Chapter 25 Amino Acids, Peptides and Proteins Organic Chemistry, 6th Edition L. G. Wade, Jr. Chapter 25 Amino Acids, Peptides and Proteins

Key points 蛋白质 较复杂的含氮生物有机大分子， 是生物体内一切细胞的重要组成 部分，是生命的重要物质基础 Classification of amino acids Preparation of amino acids Reactions of amino acids peptides Classification and structure of proteins 蛋白质 较复杂的含氮生物有机大分子， 是生物体内一切细胞的重要组成 部分，是生命的重要物质基础 氨基酸 组成蛋白质的基本成分

Sec 1: Classification and stereochemistry of amino acids Standard Amino Acids Twenty standard -amino acids. Differ in side-chain characteristics: -H or alkyl contains an -OH contains sulfur contains a nonbasic nitrogen has -COOH has a basic nitrogen

Essential Amino Acids Arginine (Arg 精) Tryptophan (Trp色) Threonine (Thr 苏) Lysine (Lys赖) Valine (Val 缬) Phenylalanine (Phe苯丙) Tryptophan (Trp色) Methionine (Met 蛋) Histidine (His组) Leucine (Leu亮) Isoleucine (Ile异亮)

Complete Proteins Provide all the essential amino acids. Examples: those in meat, fish, milk, eggs. Plant proteins are generally incomplete. Vegetarians should eat many different kinds of plants, or supplement diet with milk or eggs.

天冬氨酸 谷氨酸 二个羧基 一个氨基 酸性氨基酸 赖氨酸 精氨酸 组氨酸 一个羧基 二个或二个以上氨基 碱性氨基酸 一个羧基 一个氨基 中性氨基酸

Amino Acids R结构 英文名 中文名 缩写 PI (等电点) 中性氨基酸 -H Glycine 甘氨酸 G,Gly (甘) 6.0 -CH3 Alanine 丙氨酸 A,Ala (丙)

R结构 英文名 中文名 缩写 PI (等电点) -CH3(CH3)2 Valinea 缬氨酸 V,Val (缬) 6.0 -CH2CH(CH3)2 Leucinea 亮氨酸 L,Leu (亮) Isoleucine 异亮氨酸 I,Ile (异亮 ) 6.1 -CH2Ph Phenylalanine a 苯丙氨酸 F,Phe (苯丙) 5.5 -CH2CONH2 Asparagine 天冬酰胺 N,Asn 5.4 Tryptophan a 色氨酸 W,Trp (色) 5.9 -(CH2)2CONH2 Glutamine 谷氨酰胺 Q或Gln 5.7 Proline 脯氨酸 P,Pro (脯) 6.3

R结构 英文名 中文名 缩写 PI (等电点) -CH2OH Serine 丝氨酸 S,Ser (丝) 5.7 Threonine a 苏氨酸 T,Thr (苏) 6.5 Tyrosine 酪氨酸 Y,Tyr (酪) Hydroxyproline 羟脯氨酸 Hyp (羟脯) 6.3 -CH2SH Cysteine 半胱氨酸 C,Cys (半胱) 5.0 -CH2SSCH2- Cystine 胱氨酸 Cys-Cys 5.1 -(CH2)2SCH3 Methionine 蛋氨酸,甲硫氨酸 M,Met (蛋) 5.8

R结构 英文名 中文名 缩写 PI (等电点) 酸性氨基酸 -CH2COOH Aspartic acid 天冬氨酸 D,Asp (天冬) 3.0 -(CH2)2COOH Glutamic acid 谷氨酸 E,Glu (谷) 3.2 碱性氨基酸 -(CH2)4NH2 Lysine a 赖氨酸 K,Lys (赖) 9.8 Arginine 精氨酸 R,Arg (精) 10.8 Histidine 组氨酸 H,His (组) 7.6

Rare Amino Acids 4-Hydroxyproline, 5-hydroxylysine found in collagen胶原 . D-Glutamic acid in cell walls of bacteria. D-Serine in earthworms. -Aminobutyric acid, a neurotransmitter. -Alanine, constituent of the vitamin pantothenic acid泛酸 .

Stereochemistry of -Amino Acids

Sec 2: Preparation of Amino acids Hell-Volhard-Zelinsky reaction Reductive Amination Gabriel-Malonic Ester Synthesis Strecker Synthesis

1、 - 卤代酸的直接氨解(Amination) Hell-Volhard-Zelinsky reaction 2. Reductive Amination This method for synthesizing amino acids is biomimetic, mimics the biological process.

85%甘氨酸  3. 从邻苯二甲酰亚胺钾制备(Gabriel’s Gabriel-Malonic Ester Synthesis)

Gabriel-Malonic Ester Synthesis The amino group is protected as amide. The carboxylic acid group is protected as an ester. The  position is further activated by the additional temporary ester group.

 4. Strecker Synthesis First known synthesis of amino acid, 1850. One-pot RXN Aldehyde reaction with NH3 yields imine. Cyanide ion attacks the protonated imine. Resulting -amino nitrile is hydrolyzed to a carboxylic acid.

Strecker Mechanism Hydrolysis of the nitrile yields the amino acid. 19

Properties of Amino Acids High melting points, over 200C More soluble in water than in ether. Larger dipole moments than simple acids or simple amines. Less acidic than most carboxylic acids, less basic than most amines. 但实际上极少以此形式存在

Properties of Amino Acids 氨基酸的两性电离和等电点 氨基酸分子中含有NH2 和COOH, 为简化起见可表示为： Zwitterion pKa = 10 pKb = 12 实际水溶液呈弱酸性： 电离能力 COOH > NH2

它们发生分子内酸碱反应： 两性离子 +H+ 实际上,该平衡主要决定于羧基和氨基 如果羧基给出质子的能力和氨基接受质子的能力相匹配，则完全转成内盐的形式。 一般情况下，羧基给出质子能力强于氨基接受质子的能力 +H+

Zwitterion Amino acid exists as a dipolar ion. -COOH loses H+, -NH2 gains H+. Actual structure depends on pH.

两性离子 pH = pI 即有下面的平衡式： 正离子 负离子 pH<pI pH>pI OH 负离子 pH>pI H+ OH

Structure and pH

等电点Isoelectric Point ： 将氨基酸水溶液的酸碱度加以适当的调节，使氨基酸的酸性电离程度与碱性电离程度相等。此时，氨基酸带有的正电荷数目和负电荷数目相等，此溶液的pH值称为氨基酸的等电点，用 pI 表示。 氨基酸的 带电状态和在电场中的状况： 等电点 环境pH pH = pI 净电荷为零 在电场中不移动 在电场中移向负极 pH < pI 带正电荷 pH > pI 带负电荷 在电场中移向正极

Isoelectric Point pH at which amino acids exist as the zwitterion (neutral). Depends on structure of the side chain. Acidic amino acids, isoelectric pH ~3. Basic amino acids, isoelectric pH ~9. Neutral amino acids, isoelectric pH is slightly acidic, 5-6. Ala (丙) -CH3 丙氨酸 6.0 Asp (天冬) -CH2COOH 天冬氨酸 3.0 Lys (赖) -(CH2)4NH2 赖氨酸 9.8

Electrophoresis

要使氨基酸达到等电状态： pI < pH 7 加少量酸 中性氨基酸： 酸性氨基酸： pI < pH 7 加酸

问题： 判断标准在于是否大于或小于等电点 氨基酸 PH 带电荷（存在形式） 电场运动方向 酸性AA >7 - 阳极 =7 - 阳极 =7 - 阳极 <7 X 不知道 碱性AA >7 X 不知道 =7 + 阴极 <7 + 阴极 中性AA >7 － 阳极 =7 － 阳极 <7 X 不知道 判断标准在于是否大于或小于等电点

Sec 3 Reactions of amino acids Zwitterion and Isoelectric Point Reaction with Ninhydrin茚三酮 Reaction with HNO2 Esterification Acylation

Reaction with Ninhydrin 蓝紫色化合物 Used to visualize spots or bands of amino acids separated by chromatography or electrophoresis. Deep purple color formed with traces of any amino acid. 根据CO2的量或蓝紫色的深浅程度 作为α-氨基酸定量分析的依据

与 HNO2的反应 N2 利用此反应可以测定蛋白质分子中的自由氨基及其水解氨基酸分子中氨基的含量。 除脯氨酸外，其它氨基酸都具有 NH2，可与 HNO2反应，定量放出氮气 N2 。 HNO2 N2 利用此反应可以测定蛋白质分子中的自由氨基及其水解氨基酸分子中氨基的含量。

与茚三酮的显色反应 水合茚三酮 CO2

脱羧反应： Ba(OH)2 CO2 脱羧反应亦可在某些细菌的存在下，由于酶的作用发生，如： 精氨酸或鸟氨酸 腐胺 尸胺 赖氨酸

Esterification Use a large excess of alcohol and an acidic catalyst. Esters are often used as protective derivatives. Aqueous hydrolysis regenerates the acid.

Acylation The amino group is converted to amide. Acid chlorides and anhydrides are the acylating agents. Benzyl chloroformate, PhCH2OCOCl, is commonly used because it is easily removed.

Sec 4 Peptides Structure of Peptide The peptide bond is an amide bond. Amides are very stable and neutral.

Peptide Bond Geometry two conformations are possible for a planar peptide bond virtually all peptide bonds in naturally occurring proteins studied to date have the s-trans conformation

Peptide Bond Formation The amino group of one molecule condenses with the acid group of another. Polypeptides usually have molecular weight less than 5000. Protein molecular weight 6000-40,000,000.

Cysteine can form disulfide bridges. Disulfide Linkages Cysteine can form disulfide bridges. 41

自然界中的肽类化合物 2H 2 GSH G—S—S—G +2H 谷胱甘肽：广泛存在于动植物细胞和细菌的三肽

氧化型：谷胱甘肽（G—S—S—G）

Human Hormone Bradykinin缓激肽 具有心脏保护作用的9肽物质,舒血管和降血压等作用 An oligopeptide is made out of four to ten amino acids. Peptide structures are drawn with the N-terminal end at the left. Peptides are named from left to right: arginylprolylprolyl……arginine. 44

Bradykinin 缓激肽 http://en.wikipedia.org/wiki/File:Bradykinin_updated.png

Human Oxytocin 人催产素 Oxytocin is a nonapeptide with two cysteine residues (at Positions 1 & 6) linking part of the molecule in a large ring. 46

Bovine Insulin牛胰岛素 Insulin is composed of two separate peptide chains, the A chain containing 21 amino acid residues, and the B chain containing 30. 美国的V. du Vigneand于1953年合成了第一个天然多肽激素,获1955年度诺贝尔化学奖。 1955-1965年间，在世界范围内共有10个研究小组在进行胰岛素的人工合成 1953年，英国人F. Sanger由于测定了牛胰岛素的一级结构而获得1958年诺贝尔化学奖。 在1965年9月17日我国完成了结晶牛胰岛素的全合成。世界上第一个人工合成的蛋白质。获1982年中国自然科学一等奖。 47

Peptide Structure Determination Cleavage of disulfide linkages Determination of amino acid composition Sequencing from the N terminus C-Terminal residue analysis Partial hydrolysis

Disulfide Cleavage

Structure Determination of Peptides: Amino Acid Analysis The sequence of amino acids in a pure protein is specified genetically If a protein is isolated it can be analyzed for its sequence The composition of amino acids can be obtained by automated chromatography and quantitative measurement of eluted materials using a reaction with ninhydrin that produces an intense purple color

Amino Acid Composition Separate the individual peptide chains. Boil with 6 M HCl for 24 hours. Separate in an amino acid analyzer.

Amino Acid Analysis Chromatogram

Composition of Human Bradykinin缓激肽 53

Sequencing from the N Terminus Edman degradation: The reaction with phenyl isothiocyanate followed by hydrolysis removes the N terminus amino acid. The phenylthiohydantoin derivative is identified by chromatography. Use for peptides with < 30 amino acids. 54

Edman Degradation 55

Edman Degradation (Continued) In the final step (step 3) the thiazoline isomerizes to the more stable phenylthiohydantoin. 56

The Sanger Method 57

Sequencing from the C Terminus The enzyme carboxypeptidase cleaves the C-terminal peptide bond. However, since different amino acids react at different rates, it’s difficult to determine more than the original C- terminal amino acid. 58

C-Terminal Residue Analysis The C-terminal amino acid can be identified using the enzyme carboxypeptidase, which cleaves the C-terminal peptide bond. Eventually, the entire peptide is hydrolyzed to its individual amino acids. 59

Partial Hydrolysis Break the peptide chain into smaller fragments. Trypsin cleaves at the carboxyl group of lysine and arginine. Chymotrypsin cleaves at the carboxyl group of phenylalanine, tyrosine, and tryptophan. Sequence each fragment, then fit them together like a jigsaw puzzle. 60

Solution Phase Peptide Synthesis First, protect the amino group at the N terminus with benzyl chloroformate. Activate the carboxyl group with ethyl chloroformate to form anhydride of carbonic acid. Couple the next amino acid. Repeat activation and coupling until all amino acids needed have been added. Remove the protecting group. 61

Advantages of Solid Phase Synthesis Growing chain, built from C to N terminus, is attached to polystyrene beads. Intermediates do not have to be purified. Excess reagents are washed away with a solvent rinse. Process can be automated. Larger peptides can be constructed. 62

Attachment of the C-Terminal Amino Acid Once the C-terminal amino acid is fixed to the polymer, the chain is built on the amino group of this amino acid. 63

Cleavage of the Finished Peptide At the completion of the synthesis, the ester bond to the polymer is cleaved by anhydrous HF. Because this is an ester bond, it is more easily cleaved than the amide bonds of the peptide. 64

N,N’-Dicyclohexylcarbodiimide (DCC) Coupling When a mixture of an amine and an acid is treated with DCC, the amine and the acid couple to form an amide. 65

DCC-Activated Acyl Derivative The carboxylate ion adds to the strongly electrophilic carbon of the diimide, giving an activated acyl derivative of the acid. 66

Coupling 67

Sec 5 Proteins 1. 基本元素及其含量范围： C H O N S 2. 样品中蛋白质含量的测定 1. 基本元素及其含量范围： C H O N S % 50-55 6.0-7.3 19-24 13-19 0-4 2. 样品中蛋白质含量的测定 一般蛋白质中 N 含量 — 16 % ，由此，可测定 N 含量来推算蛋白质的含量： 样品中蛋白质的含量（ g % ） = 每克样品中含氮克数 6.25 100 % 系数 6.25 ：每克氮相当于 6.25 克蛋白质

Proteins Biopolymers of -amino acids. Amino acids are joined by peptide bond. Variety of functions: structure enzymes transport protection hormones

蛋白质的分类 纤维蛋白： 如丝蛋白、角蛋白等 球 蛋 白： 如蛋清蛋白、酶等 按形状分 纤维蛋白： 如丝蛋白、角蛋白等 球 蛋 白： 如蛋清蛋白、酶等  按生理作用分： 结构蛋白：如肌肉、皮肤、毛发等 酶：起催化作用 激素：生理调节作用 抗体：免疫作用  按化学成分分: 单纯蛋白：水解产物全为  - 氨基酸 结合蛋白：单纯蛋白+铺基 （脂蛋白、糖蛋白、磷蛋白、色蛋白、核蛋白、金属蛋白等）

Levels of Protein Structure Primary: the sequence of the amino acids in the chain and the disulfide links. Secondary: structure formed by hydrogen bonding. Examples are -helix and pleated sheet. Tertiary: complete 3-D conformation. Quaternary: association of two or more peptide chains to form protein.

Alpha Helix Each carbonyl oxygen can hydrogen bond with an N-H hydrogen on the next turn of the coil.

- Pleated Sheet Each carbonyl oxygen hydrogen bonds with an N-H hydrogen on an adjacent peptide chain.

蛋白质空间结构 二级结构 三级结构是指蛋白质分子中原子和原子团在三维空间的排列分布及肽链走向 多肽链在形成二级结构的基础上，相隔较远的氨基酸残基通过氢键、二硫键、酯键和盐键以及疏水交互作用(统称做副键)等分子内的相互作用而形成的卷 曲状、折叠状和盘绕状的较复杂的空间构象 四级结构：数个亚单位（肽链）之间在空间的相互作用缔合成更为复杂的空间结构

1. 氢键： 存 主链上肽键之间 在 形 式 主链与侧链之间 侧链与侧链之间 2. 二硫键： 1. 氢键： 主链与侧链之间 侧链与侧链之间 2. 二硫键： 蛋白质分子中两个半胱氨酸分子之间 —SH 氧化而成 —S—S—。 3. 疏水键： 侧链大的非极性基团（即疏水基团）为避开水相而群聚在一起的作用力。因此，在水相情况下，蛋白质外部表面多为亲水基团，内部多为疏水基团。 4. 范德华力： 较小的非极性基团或极性基 团间的作用力 5. 盐键： 多肽链间游离的羧基和游离氨基之间的相互作用力 6. 配位键： 蛋白质分子内金属原子与多肽链之间形成的配位键

Quaternary Structure More than one peptide chain can join to form protein structure hydrophobic interactions join chains E.g. -lactoglobulin乳球蛋白 Major serum protein in bovine milk pH 3-5 octomer pH 5-8 dimer pH > 8 monomer

Summary of Structure

Denaturation Disruption of the normal structure of a protein, such that it loses biological activity. Usually caused by heat or changes in pH. Usually irreversible. A cooked egg cannot be “uncooked.”

The End Of Chap 25 Homework: