Amino Acids, Peptides and Proteins AMINO ACIDS ‘Amino acids are the organic compounds containing both amino and carboxylic group in their molecules. They are represented by the general formula Carboxylic group Ris different for different amino acids R =Hin glycine ‘Amino group Some common examples of amino acids are NH,CH,COOH Aminoncetic aid (Glycine) CH,-CH-COOH —_-H,N- CH, ~ CH, - COOH 1 B—Aminopropionic acid NH, (B-Alanine) «2-Aminopropionic acid (a-Alanine) Depending upon the position of the amino group with respect to the carboxyl group, amino acids are classed as « -, 8 -, y or J-amino acids. The carbon atom next to the carboxyl group is regarded as a-carbon atom. Some more amino acids are : CH; ~CH-CH-COOH CH ~ CH ~ CH - COOH 1 I CH, NH, NH 2—Amino—3—methyl butanoic acid (Valine) Phenyl: aitMODERN COLLEGE CHEMISTRY (SEMESTER-y; \—CH—COOH HO [NH; - CH - COOHJCI~ + NHj-CH - COO" + | (acid) R (Base) (©) The acidic unit is NH in amino acids. Thus, they form salts with NaOH. NH3-CH - COO” + Na*OH™ —+ (NH ~ CH - COO) Na* +20 I ase) 1 R (acid) (Salt) Why low values of pK, and pK; for Amino acids. The exceptionally low values of K, and Ky speak of the dipolar structure of amino acids. According to the dipolar structures, the substituted=a MODERN COLLEGE CHEM nESTERSY ammonium ion is an acidic centre while a carboxylate ion is basic centre in amino acids. =cooH group and — NHp group are not responsible for the acidic and the basic character of amino acids. The values of K, and K, can be calculated from the following : NH - CHR) - COO™ + H,0 <=> Hx0" + HaN - CHIR) ~ COO™ + - Thus, K,= [H,0*] [NH, - CH(R) - COO J. pk, =-log Ky [H,N* - CH(R) - COO™] For this, pK, value of amino acid centre calculated N ee i NH, - CH(R) — COO” + HO <= HN‘ - CH(®) ~ COOH + OFF + - and Kiet BEsN 2 CH) = COOH] [OH] pKy=-log Ky 5s = [HN — CH(R) - COO] It may be noted that in aqueous solution, the product of the acidity constant of an acid and the basicity constant of its conjugate base is equal to (1.0 x 10-4. Thus, K, X Ky=1 x 10-4. In case of glycine, if acidity constant (Ky = 1.6 X 10-!°) of glycine is due to the substituted + ammonium ion (— NH3), then K, of conjugate base, ~ NH should be 6.3 X 10°. It is close to aliphatic amines. In the same way, if basicity constant (Kb = 2.5 x 10-12) is due to — COO, then K, of its conjugate acid (- COOH) should be 4 x 10-3. It is close to aliphatic acid carrying electron + withdrawing — NH, group. Clearly, the measured value of K, and K, of amino acids can be explained in terms of dipolar, ionic structures (Zwitter ions). In aqueous solution, the acidity and basicity of a base and an acid and its conjugate acid and its conjugate base (i.e., CHyCOOH andCH,COO orCH3NHg and CH;NH;] are related to the expression Ky x K, = 1074. From this, it can be calculated that K, is 1.6 x 10~!° for NH, of glycine and Ky = 6.3 X 10° for ~ NHb. This is a quite reasonable value for an aliphatic amine. In the same way, Ky of 2.5 x 10" for— COO of glycine means K, of 4 x 10° for — COOH. It is quite reasonable value for carboxylic acid + containing a strongly electron withdrawing - NH group. Consider the following equilibrium when a little alkali is added. + = FA - H,N-CH(R)COC+ OH = H,NCH(R)COO+ H,0 3 i ‘Strong base 2! Strong acid I ‘Weaker base II Weaker acid pS + In presence of OH ions, NH3 ion displaces amine which is a weak base, If a few drops of acid are added to I, we get + _ ik + H,N-CH(R)COO" + H3;0 <= * H,N-CH(R)COOH + H,0 ‘Stronger base ‘Stronger base Weaker acid ‘Weaker base\CIDS, PEPTIDES AND PROTEINS a In this case, the stronger acid gives up a proton to the carboxylate ion and displaces the weaker carboxylic acid. Effect of pH on the structure of c-Amino acids The structure of amino acids mainly depends upon the pH of the medium containing amino acid. In the acidic medium, (when pH is less than 1), the dipolar ion gets converted to a cation. : NH; ~CH(®)-COO- + H,0* = NH, —CH(R)COOH + H,0 Dipolar ion I (Stronger base) Weaker acid II Tl is formed when stronger acid (HyO+) donates to proton to dipolar ion (1). But when the solution of amino acid is made alkaline (pH more than 12), then the stronger base abstracts a proton from the dipolar ion I and forms the weaker base III shown below : : NH; - CH(R) - COO™ + OH” ==» H>N - CH(R)COO™ + H,0 Dipolar ion I Weaker base (IIT) At some pH value, which lies between two extremes, the Zwitter ion (dipolar ion) will be in equilibrium with varying amounts of II and II. + ei z NH ~ CH(R) - COOH == HAN - CH(R)COO- == HyN-CHRYCOO- Ir Ht I Ht mm Since structures II and III contain free - COOH and ~ NH; groups, they show the typical reactions of acids and amines. With reagents like C;H;OH (for esterification) for CHyCOCI (for acylation of — NHj), the equilibrium is disturbed and reaction continues till the whole of form II or III gets consumed. ISOELECTRIC POINT OF c-AMINO ACIDS When an electric current is passed through the strongly acidic solution of an amino acid, then the amino acid existing as cation moves towards the cathode. Similarly, when this solution is strongly basic, then the amino acid existing as anion migrates towards the anode. R-CH-COO” == R-CH-COO™ == R-CH-COOH | OH | on™ ' NH ®NH, ®NH, (anion) (Dipolar ion) (Cation) ® @ ay Thus, the exact position of equilibrium depends upon the pH of the solution. But at some intermediate value of pH, the concentration of cationic form III and anionic form II are minimum and equal to each other. In other words, we say that the concentration of dipolar ion form I will be maximum. Thus, at intermediate pH, amino acid mainly exists as dipolar ion I. Clearly, at this pH, the passage of current will not result in the migration of amino acid. The pH at which there is no net migration of the amino acid under the influence of applied field is called isoelectric point (pl) of that amino acid. It may be noted that (@ at a pH lower than pl, there will be net migration of amino acid towards cathode as the amino acid mainly exists as cation. (ii) at a pH higher than pl, there will be net migration of amino acid towards anode as the amino acid mainly exists as anion.MODERN COLLEGE CHEMISTRY (SEMESTER-y) Table 3.2 Isoelectric points of some Amino acids Amino acid Isoelectric points Amino acid Isoelectric points Neutral Amino acids Acidic Amino acids Alanine 61 Aspartic acid 28 Valine 6.0 Glutamic acid 32 Leucine 6.0 Basic Amino acids Serine 57 Lysine 9.7 Threonone 56 Arginine 108 Each amino acid has a characteristic value of isoelectric point. —— The pH of the isoelectric point depends upon other functional groups in the amino acid structure, Neutral amino acids such as glycine, alanine etc. are little more acidic than basic. (In aqueous solutions, K, of - COOH is more than K, of - NHp group). Clearly, the aqueous solution of such an amino acid contains more of anion than of cation. Thus, to supress the ionisation of dipolar ion I into anion II, some acid is added to reach the isoelectric point. Hence, the isoelectric point of some amino acids lie in the acidic range (i.e. around pH 6). Acidic amino acids have isoelectric points at a low pH, around 3. For example, aspartic acid and glutamic acid have isoelectric points 2.8 and 3.2 respectively. Basic amino acids have isoelectric points at a high pH, around 10. For example, the isoelectric points of Lysine and Arginine are 9.7 and 10.8 respectively. ELECTROPHORESIS It is defined as a technique which separates and purify compounds on the basis of differential movement of charged particles in an electric field. In paper electrophoresis, a strip of paper or cellulose acetate is used as a solid support. A mixture of amino acids is placed in the form of a spot in the centre of the strip of paper. The strip of paper is then soaked with an aqueous buffer of a particular pH which depends upon the isoelectric points of amino acids in the mixture. The two ends of the paper are then dipped into the buffer solution having electrodes (see fig. 3.1). Now electric field is applied. The following changes are observed. Glycine Aspartic acid (@) The amino acids with low isoelectric | _ Fig- 3.1 Separation of mixture after paper point compared to the pH of the buffer solution electrophoresis, start moving towards anode since they exist mainly in the anionic form, (b) The amino acids with higher isoelectric point comy moving towards cathode because they mainly exist in the cationic form. (c) The amino acids with isoelectric point equal to o} : wigs fom the origin. Thus, depending upon the isoelectric pono '9 the buffer solution do not : : tric points and the page amino acids migrate at different rates and thus, amino acids get separated from tes desity, pate Cathode Lysine pared to the pH of the buffer solution start[AMINO ACIDS, PEPTIDES AND PROTEINS the separation of a mixture containing glycine (pI = 6.0), Lysine (pl = 9.7) and aspartic acid (pl = 3.0). In paper electrophoresis, lysine moves towards cathode whereas aspartic acid moves towards anode. Glycine molecules do not move and remain at the origin. SYNTHESIS OF AMINO ACIDS Following are some important methods of preparation of amino acids: 1, By the amination of a-Halo acids. a-Chlorocarboxylic acids react with excess of liquid ammonia to form ammonium salt of an amino acid. The ammonium salt so formed on hydrolysis yields an amino acid. 2 CICH, COOH +3NH, 2°S> H,N - CH, - COONH, 122" HN - CH, - COOH Chloroacetic acid ~NH,CI Glycine 50°C. H0/H* CH, - CH(Br) - COOH + 3NH; “25> CH, - CH(NH) - COONH, 2° CHCH(NH, )COOH @-Bromopropionic acid -NHyBr ‘Alanine (i) NH3 HO- ‘CH2—CH{(Cl) - COOH —-————® HO- (CH2—CH(NH; ‘COOH HN-CH-COOH én HO GLYCINE FORMALDEHYDE + NHs Ne HOV NHe CHoH + HCN > CH CH ao ~CH-on OS cu, SH-coon Cel ACETALDEHYDE on 2-AMINO PROPANOIC ACID (-Alanine) In practice, the aldehyde is treated with a mixture of ammonium chloride and potassium cyanide in aqueous solution NH,CI + KCN ~A%422%%5 NH,CN + KCI; NH,CN A428. NH + HCN 3. Gabriel Synthesis. An ester of a-haloacid is treated with phthalimide to form the corresponding substituted phthalimide. The compound formed on hydrolysis gives phthalic acid and an amino acid. COOH -Kc! Hoon a Nii scictscoocaHe > oe = + NHgCH2 COOH 4 oats OF OH Il COOH — GLYCINE PTHALIC ACID PHTHALIMIDE4. By Azlactone synthesis, Hippuric acid (Benzoyl glycine) is treated with acetic anhydride in Presence of sodium acetate to form azlactone. This, on condensation with aldehydes followed by reduction and subsequent hydrolysis yields a-amino acid. . NHCOCeHs _(CHgCO,)0. 4 noc NMe CgHsCOC! Che ACHg002)0,, coon \cooH ‘CHgCOONa’ Glycine (Hippuric acid) CoHe—C= NL CoHs—C===N 3 | CHe I > CH, te, o-c% OH Hove ll ° azlactone 4|CeHscHO CeHs—C=N, lag CoHs—C=N Soti—ctip— cots « Me Dos OH Colts o-c~ reduction Os ¢ y oO azlactone aziactone 2H20 <> Cghs—CH—CH—COOH + CgHsCOOH 1 NHp (Phenylalanine) 5. Phthalimidomalonic ester synthesis. In this method, phthalimide is treated with bromomalonic ester to form phthalimido malonic ester. It is then treated with sodium ethoxide to get a carbanion which in turn is treated with alkyl or aralkyl halide. The product formed is then hydrolysed to get a-amino acid. Oo ° Il ll CN ns =Ker oN 1 DK + Br GH (COO Hp HBry N—CH(COOC Hsp Cat, Bromomalonic ester a ont f ri CoHsONa fo} oo Pot. phthalimide Phthalimidomalonic ester - ° ° i i N CeHsCH2C! N\, 2 —o( CH N= OL. oot (COOCeI Cen, “| C(COOCeHs)aNa*} fo OneCots i ° ° () Kona COOH (Wy DIHCH ian Tela 423-473K ‘COOH CHeOsHs Phthalle acid Phenylalanine 6, Curtlus Reaction. In this method, curtius rearrangement of malonic aci ide (1) in presence of ethyl alcohol yields urethane which on acid hy. ire acid monoszide,() drolysis forms c-amino acid,Hs . COOC,H. ‘ R KOH Um), ROH 2 Yooc,H, partial hydrolysis Yooc, Hs C,HsOH Malenic ester COOH IK R- cH- cooH -#&LA_RcHe + CHsOH ncn eot HNO Rone va EN ectys “NHic00CH cae con; CONHNH, iret Malonic srAmino seid acid mononzide 7, Hydantoin Synthesis. In this method, an aromatic aldehyde is condensed with hydantoin in presence of acetic anhydride and sodium acetate. The condensation product formed is reduced with sodium amalgam and then hydrolysed with acid. Consider the synthesis of Tryptophan. CHO Hoo —N CH= C—NH . 4 YN po {2120100 I Ye ay y o=c_nw” ‘CHsCOONa”™ ye) O=S—NH H H Indole-3-carbaldehyde Hydantoin ‘Condensation product NalHg Reduction CHp CH, Cry Nee oe ae Ow aye “Noe iydrolysi % Coon o= = uno ‘Tryptophan 8. The Darapsky Synthesis. This method involves the condensation of aldehyde with ethyl cyano acetate with simultaneous hydrogenation. The product formed is converted into an acid azide. The azide formed on curtius rearrangement forms @-amino acid. UN cN LN RCHO + H,C© ABM, R - CH, - CHO MEN, RCH, - CHX Aldehyde ‘COOC;Hs (COOC, He ~ C2Hs0H ‘CONHNH, Ethyleyanoacetate Alkyl cyanoester 1 HNO, COOH CN RCH, — CH - COOH +42 Rew, - cH SEO tee, 2 cut Hydrolysis “NHCOOC,H, ___Cuttus ‘CON, ‘a rearrangement a-Amino acid 9, By reductive amination of keto acid. The keto acid is treated with ammonia to form an imine. The imine is catalytically reduced to a-amino acid, ° NH il (CHy),CH-CH,-C - Coon Ns (CH,),CH - CH, - C - COOH Sak Keo ada Ho 32 2 Hy /Pt. (CH), CH - CH,CH(NH;)COOH— (Leucine) 10. Schmidt Synthesis. Alkylacetoacetic ester is converted to a-amino acid by Schmidt reaction followed by hydrolysisMODERN COLLEGE CHEMISTRY (SEMESTER.V)) R CH, - C - CH- Cooc, Hy —"4—+ cH, C - NH- CH co0c;H; 2% R- CH ~ CooR Alkyl acetoacetic ester 5 Schmit reaction Ni, @-Amino acid PROPERTIES OF c-AMINO ACIDS (@) Physical Properties. 1. All amino acids are crystalline solids with high melting and boiling points. 2. These are soluble in polar solvents like water but insoluble in organic solvents like benzene, 3. They have high values of the dipolemoments and exist as dipolar molecules. 4. Except glycine all @-amino acids are optically active. (b) Chemical Properties of Amino acids. We know that c-amino acids exist in equilibrium with the cationic as well as anionic forms having free-COOH and — NH group respectively. SNH ONHS NH OWL OWL R- cH - COOH == R- cH - coo- == R- cH - coo (Cation form) ut (Dipolar ion) Ht (Anionic form) From this, we say that amino acids show characteristic reactions of - NH2 group as well as —COOH group. Also in acidic medium, the equilibrium lies mainly towards cationic side and thus reaction of — COOH group are carried in the acidic medium. Similarly, reactions of amino group are carried in the basic medium. A. Reactions due to amino group (@ Basic character. Amino acids also react with strong acids like HCl to form salts. + + H3N—CH,COO” +HCI— (H3N -CH)- COOH)CI- Glycine Glycine hydrochloride The basic character of glycine is due to - COO- group as it acts as a proton acceptor. Thus, R Han cf coo} roo, [ts —}-> BASIC PART ‘Amino acids show some characteristic reactions of the functional groups. (ii) Alkylation. In basic medium, it forms N-alkyl amino acid anion when treated with alkyl halide. With excess of alkyl halide, an internal quarternary ammonium salt is formed. These salts have awitter ion character and are known as betaines. R-CH-COO- +R'x 85 R- CH- COO- +HX 1 1 NH) NHR’ N-alkyl amino acid\MINO ACIDS, PEPTIDES AND PROTEINS : 5 NH, -CH, - COO- ae cH sNH, ~ CH,COO~ Glycine a (CHy),NH — CH,COO- a (CHy)3N - CH - COO™ N,N, N-Trimethyl glycine (A Betaine) (iii) Acylation. With acetyl chloride, amino acids undergo acylation Fe NH, - CH) -COO~ + CH,Coc!] —#“ cH,CONHCH,COOH Glycine Acetyl glycine (iv) Reaction with Nitrous acids. Amino acids with primary c-amino group react with nitrous acid to form a-hydroxy acids. NH, -CH, ~COO~ +HONO =H“, Ho - CH, - COOH +N, +H,0 Giyeine 2-Hydroxy acetic acid (v) Reaction with Formaldehyde. N-methylene amino acids result. e NH ~ CH - COO + HCHO — Cz = N - CH - COO™ + H0 I R @~amino acod 'N-methylene amino acid Methylene amino acid is acidic in nature and can be titrated with an alkali. It forms the basis of Sorensonformol titration method for the estimation of amino acids. (vi) Reaction with nitrosyl chloride. With nitrosyl chloride, they form corresponding chloro acids. R - CH - COOH + NOCI — R - CH - COOH +N, +H,0 | 1 NH, ca @—amino acid a—chloro acid (vii) Reaction with 2, 4-Dinitrofluoro benzene (Sanger’s reagent). In the basic medium, they react with 2, 4-Dinitrofluorobenzene to form 2, 4-Dinitrophenyl amino acids. ox-{Oy- + HeN — CH coo” E2825 oa (Oppo + HE R R NOg NOg 2,4-Dinitrofluorobenzene amino acid anion 2, 4-Dinitrophenyl amino acid anion (Crystalline salt) Sanger’s reagent is used in the determination of N-amino acid residue of a peptide or protein. B. Reactions due to Carboxyl group (@ Reaction with bases. Amino acids react with strong bases like NaOH, KOH to form salts. + HN - CH, - COO~ + NaOH —> NH,CH2COONa + H,0 Glycine Sod. amino acetate . * + i‘ In this reaction, ~ NH, part is proton donor and is thus, an acidic part.fF CHEMISTRY (ete o — varereen, (ii) Esterification. Reaction with alcohol occurs on heating in presence of dry hydrogen Chloride gas, ester results, 5 ; This on treatment with aqueous sodium carbonate or moist silver oxide forms the corres ut HN - CH, COO- +.C,H,OH==SNH, ~ CH, - COOC;H; + H,0 Glycine Ethyl glycinate Actually this reaction takes place in two steps : HyN* - CH) - COO + HCI — Cl [HN* - CH, — COOH] C)H;0H | -H,0 + AgCl + NH ~ CH, - COOC)H, Sara Cl” [HN - CH, - COOC,H,] ~ Hy Ethyl a-amino acetate Similarly, CH3 ~CH -COO + HC1—> CH, - CH- Coon Sst. i ' = Hy ®NH; CINH; H20 + AgCl + CH, ~ CH - COOC,H, 2H [CH;CHCOOC,H, Cl I I NH, “NH A Ethyl, a-amino propionate (Gi) Decarboxylation. On heating with barium oxide or sodalime, dioxide to form an amine, a-amino acid loses carbon N Heat NH3 ~ CH ~ COO” + BaO H's RCH,NH, + BaCO, 1 P-amine R ‘@-amino acid (@) Reduction. On reduction with lithium aluminium hydride, c-amino alcohol results, nN iA NH3 ~ CH - Coo- 4A. Ny, — CH - CH 0H I ! R R ‘@-amino acid @-amino alcohol ©) Formation of acid chloride, The amino acid is first protected by acylation, The product formed is then treated with PCls or SOCI; to form acid chloride + NH, ~ CH -Coo~ ~43920, cy co _ NH- CH COOH 022, CHACONH - CH -cocl 1 Pyridine I 1 R R R @-amino acid N-acetylamino acid chloride[AMINO ACIDS, PEPTIDES AND PROTEINS C. Reaction of both Carboxyl and Amino groups (@ Action of heat. (a) a-Amino acids undergo dehydration by interaction between two molecules in such a way that — NH group of one molecule reacts with - COOH group of another. RL a CH-C R Z na NoH Heat, yy Scn-c a NH 7 Se-cuc S>c- cue OF ames ® a a-Amino acid topperazine (b) B-amino acids on heating lose a molecule of ammonia. R-CH-CH,COoH Hs RCH = CHCOOH +NH3 1 i, B-unsaturated acid NH) (c)y- and 6-amino acids on heating lose a molecule of water to form cyclic amides, called lactams Oo ° ° f% = CHo—' CHp—C==O0 wo ot 4 Nou Heat 4 NS \ CH —> _ ch NH | ‘on < yNHe 120 R HgC—CH2—NH2 HoC—CHp— NH ‘CH2— CHS '‘CHp— CH3 y-Amino butyric acid y-Butyrolactam ‘d-amino acid d-valerolactam (ii) Reaction with Metallic ions. With the aqueous solution of heavy metal ions (say aqueous copper sulphate solution) amino acids form deep coloured complexes. With copper sulphate solution, they form deep blue colour. 2+ | 2NHz —CH2—COOH + Cut ————p Ho — NHe Soe DEEP BLUE COLOUR (iii) Reaction with Ninhydrin. With an alcoholic solution of triketone (ninhydrin), amino acids form a dark blue or violet coloured complex. ° I I i OH NHp—CH—COOH, nT OH Y i i 3 OH Ninhydrin Dark blue coloured complex Ninhydrin is used for testing a-amino acids. (iv) Reaction with Phenyl Isocyanate. With phenyl Isocyanate, a-amino acids react to form phenyl hydantoic acid. This on acidification lose water to give phenyl hydantoin R—CH——NH, N -HeO | R—CH—NH\ R—CH—COOH + CeHs—N=C=0 ——> Cee ‘C=O 65: COOH HN Hel ae —v7 NHp Phenylisooyanate a-amino acid CoH Colts Phenyl-hyantore acid Phenyl hydantoinCOLLEGE CHEMISTRY (seen. MODERN MEST PEPTIDES AND THEIR CLASSIFICATION f amino group of one a-amino acia With ¢ 3 ‘These are amides forn lensation o} s formed by the condensation ¢ b group of ol et sp aa wi imination of water molecule. ‘ of other amino acid with the elimin woe IH —* NH) ~ CH ~C-NH~CH_ NHy - CH - C- OH + NH - CH - ¢ ° ri 157 O8 RO Ro oe x irri The condensation products formed by the reaction of two or ae amino acid Molecule, called peptides. They are further classified as di, - tri or polypeptides as they are made from 1% tne or more amino acid molecules. The ~ CONH is known as peptide bond or peptide linkage. Lip. ating | acids, Polypeptides also have a dipolar or Zwitter ion structure. When three molecules of amino acid get condensed, a tripeptide results which carry two Pepe linkages + + - NH - CH ~ COO” + NH —CH - COO- + NH ~ CH) ~ Coo ” Glycine i Glycine CH; Alanine — NH, ~CHt C0 --NH-CH-co - NH - CH, ~ co9- CH; Glycyl alanyl glycine (A tripeptide) In general, a polypeptide results by the condensation of n molecules of amino acids _ + HH, Nit — CH — C003 n Hg — CH — C007 iis —CH —coo-—eH20 R x R” + nets faa Hn iiilice NH —CH—COO™ b v nl A polypeptide NOMENCLATURE AND GEOMETRY OF PEPTIDES (a) Nomenclature. N-terminal end and a free N-terminal amino acid (with free group) on the right. Naming of peptide is done Starting from N-terminus to C-terminus. While namin the suffix ‘ine’ of all amino terminus is replaced by the suffix ‘yl’. The peptide § named as one word. The three letter abreviations (as Gly, Ala etc.) are also used quite often. Som examples are : 5 A -—dinepil: ( NH ~ CH ~ CO -NH ~ CH ~ COO- is named as Glycylglycine or (Gly-Gly)Itis adip-?™ CH, on ! GP) NH ~ CH, - CO -NH~CH-COO™ is named as Glycylalanine (Gly — Ala)AMINO ACIDS, PEPTIDES AND PROTEINS | + (iii) NHj= CH CO | NH = CHa ~ CO — NH — CH= COO; | CH CoHs is named as alanylglycylphenylalanine. It is a tripeptide. _ (©) Geometry of Peptide Bond. The X-ray studies of depeptides and polypeptides reveal that the peptide linkage is flat. It means that carbonyl carbon, nitrogen and four hydrogen atoms attached to them lie in one plane. The bond angle and bond lengths in a polypeptide geometry are shown below : te 20 fog HOUR ‘ t RAY ue (OY —do%, BS 1 I wo yO AN, ie S H7 SR | Ize 47 SR H ) The C -N bond distance is found to be 132 pm compared to its normal value of 147 pm. It shows that C—-N bond in dipeptides and polypeptides has about 50% double bond character. As a result of this, bond angles of the bonds to nitrogen are similar to trigonal carbon atom. Q. 7 The above delocalisation is due to some double bond character in carbon-nitrogen bond. Due to this, rotation is hindered and thus, peptide bond can show geometrical isomerism, Also trans form | Will be more stable since cis-isomer experience more steric | Ne” WG repulsions. Hence, we say that - CONH ~ group lies in one plane ! | with O and H atoms in trans orientation. From this it is clear that free rotation of a peptide chain can occur only around the bonds joining the near planar amide groups to the a- carbons. Clearly, it is Planar peptide bonds possible to describe the conformation of polypeptide chain in terms of angle p between R’ - CH ~ NH bonds. These angles are called Ramachandran angles. CHR ig xr—z PEPTIDE SYNTHESIS It is not easy to synthesise polypeptides by stepwise condensation reactions in which — NH group of one amino acid is condensed with - COOH group of other till the desired product is formed. The simple reactions involve a number of problems. The problems and how these are overcome in earlier methods are being discussed. Classical Peptide Synthesis. Consider that two different amino acids are made to react in presence of a dehydrating agent. In addition to the desired product, three more dipeptides result. For example, when mixture of glycine and alanine is heated in presence of dehydrating agent, four dipeptides result. Glycine + Alanine = Gly - Gly + Ala - Ala + Gly - Ala + Ala - Gly. ~ Hy ‘Also the condensation reaction of amino acid molecules is endothermic in nature and does not occur easily. For this, an amino acid molecule is activated by converting it into acid chloride and then it is made to react with another aminoacid molecule. The problem still persists because the two molecules of acid chloride also react to form a dipeptide. Due to this, the yield of the desired dipeptide moleculeMODERN COLLEGE CHEMISTRY (SE falls. Another problem in addition to this is, that the separation of dipeptides formed is vey aoe "Y diffcyy Consider when alanyl chloride is treated with glycine. + NH) - CH - COC! + NH2 - CH; ~ COOH —yqy* HN - ou - con COO” | Glycine x CH; CH3 Alanyl chloride i)-HCl, & Also NH) - CH-COCI + H,N- cH - cool NH; - CK - CONH - CH ~Coo- 1 il CH; CH; CH; CH, Alanyl chloride Alanyl chloride Ala-Ala The above problem is overcome by protecting amino group before the carboxyl group is activated ie., converted into acid chloride. Protecting groups in Peptide Synthesis. A protecting group is one which makes a reactive group inert and thus, prevents the molecule to go an unwanted reaction. In an amino acid, - NH group or the - COOH group can be protected, for the purpose of synthesising peptides A protecting group : (@ should be stable under the reaction conditions. , (ii) should be easily removed leaving the peptide bond. (@ Protecting the amino groups. These groups convert — NH) group into same other group of low nucleophilicity. Amino group is not protected by acetylation or benzylation because the removal of such group from acylated peptide may cleave peptide bond also. The protecting groups largely used are: (@ Benzyloxycarbonyl group (CgHsCH - O - CO) and (ii) tert-Butoxycarbonyl group. (® Benzyloxy carbonyl group. To introduce this group, benzyloxycarbonyl chloride is treated with amino group in presence of NaOH at 25°C R R oo NaOH, 25°C ! (CsHsCH - O - COCI* + H,N - CH - COO SC, Ce6HsCH, - O - CONH - CH ~- COOH Benzyloxy carbonyl chloride a~amino acid e-amino acid Benzyloxy carbonyl chloride (ii) tert-Butoxy Carbonyl group. To introduce this group, Di-tert butyldicarbonate is treated with c-amino acid in presence of triethyl amine. o Cc I R fe} R (CH3)3CO -C + | MI I >0 + HyN - CH - Coo “S2#828#, (Cy,),00 - C - NH - CH - COOH (CH,);CO-C @-amino acid {ex-Butoxy carbonyl amono acid ti ° Di-tert butyl dicarbonate (6) Protecting the Carboxyl group. The carboxyl group in amino acid is protected by the formation of its methyl or ethyl ester. Benzyl ester can also be prepared by treating with benzyl alcohol. R R + I I NH, - CH - COO + CeHsCH,0H EO. NH, ~ CH - COOCH; CH, @-amino acid Benzyl alcohol A benzyl esterR I ClCH) - OCO-NH-CH(R)COOH 22. C,H,CH,-0 - CO - NH - CH - COC! ~S0 - HCI Benzyloxy carbonyl amino acid chloride Methods of Peptide Synthesis. The synthesis of a specific peptide involves the following steps: (@ The amino group of an amino acid is protected by treatment with benzyl chloroformate. \ . ereglarny, coneonp ener e ‘CH2—O—CO—Cl + HaN—CHa— COG —»> -CHp—O—CO—NH—CH2— COOH + HCI Benzyloxy carbonyl chloride ‘Carbo-benzoxylglycine (b) The above protected amino acid (glycine) is converted to the corresponding acid chloride by treatment with thionyl chloride. Oreo O—CO—NHCH2COOH + SOClz —> CgHgCHe — O—CO—NH—CH2—CO—Cl + SO2 + HCI (c) The acid chloride (above) is then treated with another amino acid (say, alanine). (Oyo O—CO—NH—CHa—CO—CI + HeN—CH—COOH ——> CHs Alanine (Oye CaN a lam +HCI CHa (@) The above compound is then subjected to reduction. HalPd ‘CHe— O—CO—NH—CHp—CO— NH—CH—COOH —2 > oy, CH NHg—CHa—CO— NH—CH—COOH + be Toluene Glycyl alanine (Dipeptide) By repeating the above steps, a tripeptide or any polypeptide can be obtained. Drawbacks. In this method, the racemisation of the product result at the stereocentre a-to the — COC! group. benzyloxy carbonyl group can be easily removed elther on catalyti (H,/Pd) or by hydrolysis with HBr in cold acetic aci Important (Remember). A peptide synthesis involves the following steps : (® Protection of amino group (ii) Conversion of - COOH to - COCI by treated the protected amino acid with thionyl chloride. (iii) Formation of the peptide bond and (iv) Removal of the protecting group. “Its prepared by passing carboxy! chloride through a solution of benzylalcohol in toluene.MODERN COLLEGE CHEMISTRY (SEMESTg | aa | Modified Peptide Synthesis. By using Di-tert.butyl dicarbonate as protecting reagent. [tig mos method and involves the following steps + (i) The - NH group of amino acid is protecte —> t-BuOCO - NHCH,COOH tert-Butoxy Carboxyl glycine (A) | in presence of dry HCI eg .d by treatment with Di-tert butyl dicarbonate x (1-BuOCO)2 0 + HN = CH,COO” Benzyl alcohol Glycine (ii) Also - COOH is protected by treating with benzylalcohol CHsCH.OH + H,N-CH Coo” P2#C, NH, - CH - COOCH,CoHs Benzyl alcohol | ba CH; x Alanine benzyl ester (B) Alanine (iii) The protected amino acids A and B are then condensed through free ~ COOH and ~ NH, by means of dicyclohexyl carbodi-imide (DCC) to get the desired peptide bond. 1-BuOCONHCH ,COOH+H NCH COOCH,, CgHs 2° t-BuO . CONHCH,CONHCHCOOCH,C,H, 1 “w CH; ®) CH; (iv) (© formed above is then treated with HBr in cold acetic acid to form Glycylalanine + z 1-BuO . CONHCH ;CONHCH - COOCH,C,H; —#2#*—- NH, - CH,CO - CH - COO © I Cold CH3COOH — Giycylalanine CH; (ipeptide) CH; + CsHsCHyBr + CO> + (CH3),C = CH) Itis a very useful method and in the same way, dipeptide can be converted into tripeptide and so on. [elise The classical method for peptide synthesis is suitable only for peptides involving a few amino acid molecules. But the synthesis of peptides involving a large number of molecules of amino acids is a difficult task. Moreover, it is a time consuming method because peptide synthesis require steps such as protection, activation, condensation and removal of protecting group which are repeated for each higher peptide. Also the yield of peptide formed is much lower. SOLID PHASE PEPTIDE SYNTHESIS RB. Meerifield (1964) devised a method for peptide synthesis, called the solid phase peptide synthesis. The synthesis is carried on the surface of an insoluble solid support which consists of polystyrene in which some of the benzene rings are chloromethylated i.e. linked to - CH,CI groups. This polymer is crosslinked with about 2% of divinylbenzene as may be designated as CICH- polymer. The polymer is porous and has gel-like structure. ‘CH— CH2— CH ~~ ~~~CH2—CH— CH CH— “S- CHeCI CHeCI CicHp—{ Polymer] “It is prepared by passing carbonyl chloride through a solution of benzyl alcohol in toluene. CeHsCHZOH + CICOC] —+ CgHsCHp - 0 - COCI seeracanol * Ceonchonte "Boron aren choreAMINO ‘ACIDS, PEPTIDES AND PROTEINS” Ea This method is suitable for larger peptides like insulin since the other methods of peptide synthesis have drawbacks of expensive starting materials. In addition they involve large staff requirement and low yields. These problems are overcome by solid phase method. The synthesis of peptide involves the following steps : (i) The — NH) group of C-terminal amino acid of the peptide is protected by tert-Butoxy carbonyl chloride (Boc). The Boc-amino acid (protected) is attached to polystyrene polymer by heating in the presence of (C)Hs)3N in suitable solvent. Ry " \ (CHg)3C - 0 - C - NH - CH - COOH + Cl - CH, - Foes te f + (CoHs)gN [Poymer] 80°CHgOH (CHg)3C - O - C - NH — cH - GO + OCH Polymer Ester tnkage with polymer When the ester linkage is formed, the excess reagents are removed by washing with suitable solvents. (ii) In this step, the protecting group (Boc) is removed by treating with trifluoroacetic acid. Then the product formed is converted into free acid by treatment with excess (C>Hs)3N followed by washing. Ry tl \ (7. GFgCOOH (CHg)gC - O — C — NH — CH - COOCH, - [Polymer | ——=———> ls a [Poymer] on CH, R, | \ CH ~ C = CHp + COp + HaN - CH - COO CH, - [Polymer (ii?) Now second Boc protected amino acid is coupled with the polymer bound amino acid by means of dicyclohexyl carbodi-imide. Re Ry i \ 1 (CHy)3C - 0 - C - NH - CH ~ COOH + H,N - CHCOOCH, - | Cth - N= C= N Opty; in CHCl, (DCC) 1 ® i t (CHg)g - 0 - C — NH - CH - CONH ~ CH COOCH, ~ Polymer + OgHy; - NH - C — NH ~ CeHy, Deyeiohoxrea (jv) Protecting reagent is removed in the same way as described in step (ii). The product formed is again coupled with the polymer bound amino acid. Tote ol (CHiglg C—O —C—NH—CH CONH—CH CoocH, — Polymer] (i) CFgCOOH (il) (CoHs)3N Hs Re Ri | | CHgC== CHp +CO2 + HzN—CH —CONH —CH —coocH,—{ Poymer] {, 82°40 and (repeated ° Ry Ri ll (CHa)3 C—C—NH—CHCO— Polypeptide resin. | I INH — CH CO|—NH — CHCOOCH, —; MODERN COLLEGE CHEMISTRY (Sey ' Deprotection is done with HBr in acetic acid resulting into formation of free Polypeptide, “Y) Bx i tt (CHa)s C—O —} ena Leo—fu = eer —CHCOOCH: Deprotection CHs HBr in CHsCOOH CHgC== CHp + CO2 + Free polypeptide + Br — CHa Advantages. (i) The yield of polypeptide is very high. (ii) The time required for the synthesis of polypeptides and proteins is much less compared to classical peptide synthesis. (ii) After each coupling, the purification of the product is not necessary. The reason is that the insoluble polymer bound peptide is thoroughly washed with suitable solvents so that excess of reagent is removed. PROBLEM for Practice Problem. A tetrapeptide has - COOH group on alanine. This produces glycine (Gly), Valine (Val), Phenylalanine (Phe) and alanine (Ala), on complete hydrolysis. For this tetrapeptide, the number of possible sequences primary structures) with — NH group attached to a chiral centre is : NH2 NHp NH; NH2 CHoZ » CH3 - CH > (CHg)eCH - CHS Ph — CHa - CHE coon “COOH 2 ‘vooH ‘cooH Glycine Alanine Valine Phenylalanine Number of possible sequences (primary structures) with -NH} group attached to the chiral centre is 4. STRUCTURE OF POLYPEPTIDES The peptide whose structure is to be found is first hydrolysed to its constituent amino acids, which are separated and identified. The amount of each amino acid is measured and hence the number of each kind of amino acid can be calculated. The next step is to determine the sequence of the various amino acids constituting the polypeptide. It is a very difficult task since there are a large number of possibilities in which the constituent amino acids may be linked in the peptide. For example, even in a dipeptide made of glycine and alanine, the sequence may be either of the two as shown below : CH CH; | HN - CH, ~ CONH - cu cooH HN ~ CH - CONH - CH COOH Glycylalanine Alanyl glycine The two structures differ in the respect that in the first, the N-terminal amino acid is glycine (ie., the amino acid group of glycine is free) and C-terminal amino acids is alanine while in the latter (II) the N-terminal amino acid is alanine and C-terminal amino acid is glycine. Various chemical methods have been developed to remove either of the two terminal amino acid residue of a polypeptide in a step wise manner and hence the arrangement of various amino acids in a polypeptide can be established.ma oe | 1, N-terminal residue analysis. The analysis of the N-terminal residue can be done by the following methods: (a) Sanger’s method. In this method, the peptide is treated with 2, 4-dinitrofluorobenzene, called Sanger’s reagent in presence of slightly basic solution at room temperature, The presence of nitro groups with ~ I effect activates the fluorine atom for nucleophilic attack by - NH2 group of the N-terminal amino acid residue, The resulting 2, 4-dinitro phenyl peptide is hydrolysed with dilute HCI to get constituent amino acids. The yellow 2, 4-dinitrophenylamino acid is separated and then identified. NO2 R R NOz A R I I ‘Ag.NagCOg, I ! ON F + HgN—CH—CO—NH—CH—CO- = ————OoN: ‘NH—CH—CO—NH—CH —CO.... Peptide 2, 4-Dinitrofluorobenzene faa HCI NO2 i OoN: ‘NH—CH—COOH + Mixture of amino acids N-(2,4-dinitrophenyl) amino acid In this method, only the terminal amino acid is identified. On the other hand, if the polypeptide is first put to hydrolysis into a number of smaller peptides and each peptide is put to DNP analysis, it is possible to know the sequences of all the amino acids in the polypeptide chain. (6) Edman’s method. In this method, the polypeptide is treated with Edman reagent (CoHsN = C = S) in the presence of dilute alkali. The various steps are described below : CgHsN = C =S +H,N - CH - CONH - CH - CONH - CH - CO ~ etc. | | | Ri R2 R3 SoH) ol io.) YOHT C¢Hs - NH- C-N —CH-CONH - CH - CONH - CO - ete. I 1 Ry R, $ i “aN HHtMH0 Cots Ww NH BalOHe at | H ——> HoN — CH — COOH selective hydrolysis o=6— 0K I 4 i Phenylthiohydantoin of N-terminal amino acid Thus, the terminal acid residue having free - NH group has been picked up by the reagent as phenyl thiohydantoin (PTH) while the remainder of the polypeptide or protein is left intact at the end of the reaction. The PTH may be separated and identified by paper chromatography. ‘The reagent can be used again on the newly formed degraded polypeptide to give the second end amino acid and so on. The method has now been automated and thus, can be used to determine the amino acid Sequence in polypeptides. (c) Dansyl method. It is a modification of Sanger’s - DNP method. In this method, 5-dimethyl aminonaphthalene 1-sulphony! chloride (Dansyl chloride) is used and Dansyl-amino acidga MODERN COLLEGE CHEMISTRY (SEMESTs5 Pry a is hi t, it helps in easy detection . formed is identified. Since the dansyl group is highly fluorescent, it help: y and esting of dansylamino acids. R I .NH—CH—CO—NH. oxi 302. 1 —CHc9_ i i i CO barb -—-OO Reet) Jaana (CHs)2N hee {—COOH .NH—CH Dansyi chloride S02 Nor | Mixture of CO R amino acids N(CHs)2 2. C-Terminal residue analysis. Following are the methods for the analysis of C-Terminal residue: (a) Carboxyl-end degradation. In this method, the amino group is first protected by benzoylation and then the C-terminal amino acid is converted into thiohydantoin which is hydrolysed ultimately to the amino acid exactly in the same manner as in Edman method. R Ry R3 1 1 1 HN - CH - CONH ~ CH - CONH - CH - CooH -S##sOC1, * B re CoH - CONH - CH ~ CONH - CH - CONH - CH - COOH | Heat with NH NCS | and (CH3CO),0 Ri Ry R3 Cali CONH - CH - CONCH -CO-N - 6 -Ns0H, S=c C=0 \F N H R, Ry R; \ HN — CH - COOH 1 1 CHsCONH - CH - CONH ~ CH ~ COOH + HN ~ CHR, 2202, I] ‘C-terminal amino acids S=C c=0 \7 N ‘Thiohydantion of C-terminal amino acid The amino acid so formed is identified, and the procedure is repeated on the degraded polypeptide. (b) Enzymatic method (Selective Hydrolysis). For C-terminal aminoacid residue analysis, enzymatic method is quite useful. The enzyme carboxypeptide cleaves the peptide linkage next to the alaun , PEPTIDES AND (AMINO ACIDS, PROTEINS q-carboxyl group in the peptide. The enzyme is treated with the enzyme when free amino acid Aongwith the degraded peptide results R’ R” R” 1 | | _..NH ~ CH - CO — NH ~ CHCO ~ NH ~ CHCOOH Sabot peptidase , H2,0 w Br Rt | 1 1 ... NH - CH - CO- NH - CHCOOH +H,N - CH - COOH Degraded peptide C-Terminal amino acid The amino acid so obtained is identified. Now the degraded peptide with a new C-terminal residue is again treated with enzyme to get second free amino acid. The process is repeated again and again till the sequence of all the amino acid residues in the proton is made known. (i) The enzyme leucine amino peptidase attacks peptides (or proteins) only at the end which contains the free amino group. When this terminal amino acid is liberated, the new terminal having free amino acid is attacked by the enzyme. (ii) Like the amino peptidase, there is another enzyme. Carboxyl-peptidase which attacks peptides, only at the end which contains a free a-carboxyl group. Hence, after a given time of hydrolysis, estimation of the amounts of free amino acid will give their sequence. (c) Hydrazinolysis method. In this method, a peptide is heated with anhydrous hydrazine. Due to this, all amino acid residues of peptide chain are converted into hydrazides. R R R' 1 I I HNCH ~ CO - NH - CH - CONH - CH .cooH 2M#2 A R R’ R" | | | H)NCH - CONHNH, + H,NCHCONHNH) + NH) - CH - COOH Hydrazide C-Terminal amino acid The free amino acid (free C-terminal residue) is separated from hydrazides and is identified. PROTEINS The word ‘Protein’ comes from the Greek word proteios which means the first. Proteins are most for life. These are the building materials for the body in all living cells. Skin, muscles, nerves, enzyi etc. forgp-the principal component of proteins. Proteins regulate metabolism and also pl: tant rofé in the functioning of the nervous system. Proteins are polymers with high molecular mass and are polyamides in nature. The monomer units of these polyamides are a-amino carboxylic acids. A single protein molecule is composed of thousands of a-aminoacids. It is found as a result of the hydrolysis of proteins that there are about twenty two c-amino acids in protein molecules. Proteins are extremely complex nitrogeneous polymeric substances present in all forms of living matter, These are natural polymers and are essential for the growth and maintenance of life. These are Present in all living cells. Proteins are main constituents of skin, hair, muscles, nails, tendons etc. All proteins contain the elements such as carbon, hydrogen, nitrogen and sulphur. Some proteins contain phosphorus, iodine and traces of metals like zinc, copper, iron etc. These are formed by the condensation of more than 100 molecules of aminoacids. All proteins form a-amino acids on hydrolysis. About twenty six @-amino acids result on the hydrolysis of proteins. The number of proteins are much largerEa MODERN COLLEGE CHEMISTRY (SEMEg in number. The polypeptides with molecular mass more than 10,000 are termed aS proteins, Play synthesise proteins from carbon dioxide, water and inorganic compounds. Animals get their Prowse requirement mainly from plants. in Classification of Proteins. Proteins are classified on the basis of their physical and Chemica, properties and on physiological functions. A. Classification on the basis of physical and chemical properties. Depending upon the structure and their functions, proteins are divided into two main classes : (i) Fibrous proteins. Such proteins are linear thread Table 3.3 Molecular mass of some like molecules which tend to lie side by side to form fibres. Proteins The peptide chain in them are held by strong inter molecular hydrogen bonding. Due to this, they are insoluble in water. Proteins These are stable to moderate changes in temperature and ra Saas oe PH. They serve as chief structural material of animal tissues. Haemoglobin 65,000-70,000 Examples of fibrous proteins include Keratin in hair, nails, | Serum albumin (horse)| 60,000-70.000 feathers...... fibroin in silk and myosin in muscles. caeuin (i) Globular Proteins. The polypeptide molecules in | Insulin the proteins are folded into compact units and attain almost spheroidal shapes. The folding take place in a manner that hydrophilic parts are pushed outwards. Due to this, water molecules interact and thus, these proteins are soluble in water. The proteins are very sensitive to small changes in temperature and pH. Examples of such proteins include enzymes, some hormones (pancreas, thyroglobulin), haemoglobin, albumin etc. Each globular protein has its definite geometry which is due to interaction between different sites in the same molecule. These interactions may cause disulphide bridging, dipolar interactions, intermolecular hydrogen bonding and van der Waal’s interactions. B. Classification on the basis of hydrolysis products. Depending upon the composition and the hydrolysis products, proteins are classified as under : 1. Simple proteins. These are the proteins which on hydrolysis yield only amino acids. Some examples are egg albumin, globulins, glutelins etc. 2. Conjugated proteins. These are the proteins in which a peptide part is mixed into some non- protein part (prosthetic group) such as carbohydrate, phosphate group, lipids (esters of higher fatty acids) etc. For example, casein of milk and haemoglobin of blood. The conjugated proteins are further divided into classes depending upon the nature of the prosthetic group. (i) Nucleo proteins. They include nuclei of glandular tissue. The prosthetic group in them is nucleic acid. (ii) Glyco proteins, They include mucin which is a constituent of saliva. (iii) Chrome proteins. They include haemoglobin and chlorophyll etc. (contain some metal atom like iron, cobalt, manganese etc. in their structure). C. Derived proteins. These are the proteins which are produced by the metabolic degradation of some higher proteins in the. animal serum. For example, proteoses and peptons. Characteristics of Proteins. Some important characteristics of proteins are : 1, Most of the proteins are colourless, amorphous substances. However, some of the proteins are also coloured. Blood haemoglobin is red in colour.(ACIDS, PEPTIDES AND PROTEINS 2. They have high anne ve masses. For example, the molecular mass of egg albumin lies between 34000-44000, that of Haemoglobin is 65000-70000 and that of casein lies inbetween 75000 to 100,000 and so on. 3, They do not possess sharp melting points. 4, Proteins, being polypeptides, exist as dipolar ion or Zwitter ion. 5, All proteins are optically active. 6, Proteins on hydrolysis with acids, alkalies or enzymes yield amino acids. 1 Proteins are generally insoluble in water. However, albumin is soluble in water. They form colloidal solutions with water. The colloidal solution of proteins can be co-agulated by any of the following methods : (i Action of bacteria (ii) Action of heat (iii) By shaking with alcohol and (iv) By treatment with concentrated organic acids. After co-agulation, proteins lose their physiological activity and certain other properties. The phenomenon is known as denaturation. A very common example of denaturation is the coagulation of white of an egg by the action of heat. Analysis of Proteins. Proteins are complex nitrogeneous substances present in all forms of the living matter. On hydrolysis, they yield amino acids. On treatment with an acid or alkali or on heating, proteins get coagulated. Some important tests of proteins are described below : 1. Coagulation test. Heat a small portion of the egg albumin, it gets coagulated. 2, Biuret test. This test consists in adding a drop of copper sulphate solution to an alkaline solution of protein when bluish violet colour develops. Some bigger hydrolytic products of proteins also respond to this test. 3. Millon’s test. Take about 5 mL of egg albumin (a protein) dispersion in a test tube. Add to it an equal volume of Millon’s* reagent. Heat the test-tube by placing it in a beaker of boiling water. A reddish precipitate indicates the presence of a protein having a - OH group. Gelatin does not respond to this test. 4, Ninhydrin test. Take about 1 mL of egg albumin dispersion in a test tube. Add to it about 4 drops of 0.5% ninhydrin solution. Boil the contents for about 1 minute. A deep blue to violet pink or even red colour in certain cases indicates the presence of a protein. Biological Importance of Proteins. The biological importance of proteins can be described as under : 1. Enzymes. These proteins act as catalysts for the various biological reactions. Their action is highly specific and efficient. Enzyme is called intracellular when it operates within the cell while it is extracellular when it operates outside the cell as in the case of gastric juices. Enzymes are proteins which may be simple or conjugated. Enzymes enable synthetic and breakdown reactions to be carried out much more rapidly by the living organisms at body temperature. For example, the enzyme, carbonic anhydrate present in red blood cells can catalyse the decomposition of over 30 million molecules of carbonic acid in one minute. It is a reversible reaction and can be described as . Carbonic anhydrate H,CO; <== = H,0+CO) * Millon’s reagent is prepared by dissolving 5 gram each of mercuric and mercurous nitrates in 100 mL of distilled water. Add a drop of dilute sulphuric acid and KNOz Solution. + Ninhydrin solution in generally 0.1% solution of ninhydrin in pyridine.MODERN COLLEGE CHEMISTRY (SEMESTER This reaction helps in maintaining carbon dioxide level in body fluids and tissues. In the same way, pepsin, trypsin and chrymotrypsin catalyse the hydrolysis of peptide linkages _ protein molecules in the digestive tract. The enzyme present in Saliva, Ptylem, catalyses the decompo : sito of starch to maltose during chewing of food. Maltase then converts maltose into monosaccharides » 2. Antibodies. These proteins are the body defences against foreign organism. These are prog . * Produced during the invasion of body by infectious species which release foreign substance, called antigeny The newly formed protein of antibody specifically combines with the antigen which induce its synthesis, Gamma globulins found in blood are examples of antibodies. 3. Transport agents. There are certain proteins which Haemoglobin present in blood, transports oxygen from lun; simple protein, globin, which is linked to non-protein mol haemoglobin is responsible for the red colour of blood and it 4, Structural materials. Proteins serve as structural materials of animal tissues. These proteins constitute about one half of body’s total Proteins. Some common structural proteins are keratin in skin, hair, nails; myosin in muscles and collagen in tendons. Structure of Proteins. The elucidation of the detailed structure of proteins is carried out as follows : act as transport agents in the body g5 10 all parts of the body. It consists of ¢ lecule, called haema. The haema portion of tis capable of holding molecules of oxygen, 1, Elemental analysis. On elemental anal ysis, most of the proteins are found to have the following Percentage compositions. C=51-55;H=65-7;0=22-24:N=15-18, Sulphur and phosphorus when present, occur to the extent of 1% and 0.5% respectively. 2. Molecular mass determination. Molecular m: available for determination of molecular mass of structure, any particular protein structure is divided and tertiary structures of a protein. ‘ass of the protein is determined by methods polymers. Depending upon-the complexity of the into different levels. These are primary, secondary A. Primary structure. The covalent | i mt i i Structure of a protein is called its primary | _-N: c. a structure. It show how the atoms in protein |" eS ™ a ae ae molecule are joined to one another through By 8 a covalent bonds to form chains. All proteins Fig. 32 Primary SHUGUe GF PSS on hydrolysis givec-amino acids. This shows that these are polypeptides and have a structure as shown in (fig. 3.2). The primary structure of proteins dissolved in water is not disrupted by heating above 80°C. The differences in chemical and biological properties of various proteins and peptides arise due to the differences in their primary structure. For example, in haemoglobin, the protein in blood which carries oxygen, replacement of only one a-amino acid results in defective haemoglobin, This is the cause of a disease called sickle cell anaemia. In patients having this disease, the red blood cells precipitate, causing the cells to sickle and sometimes burst. Normal haemoglobin Val — His ~ Leu ~ Thr ~ Pro + Giu + Glu - Lys - t+ (Change of one amino acid) Sickle cell haemoglobin — Val — His ~ Leu ~ Thr ~ Pro + Val + Glu—Lys —fMINo ACIDS, PEPTIDES AND PROTEINS)" >) rmination of primary structure of proteins @ Hydrolysis. The protein can be hydrolysed by aci a hae, sed by acids, bases or enz; i which in turn get hydrolysed to amino acids. It shows peptide tinkeaged in thelr ble a rae confirms the presence of peptide linkages, eae nent (b) SET aoe acids. The products of hydrolysis of proteins i.¢., amino acids are separated by chromatography on an ion exchange resin and the molecular mass of each amino acid is found out. By this method, the number of various amino acids is found out. By this method, the number of various amino acid residues in the protein molecule can be estimated. , © Sequence of the arrangement of amino acids. Now we are to determine the sequence of the arrangement of various amino acids in a protein molecule. For this, a large protein molecule is broken into smaller polypeptide pieces by controlled partial hydrolysis. After this, the sequence of amino acids inthese simpler subunits is determined by a combination of terminal residue analysis and partial hydrolysis. Terminal residue analysis. Leaving lower cyclic peptides, all polypeptide molecules contain a free carboxyl group belonging to some amino acid at the end of the chain (the C-terminal amino acid) and one free amino group belonging to another amino acid at the other end (N-terminal). By some chemical method, it is now possible to identify which amino acid is at which end of the peptide chain. 1. C-Terminal residue analysis. It involves the identification of C-terminal amino acid residue of the polypeptide. The commonly employed method is: Hydrazinolysis method. In this method, a peptide is heated with anhydrous hydrazine. Due to this, all amino acid residue of peptide chain are converted into hydrazides. R Re - 1 | HNCH — CO — NH - CH - CONH - CH.coon “22, a R R' R" 1 I \ HNCH - CONHNH; + H)NCHCONHNH + NH - CH ~ COOH Hydrazide C-Terminal amino acid The free amino acid (free C-terminal residue) is separated from hydrazides and is identified. 2. N-terminal residue analysis. It involves the identification of N-terminal amino acid residue of the polypeptide. The commonly employed method is described below : Sanger’s method. In this method, the peptide is treated with 2, 4- Dinitrofluoro benzene. In this reaction, fluorine undergoes nucleophilic substitution by the free amino group of the peptide to form an N-dinitrophenyl derivative. The derivative when put to hydrolysis show cleavage of the amide linkages which bind the amino acid residues together. The isolation and identification of the amino acid fragment carrying the dinitrophenyl group shows the N-terminal amino acid residue of the peptide. F So + HaNoH— CONH—GH— CO Onn NOp R R 2, 4-Dinitrofiuoro benzene + 7" NH—GH—CO NHCH—CO-- _H,0* NH GH COOH + HgN—CH—COO ete. 7 | =— 1 R Ry R Re OaN: OnN NK NO2, On Amino acid DNP derivative ‘N-terminal amino acid residue LOB. Secondary Structure. This structure of protein shows how the polypeptide chains are arranged in space with respect to each other, The secondary structure is related to te a es al of polypeptide chains that may be linear or they may get coiled to form helical structures, structure has been confirmed by X-ray diffraction method, The helix is right handed and is known as a-helix, The bond strength of H-bonds in the secondary structure is 30-40 kJ per mole. Hydrogen bonds exist between the amide hydrogen of one peptide linkage and amide carbonyl group of another peptide linkage as follows : oN-H.... | | -N-H =C- The secondary structure of a protein must satisfy the following conditions. @ It should allow for the formation of maximum number of hydrogen bonds. (ii) It should account for the characteristic X-ray diffraction pattern of the protein. (ii) It should be in accordance with what is known about the primary structure, viz. bond lengths, bond angles and planar nature of amide group. 9 C. \ ef \ I- if \, { \ »/\ Types of secondary structures Important types of secondary structures of protein molecules are: (@ B- or Pleated sheet structure. An apparent arrangement of the peptide chains is such that they are fully extended to form flat zig-zags. Each such chain lie side by side to form a flat sheet. In each flat sheet, there is a repeated distance of 7.2 A. Such a struucture appears impossible in actual practice since crowding between side chains may bring some deviation in coplanarity (see below). HYDROGEN BOND _Fig. 3.3 p-pleated) sheet structure of a protein.The formation of flat structure is quite possible in case of small or medium size side chain (R-group). For example, silk fibroin having about 40% glycine residues has a B-structure which is quite close to elongated full sheet structure, t q-Helix Structure. In this arrangement, the polypeptide chain: ' coiled up to form a helix. The helical pattern wiih fe of faadaouel 1 ONS importance in protein structure is found to be right handed (see figure | 1 y 3.4). Itis called an q-helical structure, In this structure, an amide group in one location is hydrogen bonded with another amide function as shown. The left handed helix is known as the ct-helix. It may be noted that a-helix represents a more stable arrangement. The spiral is held together by hydrogen bonds between N — H and C = O groups vertically adjacent to one another in the helix. In the structure of a-helix, there are 3-6 amino acid residues per turn of the helix. Unstretched wool has a-helix structure. But when it is stretched, it gets converted into B- Keratin. The reason is 5 = Fig. 3.4 Helical that on stretching, the helixes are uncoiled and the chains extend side by side to give B-type sheet structure. structure of protein. Difference between a-helix and f-pleated configuration a-helix configuration Pleated configruation itis a spiral structure which is formed by the coiling | This structure is formed by the side by side of polypeptide chain formed through hydrogen | arrangement of different polypeptide chains which bonding between — NH — and - CO — groups of the | are held together by intermolecular hydrogen bonds same chain. between — NH — and — CO — group of neghbouring chains. —_I (©) Tertiary Structure. It is the three dimensional shape of a protein which arises from further foldings of polypeptide chains to give it a complex structure. Although complex, itis highly characteristic of a particular protein. The tertiary structure of protein is due to the different types of interactions of various parts of the protein with each other through amino acid side chains. The different types of interactions involved are. (i) Hydrogen bonds. In tertiary structure, there exists additional hydrogen bonds between OH and -NH group. In secondary structure, - NH and — OC- groups join by hydrogen bonds. Examples are Serine and Histidine. (ii) Disulphide linkages. The polypeptide chains can join together by oxidation coupling of SH groups to form — S — S — bond. Cysteine contains — $ — S — bonds. (iii) Dipolar electrostatic interactions. Such interactions in tertiary structure also occur between the polar substituents along the polypeptide chains. Examples showing such interactions include Lysine and aspartic acid. - (iv) van der Waal’s interactions. These are defined as the weak forces of attraction between hydrocarbon like side chains which are non-polar (alkyl or phenyl groups) in nature, Such interactions are much operative in tryptophan and phenylalanine residues. In tertiary structure, foldings take place in such a manner so that maximum number of polar side chains tend to remain on the exterior of the molecule so that they can interact with the aqueous system. whereas the nonpolar part tend to remain in the interior of the molecule.. MODERN COLLEGE CHEMISTRY (SEMESTER, | (D) Quarternary structure of proteins, When a protein has two more individual polypeptide chains called subsnits, they get arranged in different manner with respect to each other leading to formation of their quarternary structure. The quarternary structure 1s stabilised by ionic bonds, hydrogen bonds and hydrophobic interactions. Hoemoglobin, e.g., is such a protein with four polypeptide chains, two identical — alpha chains each containing 141 amino acid residues and two identical b a amino acid residues. These four subunits make almost a regular tetrahedron with each subunit carrying a heme group at its and. Difference Between Carbohydrates and Proteins. The main points of difference between carbohydrates and proteins are : 1. Action of heat. Carbohydrates get charred on heating while proteins get co-agulated on heating. 2. Hydrolysis. On acid hydrolysis, carbohydrates produce glucose while proteins form amino-acids. Salt Hydrophobic _Disulphide bridge infractions bond Hydrogen Fig. 3.5 Forces stabilising a protein structure, eta chains, each containing 145 DENATURATION AND RENATURATION OF PROTEINS (a) Denaturation of Proteins. A process after which proteins lose their physiological activity and certain other properties is called denaturation. The denaturation of protein can be caused by any one of the following: (@ Action of Bacteria (ii) Action of heat (iii) By shaking with alcohol and (iv) By treatment with acids Examples of denaturation of proteins 1. A very common example of denaturation is the coagulation of the white of an egg by the action of heat. 2. The coagulation of milk in the presence of an acid (lemon juice) to form cheese is another example of denaturation. The denaturation occurs due to the disruption of the tertiary structure. Heating increases the thermal vibration of the molecule resulting in the disruption of hydrogen bonds and salt bridges. Changes in pH have the greatest disruptive effect on hydrogen bonding and salt bridges in proteins. For example, Random coil Denaturing agent Sd Denatured protein Natural protein Fig, 3.6 Denaturation of protein.PEPTIDES AND PROTEINS —~ the polypeptide polylysine is a polymer of amino acid lysine molecules which have amino acid group on the side of the chain. In acidic medium, all side chains get positively charged due to protonation. Due to the presence of similar charge, they tepel each other leading to uncoiling of the protein molecule. Denaturation does not change the primary structure of protein. However, there is rearrangement of its Dempdary and tertiary structures. During denaturation, the protein molecule uncoils from an ordered and specific conformation into a more random conformation resulting into its coagulation from solution. (b) Renaturation. Protein denaturation may or may not be reversible. For example, when egg is hard boiled, the globular proteins present in it change to a rubber like insoluble mass. This is irreversible denaturation. The protein cannot regain its original shape. In case of reversible denaturation, the protein can pe coagulated from their colloidal solution by saturating the solution with soluble salts like ammonium sulphate, magnesium sulphate or by the addition of water, alcohol, etc. This is a reversible process and the original form can be obtained again. The reverse of denaturation is known as renaturation. A suont Questions Wits ANSWER 1. Name the building blocks of proteins. ‘Ans, a-amino acids. 2, Name one fibrous and one globular protein. ‘Ans, Keratin is fibrous whereas Haemoglobin is globular. What is a prosthetic group ? ‘Ans. A prosthetic group is a non-protein portion obtained by the hydrolysis of conjugated proteins. 4. What causes the disease sickle cell anaemia ? ‘Ans, It is caused by defective haemoglobin. Give one example of denatured protein. ‘Ans. Cheese is an example of denatured protein. 6. Name a polypeptide hormone which maintains glucose level in the blood. ‘Ans, Insulin, Frederia Sanger was awarded nobel prize for the synthesis of insulin. 1. A tripeptide on complete hydrolysis gives glycine, alanine and valine as the only amino acids but on partial hydrolysis, it gives Gly-Ala and Ala-Val as the only dipeptides. Write the structure of the tripeptide. Ans. Three possible structures of the tripeptide are 3, 5. (a) Gly - Ala- Val (b) Ala—Val-Gly and _(c) Val-Gly~Ala but structure () only gives Gly-Ala and Ala-Val as the only dipeptides. Thus structure (i) is the only tripeptide. 8. Give the best known example of a protein possessing quarternary structure. Ans. Haemoglobin. 9. What happens when glycine is heated ? Ans. Diketopiperazine is formed. 10. What happens when Alanine is treated with nitrous acid a Ans. Lactic acid is formed. CHCH(NH,)COOH + HONO —> CH;CH(OH)COOH + Nz +H20 q-alanine Lactic acidMODERN COLLEGE CHEMISTRY’ (spy, Alanine from Ethyl chloride ? i) 11, How will you synthesise ‘Ans, It involves the following steps : if Br2/P. cH,CH,c1 2% cHyCH2CN HOM, CHyCH,COOH 2". cH,- CHIBYCOO Ethyl cyanide aL */H20 CHCH(NH,)COOH #429 cH, _ Cro Alanine Write the name of the following peptides Ala, Gly, Val, Pro Ans, Alanine, Glycine, Valine, Proline. ‘What kind of bonding is responsible for the tertiary structure of proteins 2 Ans, Four main types of bonds are responsible for the formation of a tertiary stry proteins. These are hydrogen, ionic, disulphide and hydrophobic. ture of 14. What do you mean by the mixed secondary structure of proteins ? Ans, It means that they combine the helical pattern of a-type structure with the interchain hydrogen bonding of the c-type structure. 15. What do you mean by antibodies ? ‘Ans. Anti bodies are the proteins which defend the body against the invasion of foreign organisms They fight against the infectious species such as virus, bacteria etc. Gamma globulins present in blood plasma are the examples of antibodies. IC 2 OONH, 12. 13. 1. Write the formulae and names of three a-amino acids. 2. Describe Gabriel phthalimide synthesis for amino acids. 3. Give a brief account of the following : (i Isoelectric point in a-amino acids and proteins (ii) Peptide linkage. 4. Describe Azlactone synthesis for amino acids, 5. Describe two methods for the preparation of camino acids. 6. Write notes on : (i) Iso-electric point in @-amino acids (ii) Biological importance of proteins, 7. What evidence you can offer in favour of Zwitter ion formation ? 8, What is the effect of pH on the structure of amino acids ? 9. Describe the following : (® Zwitter ion structure of amino acids (ii) Iso-electric point of amino acid. 10. Write the names of the following peptide: Ala, Gly, Val, Pro. 11. What are proteins ? Discuss the diagram A-pleated sheet structure of proteins. 12, Write products expected from partial hydrolysis of the tetrapeptide: Met, Gly, Ala, Val- 13. Discuss in detail the structure of proteins,14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 25. 27, 29. 30. 31 32. 33. i a ah ‘What are proteins ? Discuss their primary, Secondary and tertiary structures ? Discuss the classification and biological importance of proteins, ‘What are amino acids and how are in the human body. (a) What is a Zwitter ion ? Discuss its physical and chemical Properties. (b) paar Peptides ? Discuss with special reference to the structure and properties of a peptide. What is a peptide linkage ? Write the structure of proteins. (a) How will you synthesise amino acids ? (i) by the amination of a-Halo acids, (ii) by Strecker synthesis (iii) by Gabriel synthesis (b) Write a note on Iso-electric point. Write what you know about peptide bond formation and its geometry. What leads to primary, secondary and tertiary structures of proteins? What are the main products of degradation of proteins which help in arriving at this structure. Define quarternary structure of proteins. Discuss factors responsible for this. How are c-amino acids, peptides and proteins related to each other? Illustrate the synthesis of dipeptide and give methods of protecting amino and carboxyl groups. Proteins formed from them ? Give the importance of proteins (iv) by azlactone synthesis. |. What are peptides ? Using carboxy, benzyloxy chloride, sketch the synthesis of dipeptide. (a) What are peptides ? What are the difficulties encountered in their synthesis ? Discuss one method used for the synthesis of peptides. (b) Why are amino acids amphoteric ? What are proteins ? Discuss the secondary structure of proteins ? How a peptide bond is formed ? Discuss its hydrolysis. Give the products formed from (i) complete and (ii) partial hydrolysis of pentapeptide. Gly. Ala. Met. Val. Gly. (a) What are peptides ? Discuss the carboxy-benzyloxy method for their synthesis. (b) At the isoelectric point, the solubility of amino acids is minimum. Explain. (a) Explain the Zwitter ion structure of proteins. (b) Write notes on : ( Gabriel phthalimide synthesis (ii) Strecker Synthesis (a) Write a short note on secondary structure of proteins. (8) Classify proteins according to their hydrolysis products. (a) Give the methods to synthesise a-Amino acids. (b) Why are amino acids called amphoteric compounds. (a) How does the dipolar amino acid change its equilibrium in acidic or alkaline medium ? (b) How will you synthesise phenylalanine from potassium phthalimide. (c) What are peptides ? Describe a method for the synthesis of a dipeptide, Explain the biological importance of proteins. (a) Write Erlenmeyer Azlactone synthesis of amino acids. (b) Give DNP and Edman method of N-terminal analysis in case of proteins. (c) Give evidences supporting dipolar nature of amino acids,= 35, 36. 37. 38. MODERN COLLEGE CHEMISTRY (SEMESTER-v) (a) Give two methods for the synthesis of a-Amino acid (b) Explain isoelectric point with respect to @-Amino acid (c) Write a short note on primary structure of proteins. (a) Describe Erlenmeyer Azalactone synthesis of a-Amino acid (b) Write a short note on secondary structure of proteins. (c) Explain dipolar ionic structure of a-Amino acid (a) Amino acids are weaker acids than carboxylic acids. Explain why ? (b) Write Erlenmeyer Azlactone synthesis of Amino acids. (c) What do you mean by N-terminal residue and C-terminal residue in proteins ? How are these identified ? (d) What function do protein perform in human body ? (a) Explain the terms : (i) Zwitter ion (b) Give two methods to synthesise a-Amino acids (c) What do you mean by Isoelectric point ? Explain. (ii) Denaturation of proteins