Dr. Wolf's CHM 424 27- 1 Chapter 27 Amino Acids, Peptides, and Proteins

Dr. Wolf's CHM 424 27- 1

Chapter 27Chapter 27Amino Acids, Peptides, Amino Acids, Peptides,

and Proteinsand Proteins


27.127.1Classification of Amino AcidsClassification of Amino Acids


FundamentalsFundamentals

While their name implies that amino acids are While their name implies that amino acids are compounds that contain an —NHcompounds that contain an —NH2 2 group and a group and a

—CO—CO22H group, these groups are actually H group, these groups are actually

present as —NHpresent as —NH33++ and —CO and —CO22

–– respectively. respectively.

They are classified as They are classified as , , , , , , etcetc. amino acids . amino acids according the carbon that bears the nitrogen.according the carbon that bears the nitrogen.


Amino AcidsAmino Acids NNHH33

++

CCOO22––

an an -amino acid that is an-amino acid that is anintermediate in the biosynthesisintermediate in the biosynthesisof ethyleneof ethylene

++HH33NNCHCH22CHCH22CCOO22

––a a -amino acid that is one of-amino acid that is one ofthe structural units present inthe structural units present incoenzyme Acoenzyme A

++HH33NNCHCH22CHCH22CHCH22CCOO22

–– a a -amino acid involved in-amino acid involved inthe transmission of nervethe transmission of nerveimpulsesimpulses


The 20 Key Amino AcidsThe 20 Key Amino Acids

More than 700 amino acids occur naturally, but More than 700 amino acids occur naturally, but 20 of them are especially important.20 of them are especially important.

These 20 amino acids are the building blocks of These 20 amino acids are the building blocks of proteins. All are proteins. All are -amino acids.-amino acids.

They differ in respect to the group attached to They differ in respect to the group attached to the the carbon. carbon.

These 20 are listed in Table 27.1.These 20 are listed in Table 27.1.


Table 27.1Table 27.1

CC CC

OO

OO––

RRRR

HH

HH33NN++

The amino acids obtained by hydrolysis of The amino acids obtained by hydrolysis of proteins differ in respect to proteins differ in respect to R R (the side chain).(the side chain).

The properties of the amino acid vary as the The properties of the amino acid vary as the structure of structure of RR varies. varies.



CC CC

OO

OO––

RRRR

HH

HH33NN++

The major differences among the side chains The major differences among the side chains concern:concern:

Size and shapeSize and shapeElectronic characteristicsElectronic characteristics



General categories of General categories of -amino acids-amino acids

nonpolar side chainsnonpolar side chainspolar but nonionized side chainspolar but nonionized side chainsacidic side chainsacidic side chainsbasic side chainsbasic side chains







CC CC

OO

OO––

HH

HH

HH33NN++

Glycine is the simplest amino acid. It is the only Glycine is the simplest amino acid. It is the only one in the table that is achiral.one in the table that is achiral.

In all of the other amino acids in the table the In all of the other amino acids in the table the carbon is a chirality center.carbon is a chirality center.

GlycineGlycine

(Gly or G)(Gly or G)



CC CC

OO

OO––

CHCH33

HH

HH33NN++

AlanineAlanine

(Ala or A)(Ala or A)

Alanine, valine, leucine, and isoleucine have Alanine, valine, leucine, and isoleucine have alkyl groups as side chains, which are nonpolar alkyl groups as side chains, which are nonpolar and hydrophobic.and hydrophobic.



CC CC

OO

OO––

CH(CHCH(CH33))22

HH

HH33NN++

ValineValine

(Val or V)(Val or V)



CC CC

OO

OO––

CHCH22CH(CHCH(CH33))22

HH

HH33NN++

LeucineLeucine

(Leu or L)(Leu or L)



CC CC

OO

OO––

CHCH33CHCHCHCH22CHCH33

HH

HH33NN++

IsoleucineIsoleucine

(Ile or I)(Ile or I)



CC CC

OO

OO––

CHCH33SCHSCH22CHCH22

HH

HH33NN++

MethionineMethionine

(Met or M)(Met or M)

The side chain in methionine is nonpolar, but The side chain in methionine is nonpolar, but the presence of sulfur makes it somewhat the presence of sulfur makes it somewhat polarizable.polarizable.



ProlineProline

CC CC

OO

OO––

CHCH22

HH

HH22NN++

HH22CCCCHH22

(Pro or P)(Pro or P)

Proline is the only amino acid that contains a Proline is the only amino acid that contains a secondary amine function. Its side chain is secondary amine function. Its side chain is nonpolar and cyclic.nonpolar and cyclic.



PhenylalaninePhenylalanine

CC CC

OO

OO––

CHCH22

HH

HH33NN++

(Phe or F)(Phe or F)

The side chain in phenylalanine (a nonpolar The side chain in phenylalanine (a nonpolar amino acid) is a benzyl group.amino acid) is a benzyl group.



CC CC

OO

OO––

CHCH22

HH

HH33NN++

NN

HH

TryptophanTryptophan

(Trp or W)(Trp or W) The side chain in The side chain in tryptophan (a nonpolar tryptophan (a nonpolar amino acid) is larger amino acid) is larger and more polarizable and more polarizable than the benzyl group than the benzyl group of phenylalanine.of phenylalanine.







CC CC

OO

OO––

CHCH22OHOH

HH

HH33NN++

SerineSerine

(Ser or S)(Ser or S)

The —CHThe —CH22OH side chain in serine can be OH side chain in serine can be

involved in hydrogen bonding.involved in hydrogen bonding.



CC CC

OO

OO––

CHCH33CHOHCHOH

HH

HH33NN++

ThreonineThreonine

(Thr or T)(Thr or T)

The side chain in threonine can be involved in hydrogen The side chain in threonine can be involved in hydrogen bonding, but is somewhat more crowded than in serine.bonding, but is somewhat more crowded than in serine.



CC CC

OO

OO––

CHCH22SSHH

HH

HH33NN++

CysteineCysteine

(Cys or C)(Cys or C)

The side chains of two remote cysteines can be The side chains of two remote cysteines can be joined by forming a covalent S—S bond.joined by forming a covalent S—S bond.



TyrosineTyrosineCC CC

OO

OO––

CHCH22

HH

HH33NN++

OHOH

(Tyr or Y)(Tyr or Y) The side chain of The side chain of tyrosine is similar to tyrosine is similar to that of phenylalanine that of phenylalanine but can participate in but can participate in hydrogen bonding.hydrogen bonding.



AsparagineAsparagine

CC CC

OO

OO––

HH

HH33NN++

HH22NNCCCHCH22

OO(Asn or N)(Asn or N)

The side chains of asparagine and glutamine The side chains of asparagine and glutamine (next slide) terminate in amide functions that are (next slide) terminate in amide functions that are polar and can engage in hydrogen bonding.polar and can engage in hydrogen bonding.



GlutamineGlutamine

CC CC

OO

OO––

HH

HH33NN++

HH22NNCCHCCH22CHCH22

OO(Gln or Q)(Gln or Q)







Aspartic AcidAspartic Acid

CC CC

OO

OO––

HH

HH33NN++

OOCCCHCH22

OO

––

(Asp or D)(Asp or D)

Aspartic acid and glutamic acid (next slide) exist Aspartic acid and glutamic acid (next slide) exist as their conjugate bases at biological pH. They as their conjugate bases at biological pH. They are negatively charged and can form ionic are negatively charged and can form ionic bonds with positively charged species.bonds with positively charged species.



Glutamic AcidGlutamic Acid

CC CC

OO

OO––

HH

HH33NN++

OOCCHCCH22CHCH22

OO

––

(Glu or E)(Glu or E)







CC CC

OO

OO––

CHCH22CHCH22CHCH22CHCH22NNHH33

HH

HH33NN++

LysineLysine++(Lys or K)(Lys or K)

Lysine and arginine (next slide) exist as their Lysine and arginine (next slide) exist as their conjugate acids at biological pH. They are conjugate acids at biological pH. They are positively charged and can form ionic bonds positively charged and can form ionic bonds with negatively charged species.with negatively charged species.



CC CC

OO

OO––

CHCH22CHCH22CHCH22NNHHCCNNHH22

HH

HH33NN++

ArginineArginine

++ NNHH22

(Arg or R)(Arg or R)



HistidineHistidine CC CC

OO

OO––

HH

HH33NN++

CHCH22

NHNHNN

(His or H)(His or H) Histidine is a basic Histidine is a basic amino acid, but less amino acid, but less basic than lysine and basic than lysine and arginine. Histidine can arginine. Histidine can interact with metal ions interact with metal ions and can help move and can help move protons from one site protons from one site to another.to another.


27.227.2Stereochemistry of Amino Stereochemistry of Amino

AcidsAcids


Configuration of Configuration of -Amino Acids-Amino Acids

Glycine is achiral. All of the other amino acids Glycine is achiral. All of the other amino acids in proteins have the in proteins have the LL-configuration at their -configuration at their carbon.carbon.

HH33NN++

HH

RR

COCO22––


27.327.3Acid-Base Behavior of Amino Acid-Base Behavior of Amino

AcidsAcids


RecallRecall

While their name implies that amino acids are While their name implies that amino acids are compounds that contain an —NHcompounds that contain an —NH2 2 group and a group and a

—CO—CO22H group, these groups are actually H group, these groups are actually

present as —NHpresent as —NH33++ and —CO and —CO22

–– respectively. respectively.

How do we know this?How do we know this?


Properties of GlycineProperties of Glycine

The properties of glycine:The properties of glycine:

high melting point:high melting point: (when heated to 233°C (when heated to 233°C it decomposes before it melts)it decomposes before it melts)solubility:solubility: soluble in water; not soluble in soluble in water; not soluble in nonpolar solventnonpolar solvent

OO

OHOHHH22NNCHCH22CC••••

••••

••••

•••• ••••

––••••

OO

OOHH33NNCHCH22CC ••••

••••

•••• ••••++

more consistent with thismore consistent with this than thisthan this


Properties of GlycineProperties of Glycine

The properties of glycine:The properties of glycine:

high melting point:high melting point: (when heated to 233°C (when heated to 233°C it decomposes before it melts)it decomposes before it melts)solubility:solubility: soluble in water; not soluble in soluble in water; not soluble in nonpolar solventnonpolar solvent

––••••

OO


••••

•••• ••••++

more consistent with thismore consistent with this

called a called a zwitterionzwitterion or or dipolar iondipolar ion


Acid-Base Properties of GlycineAcid-Base Properties of Glycine

The zwitterionic structure of glycine also follows The zwitterionic structure of glycine also follows from considering its acid-base properties.from considering its acid-base properties.

A good way to think about this is to start with the A good way to think about this is to start with the structure of glycine in strongly acidic solution, structure of glycine in strongly acidic solution, say pH = 1.say pH = 1.

At pH = 1, glycine exists in its protonated form At pH = 1, glycine exists in its protonated form (a monocation).(a monocation).

OO

OHOHHH33NNCHCH22CC++

••••

••••

•••• ••••



Now ask yourself "As the pH is raised, which is Now ask yourself "As the pH is raised, which is the first proton to be removed? Is it the proton the first proton to be removed? Is it the proton attached to the positively charged nitrogen, or is attached to the positively charged nitrogen, or is it the proton of the carboxyl group?"it the proton of the carboxyl group?"

You can choose between them by estimating You can choose between them by estimating their respective ptheir respective pKKaas.s.

OO


••••

••••

•••• ••••

typical typical ammonium ammonium ion: pion: pKKaa ~9 ~9

typical typical carboxylic carboxylic acid: pacid: pKKaa ~5 ~5



The more acidic proton belongs to the COThe more acidic proton belongs to the CO22H H

group. It is the first one removed as the pH is group. It is the first one removed as the pH is raised.raised.

OO


••••

••••

•••• ••••




Therefore, the more stable neutral form of Therefore, the more stable neutral form of glycine is the zwitterion.glycine is the zwitterion.

OO


••••

••••

•••• ••••


––••••

OO


••••

•••• ••••++


The measured pThe measured pKKaa of glycine is 2.34. of glycine is 2.34.

Glycine is stronger than a typical carboxylic acid Glycine is stronger than a typical carboxylic acid because the positively charged N acts as an because the positively charged N acts as an electron-withdrawing, acid-strengthening electron-withdrawing, acid-strengthening substituent on the substituent on the carbon. carbon.


OO


••••

••••

•••• ••••




––••••

OO


••••

•••• ••••++

The pThe pKKaa for removal of this proton is 9.60. for removal of this proton is 9.60.

This value is about the same as that for NHThis value is about the same as that for NH44++

(9.3).(9.3).

HOHO––––••••

OO


••••

•••• ••••

••••

A proton attached to N in the zwitterionic form of A proton attached to N in the zwitterionic form of nitrogen can be removed as the pH is increased nitrogen can be removed as the pH is increased further. further.


Isoelectric Point pIsoelectric Point pII

––••••

OO


••••

•••• ••••++

––••••

OO


••••

•••• ••••

••••

OO


••••

••••

•••• ••••

ppKKaa = 2.34 = 2.34

ppKKaa = 9.60 = 9.60

The pH at which the The pH at which the concentration of the concentration of the zwitterion is a zwitterion is a maximum is called the maximum is called the isoelectric pointisoelectric point. Its . Its numerical value is the numerical value is the average of the two average of the two ppKKaas.s.

The pThe pII of glycine is of glycine is 5.97.5.97.


Acid-Base Properties of Amino AcidsAcid-Base Properties of Amino Acids

One way in which amino acids differ is in One way in which amino acids differ is in respect to their acid-base properties. This is the respect to their acid-base properties. This is the basis for certain experimental methods for basis for certain experimental methods for separating and identifying them.separating and identifying them.

Just as important, the difference in acid-base Just as important, the difference in acid-base properties among various side chains affects properties among various side chains affects the properties of the proteins that contain them.the properties of the proteins that contain them.

Table 27.2 gives pTable 27.2 gives pKKaa and p and pII values for amino values for amino

acids with neutral side chains.acids with neutral side chains.


Table 27.2Table 27.2 Amino Acids with Neutral Side ChainsAmino Acids with Neutral Side Chains

CC CC

OO

OO––

HH

HH

HH33NN++

GlycineGlycineppKKa1a1 = = 2.342.34

ppKKa2a2 == 9.609.60

ppI I == 5.975.97



AlanineAlanineppKKa1a1 = = 2.342.34

ppKKa2a2 == 9.699.69

ppI I == 6.006.00

HH33NN CC CC

OO

OO––

CHCH33

HH++



ValineValineppKKa1a1 = = 2.322.32

ppKKa2a2 == 9.629.62

ppI I == 5.965.96

HH33NN CC CC

OO

OO––

CH(CHCH(CH33))22

HH++



LeucineLeucineppKKa1a1 = = 2.362.36

ppKKa2a2 == 9.609.60

ppI I == 5.985.98

HH33NN CC CC

OO

OO––

CHCH22CH(CHCH(CH33))22

HH++



IsoleucineIsoleucineppKKa1a1 = = 2.362.36

ppKKa2a2 == 9.609.60

ppI I == 5.985.98

HH33NN CC CC

OO

OO––

CHCH33CHCHCHCH22CHCH33

HH++



MethionineMethionineppKKa1a1 = = 2.282.28

ppKKa2a2 == 9.219.21

ppI I == 5.745.74

HH33NN CC CC

OO

OO––

CHCH33SCHSCH22CHCH22

HH++



ProlineProlineppKKa1a1 = = 1.991.99

ppKKa2a2 == 10.6010.60

ppI I == 6.306.30

HH22NN CC CC

OO

OO––

HH++

CHCH22HH22CCCCHH22



PhenylalaninePhenylalanineppKKa1a1 = = 1.831.83

ppKKa2a2 == 9.139.13

ppI I == 5.485.48

HH33NN CC CC

OO

OO––

HH++

CHCH22



TryptophanTryptophanppKKa1a1 = = 2.832.83

ppKKa2a2 == 9.399.39

ppI I == 5.895.89

HH33NN CC CC

OO

OO––

HH++

CHCH22

HH

NN



AsparagineAsparagineppKKa1a1 = = 2.022.02

ppKKa2a2 == 8.808.80

ppI I == 5.415.41

HH33NN CC CC

OO

OO––

HH++

HH22NNCCCHCH22

OO



GlutamineGlutamineppKKa1a1 = = 2.172.17

ppKKa2a2 == 9.139.13

ppI I == 5.655.65

HH33NN CC CC

OO

OO––

HH++

HH22NNCCHCCH22CHCH22

OO



SerineSerineppKKa1a1 = = 2.212.21

ppKKa2a2 == 9.159.15

ppI I == 5.685.68

HH33NN CC CC

OO

OO––

CHCH22OHOH

HH++



ThreonineThreonineppKKa1a1 = = 2.092.09

ppKKa2a2 == 9.109.10

ppI I == 5.605.60

HH33NN CC CC

OO

OO––

CHCH33CHOHCHOH

HH++



TyrosineTyrosineppKKa1a1 = = 2.202.20

ppKKa2a2 == 9.119.11

ppI I == 5.665.66

HH33NN CC CC

OO

OO––

HH++

CHCH22 OHOH



CysteineCysteine HH33NN CC CC

OO

OO––

CHCH22SHSH

HH++ ppKKa1a1 = = 1.961.96

ppKKa2a2 == 8.188.18

ppI I == 5.075.07


Table 27.3Table 27.3 Amino Acids with Ionizable Side ChainsAmino Acids with Ionizable Side Chains

Aspartic acidAspartic acidppKKa1a1 = = 1.881.88

ppKKa2a2 == 3.653.65

ppKKa3a3 == 9.60 9.60

ppI I == 2.772.77

HH33NN CC CC

OO

OO––

HH++

OOCCCHCH22

OO

––

For amino acids with acidic side chains, pI is the For amino acids with acidic side chains, pI is the average of paverage of pKKa1a1 and p and pKKa2a2..



Glutamic acidGlutamic acidppKKa1a1 = = 2.192.19

ppKKa2a2 == 4.254.25

ppKKa3a3 == 9.67 9.67

ppI I == 3.223.22

HH33NN CC CC

OO

OO––

HH++

OO

OOCCCHCH22CHCH22––



LysineLysine

ppKKa1a1 = = 2.182.18

ppKKa2a2 == 8.958.95

ppKKa3a3 == 10.5310.53

ppI I == 9.749.74

HH33NN CC CC

OO

OO––

HH++

CHCH22CHCH22CHCH22CHCH22NNHH33

++

For amino acids with basic side chains, pI is the For amino acids with basic side chains, pI is the average of paverage of pKKa2a2 and p and pKKa3a3..



ArginineArginine

ppKKa1a1 = = 2.172.17

ppKKa2a2 == 9.049.04

ppKKa3a3 == 12.4812.48

ppI I == 10.7610.76

HH33NN CC CC

OO

OO––

HH++

CHCH22CHCH22CHCH22NNHHCCNNHH22

++ NNHH22



HistidineHistidine

ppKKa1a1 = = 1.821.82

ppKKa2a2 == 6.006.00

ppKKa3a3 == 9.17 9.17

ppI I == 7.597.59

HH33NN CC CC

OO

OO––

HH++

CHCH22

NHNHNN


27.427.4Synthesis of Amino AcidsSynthesis of Amino Acids


From From -Halo Carboxylic Acids-Halo Carboxylic Acids

CHCH33CHCOHCHCOH

BrBr

OO

22NNHH33++HH22OO

CHCH33CHCOCHCO

NNHH33

OO

++

––

(65-70%)(65-70%)

++ NNHH44BrBr


Strecker SynthesisStrecker Synthesis

NNHH44ClCl

NaNaCNCNCHCH33CHCH

OO

CHCH33CHCHCC

NNHH22

NN

CHCH33CHCHCCOO

NNHH33

OO

++

–– (52-60%)(52-60%)

1. H1. H22O, HCl, heatO, HCl, heat

2. HO2. HO––


Using Diethyl AcetamidomalonateUsing Diethyl Acetamidomalonate

CCCC

CCOCHOCH22CHCH33

HH

OO OO

CHCH33CHCH22OO

CHCH33CCNNHH

OO

Can be used in the same manner as diethyl Can be used in the same manner as diethyl malonate (Section 21.7).malonate (Section 21.7).


ExampleExample

1. NaOCH1. NaOCH22CHCH33

2. 2. CC66HH55CHCH22ClCl

OO OO

CHCH33CHCH22OCCCOCHOCCCOCH22CHCH33

HHCHCH33CCNNHH

OO

OO OO


CHCH22CC66HH55CHCH33CCNNHH

OO

(90%)(90%)


ExampleExample

OO OO


CHCH22CC66HH55CHCH33CCNNHH

OO

HBr, HHBr, H22O, heatO, heat

OO OO

HOCCCOHHOCCCOH

CHCH22CC66HH55HH33NN++

OO

HCCOHHCCOH

CHCH22CC66HH55HH33NN++(65%)(65%)

––COCO22


27.527.5Reactions of Amino AcidsReactions of Amino Acids


Acylation of Amino GroupAcylation of Amino Group

The amino nitrogen of an amino acid can be The amino nitrogen of an amino acid can be converted to an amide with the customary converted to an amide with the customary acylating agents.acylating agents.

OO

HH33NNCHCH22COCO––++++ CHCH33COCCHCOCCH33

OO OO

CHCH33CCNNHCHHCH22COHCOH

OO OO

(89-92%)(89-92%)


Esterification of Carboxyl GroupEsterification of Carboxyl Group

The carboxyl group of an amino acid can be The carboxyl group of an amino acid can be converted to an ester. The following illustrates converted to an ester. The following illustrates Fischer esterification of alanine. Fischer esterification of alanine.

++ CHCH33CHCH22OHOH

HClHCl

OO

HH33NNCHCOCHCO––++

CHCH33

(90-95%)(90-95%)

OO

HH33NNCHCOCHCHCOCH22CHCH33

++

CHCH33

––ClCl


Ninhydrin TestNinhydrin Test

Amino acids are detected by the formation of a purple Amino acids are detected by the formation of a purple color on treatment with color on treatment with ninhydrinninhydrin..

OHOH

OO

OO

OHOH++

OO

HH33NNCHCOCHCO––++

RR

OO OO

OO

NN

OO

––

OO

RCHRCH ++ COCO22 ++ HH22OO ++


27.627.6Some Biochemical ReactionsSome Biochemical Reactions

of Amino Acidsof Amino Acids


Biosynthesis of Biosynthesis of LL-Glutamic Acid-Glutamic Acid

This reaction is the biochemical analog of reductive This reaction is the biochemical analog of reductive amination (Section 22.10).amination (Section 22.10).

HOHO22CCHCCH22CHCH22CCOCCO22HH

OO

NNHH33++

enzymes andenzymes andreducing coenzymesreducing coenzymes

HOHO22CCHCCH22CHCH22CHCOCHCO22––

NNHH33++


Transamination Transamination viavia LL-Glutamic Acid-Glutamic Acid

LL-Glutamic acid acts as a source of the amine -Glutamic acid acts as a source of the amine group in the biochemical conversion of group in the biochemical conversion of -keto-ketoacids to other amino acids. In the example to beacids to other amino acids. In the example to beshown, pyruvic acid is converted to shown, pyruvic acid is converted to LL-alanine.-alanine.


NNHH33++

++ CHCH33CCOCCO22HH

OO


Transamination Transamination viavia LL-Glutamic Acid-Glutamic Acid


NNHH33++

++ CHCH33CCOCCO22HH

OO

enzymesenzymes

HOHO22CCHCCH22CHCH22CCOCCO22HH

OO

++ CHCH33CHCOCHCO22

––

NNHH33++


MechanismMechanism

HOHO22CCHCCH22CHCH22CCHHCOCO22––

NNHH33++

++ CHCH33CCOCCO22HH

OO

The first step is imine formation between theThe first step is imine formation between theamino group of amino group of LL-glutamic acid and pyruvic-glutamic acid and pyruvicacid.acid.


MechanismMechanism

HOHO22CCHCCH22CHCH22CCHHCOCO22––

NNHH33++

++ CHCH33CCOCCO22HH

OO

CHCH33CCOCCO22––

HOHO22CCHCCH22CHCH22CCHHCOCO22

NN

––

Dr. Wolf's CHM 424 27- 83CHCH33CCOCCO22

––

HOHO22CCHCCH22CHCH22CCOCCO22

NN

––

HH

Formation of the imine is followed by Formation of the imine is followed by protonproton removal at one carbon and removal at one carbon and protonationprotonation of another of another carbon.carbon.

Dr. Wolf's CHM 424 27- 84CHCH33CCOCCO22

––


NN

––

HH



NN

HH

––


––



NN

HH

Hydrolysis of the imine function givesHydrolysis of the imine function gives-keto glutarate and -keto glutarate and LL-alanine.-alanine.


––



NN

HH


OO

––CHCH33CCOCCO22

––

NNHH33

HH

++

++

HH22OO


LL--Tyrosine is biosynthesized from Tyrosine is biosynthesized from LL--phenylalanine.phenylalanine.A key step is epoxidation of the aromatic ring to A key step is epoxidation of the aromatic ring to give an give an arene oxidearene oxide intermediate. intermediate.

Biosynthesis of Biosynthesis of LL-Tyrosine-Tyrosine

CHCH22CHCOCHCO22

––

NNHH33++



CHCH22CHCOCHCO22

––

NNHH33++

CHCH22CHCOCHCO22

––

NNHH33++

OO

OO22, enzyme, enzyme



CHCH22CHCOCHCO22

––

NNHH33++

OO

enzymeenzyme

CHCH22CHCOCHCO22

––

NNHH33++

HHOO



Conversion to Conversion to LL-tyrosine is one of the major -tyrosine is one of the major metabolic pathways of metabolic pathways of LL-phenylalanine.-phenylalanine.

Individuals who lack the enzymes necessary to Individuals who lack the enzymes necessary to convert convert LL-phenylalanine to -phenylalanine to LL-tyrosine can suffer -tyrosine can suffer from PKU disease. In PKU disease, from PKU disease. In PKU disease, LL--phenylalanine is diverted to a pathway leading phenylalanine is diverted to a pathway leading to phenylpyruvic acid, which is toxic.to phenylpyruvic acid, which is toxic.

Newborns are routinely tested for PKU disease. Newborns are routinely tested for PKU disease. Treatment consists of reducing their dietary Treatment consists of reducing their dietary intake of phenylalanine-rich proteins.intake of phenylalanine-rich proteins.


DecarboxylationDecarboxylation

Decarboxylation is a common reaction of Decarboxylation is a common reaction of --amino acids. An example is the conversion of amino acids. An example is the conversion of LL-histidine to histamine. Antihistamines act by -histidine to histamine. Antihistamines act by blocking the action of histamine.blocking the action of histamine.

CHCH22CHCOCHCO22

––

NNHH33++NNHH

NN


DecarboxylationDecarboxylation

CHCH22CHCOCHCO22

––

NNHH33++NNHH

NN

––COCO22, enzymes, enzymes

CHCH22CHCH2 2 NNHH22

NNHH

NN


NeurotransmittersNeurotransmitters

The chemistry of the The chemistry of the brain and central brain and central nervous system is nervous system is affected by affected by neurotransmitters.neurotransmitters.

Several important Several important neurotransmitters neurotransmitters are biosynthesized are biosynthesized from from LL-tyrosine.-tyrosine.

OOHH

COCO22––

HHHH

HHHH33NN

++

LL-Tyrosine-Tyrosine



The common name The common name of this compound is of this compound is LL-DOPA. It occurs -DOPA. It occurs naturally in the naturally in the brain. It is widely brain. It is widely prescribed to reduce prescribed to reduce the symptoms of the symptoms of Parkinsonism.Parkinsonism.

OOHH

COCO22––

HHHH

HHHH33NN

++

LL-3,4-Dihydroxyphenylalanine-3,4-Dihydroxyphenylalanine

HHOO



Dopamine is formed Dopamine is formed by decarboxylation by decarboxylation of of LL-DOPA.-DOPA.

OOHH

HH

HHHH

HHHH22NN

HHOO

DopamineDopamine



OOHH

HH

HHHH

OHOHHH22NN

HHOO

NorepinephrineNorepinephrine



OOHH

HH

HHHH

OHOHCHCH33NNHH

HHOO

EpinephrineEpinephrine


27.727.7PeptidesPeptides


PeptidesPeptides

Peptides are compounds in which an amide Peptides are compounds in which an amide bond links the amino group of one bond links the amino group of one -amino acid -amino acid and the carboxyl group of another.and the carboxyl group of another.

An amide bond of this type is often referred to An amide bond of this type is often referred to as a peptide bond.as a peptide bond.

Dr. Wolf's CHM 424 27- 100

Alanine and GlycineAlanine and Glycine

CHCH33

OO

CC++

HH

CC OO––

HH33NN

OO

CC

HH

HH

CCHH33NN++

OO––

Dr. Wolf's CHM 424 27- 101

AlanylglycineAlanylglycine

CHCH33

OO

CCHH33NN++

HH

CC

OO

CCNN

HH

HH

CC OO––

HH

Two Two -amino acids are joined by a peptide bond -amino acids are joined by a peptide bond in alanylglycine. It is a in alanylglycine. It is a dipeptidedipeptide..

Dr. Wolf's CHM 424 27- 102


CHCH33

OO

CCHH33NN++

HH

CC

OO

CCNN

HH

HH

CC OO––

HH

Ala—GlyAla—Gly

AGAG

N-terminusN-terminus C-terminusC-terminus

Dr. Wolf's CHM 424 27- 103

Alanylglycine and glycylalanine are Alanylglycine and glycylalanine are constitutional isomersconstitutional isomers

CHCH33

OO

CCHH33NN++

HH

CC

OO

CCNN

HH

HH

CC OO––

HH

HH

OO

CCHH33NN++

HH

CC

OO

CCNN

HH

CHCH33

CC OO––

HH

AlanylAlanylglycineglycineAlaAla——GlyGly

AAGG

GlycylGlycylalaninealanineGlyGly——AlaAla

GGAA

Dr. Wolf's CHM 424 27- 104


CHCH33

OO

CCHH33NN++

HH

CC

OO

CCNN

HH

HH

CC OO––

HH

The peptide bond is The peptide bond is characterized by a characterized by a planar geometry.planar geometry.

Dr. Wolf's CHM 424 27- 105

Higher PeptidesHigher Peptides

Peptides are classified according to the number Peptides are classified according to the number of amino acids linked together.of amino acids linked together.

dipeptides, tripeptides, tetrapeptides, etc.dipeptides, tripeptides, tetrapeptides, etc.

Leucine enkephalin is an example of a Leucine enkephalin is an example of a pentapeptide.pentapeptide.

Dr. Wolf's CHM 424 27- 106

Leucine EnkephalinLeucine Enkephalin

Tyr—Gly—Gly—Phe—LeuTyr—Gly—Gly—Phe—LeuYGGFLYGGFL

Dr. Wolf's CHM 424 27- 107

OxytocinOxytocin

Oxytocin is a cyclic nonapeptide.Oxytocin is a cyclic nonapeptide.

Instead of having its amino acids linked in an Instead of having its amino acids linked in an extended chain, two cysteine residues are extended chain, two cysteine residues are joined by an joined by an S—S S—S bond.bond.

N-terminusN-terminus

C-terminusC-terminusIle—Gln—AsnIle—Gln—Asn

TyrTyr

CysCys SS SS

Cys—Pro—Leu—GlyNHCys—Pro—Leu—GlyNH22

11

22

3344 55

66 77 88 99

Dr. Wolf's CHM 424 27- 108

OxytocinOxytocin

S—S bond

An S—S bond between two cysteines isAn S—S bond between two cysteines isoften referred to as a often referred to as a disulfide bridgedisulfide bridge..

Dr. Wolf's CHM 424 27- 109

27.827.8Introduction to Peptide Introduction to Peptide Structure DeterminationStructure Determination

Dr. Wolf's CHM 424 27- 110

Primary StructurePrimary Structure

The primary structure is the amino acid The primary structure is the amino acid sequence plus any disulfide links.sequence plus any disulfide links.

Dr. Wolf's CHM 424 27- 111

Classical Strategy (Sanger)Classical Strategy (Sanger)

1.1. Determine what amino acids are present and Determine what amino acids are present and their molar ratios.their molar ratios.

2. 2. Cleave the peptide into smaller fragments, and Cleave the peptide into smaller fragments, and determine the amino acid composition of these determine the amino acid composition of these smaller fragments.smaller fragments.

3. 3. Identify the N-terminus and C-terminus in the Identify the N-terminus and C-terminus in the parent peptide and in each fragment.parent peptide and in each fragment.

4.4. Organize the information so that the sequences Organize the information so that the sequences of small fragments can be overlapped to reveal of small fragments can be overlapped to reveal the full sequence.the full sequence.

Dr. Wolf's CHM 424 27- 112

27.927.9Amino Acid AnalysisAmino Acid Analysis

Dr. Wolf's CHM 424 27- 113

Amino Acid AnalysisAmino Acid Analysis

Acid-hydrolysis of the peptide (6 M HCl, 24 hr) Acid-hydrolysis of the peptide (6 M HCl, 24 hr) gives a mixture of amino acids.gives a mixture of amino acids.

The mixture is separated by ion-exchange The mixture is separated by ion-exchange chromatography, which depends on the chromatography, which depends on the differences in pdifferences in pII among the various amino among the various amino acids.acids.

Amino acids are detected using ninhydrin.Amino acids are detected using ninhydrin.

Automated method; requires only 10Automated method; requires only 10-5-5 to 10 to 10-7 -7 g g of peptide.of peptide.

Dr. Wolf's CHM 424 27- 114

27.1027.10Partial Hydrolysis of ProteinsPartial Hydrolysis of Proteins

Dr. Wolf's CHM 424 27- 115

Partial Hydrolysis of Peptides and ProteinsPartial Hydrolysis of Peptides and Proteins

Acid-hydrolysis of the peptide cleaves all of the Acid-hydrolysis of the peptide cleaves all of the peptide bonds.peptide bonds.

Cleaving some, but not all, of the peptide bonds Cleaving some, but not all, of the peptide bonds gives smaller fragments.gives smaller fragments.

These smaller fragments are then separated These smaller fragments are then separated and the amino acids present in each fragment and the amino acids present in each fragment determined.determined.

Enzyme-catalyzed cleavage is the preferred Enzyme-catalyzed cleavage is the preferred method for partial hydrolysis.method for partial hydrolysis.

Dr. Wolf's CHM 424 27- 116

Partial Hydrolysis of Peptides and ProteinsPartial Hydrolysis of Peptides and Proteins

The enzymes that catalyze the hydrolysis of The enzymes that catalyze the hydrolysis of peptide bonds are called peptide bonds are called peptidasespeptidases, , proteasesproteases, , or or proteolyticproteolytic enzymesenzymes..

Dr. Wolf's CHM 424 27- 117

TrypsinTrypsin

Trypsin is selective for cleaving the peptide bond Trypsin is selective for cleaving the peptide bond to the carboxyl group of lysine or arginine.to the carboxyl group of lysine or arginine.

NNHCHCHCHC

OO

R'R'

NNHCHCHCHC

OO

R"R"

NNHCHCHCHC

OO

RR

lysine or argininelysine or arginine

Dr. Wolf's CHM 424 27- 118

ChymotrypsinChymotrypsin

Chymotrypsin is selective for cleaving the peptideChymotrypsin is selective for cleaving the peptidebond to the carboxyl group of amino acids withbond to the carboxyl group of amino acids withan aromatic side chain.an aromatic side chain.

NNHCHCHCHC

OO

R'R'

NNHCHCHCHC

OO

R"R"

NNHCHCHCHC

OO

RR

phenylalanine, tyrosine, tryptophanphenylalanine, tyrosine, tryptophan

Dr. Wolf's CHM 424 27- 119

CarboxypeptidaseCarboxypeptidase

proteinproteinHH33NNCHCCHC

OO

RR

++NNHCHCOHCHCO

OO

RR

––CC

OO

Carboxypeptidase is selective for cleavingCarboxypeptidase is selective for cleavingthe peptide bond to the C-terminal amino acid.the peptide bond to the C-terminal amino acid.

Dr. Wolf's CHM 424 27- 120

27.1127.11End Group AnalysisEnd Group Analysis

Dr. Wolf's CHM 424 27- 121

End Group AnalysisEnd Group Analysis

Amino sequence is ambiguous unless we know Amino sequence is ambiguous unless we know whether to read it left-to-right or right-to-left.whether to read it left-to-right or right-to-left.

We need to know what the N-terminal and C-We need to know what the N-terminal and C-terminal amino acids are.terminal amino acids are.

The C-terminal amino acid can be determined The C-terminal amino acid can be determined by carboxypeptidase-catalyzed hydrolysis.by carboxypeptidase-catalyzed hydrolysis.

Several chemical methods have been Several chemical methods have been developed for identifying the N-terminus. They developed for identifying the N-terminus. They depend on the fact that the amino N at the depend on the fact that the amino N at the terminus is more nucleophilic than any of the terminus is more nucleophilic than any of the amide nitrogens.amide nitrogens.

Dr. Wolf's CHM 424 27- 122

Sanger's MethodSanger's Method

The key reagent in Sanger's method for The key reagent in Sanger's method for identifying the N-terminus is 1-fluoro-2,4-identifying the N-terminus is 1-fluoro-2,4-dinitrobenzene.dinitrobenzene.

1-Fluoro-2,4-dinitrobenzene is very reactive 1-Fluoro-2,4-dinitrobenzene is very reactive toward nucleophilic aromatic substitution toward nucleophilic aromatic substitution (Section 23.5).(Section 23.5).

FFOO22NN

NONO22

Dr. Wolf's CHM 424 27- 123


1-Fluoro-2,4-dinitrobenzene reacts with the 1-Fluoro-2,4-dinitrobenzene reacts with the amino nitrogen of the N-terminal amino acid.amino nitrogen of the N-terminal amino acid.

FFOO22NN

NONO22

NNHCHHCH22CC NNHCHCOHCHCO

CHCH33

NNHCHCHCHC

CHCH22CC66HH55

HH22NNCHCCHC

OO OOOOOO

CH(CHCH(CH33))22

––++

OO22NN

NONO22


CHCH33

NNHCHCHCHC

CHCH22CC66HH55

NNHCHCHCHC

OO OOOOOO

CH(CHCH(CH33))22

––

Dr. Wolf's CHM 424 27- 124


Acid hydrolysis cleaves all of the peptide bonds Acid hydrolysis cleaves all of the peptide bonds leaving a mixture of amino acids, only one of leaving a mixture of amino acids, only one of which (the N-terminus) bears a 2,4-DNP group.which (the N-terminus) bears a 2,4-DNP group.

OO22NN

NONO22


CHCH33

NNHCHCHCHC

CHCH22CC66HH55

NNHCHCHCHC

OO OOOOOO

CH(CHCH(CH33))22

––

HH33OO++

OO

OO22NN

NONO22

NNHCHCOHHCHCOH

CH(CHCH(CH33))22

HH33NNCHCOCHCO––

CHCH33

++HH33NNCHCH22COCO––

OO OO

++

OO

HH33NNCHCOCHCO––

CHCH22CC66HH55

++++ ++

++

Dr. Wolf's CHM 424 27- 125

27.1227.12InsulinInsulin

Dr. Wolf's CHM 424 27- 126

InsulinInsulin

Insulin is a polypeptide with 51 amino acids.Insulin is a polypeptide with 51 amino acids.

It has two chains, called the A chain (21 amino It has two chains, called the A chain (21 amino acids) and the B chain (30 amino acids).acids) and the B chain (30 amino acids).

The following describes how the amino acid The following describes how the amino acid sequence of the B chain was determined.sequence of the B chain was determined.

Dr. Wolf's CHM 424 27- 127

The B Chain of Bovine InsulinThe B Chain of Bovine Insulin

Phenylalanine (F) is the N terminus.Phenylalanine (F) is the N terminus.

Pepsin-catalyzed hydrolysis gave the four peptides:Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHLFVNQHLCGSHL VGAL VGAL VCGERGF VCGERGF YTPKA YTPKA

Dr. Wolf's CHM 424 27- 128


FVNQHLCGSHLFVNQHLCGSHL

VGALVGAL

VCGERGFVCGERGF

YTPKAYTPKA

Dr. Wolf's CHM 424 27- 129



Pepsin-catalyzed hydrolysis gave the four peptides:Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL FVNQHLCGSHL VGAL VGAL VCGERGF VCGERGF YTPKA YTPKA

Overlaps between the above peptide sequences were Overlaps between the above peptide sequences were found in four additional peptides:found in four additional peptides:

SHLVSHLVLVGALVGAALTALTTLVCTLVC

Dr. Wolf's CHM 424 27- 130



SHLVSHLVLVGALVGA

VGALVGAL

ALTALT

TLVCTLVCVCGERGFVCGERGF

YTPKAYTPKA

Dr. Wolf's CHM 424 27- 131



Pepsin-catalyzed hydrolysis gave the four peptides:Pepsin-catalyzed hydrolysis gave the four peptides: FVNQHLCGSHL FVNQHLCGSHL VGAL VGAL VCGERGF VCGERGF YTPKA YTPKA

Overlaps between the above peptide sequences were Overlaps between the above peptide sequences were found in four additional peptides:found in four additional peptides:

SHLVSHLVLVGALVGAALTALTTLVCTLVC

Trypsin-catalyzed hydrolysis gave GFFYTPK which Trypsin-catalyzed hydrolysis gave GFFYTPK which completes the sequence.completes the sequence.

Dr. Wolf's CHM 424 27- 132



SHLVSHLVLVGALVGA

VGALVGAL

ALTALT


GFFYTPKGFFYTPK

YTPKAYTPKA

Dr. Wolf's CHM 424 27- 133



SHLVSHLVLVGALVGA

VGALVGAL

ALTALT


GFFYTPKGFFYTPK

YTPKAYTPKA

FVNQHLCGSHLVGALTLVCGERGFFYTPKAFVNQHLCGSHLVGALTLVCGERGFFYTPKA

Dr. Wolf's CHM 424 27- 134

InsulinInsulin

The sequence of the A chain was determined The sequence of the A chain was determined using the same strategy.using the same strategy.

Establishing the disulfide links between cysteine Establishing the disulfide links between cysteine residues completed the primary structure.residues completed the primary structure.

Dr. Wolf's CHM 424 27- 135

Primary Structure of Bovine InsulinPrimary Structure of Bovine Insulin

N terminus N terminus of of A chainA chain

N terminus N terminus of B chainof B chain

C terminus C terminus of of B chainB chain

C terminus C terminus of of A chainA chain55

55

1515

1010

1515

2020

2020

25253030

SS SS

1010

SSSS

SSSSF

F F

F

V N Q H L

C C

C

C

C

VV

VVG

G

G

S

S

S

H L L

L

G A

A

AC

L Y

Y

E

E L E

R

Y

YI Q

K P T

QN

N

Dr. Wolf's CHM 424 27- 136

27.1327.13The Edman Degradation and The Edman Degradation and

Automated Sequencing of Automated Sequencing of PeptidesPeptides

Dr. Wolf's CHM 424 27- 137

Edman DegradationEdman Degradation

1. 1. Method for determining N-terminal amino Method for determining N-terminal amino acid.acid.

2.2. Can be done sequentially one residue at a Can be done sequentially one residue at a time on the same sample. Usually one can time on the same sample. Usually one can determine the first 20 or so amino acids from determine the first 20 or so amino acids from the N-terminus by this method.the N-terminus by this method.

3. 103. 10-10 -10 g of sample is sufficient.g of sample is sufficient.

4. Has been automated.4. Has been automated.

Dr. Wolf's CHM 424 27- 138


The key reagent in the Edman degradation is The key reagent in the Edman degradation is phenyl isothiocyanate.phenyl isothiocyanate.

NN CC SS

Dr. Wolf's CHM 424 27- 139


Phenyl isothiocyanate reacts with the amino Phenyl isothiocyanate reacts with the amino nitrogen of the N-terminal amino acid.nitrogen of the N-terminal amino acid.

peptidepeptideHH33NNCHCCHC

OO

RR

++NNHHCC66HH55NN CC SS ++

Dr. Wolf's CHM 424 27- 140


peptidepeptideHH33NNCHCCHC

OO

RR

++NNHHCC66HH55NN CC SS ++

peptidepeptideCC66HH55NNHCHCNNHHCHCCHC

OO

RR

NNHH

SS

Dr. Wolf's CHM 424 27- 141



OO

RR

NNHH

SS

The product is a phenylthiocarbamoyl (PTC)The product is a phenylthiocarbamoyl (PTC)derivative.derivative.

The PTC derivative is then treated with HCl in The PTC derivative is then treated with HCl in an anhydrous solvent. The N-terminal amino an anhydrous solvent. The N-terminal amino acid is cleaved from the remainder of the acid is cleaved from the remainder of the peptide.peptide.

Dr. Wolf's CHM 424 27- 142



OO

RR

NNHH

SS

HClHCl

peptidepeptideHH33NN++

++CC66HH55NNHH CC

SSCC

NN CHCH

RR

OO

Dr. Wolf's CHM 424 27- 143


CC66HH55NNHH CCSS

CC

NN CHCH

RR

OO

The product is a thiazolone. Under theThe product is a thiazolone. Under theconditions of its formation, the thiazoloneconditions of its formation, the thiazolonerearranges to a phenylthiohydantoin (PTH)rearranges to a phenylthiohydantoin (PTH)derivative.derivative.


++

Dr. Wolf's CHM 424 27- 144


CC66HH55NNHH CCSS

CC

NN CHCH

RR

OO

CCCCNN

HNHN CHCH

RR

OOSS

CC66HH55The PTH derivative is The PTH derivative is isolated and identified. isolated and identified. The remainder of the The remainder of the peptide is subjected to peptide is subjected to a second Edman a second Edman degradation.degradation.


++

Dr. Wolf's CHM 424 27- 145

27.1427.14The Strategy of Peptide SynthesisThe Strategy of Peptide Synthesis

Dr. Wolf's CHM 424 27- 146

General ConsiderationsGeneral Considerations

Making peptide bonds between amino acids is Making peptide bonds between amino acids is not difficult.not difficult.

The challenge is connecting amino acids in the The challenge is connecting amino acids in the correct sequence.correct sequence.

Random peptide bond formation in a mixture of Random peptide bond formation in a mixture of phenylalaninephenylalanine and and glycineglycine, for example, will give , for example, will give four dipeptides.four dipeptides.

PhePhe——PhePhe GlyGly——GlyGly PhePhe——Gly Gly GlyGly——Phe Phe

Dr. Wolf's CHM 424 27- 147

General StrategyGeneral Strategy

1.1. Limit the number of possibilities by Limit the number of possibilities by "protecting" the nitrogen of one amino acid "protecting" the nitrogen of one amino acid and the carboxyl group of the other.and the carboxyl group of the other.

N-ProtectedN-Protectedphenylalaninephenylalanine

C-ProtectedC-Protectedglycineglycine

NNHCHCOHHCHCOH

CHCH22CC66HH55

OO

XX HH22NNCHCH22CC

OO

YY

Dr. Wolf's CHM 424 27- 148


2.2. Couple the two protected amino acids.Couple the two protected amino acids.

NNHCHHCH22CC

OO

YYNNHCHCHCHC

CHCH22CC66HH55

OO

XX

NNHCHCOHHCHCOH

CHCH22CC66HH55

OO

XX HH22NNCHCH22CC

OO

YY

Dr. Wolf's CHM 424 27- 149


3.3. Deprotect the amino group at the N-terminus Deprotect the amino group at the N-terminus and the carboxyl group at the C-terminus.and the carboxyl group at the C-terminus.

NNHCHHCH22COCO

OO

HH33NNCHCCHC

CHCH22CC66HH55

OO++ ––

Phe-GlyPhe-Gly

NNHCHHCH22CC

OO

YYNNHCHCHCHC

CHCH22CC66HH55

OO

XX

Dr. Wolf's CHM 424 27- 150

27.1527.15Amino Group ProtectionAmino Group Protection

Dr. Wolf's CHM 424 27- 151

Amino groups are normally protected by Amino groups are normally protected by converting them to amides.converting them to amides.

Benzyloxycarbonyl (CBenzyloxycarbonyl (C66HH55CHCH22O—) is a common O—) is a common

protecting group. It is abbreviated as protecting group. It is abbreviated as ZZ..

ZZ-protection is carried out by treating an amino -protection is carried out by treating an amino acid with benzyloxycarbonyl chloride.acid with benzyloxycarbonyl chloride.

Protect Amino Groups as AmidesProtect Amino Groups as Amides

Dr. Wolf's CHM 424 27- 152

Protect Amino Groups as AmidesProtect Amino Groups as Amides CHCH22OCClOCCl

OO

++ HH33NNCHCOCHCO

CHCH22CC66HH55

OO

––++

1. NaOH, H1. NaOH, H22OO

2. H2. H++

NNHHCHCOHCHCOH

CHCH22CC66HH55

OO CHCH22OCOC

OO

(82-87%)(82-87%)

Dr. Wolf's CHM 424 27- 153

Protect Amino Groups as AmidesProtect Amino Groups as Amides

NNHHCHCOHCHCOH

CHCH22CC66HH55

OO CHCH22OCOC

OO

is abbreviated as: is abbreviated as:

ZZNNHHCHCOHCHCOH

CHCH22CC66HH55

OO

or Z-Pheor Z-Phe

Dr. Wolf's CHM 424 27- 154

An advantage of the benzyloxycarbonyl An advantage of the benzyloxycarbonyl protecting group is that it is easily removed by:protecting group is that it is easily removed by:

a) hydrogenolysisa) hydrogenolysis

b) cleavage with HBr in acetic acidb) cleavage with HBr in acetic acid

Removing Z-ProtectionRemoving Z-Protection

Dr. Wolf's CHM 424 27- 155

Hydrogenolysis of Z-Protecting GroupHydrogenolysis of Z-Protecting Group

NNHHCHCCHCNNHCHHCH22COCO22CHCH22CHCH33

CHCH22CC66HH55

OO CHCH22OCOC

OO

HH22, Pd, Pd

HH22NNCHCCHCNNHCHHCH22COCO22CHCH22CHCH33

CHCH22CC66HH55

OO CHCH33 COCO22

(100%)(100%)

Dr. Wolf's CHM 424 27- 156

HBr Cleavage of Z-Protecting GroupHBr Cleavage of Z-Protecting Group


CHCH22CC66HH55

OO CHCH22OCOC

OO

HBrHBr


CHCH22CC66HH55

OO CHCH22BrBr COCO22

(82%)(82%)

++

BrBr ––

Dr. Wolf's CHM 424 27- 157

The The tert-tert-Butoxycarbonyl Protecting GroupButoxycarbonyl Protecting Group

NNHHCHCOHCHCOH

CHCH22CC66HH55

OO

(CH(CH33))33COCCOC

OO

is abbreviated as: is abbreviated as:

BocBocNNHHCHCOHCHCOH

CHCH22CC66HH55

OO

or Boc-Pheor Boc-Phe

Dr. Wolf's CHM 424 27- 158

HBr Cleavage of Boc-Protecting GroupHBr Cleavage of Boc-Protecting Group


CHCH22CC66HH55

OO

(CH(CH33))33CCOCOC

OO

HBrHBr


CHCH22CC66HH55

OO

COCO22

(86%)(86%)

++

BrBr ––CHCH22CC

HH33CC

HH33CC

Dr. Wolf's CHM 424 27- 159

27.1627.16Carboxyl Group ProtectionCarboxyl Group Protection

Dr. Wolf's CHM 424 27- 160

Carboxyl groups are normally protected as Carboxyl groups are normally protected as esters.esters.

Deprotection of methyl and ethyl esters isDeprotection of methyl and ethyl esters isby hydrolysis in base.by hydrolysis in base.

Benzyl esters can be cleaved byBenzyl esters can be cleaved byhydrogenolysis.hydrogenolysis.

Protect Carboxyl Groups as EstersProtect Carboxyl Groups as Esters

Dr. Wolf's CHM 424 27- 161

Hydrogenolysis of Benzyl EstersHydrogenolysis of Benzyl Esters

NNHHCHCCHCNNHCHHCH22COCOCHCH22CC66HH55

CHCH22CC66HH55

OO

CC66HH55CHCH22OCOC

OO OO

HH22, Pd, Pd

HH33NNCHCCHCNNHCHHCH22COCO

CHCH22CC66HH55

OO

CC66HH55CHCH33 COCO22

(87%)(87%)

++ ––CHCH33CC66HH55

Dr. Wolf's CHM 424 27- 162

27.1727.17Peptide Bond FormationPeptide Bond Formation

Dr. Wolf's CHM 424 27- 163

The two major methods are:The two major methods are:

1. coupling of suitably protected amino acids 1. coupling of suitably protected amino acids using using NN,,N'N'-dicyclohexylcarbodiimide (DCCI)-dicyclohexylcarbodiimide (DCCI)

2. via an 2. via an active esteractive ester of the N-terminal amino of the N-terminal amino acid.acid.

Forming Peptide BondsForming Peptide Bonds

Dr. Wolf's CHM 424 27- 164

DCCI-Promoted CouplingDCCI-Promoted Coupling

ZZNNHHCHCOHCHCOH

CHCH22CC66HH55

OO

++ HH22NNCHCH22COCHCOCH22CHCH33

OO

DCCI, chloroformDCCI, chloroform

ZZNNHHCHCCHC

CHCH22CC66HH55

OO

NNHCHHCH22COCHCOCH22CHCH33

OO

(83%)(83%)

Dr. Wolf's CHM 424 27- 165

Mechanism of DCCI-Promoted CouplingMechanism of DCCI-Promoted Coupling

ZZNNHHCHCOHCHCOH

CHCH22CC66HH55

OO

++ CC66HH1111NN CC NCNC66HH1111

CHCH22CC66HH55

OO

CC66HH1111NN CC

CC66HH1111NN

HH

OCCHOCCHNNHZHZ

Dr. Wolf's CHM 424 27- 166


CHCH22CC66HH55

OO

CC66HH1111NN CC

CC66HH1111NN

HH

OCCHOCCHNNHZHZ

The species formed by addition of the Z-The species formed by addition of the Z-protected amino acid to DCCI is similar in protected amino acid to DCCI is similar in structure to an acid anhydride and acts as an structure to an acid anhydride and acts as an acylating agent.acylating agent.

Attack by the amine function of the carboxyl-Attack by the amine function of the carboxyl-protected amino acid on the carbonyl group protected amino acid on the carbonyl group leads to nucleophilic acyl substitution.leads to nucleophilic acyl substitution.

Dr. Wolf's CHM 424 27- 167


HH22NNCHCH22COCHCOCH22CHCH33

OO

CC66HH1111NN CC

CC66HH1111NHNH

HH

OO ++ ZZNNHHCHCCHC

CHCH22CC66HH55

OO


OO

CHCH22CC66HH55

OO

CC66HH1111NN CC

CC66HH1111NN

HH

OCCHOCCHNNHZHZ

Dr. Wolf's CHM 424 27- 168

A A pp-nitrophenyl ester is an example of an "active -nitrophenyl ester is an example of an "active ester."ester."

pp-Nitrophenyl is a better leaving group than -Nitrophenyl is a better leaving group than methyl or ethyl, and methyl or ethyl, and pp-nitrophenyl esters are -nitrophenyl esters are more reactive in nucleophilic acyl substitution.more reactive in nucleophilic acyl substitution.

The Active Ester MethodThe Active Ester Method

Dr. Wolf's CHM 424 27- 169

The Active Ester MethodThe Active Ester Method

ZZNNHHCHCCHCOO

CHCH22CC66HH55

OO

++ HH22NNCHCH22COCHCOCH22CHCH33

OO NONO22

chloroformchloroform

ZZNNHHCHCCHC

CHCH22CC66HH55

OO


OO

(78%)(78%)

++ HHOO

NONO22

Dr. Wolf's CHM 424 27- 170

27.1827.18Solid-Phase Peptide Synthesis:Solid-Phase Peptide Synthesis:

The Merrifield MethodThe Merrifield Method

Dr. Wolf's CHM 424 27- 171

Solid-Phase Peptide SynthesisSolid-Phase Peptide Synthesis

In solid-phase synthesis, the starting material is In solid-phase synthesis, the starting material is bonded to an inert solid support.bonded to an inert solid support.

Reactants are added in solution.Reactants are added in solution.

Reaction occurs at the interface between the Reaction occurs at the interface between the solid and the solution. Because the starting solid and the solution. Because the starting material is bonded to the solid, any product from material is bonded to the solid, any product from the starting material remains bonded as well.the starting material remains bonded as well.

Purification involves simply washing the Purification involves simply washing the byproducts from the solid support.byproducts from the solid support.

Dr. Wolf's CHM 424 27- 172

The Solid SupportThe Solid Support

The solid support is a copolymer of styrene and The solid support is a copolymer of styrene and divinylbenzene. It is represented above as if it divinylbenzene. It is represented above as if it were polystyrene. Cross-linking with were polystyrene. Cross-linking with divinylbenzene simply provides a more rigid divinylbenzene simply provides a more rigid polymer.polymer.

CHCH22 CHCH22 CHCH22 CHCH22CHCH CHCH CHCH CHCH

Dr. Wolf's CHM 424 27- 173


Treating the polymeric support with Treating the polymeric support with chloromethyl methyl ether (ClCHchloromethyl methyl ether (ClCH22OCHOCH33) and ) and

SnClSnCl44 places places ClCHClCH22 side chains on some of the side chains on some of the

benzene rings.benzene rings.


Dr. Wolf's CHM 424 27- 174



CHCH22ClCl

The side chain chloromethyl group is a benzylic The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution halide, reactive toward nucleophilic substitution (S(SNN2).2).

Dr. Wolf's CHM 424 27- 175



CHCH22ClCl

The chloromethylated resin is treated with the Boc-The chloromethylated resin is treated with the Boc-protected C-terminal amino acid. Nucleophilic protected C-terminal amino acid. Nucleophilic substitution occurs, and the Boc-protected amino substitution occurs, and the Boc-protected amino acid is bound to the resin as an ester.acid is bound to the resin as an ester.

Dr. Wolf's CHM 424 27- 176

The Merrifield ProcedureThe Merrifield Procedure


CHCH22ClCl

BocBocNNHHCHCOCHCO

RR

OO––

Dr. Wolf's CHM 424 27- 177


BocBocNNHHCHCOCHCO

RR

OO


CHCH22

Next, the Boc Next, the Boc protecting group is protecting group is removed with HCl.removed with HCl.

Dr. Wolf's CHM 424 27- 178


HH22NNCHCOCHCO

RR


CHCH22OO

DCCI-promoted DCCI-promoted coupling adds the coupling adds the second amino acidsecond amino acid

Dr. Wolf's CHM 424 27- 179


NNHCHCOHCHCO

RR

OO


CHCH22

BocBocNNHCHCHCHC

R'R'

OO

Remove the Boc Remove the Boc protecting group.protecting group.

Dr. Wolf's CHM 424 27- 180



CHCH22

NNHCHCOHCHCO

RR

OO

HH22NNCHCCHC

R'R'

OO

Add the next amino Add the next amino acid and repeat.acid and repeat.

Dr. Wolf's CHM 424 27- 181


Remove the peptide Remove the peptide from the resin with from the resin with HBr in CFHBr in CF33COCO22HH


CHCH22

NNHCHCOHCHCO

RR

OO

NNHCHCHCHC

R'R'

OO

CC

OO++

HH33NN peptide

Dr. Wolf's CHM 424 27- 182

The Merrifield ProcedureThe Merrifield Procedure CHCH22 CHCH22 CHCH22 CHCH22CHCH CHCH CHCH CHCH

CHCH22BrBr

NNHCHCOHCHCO

RR

OO

NNHCHCHCHC

R'R'

OO

CC

OO++

HH33NN peptide––

Dr. Wolf's CHM 424 27- 183

The Merrifield MethodThe Merrifield Method

Merrifield automated his solid-phase method.Merrifield automated his solid-phase method.

Synthesized a nonapeptide (bradykinin) in 1962 Synthesized a nonapeptide (bradykinin) in 1962 in 8 days in 68% yield.in 8 days in 68% yield.

Synthesized ribonuclease (124 amino acids) in Synthesized ribonuclease (124 amino acids) in 1969.1969.

369 reactions; 11,391 steps369 reactions; 11,391 steps

Nobel Prize in chemistry: 1984Nobel Prize in chemistry: 1984

Dr. Wolf's CHM 424 27- 184

27.1927.19Secondary StructuresSecondary Structures

of Peptides and Proteinsof Peptides and Proteins

Dr. Wolf's CHM 424 27- 185

Levels of Protein StructureLevels of Protein Structure

Primary structure = the amino acid sequence Primary structure = the amino acid sequence plus disulfide linksplus disulfide links

Secondary structure = conformational Secondary structure = conformational relationship between nearest neighbor amino relationship between nearest neighbor amino acidsacids

helixhelixpleated pleated sheet sheet

Dr. Wolf's CHM 424 27- 186

Levels of Protein StructureLevels of Protein Structure

planar geometry of peptide bondplanar geometry of peptide bondanti conformation of main chainanti conformation of main chainhydrogen bonds between N—H and O=Chydrogen bonds between N—H and O=C

The The -helix and pleated -helix and pleated sheet are both sheet are both characterized by:characterized by:

Dr. Wolf's CHM 424 27- 187

Pleated Pleated Sheet Sheet

Shown is a Shown is a sheet of protein chains composed of sheet of protein chains composed of alternating glycine and alanine residues.alternating glycine and alanine residues.

Adjacent chains are antiparallel.Adjacent chains are antiparallel.

Hydrogen bonds between chains.Hydrogen bonds between chains.

van der Waals forces produce pleated effect.van der Waals forces produce pleated effect.

Dr. Wolf's CHM 424 27- 188

Pleated Pleated Sheet Sheet

Sheet is most commonly seen with amino acids Sheet is most commonly seen with amino acids having small side chains (glycine, alanine, serine).having small side chains (glycine, alanine, serine).

80% of fibroin (main protein in silk) is repeating 80% of fibroin (main protein in silk) is repeating sequence of —Gly—Ser—Gly—Ala—Gly—Ala—.sequence of —Gly—Ser—Gly—Ala—Gly—Ala—.

Sheet is flexible, but resists stretching.Sheet is flexible, but resists stretching.

Dr. Wolf's CHM 424 27- 189

HelixHelix

Shown is an Shown is an helix of a protein helix of a protein in which all of the amino acids in which all of the amino acids are are LL-alanine.-alanine.

Helix is right-handed with 3.6 Helix is right-handed with 3.6 amino acids per turn.amino acids per turn.

Hydrogen bonds are within a Hydrogen bonds are within a single chain.single chain.

Protein of muscle (myosin) and Protein of muscle (myosin) and wool (wool (-keratin) contain large -keratin) contain large regions of regions of -helix. Chain can -helix. Chain can be stretched.be stretched.

Dr. Wolf's CHM 424 27- 190

27.2027.20Tertiary StructureTertiary Structure

of Peptides and Proteinsof Peptides and Proteins

Dr. Wolf's CHM 424 27- 191

Tertiary StructureTertiary Structure

Refers to overall shape (how the chain is folded)Refers to overall shape (how the chain is folded)

Fibrous proteins (hair, tendons, wool) have Fibrous proteins (hair, tendons, wool) have elongated shapeselongated shapes

Globular proteins are approximately sphericalGlobular proteins are approximately spherical

most enzymes are globular proteinsmost enzymes are globular proteins

an example is carboxypeptidasean example is carboxypeptidase

Dr. Wolf's CHM 424 27- 192


Carboxypeptidase is an enzyme that catalyzes Carboxypeptidase is an enzyme that catalyzes the hydrolysis of proteins at their C-terminus.the hydrolysis of proteins at their C-terminus.

It is a metalloenzyme containing ZnIt is a metalloenzyme containing Zn2+2+ at its at its active site.active site.

An amino acid with a positively charged side An amino acid with a positively charged side chain (Arg-145) is near the active site.chain (Arg-145) is near the active site.

Dr. Wolf's CHM 424 27- 193


Disulfide bondDisulfide bond


C-terminusC-terminus

Zn2+

Arg-145

tube modeltube model ribbon modelribbon model

Dr. Wolf's CHM 424 27- 194

What happens at the active site?What happens at the active site?

HH33NN peptidepeptide

OO

NNHCHCHCHC++

CC

•••• ••••

RR

OO

OO

––

HH22NN

HH22NN

CC Arg-145Arg-145++

Dr. Wolf's CHM 424 27- 195



OO

NNHCHCHCHC++

CC

•••• ••••

RR

OO

OO

––

HH22NN

HH22NN

CC Arg-145Arg-145++

The peptide or protein is bound at the active site The peptide or protein is bound at the active site by electrostatic attraction between its negatively by electrostatic attraction between its negatively charged carboxylate ion and arginine-145.charged carboxylate ion and arginine-145.

Dr. Wolf's CHM 424 27- 196


HH33NN

ZnZn2+2+

peptidepeptide

OO

NNHCHCHCHC++

CC

•••• ••••

RR

OO

OO

––

HH22NN

HH22NN

CC Arg-145Arg-145++

ZnZn2+2+ acts as a Lewis acid toward the carbonyl acts as a Lewis acid toward the carbonyl oxygen, increasing the positive character of the oxygen, increasing the positive character of the carbonyl carbon.carbonyl carbon.

Dr. Wolf's CHM 424 27- 197


HH33NN

ZnZn2+2+

peptidepeptide

OO

NNHCHCHCHC++

CC

•••• ••••

RR

OO

OO

––

HH22NN

HH22NN

CC Arg-145Arg-145++

Water attacks the carbonyl carbon. Nucleophilic Water attacks the carbonyl carbon. Nucleophilic acyl substitution occurs.acyl substitution occurs.

OO•••• ••••

HH

HH

Dr. Wolf's CHM 424 27- 198


ZnZn2+2+

HH22NN

CC Arg-145Arg-145++


OO++

CC

•••• ••••

OO ••••••••

••••

––

HH33NNCHCCHC

RR

OO

OO

––

HH22NN

++

Dr. Wolf's CHM 424 27- 199

27.2127.21CoenzymesCoenzymes

Dr. Wolf's CHM 424 27- 200

CoenzymesCoenzymes

The range of chemical reactions that amino acid The range of chemical reactions that amino acid side chains can participate in is relatively side chains can participate in is relatively limited.limited.

acid-base (transfer and accept protonsacid-base (transfer and accept protons))nucleophilic acyl substitutionnucleophilic acyl substitution

Many other biological processes, such as Many other biological processes, such as oxidation-reduction, require oxidation-reduction, require coenzymescoenzymes, , cofactorscofactors, or , or prostheticprosthetic groupsgroups in order to occur. in order to occur.

Dr. Wolf's CHM 424 27- 201

CoenzymesCoenzymes

NADH, coenzyme A and coenzyme BNADH, coenzyme A and coenzyme B1212 are are

examples of coenzymes.examples of coenzymes.

Heme is another example.Heme is another example.

Dr. Wolf's CHM 424 27- 202

HemeHeme NN

NN NN

NN

Fe

HH33CC

HH33CC CHCH33

CHCH33

CHCH22CHCH22COCO22HH

CHCH CHCH22

HH22CC CHCH

HOHO22CCHCCH22CHCH22

Molecule surrounding iron is a Molecule surrounding iron is a type of porphyrin.type of porphyrin.

Dr. Wolf's CHM 424 27- 203

MyoglobinMyoglobin


C-terminusC-terminus Heme

Heme is the coenzyme that binds oxygen in myoglobin Heme is the coenzyme that binds oxygen in myoglobin (oxygen storage in muscles) and hemoglobin (oxygen (oxygen storage in muscles) and hemoglobin (oxygen transport).transport).

Dr. Wolf's CHM 424 27- 204

27.2227.22Protein Quaternary Structure:Protein Quaternary Structure:

HemoglobinHemoglobin

Dr. Wolf's CHM 424 27- 205

Protein Quaternary StructureProtein Quaternary Structure

Some proteins are assemblies of two or more Some proteins are assemblies of two or more chains. The way in which these chains are chains. The way in which these chains are organized is called the quaternary structure.organized is called the quaternary structure.

Hemoglobin, for example, consists of 4 Hemoglobin, for example, consists of 4 subunits.subunits.

There are 2 There are 2 chains (identical) and 2 chains (identical) and 2 chains chains (also identical).(also identical).

Each subunit contains one heme and each Each subunit contains one heme and each protein is about the size of myoglobin. protein is about the size of myoglobin.

Dr. Wolf's CHM 424 27- 206

End of Chapter 27End of Chapter 27

Documents

Dr. Wolf's CHM 424 27- 1 Chapter 27 Amino Acids, Peptides, and Proteins