Chapter 18

Amino Acids, Peptides, and Proteins

(氨基酸、肽、蛋白质 )

Amino acids

NH2

CH CR

O

OH CH2C

O

OHCHR

NH2

-amino acid -amino acid

L-amino acids

Most of the chiral centers in natural α-amino acids are (S)-configurations.

H2N H

CO2H

R

The relative configuration of most α-Amino Acids are L-.

Peptides and proteins are polymers of amino acids linked together

by amide bonds

H2N CH C

R

O

OH

Glycine, Gly 甘氨酸

Alanine, Ala 丙氨酸

Valine, Val 缬氨酸

Leucine, Leu 亮氨酸

Isoleucine, Ile异亮氨酸

The 20 standard α-amino acids

H2N CHC

H

OH

OH2N CHC

CH3

OH

OH2N CHC

CH

OH

O

CH3

CH3

H2N CHC

CH2

OH

O

CHCH3

CH3

H2N CHC

CH

OH

O

CH3

CH2

CH3

H2N CHC

CH2

OH

O

Phenylalanine, Phe 苯丙氨酸

R = hydrocarbyl R 为烃基的氨基酸

Hydroxy-Containing Amino Acids 含羟基氨基酸

Serine, Ser 丝氨酸

Threonine, Thr苏氨酸

H2N CHC

CH2

OH

O

OH

H2N CHC

CH

OH

O

OH

CH3

H2N CHC

CH2

OH

O

OH

Tyrosine, Tyr酪氨酸

Sulfur-Containing Amino Acids 含硫氨基酸

Cysteine, Cys 半胱氨酸 Methionine, Met

蛋氨酸

H2N CHC

CH2

OH

O

SH

H2N CHC

CH2

OH

O

CH2

S

CH3

Acidic Amino Acids 酸性氨基酸

Amides of Acidic Amino Acids 酸性氨基酸的酰胺

Aspartatic acid, Asp, 门冬氨酸

Glutamic acid, Glu, 谷氨酸

Asparagine,Asn, 门冬酰胺

Glutamine,Gln, 谷氨酰胺

H2N CHC

CH2

OH

O

CH2

C

OH

O

H2N CHC

CH2

OH

O

C

OH

O

H2N CHC

CH2

OH

O

CH2

C

NH2

O

H2N CHC

CH2

OH

O

C

NH2

O

Basic Amino Acids 碱性氨基酸

Lysine, Lys 赖氨酸

Arginine, Arg 精氨酸

H2N CHC

CH2

OH

O

CH2

CH2

NH

C

NH2

NH

H2N CHC

CH2

OH

O

CH2

CH2

CH2

NH2

H2N CHC

CH2

OH

O

NNH

Histidine, His组氨酸

HN

C OH

OH2N CHC

CH2

OH

O

HN

Tryptophan, Try色氨酸

Proline, Pro 脯氨酸

The 8 essential amino acids ( 必需氨基酸，八种 )

Acid–Base Properties of Amino Acids

H3N+CHCO2-

R

Dipolar ion(Á½¼«Àë×Ó£©

H2NCHCO2H

R

H3N+CHCO2H

RH+

Cationic form (ÑôÀë×ÓÐÎʽ£©

OH-

+ H+H2NCHCO2

-

R

Anionic form (ÒõÀë×ÓÐÎʽ£©

OH-

The isoelectric point (pI，等电点 ) of an amino acid is the pH at which it has no net charge.

pI =pKa1 + pKa2

2

pKa1 pKa2

isoelectric point (pI ，等电点 )

Acidic amino acids: aspartic acid 门 冬氨酸 (2.8),

glutamic acid 谷氨酸 (3.2),

Neutral amino acids: (5.0~6.3)

Basic amino acids: lysine 赖氨酸 (9.7)

arginine 精氨酸 (10.8)

histidine 组氨酸 (7.6)

Problem 24-4:

Draw the structure of the predominant form of

(a)Valine at pH 11

(b)Proline at pH 2

(c)Arginine at pH 7

(d)Glutamic acid at pH 7

A mixture of amino acids can be separated by Electrophoresis ( 电泳 ) on the basis of their pI values

Ninhydrin( 茚三酮 ) is used to detect the individual amino acidsO

O

O

Ninhydrin( 茚三酮 )氨基酸的显色剂

pH 6 buffer solution 缓冲溶液

A mixture of amino acids can also be separated on thebasis of polarity

1. Acylation of amino group

Reactions of α-amino acids

RCHCOOH

NH2

R'COCl or anhydrideRCHCOOH

NHCOR'

PhCH2OCOCl, CbzCl: benzyl chloroformate, 氯甲酸苄酯 , 保护氨基

PhCH2OCOClPhCH2OCONHCHCOOH

R

RCHCOOH

NH2

+

H2, Pd

RCHCOOH

NH2

+ CO2 + PhCH3

2. Alkylation

F

NO2

O2N RCHCOOH

NH2

+ NHCHCOOH

NO2

O2N

R

Application ：氨基酸的 N- 端的测定

RCHCOOH

NH2

R'X+ RCHCOOH

NHR'

3. Reaction with HNO2

RCHCOOH

NH2

+ HNO2 RCHCOOH

N2

RCHCOOH

OH+

4. Reaction with ninhydrin 茚三酮

O

O

O CHCOOHH2N+

O

O O

O

N

R

OH

O O

O

N2

+ RCHO + CO2

deep purple深紫色

Note: proline can not give this phenomenon.

O

O

O

O

O

N CH

R

C

O

OHCHCOOHH2N+ :

H+

O

O

N CH R

H+

H2O

: O

O

HN CH R

OH

:

H+

-RCHO

O

O

NH2

ninhydrinO

O O

O

N

OH

O O

O

N

R

5. Esterification of the carboxy group

Application ：保护氨基酸的羧基

RCHCOOH

NH2

R'OH+ H+RCHCOOR'

NH3+

RCHCOOH

NH2

+SOCl2

RCHCOOCH3

NH3+

CH3OH

Synthesis of α-amino acids

1. Reductive amination of α-ketoacid 24-5A

R C

O

COOHNH3, H2/Pd

R CH

NH2

COOH

α-ketoacid (酮酸 ) α-amino acid

A biomimetic synthesis 生物模拟合成

HOOC CH2CH2C COO

O+ NH4

+ +

N

H H

NH2

O

sugar

enzyme

HOOC CH2CH2CH COO

NH3

NADH

+ H+

+N

sugar

NH2

O

+ H2O

NAD+

NADH ：烟酰胺腺嘌呤二核苷酸，还原态NAD+ ：氧化态

α-ketoglutaric acidα- 氧代戊二酸

L-glutamic acidL- 谷氨酸

2. Amination of α-halo acids

HVZ reaction

A)A large excess of amine was used;B)The adjacent carboxylate ion in the product reduces the

nucleophilicity of the amino group.

CH2CO2HR(1) Br2, PBr3

(2) H2ORCHCO2H

Br

NH3 (excess)RCHCO2

-

NH2

NH4+

Acceptable yield

N-K+

O

O

+ ClCH2CO2C2H5 N

O

O

CH2CO2C2H5

COOH

COOH

+ C2H5OHNH3+

+CH2CO2

- HCl

This method is a modification of the Gabriel synthesis of amines.The yields are usually high and the products are easily purified.

3. The Gabriel-malonic ester synthesis

N-K+

O

O

+ BrCHCO2C2H5

COOC2H5

N

O

O

CHCO2C2H5

COOC2H5

(1) EtONa

(2) RXN

O

O

CCO2C2H5

COOC2H5

R

H3O+

heatH3N CCO2H

H

R

4. The Strecker synthesis

RCHO + NH3 + HCN RCHCN

NH2

H3O+, heat

NH3+

RCHCO2-

¦Á-Amino nitrile ¦Á-Amino acid

Resolution of D,L-amino acids ( 氨基酸的拆分 )

RCHCO2-DL

NH3+

(CH3CO)2ORCHCO2HDL

NHCOCH3

acylase

+H3N H

CO2-

R

NHCOCH3H

CO2-

R

+

Easily separated

L-Amino acid (L-°±»ùËᣩ

D-N-Acylamino acid (D-N-乙õ£°±»ùËᣩ

CH3COOH

Acylase 酰化酶，只使 L- 乙酰氨基酸脱乙酰基

Peptides 肽

N-terminusN-端 C-terminus

C-端

Peptide Bond (肽键 )

CH3CHCONHCH2CONHCHCOOHNH2 CH3

alanylglycylalanine; ala-gly-ala; AGA丙氨酰甘氨酰丙氨酸 , 丙甘丙

Formation of Disulfide Bonds

Disulfides can be reduced to thiols

半胱氨酸 胱氨酸

The disulfide bridge in proteins contributes to the overallshape of a protein

牛胰岛素

Synthesis of peptides (24-10, 11)

Problem: Synthesis of ala-gly

NH3+

+ C6H5CH2OC

O

Cl CH3CH CO2H

NH

C

OCH2C6H5

O

OH-25¡æ

AlaZ-Ala

CH3CHCO2-

(1) (C2H5)3NCH3CHCOCOC2H5

NH

Z

(2) ClCO2C2H5

O O

CO2 + C2H5OH

CH3CH C

O

NHCH2COOH

NH

Z

CHCO2-H3N

H2 / Pd CH3CH C

O

NHCH2CO2-

NH3+

+ CH3 + CO2

ala-gly

Solution-phase Peptide Synthesis 液相合成

Solid-phase Peptide Synthesis 固相合成

Merifield automated solid-phase synthesis of a tripeptide

NH3CHCOO

R

+ (CH3)3COCOCOC(CH3)3

O ONCCOOH

R

CO

OButNaOH

di-tert-butyldicarbonate ， Boc2O二碳酸二叔丁酯； Boc 酸酐

Amino acids can be added to the growing C-terminal endby repeating these two steps

…………………

Peptide structure determination

Step 1: cleavage of the disulfide bridges

Step 2: determine the number and kinds of amino acids in the peptide or protein

protein amino acids6 N HCl

100°C24 h

Step 3: sequencing the peptides

----terminal residue analysis

O2N

NO2

F + NH2CHCO-NHCHCO

R R'

etc

HCO3-

(-HF)

O2N

NO2

NHCHCO-NHCHCO

R R'

etcH3O+

2,4-Dinitrofluorobenzene (DNFB) (2,4-¶þÏõ »ù·ú±½£©

Polypeptide (¶àëÄ£©

Labeled polypeptide (ÓбêÖ¾µÄ¶àëÄ£©

O2N

NO2

NHCHCO2H

R

+ H3N+CHCO2-

R'

Labeled N-terminal amino acid(ÓбêÖ¾µÄN-¶Ë°±»ùËᣩ

Mixture of amino acids (»ì ºÏ °±»ùËᣩ

Separated and identify

O2N

NO2

F + NH2CHCO-NHCHCO

R R'

etc

HCO3-

(-HF)

O2N

NO2

NHCHCO-NHCHCO

R R'

etcH3O+

2,4-Dinitrofluorobenzene (DNFB) (2,4-¶þÏõ »ù·ú±½£©

Polypeptide (¶àëÄ£©

Labeled polypeptide (ÓбêÖ¾µÄ¶àëÄ£©

O2N

NO2

NHCHCO2H

R

+ H3N+CHCO2-

R'

Labeled N-terminal amino acid(ÓбêÖ¾µÄN-¶Ë°±»ùËᣩ

Mixture of amino acids (»ì ºÏ °±»ùËᣩ

Separated and identify

Sequencing from the N-terminus: the Edman degradation

NH2 CH C

R

O

NH CH C

R1

O

NH peptidePh N C S

H3O+

(1)

(2)

NH2CH C

R1

O

NH peptideHN

N

S

Ph

OR

+

PhNCS

Ph N C S

HNN

S

Ph

OR1

+ NH2-peptide

……………

NH2 CH C

R

O

NH CH C

R1

O

NH peptidePh N C S

H3O+

(1)

(2)

NH2CH C

R1

O

NH peptideHN

N

S

Ph

OR

+

NH2 CH C

R

O

NH CH C

R1

O

NH peptidePh N C S

H3O+

(1)

(2)

NH2CH C

R1

O

NH peptideHN

N

S

Ph

OR

+

The C-terminal amino acid can be identified by treating the protein with carboxypeptidase （羧肽酶）

Partial hydrolysis ( 部分水解 )Problem: acid-catalysed hydrolysis of oxytocin (after cleavage of the disulfide bond) gives a mixture that include the following peptides:

Ile-Gln-Asn-Cys

Gln-Asn-Cys-Pro

Pro-Leu-gly.NH2

Cys-Tyr-Ile-Gln-Asn

Cys-Pro-Leu-Gly

Propose the complete sequence of oxytocin.

Answer: Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly.NH2

Secondary Structure of Proteins蛋白质的二级结构

Describe the conformation of segments of the backbonechain of a peptide or protein.

Three factors determine the choice of secondarystructure:

• the regional planarity about each peptide bond

• maximization of the number of peptide groups that engage in hydrogen bonding

• adequate separation between nearby R groups

The -Helix ( 螺旋结构 ) Is Stabilized by Hydrogen Bonds

Prolines are helix breakers

Two Types of -Pleated Sheets ( 折叠结构 )

Most globular proteins( 球蛋白 ) have random coil ( 线团 ) conformations

The tertiary structure is （三级结构） the three-dimensional

arrangement of all the atoms in the protein

The stabilizing interactions include covalent bonds, hydrogen bonds, electrostatic attractions, and hydrophobic interactions

The tertiary structure is defined by the primary structure

Disulfide bonds are the only covalent bonds that can form when a protein folds

Proteins that have more than one peptide chain arecalled oligomers （低聚物）

The quaternary structure （四级结构）

The quaternary structure refers to the association of two or more peptides chains in the complete protein.

Summary• 20 standard amino acids• Isoelectric point, acid–base properties• Protection of amino and carboxyl groups• Reaction with ninhydrin• Lab synthesis of amino acids (4 methods)• Structure of peptides• Synthesis of peptides (solution and solid

synthesis)• Analysis of peptides• Structure of proteins

assignment

• 24-34, 35, 36, 43, 44