● CHAPTER 27 AMINO ACIDS, PEPTIDES, AND PROTEINS NUCLEIC ACIDS SOLUTIONS TO TEXT PROBLEMS 27.1(b) L-Cysteine is the only amino acid in Table 27. 1 that has the r configuration at its stereogenic CO H CH, HN+H=—CO2= 一CO CH SH HSCH The order of decreasing sequence rule pre 一>HSCH When the molecule is oriented so that the lowest ranked substituent(H)is held away from us, the order of decreasing precedence traces a clockwise path Han CO2 Clockwise: therefore R The reason why L-cysteine has the R configu while all the other L-amino acids have the S configuration lies in the fact that the sH substituent is the only sic outranks-CO, according to the sequence rule. Remember, rank order is determined by 752 Back Forward Main Menu TOC Study Guide Toc Student OLC MHHE Website
752 CHAPTER 27 AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS SOLUTIONS TO TEXT PROBLEMS 27.1 (b) L-Cysteine is the only amino acid in Table 27.1 that has the R configuration at its stereogenic center. The order of decreasing sequence rule precedence is When the molecule is oriented so that the lowest ranked substituent (H) is held away from us, the order of decreasing precedence traces a clockwise path. The reason why L-cysteine has the R configuration while all the other L-amino acids have the S configuration lies in the fact that the —CH2SH substituent is the only side chain that outranks —CO2 according to the sequence rule. Remember, rank order is determined by CH2SH H3N CO2 Clockwise; therefore R H3N HSCH2 CO2 H L-Cysteine CO2 H CH2SH H3N NH3 CO2 HSCH2 H C H3N CO2 HSCH2 H C Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS 753 atomic number at the first point of difference, and-C-S outranks-C-O. In all the other amino acids-CO, outranks the substituent at the stereogenic center. The reversal in the Cahn-Ingold-Prelog descriptor comes not from any change in the spatial arrangement of substituents at the stereogenic center but rather from a reversal in the relative ranks of the carboxylate group and the side che (c) The order of decreasing sequence rule precedence in L-methionine is H,N->-CO.>>-CHCHSCH >H- Sulfur is one atom further removed from the stereogenic center, and so C-O outranks CO, H3N CH,,SCH CHSCHCH The absolute configuration is s 27. 2 The amino acids in Table 27. 1 that have more than one stereogenic center are isoleucine and threo- nine. The stereogenic centers are marked with an asterisk in the structural formulas shown. CH3CH,CH--CHCO CH NH OH NH Isoleucine Threonine 27.3(b The zwitterionic form of tyrosine is the one shown in Table 27.1 HO CHCHCO NH3 (c) As base is added to the zwitterion, a proton is removed from either of two positions, the am- monium group or the phenolic hydroxyl. The acidities of the two sites are so close that it is not possible to predict with certainty which one is deprotonated preferentially. Thus two struc- tures are plausible for the monoanion: HO -CH CHCO CH, CHCO, NH NH In fact, the proton on nitrogen is slightly more acidic than the phenolic hydroxyl, as measured by the pka values of the following model compounds pk,9.75 CHCHCO CHO -CH,CHCO, N(CH3)3 Back Forward Main Menu TOC Study Guide Toc Student OLC MHHE Website
atomic number at the first point of difference, and —C—S outranks —C—O. In all the other amino acids —CO2 outranks the substituent at the stereogenic center. The reversal in the Cahn–Ingold–Prelog descriptor comes not from any change in the spatial arrangement of substituents at the stereogenic center but rather from a reversal in the relative ranks of the carboxylate group and the side chain. (c) The order of decreasing sequence rule precedence in L-methionine is Sulfur is one atom further removed from the stereogenic center, and so C—O outranks C—C—S. The absolute configuration is S. 27.2 The amino acids in Table 27.1 that have more than one stereogenic center are isoleucine and threonine. The stereogenic centers are marked with an asterisk in the structural formulas shown. 27.3 (b) The zwitterionic form of tyrosine is the one shown in Table 27.1. (c) As base is added to the zwitterion, a proton is removed from either of two positions, the ammonium group or the phenolic hydroxyl. The acidities of the two sites are so close that it is not possible to predict with certainty which one is deprotonated preferentially. Thus two structures are plausible for the monoanion: In fact, the proton on nitrogen is slightly more acidic than the phenolic hydroxyl, as measured by the pKa values of the following model compounds: HO CH2CHCO2 N(CH3)3 CH3O CH2CHCO2 NH3 pKa 9.75 pKa 9.27 HO and CH2CHCO2 NH2 O CH2CHCO2 NH3 HO CH2CHCO2 NH3 Isoleucine CH3CH2CH CH3 CHCO2 * * NH3 Threonine CH3CH OH CHCO2 * * NH3 CH2CH2SCH3 H CO2 H3N CO2 NH3 H CH3SCH2CH2 C H3N H CO2 CH2CH2SCH3 AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS 753 Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
754 AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS (d) On further treatment with base, both the monoanions in part (c) yield the same dianion CHCHCO 27.4 At pH I the carboxylate oxygen and both nitrogens of lysine are protonated H3NCH, CH,CH,CH,CHCO,H Principal form at pH As the ph is raised, the carboxyl proton is removed first. H3 NCH,CH,CH CH, CHCO H HO HNCH..CHCO+ HO The pk value for the first ionization of lysine is 2. 18(from Table 27.3), and so this process is virtually complete when the pH is greater than this value The second pKa value for lysine is 8.95. This is a fairly typical value for the second pKa of amino acids and likely corresponds to proton removal from the nitrogen on the a carbon. The species that results is the predominant one at pH 9 H3NCH, CH, CH, CH,CHCO, HO H3NCH, CH,CH, CH- CHCO, H,O Principal form at pH 9) The pKa value for the third ionization of lysine is 10.53. This value is fairly high compared with those of most of the amino acids in Tables 27. 1 to 27.3 and suggests that this proton is removed from the nitrogen of the side chain. The species that results is the major species present at pH values greater than 10.53 H3NCH,CH,CH, CH,CHCO, HO H,NCH, CH,CH,CH,CHCO, N (Principal form at pH 13) 27.5 To convert 3-methylbutanoic acid to valine, a leaving group must be introduced at the a carbon prior to displacement by ammonia. This is best accomplished by bromination under the conditions of the Hell-Volhard-Zelinsky reaction Br2- P H3 (CH3)2CHCH2 CO2H or Br. PC(CH3)2CHCHCO2H (CH3)2CHCHCO 3-Methylbutanoi 2-Bromo-3-methylbutanoic Back Forward Main Menu TOC Study Guide Toc Student OLC MHHE Website
754 AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS (d) On further treatment with base, both the monoanions in part (c) yield the same dianion. 27.4 At pH 1 the carboxylate oxygen and both nitrogens of lysine are protonated. As the pH is raised, the carboxyl proton is removed first. The pKa value for the first ionization of lysine is 2.18 (from Table 27.3), and so this process is virtually complete when the pH is greater than this value. The second pKa value for lysine is 8.95. This is a fairly typical value for the second pKa of amino acids and likely corresponds to proton removal from the nitrogen on the carbon. The species that results is the predominant one at pH 9. The pKa value for the third ionization of lysine is 10.53. This value is fairly high compared with those of most of the amino acids in Tables 27.1 to 27.3 and suggests that this proton is removed from the nitrogen of the side chain. The species that results is the major species present at pH values greater than 10.53. 27.5 To convert 3-methylbutanoic acid to valine, a leaving group must be introduced at the carbon prior to displacement by ammonia. This is best accomplished by bromination under the conditions of the Hell–Volhard–Zelinsky reaction. (CH3)2CHCH2CO2H Br2, P or Br2, PCl3 NH3 (CH3)2CHCHCO2H Br (CH3)2CHCHCO2 NH3 3-Methylbutanoic acid 2-Bromo-3-methylbutanoic acid Valine H3NCH2CH2CH2CH2CHCO2 HO NH2 H2NCH2CH2CH2CH2CHCO2 NH2 (Principal form at pH 13) H3NCH2CH2CH2CH2CHCO2 HO NH3 H3NCH2CH2CH2CH2CHCO2 H2O NH2 (Principal form at pH 9) H3NCH2CH2CH2CH2CHCO2H HO NH3 H3NCH2CH2CH2CH2CHCO2 H2O NH3 H3NCH2CH2CH2CH2CHCO2H NH3 (Principal form at pH 1) O CH2CHCO2 NH2 Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS 755 Valine has been prepared by this method. The Hell-Volhard-Zelinsky reaction was carried out in 889 yield, but reaction of the a-bromo acid with ammonia was not very efficient, valine being nly 48% yield in this step 27.6 In the Strecker synthesis an aldehyde is treated with ammonia and a source of cyanide ion. The resulting amino nitrile is hydrolyzed to an amino acid L.H. O heat (CH3),CHCH (CH3)2 CHCHO≡N 2. HO (CH,), CHCHCO, NH NH3 valine As actually carried out, the aldehyde was converted to the amino nitrile by treatment with an aque ous solution containing ammonium chloride and potassium cyanide. Hydrolysis was achieved in aqueous hydrochloric acid and gave valine as its hydrochloride salt in 65%o overall yield 27.7 The alkyl halide with which the anion of diethyl acetamidomalonate is treated is 2-bromopropane NaOCH, CH, CHa CNHCH(CO, CH, CH3)2+( CH3), CHBr CH.CHAOH CH, CNHC(CO, CH, CH3)2 CH(CH3) Diethyl acetamidomalonate 2-Bromopropane Diethyl acetamidoisopropylmalonate This is the difficult step in the synthesis; it requires a nucleophilic substitution of the Sn2 type volving a secondary alkyl halide Competition of elimination with substitution results in only a 37%0 observed yield of alkylated diethyl acetamidomalonate. Hydrolysis and decarboxylation of the alkylated derivative are straightforward and proceed in 85% yield to give valine HBr. Ho CH_CNHC(CO, CH,CH3)2 H3 NC(CO,H) H,NCHCO CH(CH3) CH(CH3h2 Diethyl 2.Aminoisopropylmalonic valine The overall yield of valine(31%)is the product of 37%X 85%0 27.8 Ninhydrin is the hydrate of a triketone and is in equilibrium with it. OH Hydrated form of Triketo form of An amino acid reacts with this triketone to form an imine -O RCHCO2 ≥ NCHCO2 Triketo form of Imine Back Forward Main Menu TOC Study Guide Toc Student OLC MHHE Website
Valine has been prepared by this method. The Hell–Volhard–Zelinsky reaction was carried out in 88% yield, but reaction of the -bromo acid with ammonia was not very efficient, valine being isolated in only 48% yield in this step. 27.6 In the Strecker synthesis an aldehyde is treated with ammonia and a source of cyanide ion. The resulting amino nitrile is hydrolyzed to an amino acid. As actually carried out, the aldehyde was converted to the amino nitrile by treatment with an aqueous solution containing ammonium chloride and potassium cyanide. Hydrolysis was achieved in aqueous hydrochloric acid and gave valine as its hydrochloride salt in 65% overall yield. 27.7 The alkyl halide with which the anion of diethyl acetamidomalonate is treated is 2-bromopropane. This is the difficult step in the synthesis; it requires a nucleophilic substitution of the SN2 type involving a secondary alkyl halide. Competition of elimination with substitution results in only a 37% observed yield of alkylated diethyl acetamidomalonate. Hydrolysis and decarboxylation of the alkylated derivative are straightforward and proceed in 85% yield to give valine. The overall yield of valine (31%) is the product of 37% 85%. 27.8 Ninhydrin is the hydrate of a triketone and is in equilibrium with it. An amino acid reacts with this triketone to form an imine. O O O Triketo form of ninhydrin O NCHCO2 R O -Amino acid Imine RCHCO2 NH3 HO H2O O O O Triketo form of ninhydrin Hydrated form of ninhydrin O O OH OH HBr, H2O heat CO2 heat 2-Aminoisopropylmalonic acid Diethyl acetamidoisopropylmalonate CH(CH3)2 CH3CNHC(CO2CH2CH3)2 O CH(CH3)2 H3NC(CO2H)2 Valine CH(CH3)2 H3NCHCO2 NaOCH2CH3 CH3CH2OH Diethyl acetamidomalonate CH3CNHCH(CO2CH2CH3)2 O Diethyl acetamidoisopropylmalonate CH(CH3)2 CH3CNHC(CO2CH2CH3)2 O 2-Bromopropane (CH3)2CHBr NH3 HCN 1. H3O, heat 2. HO (CH3)2CHCHCO2 NH3 2-Methylpropanal Valine (CH3)2CHCH O 2-Amino-3- methylbutanenitrile (CH3)2CHCHC N NH2 AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS 755 Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
756 AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS This imine then undergoes decarboxylation R CHR The anion that results from the decarboxylation step is then protonated. The product is shown as its diketo form but probably exists as an enol x+-0 NECHR Hydrolysis of the imine function gives an aldehyde and a compound having a free amino group N=CHR H,O t RCH This amine then reacts with a second molecule of the triketo form of ninhydrin to give an imine Proton abstraction from the neutral imine gives its conjugate base, which is a violet dye violet dye 27.9 The carbon that bears the amino group of 4-aminobutanoic acid corresponds to the a carbon of an a-amino acid CH,CH, CH,CO, arises by decarboxylation of O, CCHCH,CH, CO tNH 4-Aminobutanoic acid Glutamic acid Back Forward Main Menu TOC Study Guide Toc Student OLC MHHE Website
This imine then undergoes decarboxylation. The anion that results from the decarboxylation step is then protonated. The product is shown as its diketo form but probably exists as an enol. Hydrolysis of the imine function gives an aldehyde and a compound having a free amino group. This amine then reacts with a second molecule of the triketo form of ninhydrin to give an imine. Proton abstraction from the neutral imine gives its conjugate base, which is a violet dye. 27.9 The carbon that bears the amino group of 4-aminobutanoic acid corresponds to the carbon of an -amino acid. CH arises by decarboxylation of 2CH2CH2CO2 NH3 4-Aminobutanoic acid O2CCHCH2CH2CO2 NH3 Glutamic acid H2O O O N H O O OH Violet dye O O N O O O O O O O NH2 H O O N H O O H2O O O NH2 H RCH O O O H N CHR O N H2O O CHR OH O O H N CHR O N O CH C R O O CO2 O N O CHR 756 AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS 757 27.10 (b) Alanine is the N-terminal amino acid in Ala-Phe Its carboxyl group is joined to the nitrogen of phenylalanine by a peptide bo H3NCHC--NHCHCO Alanine (c) The positions of the amino acids are reversed in Phe-Ala. Phenylalanine is the N terminus and alanine is the C terr HANCHC-NHCHCO, C6HSCH Alanine (d) The carboxyl group of glycine is joined by a peptide bond to the amino group of glutamic acid H3NCHC-NHCHCO CHCH CO The dipeptide is written in its anionic form because the carboxyl group of the side chain is ionized at pH 7. Alternatively, it could have been written as a neutral zwitterion with a CH,CH, CO, H side chain (e) The peptide bond in Lys-Gly is between the carboxyl group of lysine and the amino group of H3NCHC--NHCH, CO, KG H,NCH,CH, CH,CH Glycine The amino group of the lysine side chain is protonated at pH 7, and so the dipeptide is written here in its cationic form it could have also been written as a neutral zwitterion chain H,NCH,CH,Ch,CH (f) Both amino acids are alanine in D-Ala-D-Ala. The fact that they have the d configuration has no effect on the constitution of the dipeptide. H-NCHC-NHCHCO D-A-D-A H3 Alanine Back Forward Main Menu TOC Study Guide Toc Student OLC MHHE Website
27.10 (b) Alanine is the N-terminal amino acid in Ala-Phe. Its carboxyl group is joined to the nitrogen of phenylalanine by a peptide bond. (c) The positions of the amino acids are reversed in Phe-Ala. Phenylalanine is the N terminus and alanine is the C terminus. (d) The carboxyl group of glycine is joined by a peptide bond to the amino group of glutamic acid. The dipeptide is written in its anionic form because the carboxyl group of the side chain is ionized at pH 7. Alternatively, it could have been written as a neutral zwitterion with a CH2CH2CO2H side chain. (e) The peptide bond in Lys-Gly is between the carboxyl group of lysine and the amino group of glycine. The amino group of the lysine side chain is protonated at pH 7, and so the dipeptide is written here in its cationic form. It could have also been written as a neutral zwitterion with the side chain H2NCH2CH2CH2CH2. ( f ) Both amino acids are alanine in D-Ala-D-Ala. The fact that they have the D configuration has no effect on the constitution of the dipeptide. NHCHCO2 d-A-d-A CH3 H3NCHC CH3 O Alanine Alanine H3NCHC KG H3NCH2CH2CH2CH2 O Lysine NHCH2CO2 Glycine H3NCH2C NHCHCO2 GE CH2CH2CO2 O Glycine Glutamic acid H3NCHC FA C6H5CH2 O Phenylalanine NHCHCO2 CH3 Alanine H AF 3NCHC CH3 O Alanine NHCHCO2 CH2C6H5 Phenylalanine AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS 757 Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
758 AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS 27.11(b) When amino acid residues in a dipeptide are indicated without a prefix, it is assumed that the configuration at the a carbon atom is L. For all amino acids except cysteine, the L configura tion corresponds to S. The stereochemistry of Ala-Phe may therefore be indicated for the zigzag conformation as shown. H H C The L configuration corresponds to S for each of the stereogenic centers in Ala-Phe. (c) Similarly, Phe-Ala has its substituent at the N-terminal amino acid directed away from us, whereas the C-terminal side chain is pointing toward us, and the L configuration corresponds to s for each stereogenic center. OH,C H H-N C6HsCH, (d) There is only one stereogenic center in Gly-Glu. It has the L (or S) configuration. CH,CH,CO H (e) In order for the N-terminal amino acid in Lys-Gly to have the L (or S)configuration, its side chain must be directed away from us in the conformation indicated H-N H,NCH, CH, CH, CH, H (f) The configuration at both a-carbon atoms in D-Ala-D-Ala is exactly the reverse of the config- uration of the stereogenic centers in parts(a)through(e). Both stereogenic centers have the D H CH 27.12 Figure 27.7 in the text gives the structure of leucine enkephalin Methionine enkephalin differs from it only with respect to the C-terminal amino acid. The amino acid sequences of the two pentapep- Tyr-Gly-Gly-Phe-Leu Tyr-Gly-Gly-Phe-Met Leucine enkephalin Methionine enkephalin The peptide sequence of a polypeptide can also be expressed using the one-letter abbreviations listed in text Table 27.1. Methionine enkephalin becomes YGGFM Back Forward Main Menu TOC Study Guide Toc Student OLC MHHE Website
27.11 (b) When amino acid residues in a dipeptide are indicated without a prefix, it is assumed that the configuration at the carbon atom is L. For all amino acids except cysteine, the L configuration corresponds to S. The stereochemistry of Ala-Phe may therefore be indicated for the zigzag conformation as shown. The L configuration corresponds to S for each of the stereogenic centers in Ala-Phe. (c) Similarly, Phe-Ala has its substituent at the N-terminal amino acid directed away from us, whereas the C-terminal side chain is pointing toward us, and the L configuration corresponds to S for each stereogenic center. (d) There is only one stereogenic center in Gly-Glu. It has the L (or S) configuration. (e) In order for the N-terminal amino acid in Lys-Gly to have the L (or S) configuration, its side chain must be directed away from us in the conformation indicated. ( f ) The configuration at both -carbon atoms in D-Ala-D-Ala is exactly the reverse of the configuration of the stereogenic centers in parts (a) through (e). Both stereogenic centers have the D (or R) configuration. 27.12 Figure 27.7 in the text gives the structure of leucine enkephalin. Methionine enkephalin differs from it only with respect to the C-terminal amino acid. The amino acid sequences of the two pentapeptides are The peptide sequence of a polypeptide can also be expressed using the one-letter abbreviations listed in text Table 27.1. Methionine enkephalin becomes YGGFM. Tyr-Gly-Gly-Phe-Leu Leucine enkephalin Tyr-Gly-Gly-Phe-Met Methionine enkephalin H3N H O N H H CH3 CH3 CO2 H3N H3NCH2CH2CH2CH2 O N H H CO2 H3N CH2CH2CO2 O N H H CO2 H3N C6H5CH2 OH3C N H H H CO2 H3N H3C O N H H H CO2 CH2C6H5 758 AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS 759 27.13 Twenty-four tetrapeptide combinations are possible for the four amino acids alanine(A), glycine (G), phenylalanine(F), and valine (V). Remember that the order is important; AG is not the same peptide as GA. Using the one-letter abbreviations for each amino acid the possibilities are AGFV ACⅤF AFGV AFVG AVGE AVG GAFV GAVF GFAV GVA GVFA GVAF FAGV FAVG FVAG FVGA FGAF FGFA VAGF VAFGⅤ GAF GFAⅤFA 27 14 Chymotrypsin cleaves a peptide selectively at the carboxyl group of amino acids that have aromatic side chains. The side chain of phenylalanine is a benzyl group, CBH_ CH2. If the dipeptide isolated after treatment with chymotrypsin contains valine (V) and phenylalanine(F), its sequenc beⅤF H3 NCHC--NHCHC-—计 Rest of peptide chymotrypsin NCHC-NHCHCO+ Rest of peptide (CH3)2CH CHCH (CH3),CH H,C6H5 alanine Valinylphenylalanine(VF) The possible sequences for the unknown tetrapeptide are VFAG and VFGa 27.15 The Edman degradation removes the N-terminal amino acid, which is identified as a phenylthiohy danton derivative. The first Edman degradation of Val-Phe-Gly-Ala gives the phenylthiohydantoin derived from valine; the second gives the phenylthiohydantoin derived from phenylalar CaH CsH second Val-Phe-Gly-Ala Edman degradation Phe-Gly-Ala SAo Edman degradation. Gly-Ala CH(CH3)2 CH, CAH5 27.16 Lysine has two amino groups. Both amino functions are converted to amides on reaction with benzyloxycarbonyl chloride H2NCHCO2+ 2C6H_ CH,OCCI C6HS CH,OCNHCHCO,H H,NCH, CH, CHCH C6HSCHOCNHCH, CH,CH,CH We therefore need to protect the amino group of alanine and the carboxyl group of leucine leucine 27 17 The peptide bond of Ala-Leu connects the carboxyl group of alanine and the amino group of Protect the amino group of alanine as its benzyloxycarbonyl derivative H3NCHCO2 CHsCH,OCCl CAHCH,OCNHCHCO,H CH Alanine Benzyloxycarbonyl Z-Protected alanine Back Forward Main Menu TOC Study Guide Toc Student OLC MHHE Website
27.13 Twenty-four tetrapeptide combinations are possible for the four amino acids alanine (A), glycine (G), phenylalanine (F), and valine (V). Remember that the order is important; AG is not the same peptide as GA. Using the one-letter abbreviations for each amino acid the possibilities are AGFV AGVF AFGV AFVG AVGF AVFG GAFV GAVF GFAV GFVA GVFA GVAF FAGV FAVG FVAG FVGA FGAF FGFA VAGF VAFG VGAF VGFA VFAG VFGA 27.14 Chymotrypsin cleaves a peptide selectively at the carboxyl group of amino acids that have aromatic side chains. The side chain of phenylalanine is a benzyl group, C6H5CH2 —. If the dipeptide isolated after treatment with chymotrypsin contains valine (V) and phenylalanine (F), its sequence must be VF. The possible sequences for the unknown tetrapeptide are VFAG and VFGA. 27.15 The Edman degradation removes the N-terminal amino acid, which is identified as a phenylthiohydantoin derivative. The first Edman degradation of Val-Phe-Gly-Ala gives the phenylthiohydantoin derived from valine; the second gives the phenylthiohydantoin derived from phenylalanine. 27.16 Lysine has two amino groups. Both amino functions are converted to amides on reaction with benzyloxycarbonyl chloride. 27.17 The peptide bond of Ala-Leu connects the carboxyl group of alanine and the amino group of leucine. We therefore need to protect the amino group of alanine and the carboxyl group of leucine. Protect the amino group of alanine as its benzyloxycarbonyl derivative. H3NCHCO2 CH3 Alanine C6H5CH2OCCl O Benzyloxycarbonyl chloride C6H5CH2OCNHCHCO2H CH3 O Z-Protected alanine O H2NCHCO2 2C6H5CH2OCCl O O C6H5CH2OCNHCHCO2H C6H5CH2OCNHCH2CH2CH2CH2 H2NCH2CH2CH2CH2 Val-Phe-Gly-Ala Phe-Gly-Ala first Edman degradation second Edman degradation S HN CH(CH3)2 O Gly-Ala S HN CH2C6H5 N O C6H5 N C6H5 CH2C6H5 NHCHC O H3NCHC O (CH3)2CH Valine Phenylalanine CH2C6H5 NHCHCO O H3NCHC O (CH3)2CH Valinylphenylalanine (VF) Rest of peptide Rest of peptide chymotrypsin AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS 759 Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
760 AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS Protect the carboxyl group of leucine as its benzyl ester IH heat H,NCHCO CHCH OH H,NCHCO, CH2C6H5 CH3)2CHCH (CH3),CHCH, Benzyl alcohol Leucine benzyl ester Coupling of the two amino acids is achieved by N, N-dicyclohexylcarbodiimide(DCCI)-promoted amide bond formation between the free amino group of leucine benzyl ester and the free carboxyl group of Z-protected alanin DCCI C6HSCH2OCNHCHCO,H H,NCHCOCH,CH C6HSCHOCNHCHCNHCHCOCH,CBH CH3 (CH3)2CHCH H3 CH, CH(CH3)2 Z-Protected alanine Leucine benzyl es Both the benzyloxycarbonyl protecting group and the benzyl ester protecting group may be removed by hydrogenolysis over palladium. This step completes the synthesis of Ala-Leu CH, CH,OCNHCHCNHCHCOCH,CH H2 Pd, HNCHCNHCHCO2 CH3 CH, CH(CH3)2 CH,CH(CH3)2 Protected dipeptide 27.18 As in the DCCI-promoted coupling of amino acids, the first step is the addition of the Z-protected amino acid to DCCI to give an O-acylisourea NCH ZNHCHCOH+ CHIN=C=NCBH ZNHCHCO R NHC HIL Z-Pro DCCI cvlisourea This O-acylisourea is attacked by p-nitrophenol to give the p-nitrophenyl ester of the Z-protected o acid NChU ON +Rc--O-C C6HuNhCnHC Hul NHC6HII 27 19 To add a leucine residue to the n terminus of the ethyl ester of z-Phe-Gly, the benzyloxycarbonyl protecting group must first be removed. This can be accomplished by hydrogenolysis CBHSCH2OCNHCHCNHCH, COCH, CH H.NCHCNHCH COCH CH C6HSCH2 C6H-CH Z-Protected ethyl ester of Phe-Gly y ethyl ester Back Forward Main Menu TOC Study Guide Toc Student OLC MHHE Website
Protect the carboxyl group of leucine as its benzyl ester. Coupling of the two amino acids is achieved by N,N′-dicyclohexylcarbodiimide (DCCI)-promoted amide bond formation between the free amino group of leucine benzyl ester and the free carboxyl group of Z-protected alanine. Both the benzyloxycarbonyl protecting group and the benzyl ester protecting group may be removed by hydrogenolysis over palladium. This step completes the synthesis of Ala-Leu. 27.18 As in the DCCI-promoted coupling of amino acids, the first step is the addition of the Z-protected amino acid to DCCI to give an O-acylisourea. This O-acylisourea is attacked by p-nitrophenol to give the p-nitrophenyl ester of the Z-protected amino acid. 27.19 To add a leucine residue to the N terminus of the ethyl ester of Z-Phe-Gly, the benzyloxycarbonyl protecting group must first be removed. This can be accomplished by hydrogenolysis. C6H5CH2OCNHCHCNHCH2COCH2CH3 H2NCHCNHCH2COCH2CH3 C6H5CH2 O O O O O C6H5CH2 H2, Pd Z-Protected ethyl ester of Phe-Gly Phe-Gly ethyl ester O2N OH RC O O2N O C NHC6H11 NC6H11 H OCR C6H11NHCNHC6H11 O O Z-Protected amino acid R ZNHCHCOH O DCCI C6H11N C NC6H11 O-Acylisourea R ZNHCHCO O C NC6H11 NHC6H11 Ala-Leu CH3 CH2CH(CH3)2 H3NCHCNHCHCO2 O H2, Pd Protected dipeptide CH3 CH2CH(CH3)2 C6H5CH2OCNHCHCNHCHCOCH2C6H5 O O O Z-Protected alanine C6H5CH2OCNHCHCO2H CH3 O Leucine benzyl ester (CH3)2CHCH2 H2NCHCOCH2C6H5 O DCCI Protected dipeptide CH3 CH2CH(CH3)2 C6H5CH2OCNHCHCNHCHCOCH2C6H5 O O O H3NCHCO2 (CH3)2CHCH2 Leucine C6H5CH2OH Benzyl alcohol (CH3)2CHCH2 H2NCHCO2CH2C6H5 Leucine benzyl ester 1. H, heat 2. HO 760 AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS 761 The reaction shown has been carried out in 100% yield. Alternatively, the benzyloxycarbonyl protecting group may be removed by treatment with hydrogen bromide in acetic acid. This latter route has also been reported in the chemical literature and gives the hydrobromide salt of Phe-Gly ethyl ester in 82%o yield Once the protecting group has been removed, the ethyl ester of Phe-Gly is allowed to react with the p-nitrophenyl ester of Z-protected leucine to form the protected tripeptide. Hydrogenolysis of the Z-protected tripeptide gives Leu-Phe-Gly as its ethyl ester. C6H-CH,OCNHCHCO NO2 H,NCHCNHCH, COCH, CH3 Phe-Gly ethyl ester Z-protected leucine C6HSCHOCNHCHCNHCHCNHCH, COCH, CH3 (CH3), CHCH, CHaC6H5 H,NCHCNHCHCNHCH, COCH,CH (CH3)2CHCH2 CH,C6H5 27.20 Amino acid residues are added by beginning at the C terminus in the Merrifield solid-phase approach to peptide synthesis. Thus the synthesis of Phe-Gly requires glycine to be anchored to the solid support. Begin by protecting glycine as its tert-butoxycarbonyl (Boc)derivative (CH3)COCCI H3NCHCO2-(CH3)3COCNHCH, CO,H tert-Butoxycarbonyl Boc-Protected glycine The protected glycine is attached via its carboxylate anion to the solid support (CH3)3COCNHCH, CO,H (CH,)3COCNHCH, COCH,-resin Boc-Protected glycine Back Forward Main Menu TOC Study Guide Toc Student OLC MHHE Website
The reaction shown has been carried out in 100% yield. Alternatively, the benzyloxycarbonyl protecting group may be removed by treatment with hydrogen bromide in acetic acid. This latter route has also been reported in the chemical literature and gives the hydrobromide salt of Phe-Gly ethyl ester in 82% yield. Once the protecting group has been removed, the ethyl ester of Phe-Gly is allowed to react with the p-nitrophenyl ester of Z-protected leucine to form the protected tripeptide. Hydrogenolysis of the Z-protected tripeptide gives Leu-Phe-Gly as its ethyl ester. 27.20 Amino acid residues are added by beginning at the C terminus in the Merrifield solid-phase approach to peptide synthesis. Thus the synthesis of Phe-Gly requires glycine to be anchored to the solid support. Begin by protecting glycine as its tert-butoxycarbonyl (Boc) derivative. The protected glycine is attached via its carboxylate anion to the solid support. (CH3)3COCNHCH2CO2H O (CH3)3COCNHCH2COCH2 resin O O Boc-Protected glycine 1. HO 2. ClCH2 resin (CH3)3COCCl (CH3) H3NCH2CO2 3COCNHCH2CO2H O O tert-Butoxycarbonyl chloride Glycine Boc-Protected glycine Z-protected Leu-Phe-Gly ethyl ester C6H5CH2OCNHCHCO NO2 (CH3)2CHCH2 O O H2NCHCNHCH2COCH2CH3 O O C6H5CH2 p-Nitrophenyl ester of Z-protected leucine Phe-Gly ethyl ester C6H5CH2OCNHCHCNHCHCNHCH2COCH2CH3 (CH3)2CHCH2 O O CH2C6H5 O O Leu-Phe-Gly ethyl ester H2NCHCNHCHCNHCH2COCH2CH3 (CH3)2CHCH2 O O CH2C6H5 O H2, Pd AMINO ACIDS, PEPTIDES, AND PROTEINS. NUCLEIC ACIDS 761 Back Forward Main Menu TOC Study Guide TOC Student OLC MHHE Website
