorganic iron is a more effective way of increasing iron retention.17
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•Chan et al. in 1959Have shown that Anemia usually responds to Oral iron therapy in any ferrous salt like sulphate, fumarate, succinate or gluconate.18
•Stephen H. Robinson in 1969Has shown bilirubin formation has been increased in rats with iron deficiency anemia which can originate from hemolysis of circulating red blood cell, iron deficiency disorders heme metabolism in bone marrow which can be overcome with iron therapy.19
•Gordeuk et al. in 1986Have shown that Carbonyl iron is safe, effective, well tolerated in treatment of iron deficiency anemia and has less toxicity in children than with ferrous sulfate.20
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OXIDATIVE STRESS IN IRON DEFICIENCY ANEMIA
•Ramachandran in 1985Has shown that lipid peroxidation plays a major role in the reported decrease in red cell life-span in iron deficiency.21
•Jansson et al. in 1985Have found the higher content of superoxide dismutase in iron deficient RBC than control, which suggests an increased formation of SOD compensatory to an increased oxidative stress.22
•Mehmat et al. in 2002Have shown that Oral iron treatment improved the IDA and recovered antioxidant defense system by increasing SOD and GSH-Px activity.23
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•Farshad et al. in 2008 Have shown that the activities of erythrocyte cytoprotective enzyme decrease and lipid peroxidation increases in women with IDA which may lead to other degenerative disorders.24
•Coghetto et al. in 2009 Have shown that the patients with IDA are subjected to chronic oxidative stress.25
•Jong-Ha Yoo et al. in 2009Have shown that blood reactive oxygen species was lower and total antioxidant activity was higher after treatment which supports the higher oxidative stress hypothesis in IDA.26
•Krishnamurthy et al. in 2010Have shown that Oral iron is safe and efficacious in children with IDA.27
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ANTIOXIDANT NUTRACEUTICAL PEPTIDES
•Rodney et al. in 1996 Have shown that Cysteine and Methionine residues function in the catalytic cycle of several enzymes and constitute an important antioxidant defense mechanism.28
•Chan et al. in 1998 Have shown that in the antioxidant peptide (LLPHH) derived from proteolytic digest of a Soybean protein, HH plays a major role in antioxidant activity.29
•Kunio S. and Jiun-Rong C. in 2002Have shown that peptides having potent antioxidant activity were from the hydrolysate of Wheat gluten with amino sequences Leu-Gln-Pro-Gly-Gln-Gln-Gly and Ala-Gln-Ile-Pro-Gln-Gln.30
•Saito et al. in 2003Have constructed combinatorial libraries with histidine, tryptophan, alanin and glycin, based on antioxidative peptide isolated from a soybean protein hydrolysate and concluded their antioxidant activity against the peroxidation of linoleic acid, the reducing activity, the radical scavenging activity and the peroxynitrite scavenging activity.31
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•Zhao et al. in 2004Have developed peptide antioxidants that target the inner mitochondrial membrane which potentially reduced intracellular reactive oxygen species and can be beneficial in the treatment of ageing and diseases associated with oxidative stress.32
•E Aida et al. in 2004 Have shown that the ability of Whey protein hydrolysate fractions to delay lipid oxidation was found to be related to the prevelence of histidine and hydrophobic amino acids.33
•Hannu K. and Anne P. in 2006 Have shown that naturally formed bioactive peptides have been found in fermented dairy products such as yoghurt, sour milk and cheese and the peptide with amino acid sequence Ala-Arg-His-Pro-His-Pro-His-Leu-Ser-Phe-Met shows antioxidant activity.34
•Anne Pihlanto in 2006 Has shown that milk-derived antioxidant peptides are composed of 5-11 amino acids including hydrophobic amino acids, proline, histidine, tyrosine or tryptophan in sequence.35
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•Wakabayasi et al. in 2006Have reviewed on an iron binding glycoprotein present in milk which is considered to be an important host defence molecule and has a diverse range of physiological functions such as antimicrobial/antiviral activities, immunomodulatory activity, anticancer, antioxidant, anti-infective and anti-inflammatory activity.36
•Chen et al. in 2006Have investigated peanut protein hydrolysate for its antioxidant activities, including its ability the autooxidation of linoleic acid, the scavenging effect on the 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) free radical, the reducing power and the inhibition of liver lipid oxidation.37
•Kati et al. in 2008Have shown that bioactive peptides are found in milk, egg, meat, fish as well as in many plants and amongst them peptides with a sequence Pro-His-His showed the greatest antioxidant activity.38
•Dan et al in 2008 Have shown that brain permeable iron chelators may present therapeutic benefits and the NAP(Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) peptide showed inhibition of lipid peroxidation and hydroxyl radical formation.39
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•Minelli et al. in 2007 Have shown that neuroprotective activity of cyclo(His-Pro) deals with increase antioxidant protection and its oral administration improves a synergistic mechanism the glycaemic control in diabetes.40
•Mine Y. and Katayama S. in 2008Have shown that phosphopeptides from hen egg yolk act as inhibitor of lipid peroxidation and radical scavengers.41
•Wu-Yang et al. in 2010 Have shown that the oxygen radical-scavanging effects of egg white protein ovotransferrin might be constitutive amino acid tryptophan and the bond between tryptophan and arginine.42
•Bahareh H. Sarmadi. and Amin Ismail in 2010Have reviewed the antioxidative activity of bioactive peptides can be attributed to their radical scavanging, inhibition of lipid peroxidation and metal iron chelation properties of peptide which may be affected by amino acid sequence.43
•Anusha G. P. and Li-Chan C. Y. in June,2011 Have reviewed the food derived peptides which are being considered as potential sources to control various oxidative processes in the human body as well as in food.44
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IRON ABSORPTION AND IRON-AMINOACID CHELATE
•Adelia et al. in 2000Have shown that in whole maize meal, iron from ferrous bisglycinate is better absorbed than iron from ferrous sulfate or ferric triglycinate.45
•H. Dewayne Asmead in 2001Has reviewed on the absorption and metabolism of iron aminoacid chelate and has shown that intestinal absorption of iron from iron amino acid chelate compared to the inorganic salts and iron amino acid chelate is both a safe and effective source of iron for treatment of iron deficiency.46
•Marcos et al. in 2002Have shown that Fe+3-peptide complex is a potential compound for use as an iron source in treatment of patients with IDA.47
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•Bertille et al. in 2005 Have shown that when bound to Fe, caseinphosphopeptides derived from milk proteins resist luminal digestion, enhance Fe solubility and could improve its bioavailability.48
•Yeung et al. in 2006Have shown that casein phosphopeptides enhances the iron availability from foods with low availability.49
•Konstantina et al. in 2007Have shown that milk proteins produce a range of bioactive peptides and among those are peptides that may enhance iron absorption.50
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Objective of the work
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Iron-deficiency anemia is a common anemia (low red blood cell level) caused by insufficient dietary intake and absorption of iron, and/or iron loss from intestinal bleeding, parasitic infection, menstruation, etc. Red blood cells contain iron and are not formed when iron is deficient.
The mechanism by which iron enters the mucosal cells lining the upper gastrointestinal tract is unknown. Most cells in the rest of the body are believed to acquire iron from plasma transferrin (an iron-peptide chelate), via specific transferrin receptors and receptor-mediated endocytosis.
No more than 5% of vegetable iron is absorbed and while the absorption of iron from meat, poultry and fish may be somewhat higher, significant worldwide consumption of animal proteins is limited to the more affluent.
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A literature survey on antioxidant activity of some nutraceutical peptides, iron absorption and anemia shows that food like whole milk, soybean, egg have some bio active peptides which are useful to treat oxidative stress during anemia and increase absorption of iron in human body.
The objective of our work is
To design some small synthetic antioxidant peptides which will be useful in treatment of anemia by lowering red blood cell oxidative stress and by increasing iron absorption in body.
To synthesize the peptides using more economical liquid phase method (CPE method) and characterize by M.P, TLC, FT-IR, NMR and Mass spectral analysis.
To evaluate the synthesized compounds for antioxidant activity followed by prediction of their biological activity spectra for antianemic activity using the chemistry software server PASS for prediction of biological activity spectra (http://195.178.207.233/PASS/).
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Plan of work
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1. Designing of some nutraceutical based linear and cyclic peptide molecules based on rational structure based drug design.
2. Docking of modeled peptides with the receptor (Glutathione reductase) by using software- Glide (version: 5.0).
3. Checking the ADME profile of best docked compounds with the help of software- QikProp (version: 3.1).
4. Synthesis of selected peptides by liquid phase peptide synthesis method.
5. Synthesis of iron peptide chelates.
6. Characterization of the synthesized compounds: • Physicochemical characterization (M.P, Rf value)• Spectral analysis (FT-IR, NMR and Mass)
7. To evaluate of antioxidant activity of synthesized peptides followed by prediction of biological activity spectra.
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3. Experimental
work
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Experimental work
Designing of new molecule and docking studies
Prediction of ADME properties of compound
Prediction of biological activity Synthetic studies
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MOLECULAR MODELING AND DOCKING STUDIES
•Docking is the process of fitting of the ligand into the receptor.
•It does not only give an idea about how the ligand is going to bind with the receptors but also about the extent to which conformational changes can be brought in the receptor structure.
• A mixture of purified phthalic anhydried (2.2gm, 0.015mol) and alanine
(1.25gm, 0.015mol) was heated on sand bath, within 25 minutes the mixture
melted.
• Heating was continued for another 15 min. The reaction mixture was cooled,
the residue was dissolved in ethanol and filtered off, water was added to the
clear filtrate till turbid and then the solution was cooled at 0ºC for 24 hours.
• Fine crystals of phthaloyl alanine separated were filtered and dried and their
melting point and Rf value were found out for their physical characterization.
Protection of α-Amino Group
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Preparation of Ala-His-Cys :
Step-1: (Condensation of Phthaloyl Ala-His)
The CPE reagent was added to phthaloyl Alanine (1.095gm, 0.005mol)
and stirred to a clear solution. Then (0.078gm, 0.005 mol) of Histidine
was added to the mixture and stirred to a clear solution. To this mixture,
triethylamine was added to maintaining the pH 7 and the reaction
temperature kept below 5ºC. This mixture was then left for 6 hrs at
0oC, the product was filtered out and washed with solvent ether and
dried.
Peptide synthesis:54
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Step-2: (Condensation of second amino acid)
The CPE reagent was added to phthaloyl ala-his (0.356gm, 0.001mol) and stirred to a clear solution. Then (0.121gm, 0.001 mol) of cysteine was added to the mixture and stirred to a clear solution. To this mixture, triethylamine was added to maintaining the pH 7 and the reaction temperature kept below 5ºC.This mixture was then left for 6 hrs at 0oC, the product was filtered out and washed with solvent ether and dried. Step-3: Deprotection
Phthaloyl ala-his-cys (0.32, 0.0007mol.) was dissolved in ethanol (25 ml). Hydrazine hydrate (0.1 ml, 70 %) was added to it and the mixture was heated on a steam bath for 2 hrs. The reaction mixture was cooled, acidified with conc. HCl and again heated on water bath at 50oC for 1 hr. Phthaloyl-hydrazide was removed by filtering it out. The filtrate was neutralized with triethylamine to liberate free peptide which is held back in solution. Removal of triethylamine was carried out under vaccum distillation followed by crystallization from aqueous ethanol gave the tripeptide as colourless crystal.
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General scheme for synthesis of Iron-Peptide chelates55:
The chelate-containing solution was evaporated in a rotary evaporator, product is dried and stored at 25-270C.
Insoluble form of Fe+3-Peptide complex was obtained by adjusting the pH to 3.5 with 0.1N HCl.
Addition of FeCl3.6H2O was stopped when free Fe+3 is detected in the mixture after reaction with potassium thiocyanate(KSCN).
2.5gm of FeCl3.6H2O was slowly added with constant mixing to a solution containing the peptide (10%w/v) at 270C.
pH 7.8 maintained with 0.1N NaOH.
Fe+3 and thiocynate show intense red color.
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Determination of DPPH radical scavenging activity:
The standard (Ascorbic acid) and samples (synthesized peptides) were prepared in different concentrations 50, 100, 150, 200, 250µg/ml with methanol and then 2ml of sample was added to 2ml of 0.15mM DPPH in 100ml methanol. The mixture was allowed to stand at room temperature in the dark for 30 min. The absorbance of the mixture was measured at 517nm using a spectrophotometer (Shimadzu, Japan). The control was conducted in the same manner where methanol was used instead of sample.
DPPH radical scavenging activity was calculated as per following equation:RSA (%) = (Acontrol −Asample)/Acontrol ×100, where Asample is the absorbance of sample and Acontrol is the absorbance of the control. The IC50 value was defined as an effective concentration of peptide that is required to scavenge 50% of radical activity. All the compounds were compared with standard (Ascorbic acid) for antioxidant activity.
Antioxidant activity56,57,58:
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The PASS internet tools were used for prediction of biological activity of synthesized compounds. The input MDL Mol files (*.mol) file of compounds were drawn with the help of Chemdraw Ultra 10.0 and Chem3D ultra 10.0 softwares. This software gave Pa and Pi ratio (active and inactive ratio) in Pa > 30%, Pa > 50% and Pa > 70% levels.
Pa (probability "to be active") estimates the chance that the studied compound is belonging to the sub-class of active compounds (resembles the structures of molecules, which are the most typical in a sub-set of "actives" in PASS training set).
Pi (probability "to be inactive") estimates the chance that the studied compound is belonging to the sub-class of inactive compounds (resembles the structures of molecules, which are the most typical in a sub-set of "inactives" in PASS training set).
The PASS tools available at “http://195.178.207.233/PASS/ pre-dict.php” were used in April 2012.
PREDICTION OF BIOLOGICAL ACTIVITY SPECTRA59:
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4. Result and discussion
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We have designed some linear and cyclic small peptides
containing different amino acid combinations of Alanine, Glycine,
Asparagine, Glutamic acid, Histidine, Cysteine, Proline and Glutathione.
The designed compounds were docked into crystal structure of Glutathione
reductase (pdb: 1GRE) which is the most accurate structure available. The
interaction energy between designed molecule and receptors were
calculated and the results are presented in the Table-4.1. The score
represented in terms of Gibbs free energy (∆G).
Docking studies:
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NAME OF LIGAND CHEMICAL NAME
DOCKING SCORE
IN SP MODE
DOCKING SCORE
IN XP MODE
FAD Flavine Adanine Dinucleotide -13.86 -14.03
SSPS1 Ala-Asn-Gsh -12.58 -13.20
SSPS2 Ala-Asn-Glu-Asn -11.48 -11.74
SSPS3 Ala-Tyr-Gln-Glu -9.04 -8.97
SSPS4 Ala-Trp-Gsh -10.23 -9.98
SSPS5 Ala-Gsh-Pro-His -8.63 -9.08
SSPS6 Ala-His-Gsh -8.53 -7.55
SSPS7 Ala-Pro-Gsh -11.67 -12.32
SSPS8 Ala-His-Cys -11.96 -12.62
SSPS9 C(Ala-His-Cys) -10.69 -11.79
SSPS10 C(Ala-Asn-Gsh) -8.01 -7.98
SSPS11 C(Ala-Asn-Glu-Asn) -3.05 -5.59
Table 4.1: Docking scores of designed linear and cyclic peptides in 1GRE (Glutathione
Reductase) receptor grid:
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NAME OF LIGAND CHEMICAL NAME
DOCKING SCORE
IN SP MODE
DOCKING SCORE
IN XP MODE
SSPS12 C(Ala-Pro-Gsh) -7.89 -8.12
SSPS13 C(Ala-His-His-Gsh) -5.82 -6.03
SSPS14 Gly-Asn-Gsh -11.02 -11.59
SSPS15 Gly-His-Gsh -10.84 -9.95
SSPS16 Gly-Trp-Gsh -11.45 -11.78
SSPS17 Gly-Ser-Val-Cys-Ser -8.59 -9.87
SSPS18 Gly-Gsh-Pro-His -7.93 -8.79
SSPS19 Gly-Asn-Glu-Asn -7.86 -8.86
SSPS20 Gly-His-Pro-Glu -7.54 -8.83
SSPS 21 Gly-Pro-His-His -11.90 -12.04
SSPS22 C(Gly-Pro-His-His) -6.33 -7.02
SSPS23 C(Gly-Trp-Gsh) -4.71 -5.33
SSPS24 C(Gly-Asn-Gsh) -5.32 -5.04
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Figure 4.1: Crystal structure of Glutathione reductase (1GRE) in ribbon form
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XP Glide-predicted pose of molecule SSPS1. Active site amino acid residues and inhibitor are represented as ball and sticks. While the inhibitor SSPS1 is colored with the atoms as carbon: green, hydrogen: cyan, nitrogen: blue, and oxygen: red. Hydrogen bond interactions are represented by yellow dotted lines. SSPS1 is forming six hydrogen bonds with Histidine 52, Histidine 129, Alanine 115, Serine 51, Asparagine 294, Glutamic acid 50 and Aspartic acid 331. The distance of hydrogen bonding is 1.91A0, 2.297A0, 2.184A0, 1.978A0, 2.154A0, 2.067A0 and 2.082A0 respectively.(Fig. 4.2)
Figure 4.2: Docking of SSPS1 with 1GRE
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Figure 4.3: Docking of SSPS9 with 1GRE
XP Glide-predicted pose of molecule SSPS9. Active site amino acid residues and inhibitor are represented as sticks. While the inhibitor SSPS9 is colored with the atoms as carbon: green, hydrogen: cyan, nitrogen: blue, and oxygen: red. Hydrogen bond interactions are represented by yellow dotted lines. SSPS9 is forming three hydrogen bonds with Glycine 37, Threonine 156 and Serine 51. The distance of hydrogen bonding is 1.507A0, 1.734A0 and 2.275A0 respectively.(Fig. 4.3)
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ADME PROPERTIES PREDICTION:
CompoundMolecular
weightQPlogPo/w QPlogS
Human oral
absorption
%Oral
absorption
SSPM1 492.50 -2.115 -0.576 2 33.66
SSPM2 446.41 -3.028 +0.261 1 26.03
SSPM3 509.50 -3.440 -0.280 1 16.79
SSPM4 564.6 -3.110 -0.099 1 22.79
SSPM5 612.6 -2.870 -0.039 2 24.21
SSPM6 515.54 -1.942 -1.536 1 26.06
SSPM7 475.52 -1.814 -1.095 1 35.05
SSPM8 372.40 -1.984 -1.708 1 24.95
SSPM9 354.38 -3.712 +0.299 2 67.48
SSPM10 474.48 -2.302 -0.962 2 40.88
SSPM11 428.39 -1.841 -1.574 2 45.66
Table 4.2: Predicted ADME properties of designed molecules: