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