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Chapter 2 Bioorganic Chemistry of the Amino Acids  2.1 General Properties  If we consider the protein constituents of ourselves (hair, nails, muscles,

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Presentation on theme: "Chapter 2 Bioorganic Chemistry of the Amino Acids  2.1 General Properties  If we consider the protein constituents of ourselves (hair, nails, muscles,"— Presentation transcript:

1 Chapter 2 Bioorganic Chemistry of the Amino Acids  2.1 General Properties  If we consider the protein constituents of ourselves (hair, nails, muscles, connective tissues,etc), we might suspect that the molecules which constitute a complex organism must be of a complex nature. As such, one might investigate the nature of these “life molecules”. Upon treating a protein sample with aqueous acid or base, one would no longer observe the intact protein molecule, but instead a solution containing many simpler,much smaller molecules: the amino acids.

2  General form of α–amino acids

3  protein 蛋白质  polymer 高分子,聚合物  atom 原子  carbon 碳  monomeric unit 单体  carboxyl group 羧基  hydrogen 氢  central carbon 中心碳  amino group 氨基  substituent 取代基  primary amino group 伯氨基  secondary amino group 仲氨基  imino acid 亚氨酸

4 The groups of amino acids It is possible to divide the amino acids into three groups based on their polarity( 极性).  Acidic amino acids: 2  Basic amino acids:4  Neutral amino acids:14

5 ( 1 ) Acidic Amino Acids 酸性氨基酸  Acidic amino acids are recognized by their ability to form insoluble (不溶性的) calcium or barium ( 钡) salts in alcohol (乙 醇). The side chains of these amino acids possess a carboxyl group, giving rise to their acidity. The two acidic amino acids are:

6  (a) Aspartic acid 天冬氨酸 ( abbreviation:Asp)

7  (b) Glutamic acid 谷氨酸  (abbreviation:Glu)

8 ( 2 ) Basic Amino Acids 碱性氨基酸  Basic amino acids are recognized by their ability to form precipitates (沉淀) with certain acids. Members of this group include

9  (a) Lysine 赖氨酸  (abbreviation: Lys)  The four methylene (亚甲基) groups are expected to give a flexible( 柔性的,挠曲性的) amino function to protein molecules.

10  ( b) Hydroxylysine 羟基赖氨酸  (abbreviation:Hylys)  This amino acid is found only in the structural protein of connective tissues, collagen.

11  ( C) Arginine 精氨酸  (abbreviation:Arg)

12  This amino acid is characterized by the guanidine function (胍基), which gives rise to its high basicity. Indeed, guanidine is one of the strongest organic bases known, being comparable in strength with sodium hydroxide (氢氧化钠). Hence, at physiological PH(7.35) this group is always ionized (离子化).

13  (d)Histidine 组氨酸  (abbreviation:His)

14  This amino acid contains the heterocylic (杂环的) imidazole (咪唑) ring and possesses a unique chemistry. It is both acid and weak base as well as an excellent nucleophile (亲核试剂), and the only amino acid that has a pKa which approximates physiological pH (7.35). As such, it can both pick up and dissociate (离解) protons within the biological milieu( 周围,环境).

15  Further, it may so function simultaneously by picking up a proton on one side of the ring, and donating it to the other. It has the potential of acting as a proton-relay system( 质子传递体系).

16 ( 3 ) Neutral Amino Acids 中性氨基酸  Neutral amino acids contain organic side chains which can neither donate nor accept protons. The simplest (and the only optically inactive amino acid) is:

17  (a) Glysine 甘氨酸  (abbreviation:Gly)  Obviously, little chemistry is associated with this amino acid, and its biological role is that of a structural component where limited space (compactness) is important. A number of structural proteins (collagen, silk, wool) contain significant amounts of glycine.

18  There are a number of amino acids which are hydrophobic (疏水的) by virtue of hydrocarbon side chains( 碳氢侧链). These include:

19  ( b) Alanine 丙氨酸  (abbreviation:Ala)

20  (c) Valine 缬氨酸  (abbreviation:Val)

21  (d) Leucine 亮氨酸  (abbreviation:Leu)

22  (e) Isoleucine 异亮氨酸 (abbreviation:Ile)

23 Amino acids exist which contain aromatic ( 芳香族的) hydrocarbon side chains.  (f) Phenylalanine 苯丙氨酸  (abbreviation:Phe)  Polarizableπelectron cloud.

24  (g)Tyrosine 酪氨酸  (abbreviation: Tyr)  Hydroxylation( 羟基化) of phenylalanine

25  This amino acid possesses a dissociable (phenolic 酚) hydroxyl of pKa=10.07. The similarity between phenylalanine and tyrosine allows the former to be converted to the latter in the human.  As such, it is phenylalanine, and not tyrosine, which is an essential amino acid to the diet. These amino acids are the precursors for the synthesis of the hormone (激素 )adrenaling( 肾上腺).

26 Other neutral hydroxylic amino acids include:  (h) Serine 丝氨酸  (abbreviation: Ser)

27  The hydroxymethyl function( 羟甲基) is not dissociable under typical physiological conditions. However, serine does serve an important function in a number of biochemical reactions, because of the ability of the primary hydroxyl to act as a nucleophile under appropriate conditions.

28  (i) Threonine 苏氨酸  (abbreviation: Thr)  The secondary hydroxyl (仲羟基) is not know to participate in any biochemical reactions.

29  (j) Cysteine 半胱氨酸  (abbreviation:Cys)  Replacement of the serine oxygen with sulfur (硫) gives rise to a dissociable proton. 

30  The sulfur atom (with its polarizable or elastic electron cloud) is one of the best nucleophiles known and cysteine, like serine, can participate in a number of biochemical reactions.

31  Also, the sulfhydryl ( 巯基) of cysteine is quite oxidizable to give rise to the disulfide, cystine (胱氨酸).

32  (k) Methionine 甲硫氨酸,蛋氨酸  (abbreviation: Met)

33  This amino acid contains a center of high polarizability in the otherwise inert hydrocarbon side chain. Nucleophilic attack of this sulfur atom on the biological energy store adenosine( 腺苷) triphosphate (三磷 酸) (ATP) give rise to the cationic biochemically important methyl group donor: S-adenosylmethionine ( S- 腺苷甲硫氨酸).


35 (l) Tryptophan 色氨酸 (abbreviation:Try) indole ring( 吲哚环)

36  The indole ring is an excellentπelectron donor. In the presence of an “electron sink( 电子槽) ”,it may give rise to a charge- transfer complex, or an electron overlap ( a very weak bond ) between this source and the sink.

37  Two neutral amino acids arise from the formation of the primary amides of aspartic and glutamic acids. These are:

38  (m)Asparagine 天冬酰胺  (abbreviation: Asn)

39  (n)Glutamine 谷氨酰胺  (abbreviation: Gln)

40  Transformation of the carboxylate to amide ( 酰胺 )functions gives rise to a species which can participate in hydrogen bonding important to biological function.

41 人体必需氨基酸  苏氨酸  缬氨酸  异亮氨酸  亮氨酸  赖氨酸  甲硫氨酸  苯丙氨酸  色氨酸

42 (4)The two α-imino acids are:  (o) Proline 脯氨酸 (abbreviation: Pro)  (p) Hydroxyproline 羟基脯氨酸  (abbreviation: Hypro)

43  It is this secondary amino function which gives rigidity( 刚性), and a change in direction, to the peptide backbone of which proteins are built. Thus, the direction of the collagen helix ( 螺旋) (collagen is a “triple helix” with three separate polypeptide chains wrapped around one another) is continually changing as a result of its proline and hydroxyproline content. Collagen is the only protein in which hydroxypoline is found.

44  In addition to these common amino acids which make up the protein molecule, a number of other amino acids exists which are not present in the protein but are biochemically important. These may be α-,β-,γ-,orδ-substituted.

45  γ-aminobutyric acid γ -氨基丁酸 (GABA):  neurotransmitter 神经传递素

46  2 , 5 - diiodotyrosine 2 , 5 -二碘酪氨酸  Thyroid hormone precursor 甲状腺激素前体

47  β-alanineβ -丙氨酸  Precursor of the vitamin pantothenic acid 遍 多酸

48  β - cyanoalanineβ -氰基丙氨酸  A plant amino acid

49  penicillamine 青霉胺  A clinically useful metal chelating agent 鳌合 剂

50  While all amino acids present in proteins exist in the L-configuration, a few nonprotein amino acids do exist in the D-configuration. The importance of the absolute configuration with regard to protein structure and function will become obvious with a deeper consideration of bioorganic processes.

51 D- 氨基酸的用途 医药方面: 老年痴呆;止痛药;肿瘤治疗;类激素;生长激素抑制剂等 食品领域: 甜味剂 化妆品领域

52 2.2 Dissociation Behavior 离解行为  2.2.1 Zwitterion 两性离子  Organic salts  Amino acids are crystalline solids 结晶固体 which usually decompose 分解 or melt 融化 in the range of 200-350 ℃, and are poorly soluble in organic solvents. These properties suggest that they are organic salts and the evidence is that they exist in the crystal lattice 晶格 as a dipolar ion or zwitterion. That is, the acidic proton from the carboxyl function protonates the amino function on the same molecule.

53  This is not peculiar to amino acids but can instead be representative of any organic salt (i.e., nucleotides; organic molecules which can contain cationic, nitrogen and anionic, phosphate within the same molecule).

54  Dipolar ion or zwitterion  氨基酸可以看作有机盐。 氨基酸中,羧基离解出 质子与氨基质子化在同 一个分子里形成内盐 (inner salt) 的结构。

55  In solution two possible dissociation pathways exist for all amino acids.

56 2.2 isoionic point and isoelectric point 等离子点和等电点  In solution there will be an acidity (pH) at which the amino acids will exist in the zwitterionic form, or have no net charge.  The pH at which this occurs an a result of the proton condition is referred to as the isoionic point (pIi).  When it is observed that there is no net charge on the molecule within the system as judged by experimental conditions (i.e., no mobility during an electrophoresis experiment) the pH at which this occurs is referred to as the isoelectric point (pIe).

57  For an aqueous solution of amino acids:  PIi=pIe  However, for proteins this is not necessarily the case, since they may be binding ions other than protons which contribute to an overall charge balance (no net charge).

58  It might be expected and is observed that proteins at their respective isoelectric points will be less soluble than at pH values above or below at this point. As they will have no net charge, they will more readily aggregate ( 聚集) and precipitate (沉淀). Further, since different proteins will have different amino acid compositions, they will possess characteristic pIe values.

59  Such is the basis for protein purification by isoelectric precipitation. The protein mixture is adjusted to a pH that is equivalent to the pIe of the desired protein, allowing the latter to precipitate out of the mixture.

60 2.5 Biological Synthesis of Proteins 蛋白质的生物合成  Much the same principle of carboxylate activation is applicable to the in vivo 在体的,体内的 synthesis of proteins. Again the carboxylate of an amino acid becomes activated by reaction with ATP to form an anhydride intermediate 酐中间体. The next step does not simply involve attack of a second amino acid of this anhydride since the synthesis of a protein involves the precise sequential coupling of many (up to a few hundred) amino acids. A template or “ordered surface” must be available to ensure correct sequencing of the protein molecule. The macromolecule that serves such a function is a polynucleotide 聚核苷酸, transfer ribonucleic acid (tRNA).

61  1 Activation of Amino Acids 氨基酸的活化  Activation of the amino acid by ATP is only an intermediate step catalyzed by the enzyme 酶 aminoacyl-tRNA synthetase. The 3’- or 2’-hydroxyl of the terminal adenylic acid of the tRNA molecule then attacks the anhydride intermediate to give an aminoacyl-tRNA molecule.  氨基酸与 ATP 和 tRNA 反应生成氨酰- tRNA 分子


63  The ester linkage between the hydroxyl of the tRNA is a high energy bond (due to the adjacent 2’-hydroxyl and cationic amino functions) so that the overall enzyme catalyzed reaction has a free energy change close to zero.  Each amino acid has a specific tRNA molecule and one specific aminoacyl-tRNA synthetase enzyme. In turn, each aminoacyl-tRNA synthetase enzyme will accept only its particular amino acid as a substrate.  每一个氨基酸有其专一的 tRNA 分子和一个专一的氨 酰- tRNA 合成酶。

64  2 Ribsome 核糖体  Once synthesis of the aminoacyl-tRNA is complete, the amino acid no longer serves a recognition function. Specificity is dictated by the tRNA portion of the molecule by its interaction with the genetic message(mRNA) and another large surface upon which protein synthesis take place; a cellular organelle 细胞器 referred as the ribosome.

65  The ribosome is a large cellular organelle, composed of RNA and a number of different proteins, and built of two dissociable subunits 亚基, 亚单位. This provides the ultimate ordered surface for protein synthesis, being able to interact precisely with the large tRNA portions of the various aminoacyl - tRNA’s.  Protein synthesis is a dynamic process, but again this process occurs in an ordered fashion which the sequential coupling of amino acids demands.

66  The site to which the fMet-tRNA fMet binds on the ribosome is referred to as the peptidyl site. 肽基部位,供体部位( P)  The second amino acid (aminoacyl-tRNA) also binds on the ribosome (at the so-called aminoacyl site) in close proximity to the fMet-tRNA fMet. 氨酰基部位, 受体部位( A)

67  3 Protein synthesis 蛋白质合成 Protein synthesis begins with the N-terminal amino acid and proceeds from this point.  In some bacteria, yeast 酵母菌, and higher organisms, this first aminoacyl-tRNA is known to be N-formylmethionyl-tRNA fMet N -甲酰甲硫氨酰- tRNA.

68 HC O  fMet  甲酰化甲硫氨酸与 ATP 和 tRNA 反应生成甲酰甲硫氨酰- tRNA 分子 fMet-tRNAfMet

69  Formylation of the amino function can be considered as a protecting group to prevent participation of the amino function is peptide bond formation. The fMet-tRNA fMet is then the first aminoacyl-tRNA to bind to the ribosome and mRNA. After the protein is synthesized, the formyl group is removed by enzymatic cleavage (formylase).  fMet-tRNA fMet 结合在核糖体和 mRNA 上。

70  The amino function of the aminoacyl-tRNA now attacks the f-methionine, at which point the tRNA fMet becomes a leaving group, and the peptide bond is formed. This reaction is enzyme catalyzed.  另一分子氨酰 -tRNA 上的氨基进攻甲酰甲硫 氨酸,肽键形成。

71  While this completes peptide bond formation, obviously some physical changes must occur in order that another incoming aminoacyl-tRNA may attach to the dipeptide. The dipeptidyl-tRNA at the aminoacyl site is physically shifted to the peptidyl site, simultaneously displacing the tRNA fMet. This, very likely, results from a conformational change on the ribosome which again requires energy at the expense of a molecule of GTP.  氨酰基位置的二肽- -tRNA 移到肽基位置。

72  The aminoacyl site is now empty, and the mRNA has also shifted, (translocation) so that it can now dictate the entrance of a new aminoacyl-tRNA to the aminoacyl site.  新的氨酰- -tRNA 分子进入氨酰基位置。  Once this has occurred, peptide bond formation (to give a tripeptide) can take place, thus repeating the sequence of events as described.  新的肽键形成。



75 2.6 Chemical Synthesis of Proteins 蛋白质的化学合成  Biological synthesis of proteins is a complex process requiring ordered macromolecular surfaces, protein catalysts, energy storage forms, etc. This alone would indicate that the chemical synthesis of a protein would be difficult undertaking. Therefore, a number of considerations must be made before attempting such a project.

76 1 Considerations 要考虑的因素  (1)specificity 专一性  First, specificity or sequential coupling of the amino acids must be achieved. Unwanted side reactions must be avoided.  Consider, for example, the synthesis of the dipeptide glycylalanine (Gly-Ala). It is not possible to mix the two amino acids together and let them react as the desired sequence will not be the only product. In fact, the number of possible dipeptides is four (Gly-Gly, Gly-Ala, Ala-Gly, Ala-Ala), of which only one is the correct sequence.

77  The problem becomes worse with increasing chain length. If random reaction were allowed for the synthesis of half of the gramicidin S 短杆菌肽 S molecule (i.e. five amino acids were placed in a reaction vessel and allowed to react,) then product pentapeptides 五肽 of 3125 possible sequences would form. Only one would be the desired sequence, with the other 3124 pentapeptides having to be separated and discarded as by- products 副产物.

78  解决的方法:使用保护基( protecting group)  Clearly, a successful protein synthesis demands ordered sequential coupling of amino acids, with little by-product formation. Such is achieved by the use of protecting groups for amino functions, carboxyl functions, and potentially reactive amino acid side chains.

79  For example, returning to the synthesis of Gly-Ala, if the amino function of glycine is protected (rendered chemically unreactive ) then reaction of alanine is also protected, then the only possible reaction is that between the carboxyl (activated ) of glycine and the amino of alanine to give the desired dipeptide product.  PG-NH-CH 2 -COOH NH 2 -CH-COO-PG CH 3

80  Of course, once the peptide bond is synthesized it becomes necessary to remove the protecting groups under conditions that will not affect the product. PG-NH-CH 2 -CONH 2 -CH-COO-PG CH3

81  Consequently, protecting groups must easily be both attached to the reactants and removed form the product under mild conditions and in high yield.

82  (2) optical purity 光学纯度  Another factor peculiar to biological systems must be considered: optical purity 光学纯度. Proteins are made of L-amino acids. As such, a chemical synthesis must start with L-amino acids and racemization 外消旋作用 must be minimized during the synthesis. This is especially true in the synthesis of an enzyme, as catalytic activity is dependent upon optical integrity.

83  保护基应满足以下条件:  在接肽时能起保护作用  接肽后易于在缓和条件下除去,不引起肽键的断裂  每一步有较高的产率。  好的光学纯度。

84 2 合成步骤 ( 1 )接保护基 PG-NH-CH 2 -COOH + NH 2 -CH-COO-PG ( 2 )羧基活化 PG-NH-CH 2 -CO - ( 3 )肽键形成 PG-NH-CH 2 -CONH-CH-COO-PG ( 4 )去保护基 NH 2 -CH 2 -CONH-CH-COOH R PG

85  2.6.1 Amino Protecting Groups 氨基的保护基

86  (1)tert-Butoxycarbonyl Function (tert-BOC) 叔丁氧羰基  tert-Butoxycarbonyl function is an acyl protecting group for the amino function and is readily removed under mild acidic conditions. Once acylated, the amino function is rendered chemically unreactive or nonnucleophilic as a result of electron delocalization into the amide bond (carbamate 氨基甲酸盐 ).

87  基团的引入:  This may be introduced by reaction of the chloride with the amino acid.  The chloride is synthesized from tert- butanol( 叔丁醇) and phosgene (光气). 氯代甲酸叔丁酯

88  However, it is too unstable for convenient use and storage.

89  This has been replaced by the less reactive tert-BOC azide( 叠氮化物). Its dangerous (explosive).  Presently, the reagents of choice are “BOC- ON”[2-(ter-but-oxycarbonyloxyimino)-2- phenylacetonitrile] [2- (叔丁氧羰基氧代亚氨 基)- 2 -苯基乙 ]and di-tert-butyl dicarbonate 二叔丁基二碳酸酯.  Both give tert-BOC amino acids in high yields.


91  保护基的除去:  The tert-BOC group is readily removed under mildly acidic conditions to give gaseous isobutylene 异丁烯 and carbon dioxide.

92  Acid conditions used to remove the group include hydrogen bromide in acetic acid, hydrogen chloride in acetic acid, warm acetic acid (80%), anhydrous trifluoroacetic acid, and liquid hydrogen fluoride.  可在酸性条件下去保护基,如 HBr/HAc, HCl/HAc,80% 温热醋酸,无水三氟乙酸,液 态氟化氢。

93  (2)Carbobenzoxy Function (CBz) 苄氧羰基  Carbobenzoxy function (CBz) is also an acyl protecting group for the amino function which is removed by hydrogenolysis 氢解 or under acidic conditions.

94  基团的引入:  The chloride is prepared by reaction of benzyl alcohol( 苄醇) with phosgene:

95  The chloride may be reacted with amino acids in mildly alkaline, aqueous media.

96  保护基的除去:催化氢化;酸性条件  The CBZ function is more acid resistant than the tert-BOC group, and it will not be removed by warm acetic acid for the period of time needed to remove the trityl 三苯甲基 and p-methoxytrityl functions. It is base resistant (i.e. dilute 稀释的,淡 的 NaOH at room temperature), but not to the same extent as the tert-BOC function due to the better leaving ability of the benzyl group.

97  A The CBz function may be removed by catalytic hydrogenation 催化氢化 :

98  Hydrogenation of the benzene ring would occur to some extent only under high pressure, and in the presence of platinum 钯. However, poisoning of the metal catalyst may result from the presence of sulfur- containing amino acids. In some cases this may be overcome by the use of excess catalyst.

99  B 酸性条件  The CBz group may also be removed by HBr in anhydrous acetic acid (room temperature).  HBr/ 无水乙酸,室温

100  The mechanism involves alkyl fission 烷基分 裂,烷氧键断裂 :

101  Other acids may also be used, but these usually required elevated temperatures (i.e., boiling trifluoroacetic acid, boiling methanolic HCl)  其他的酸也可以使用,但常需加热(如沸腾 的三氟醋酸,沸腾的 HCl/ 甲醇)

102  (3)Phthaloyl Function 肽酰基 ,邻苯二甲酰基  Phthaloyl function is an acyl anhydride 酰酐 which is removed by hydrazine 肼 or phenylhydrazine 苯 肼.

103 基团的引入: A 熔融法: The classical method of preparation of N- phthaloyl amino acids involves fusing together a mixture of phthalic anhydride 邻苯 二甲酸酐 and the amino acid.

104  缺点: However, heating can be damaging to sensitive peptides and significantly racemization can result. △

105  B 邻乙氧羰基硫代苯甲酸与氨基酸酯作用,再与 HBr/ 醋酸水 溶液反应  Preparation of the N-phthaloyl amino acid has been achieved by reaction of o-carboethoxythiobenzonic acid with an amino acid ester, followed by reaction with aqueous HBr.  离去基顺序:- X> - OOCR> - OR> - NH2 活泼的硫化物

106  C 氨基酸与 N- 乙氧羰基邻苯二甲酰亚胺反应  The most general method used is the reaction of an amino acid with N-carboethoxyphthalimide.  制备: This reagent is prepared by reaction of phthalimide 邻苯二甲酰亚胺 with ethyl chloroformate 氯甲酸乙酯.

107  引入氨基酸: Reaction with amino acids takes place in dilute alkaline, aqueous solution without heat. 在常温,稀碱水溶液中与氨基酸反应

108  除去保护基:  The phthaloyol group is unaffected by catalytic hydrogenation, sodium in liquid ammonia, and is acid resistant (i.e., HCL or HBr in acetic acid water, at room temperature). However, this group is base labile 不安定的,易发生变化的.

109  A Base treatment 碱性条件下除去  The product formed upon base treatment is cleaved by slightly acidic conditions to give the free amino acid:

110  B 肼解 : 水合肼的水溶液或醇溶液  The classical method for removal of the phthaloyl function is treatment with aqueous or ethanolic hydrazine hydrate 水合肼. The hydrazide 酰肼 by-product separates out by precipitation. Hydrazine is a good nucleophile (so-called α-effect) which will attack the anhydride-like phthaloyl function under conditions that will not affect peptide linkages.


112  (4)p-Toluenesulfonyl (Tosyl) Function  对甲苯磺酰基  p-Toluenesulfonyl (tosyl) function allows protection of the amino function by sulfonylation 磺酰化作用, instead of acylation. Removal is accompllished by sodium in liquid ammonia 液氨.

113  基团引入: Sulfonylation proceeds in aqueous base or pyridine 吡啶 :

114  除去保护基:  The tosyl function is unaffected by catalytic hydrogenation, and is resistant to most conditions of acidity and alkalinity (i.e., HBr in acetic acid, at room temperature).  Removal of the tosyl function is undertaken by dissolving the protected amino acid/peptide in liquid ammonia and slowly adding sodium. Excess sodium is destroyed at the end of the reaction by the addition of ammonium chloride, iodide, or acetic acid.


116  (5) Triphenylmethyl (trityl) Function 三苯甲基  Triphenylmethyl (trityl) function may be used to protect the amino function by an SN1 alkylation. The trityl group is very acid labile. Whereas acyl and sulfonyl groups protect amino functions by rendering the nitrogen non-nucleophilic, the trityl function preserves this nucleophilicity (basicity) and protects the amino group by steric bulk 空间位阻. In fact, this can be a disadvantage, because the bulky trityl group can also interfere with peptide coupling at the (activated) carboxyl.

117  基团的引入:  The trityl function is introduced by reaction of trityl chloride with an amino acid ester in the presence of an organic base and in an organic solvent. The reaction cannot be done in aqueous media.

118  除去保护基:  酸性条件  The trityl group is stable to base, may be removed by hydrogenolysis, but is very susceptible 易受影响 的 to acid. It can be removed under conditions (i.e., 80% acetic acid, 1/2hour, 30 ℃ ) that will not affect the tert-BOC and CBz functions. A variety of acids may be used which include acetic acid, HCL in water, acetic acid, methanol and chloroform, and trifluoroacetic acid. The case of acidolysis is the result of the stabilized carbonium ion 正碳离子 :


120  An even more acid labile trityl analogue which has found some use as an amino protecting group is the p-methoxytrityl function.

121  (6)Formyl Function 甲酰基  Formyl function may be used as a simple acyl protecting group, as was observed for the N-terminal amino acid in the biological synthesis of proteins. It may be removed under mildly acidic conditions that will not affect peptide bonds.

122  基团的引入:甲酸乙酸酐与氨基酸反应  Amino acids may be formylated in formic acid-acetic anhydride ( formic anhydride is unstable).

123  除去保护基:  A 酸性条件  The formyl group is not affectd by catalytic hydrogenation and sodium in liquid ammonia, but is only resistant to gentle base ( i.e., conditions that will saponify ester linkages; the amide would be poorer leaving group than alkoxide 醇盐 ). It is easily removed by acid or hydrazine ( and AcOH) in alcohol.

124  B 肼的醇溶液或过氧化氢  It can also be removed from the amino acid by NH 2 NH 2 in 60%EtOH+HOAc or from both amino acid and peptides by 15% aq H 2 O 2, 60 ℃,2hr. However, esters are probably affected by these procedures.

125  ( 7 ) Trifluoroacetyl Function 三氟乙酰基  Trifluoroacetyl function may be used as an acyl protecting group which is removed in mild base.

126  基团的引入:三氟乙酸酐或三氟乙酸硫代乙酯与氨 基酸反应  It may be introduced upon reaction of the amino acid with trifluoroacetic anhydride or trifluoroacetic acid thioethyl ester.  缺点:易消旋  The disadvantage is that trifluoroacetyl amino acids readily racemize.

127  除去保护基:温和碱  The group may be removed by sodium hydroxide at room temperature; conditions that do not affect most other protecting groups.  氢氧化钠,室温

128  2.6.2 Carboxyl Protection Groups 羧基保护基  If two amino acids, neither of which has a protected or activated carboxyl function, are to be coupled together to form a dipeptide, then treatment with a reagent that simultaneously activates the carboxyls and brings about peptide bond formation will result in self-condensation. This may be avoided by protection of the carboxyl group of one amino acid.

129  Generally, this is accomplished by esterfication 酯化作用 of the carboxyl group to be protected.  保护的方法:使羧基酯化。

130  ( 1 ) methyl or ethyl ester 形成甲酯或乙酯  The methyl or ethyl ester may be synthesized by reaction of the amino acid with gasous HCl in mathanol or ethanol ( Fisher esterification). 氨基酸 + 甲醇或乙醇 HCl

131  除去保护基:皂化反应

132  (2)benzyl ester 形成苄酯  While esters may be saponified by base more easily than peptides ( the alkoxide 醇 盐 is a better leaving group), the alkaline conditions that are required may not be compatible to the polypeptide. Use of benzyl esters allows removal of the protecting group under neural conditions is catalytic hydrogenation.

133 基团的引入:氨基酸甲酯与苄醇反应 生成苄酯 transesterification 酯基转移作用

134  除去保护基:中性条件下催化氢化  neural conditions via catalytic hydrogenation.

135  (3)tert-butyl ester 叔丁酯  基团的引入:  A 氨基酸与异丁烯反应  B 氨基酸与醋酸叔丁酯进行酯交换反应。  The ester, which for steric reasons cannot be prepared from tert-butanol, may be synthesized from isobutylene gas in the presence of acid, or by transersterification with tert- butyl acetate.

136  氨基酸与异丁烯反应:  氨基酸与叔丁酸乙酯反应:

137  除去保护基:温和酸性条件  Preparation of the tert-butyl ester confers resistance to alkali, but allows removal of the protecting group under mildly acidic conditions.

138  ( 4 ) 2-trimethylsilylethyl function  2- (三甲硅基 ) 乙基  This may be synthesized by the reaction of the acid with 2-trimethylsilyethanol and dicyclohexylcarbodimide (DCC, a dehydration or coupling agent 脱水剂或偶连剂 )  基团的引入 : 酸与甲硅烷醇在二环己基碳二亚胺存在 下进行反应


140  除去保护基:中性条件下用氟化物处理  Removal of the protecting group may be achieved under neutral conditions by treatment with fluoride. Fluoride has found much used for the removal of silyl protecting groups on carbohydrates 碳水化合 物. In anhydrous solvent systems, it is a good nucleophile.

141  Side Chain Protecting 侧链保护  During the course of a peptide synthesis, protection of the amino and carboxyl functions may not be adequate. Under the conditions of peptide bond formation nucleophlic or other chemically reactive amino acid side chains may participate in unwanted side reactions. This may be avoided by protection of these side chains with protecting groups that meet the same general requirements of all protecting groups: introduction and removal under mild conditions, etc.

142  Example 1: Thiol group 巯基 of cysteine  引入苄基保护  The thiol group is a potent nucleophile, and is susceptible to oxidation to the disulfide form. It therefore must be protected during peptide synthesis. Use may be made of the benzyl group 苄基, which is introduced by the reaction of the thiol with benzyl chloride.

143  除去保护基: Na -液氨  Benzyl functions are susceptible to hydrogenolysis and so this group may be removed by reduction with sodium in liquid ammonia.

144  Example2 : amino functions of ornithine and lysine 鸟氨酸和赖氨酸上的侧链氨基  Discrimination of the side chain amino functions may be achieved by first forming the copper 铜 chelate 螯合物 of the amino acid.


146  除去保护基: H 2 S 处理, 使 α - NH2 游离

147  2.6.3 Peptide Bond Formation 肽键的形成  Two general approaches are possible. The first is to convert the amino protected amino acid to an activated form and then react this with the amino function of a second amino acid. As will be recalled, activation is necessary as work must be done during peptide bond formation. Alternatively, it is possible to react two amino acids together ( one amino protected, the other carboxyl protected ) in the presence of a coupling agent which activates the carboxyl function in situ 原位.

148  两种方法 :  一是羧基活化:将氨基保护的氨基酸变成活化形式, 与另一分子氨基酸反应。  PG-NH-CH 2 -COOH  PG-NH-CH 2 -COCl+NH 2 -CH 2 -COOH  二是原位活化:两分子氨基酸(一个氨基保护,一 个羧基保护)在偶联剂的作用下原位活化。 偶联剂 二肽 PG-NH-CH 2 -COCl 二肽

149  1 羧基活化法  ( 1 ) Acyl Chlorides 酰氯  Amino acids may be converted to their acyl chlorides:  The acyl chloride formed will readily react with the amino function of a second amino acid to give peptide bond formation.  氨基酸转化为酰氯,与另一分子氨基酸形成肽键。  缺点:易外消旋化

150  ( 2 ) Anhydrides 酸酐  Mixed anhydrides tend to be less reactive than acyl chlorides (poorer leaving group )gives less racemization upon their formation. Synthesis may be accomplished by nucleophilic attack of the amino acid carboxylate on an acyl halide.  混酐的活性低于酰氯,外消旋化作用较小。

151  The anhydride formed will readily react with the amino function of the second amino acid. However, the amino function can react with one of two carbonyls, so that considerable by-product formation can occur.  形成的酐易与另一分子的氨基酸的氨基反应。 但是氨基可能与两个羰基中的一个反应,从 而产生许多副产物。

152  This may be avoided by synthesis of a mixed anhydride in which nucleophilic attack will be preferred at one of the two possible carbonyls. Reaction of an amino acid with ethyl choloroformate 氯甲酸乙酯 forms such an anhydride.  解决方法:合成混酐,使得其中一个羰基更 容易被亲核进攻。  可用氨基酸与氯甲酸乙酯反应生成这种酐。

153  Only one carbonyl is attacked, as it is more electrophilic ( the other carbonyl is flanked by two oxygen atoms which can delocalize their nonbonding electrons) and it is a poorer leaving group.  第一个羰基亲电性更好,第二个羰基位于两个氧原子之间, 有离域效应

154  Racemization can still occur and synthesis of the anhydride must be undertaken at low temperature to avoid decomposition.  仍会发生外消旋化。酐的合成必须在低温下 进行以防止分解。

155  (3)Acyl Azides 叠氮酰  Use of acyl azides leads to less racemization (not significant) than acyl chlorides or anhydrides. 叠氮酰引起的外消旋 化比酰氯或酸酐少。  制备:  A 酰氯 叠氮酰  The azide 叠氮化物 could be synthesized from the acyl chloride.


157  缺点: however, this would require the conversion of the amino acid to the unfavorable acyl chloride, thus defeating the purpose of the synthesis.  操作不便

158  B 氨基酸酯 叠氮酰  Synthesis is instead undertaken with the amino acid ester.

159  Storage of the azide is difficult, and all handling of the azide must occur at low temperature to prevent conversion to the isocynate via the Curtius rearrangement:  缺点:叠氮化物储存困难,而且所有的操作必 须在低温下进行以防止通过重排转化成异氰酸 酯。 活泼氮烯中间体

160  It is possible to convert the azide to a stable form that may be conveniently handled by reaction with N- hydroxysuccinimide:  解决方法: 可把叠氮化物与 N -羟基丁二酰亚胺反应转化为 稳定的形式以方便操作。  The product is a stable, crystalline : “activated ester”  产物是稳定的晶体:活化酯。

161 2 原位活化法  In situ activation of the carboxyl function, in the presence of a nucleophilic amino function, may be achieved with a variety of coupling agents.

162  (1)The Carbodimides 碳二亚胺  The carbodimides are a family of coupling 偶联 (dehydrating 脱水, condensing 缩合 ) agents of the general structure.  The central carbon atom, flanked by two nitrogen atoms, is electrophilic and thus subject to nucleophlic attack by a variety of reagent. The most well-known reactions are those between carbodimides and acids.  中心碳有亲电性,可接受亲核进攻。

163  For example, carbodimides will undergo addition reactions with a variety of weak acids: 硫醇形式硫脲形式

164  Most important, at least for the synthesis of biologically interesting compounds, is the reaction of carboxylic, phosphoric, and sulfonic acids. Under appropriate conditions, these will add to carbodimides to form anhydride-like intermediates, and eventually acid anhydrides.  在生理上的重要反应是与羧酸,磷酸和硫酸 的反应。在适当的条件下可形成类似于酐的 中间体,最后形成酸酐。

165 This is illustrated for carboxylic acid : 以羧酸为例  邻酰基脲(类似酸酐的中间物)

166  The unstable intermediate formed can either rearrange to form a urea 脲, or a second molecule of acid can attack to give the acid anhydride product.  不稳定的中间体经过重排形成脲,或者第二分子的 酸进攻中间体形成酸酐。 邻酰基脲 重排产物:脲 酸酐

167  In anticipating the use of carbodimides for amide synthesis, thought must be given to the selection of the “R” group. The stability of aliphatic 脂肪族的 and aromatic 芳香族的 carbodimides is a function of the substituting groups, so that decomposition and polymerization can occur upon standing. Alkyl chain length has little influence upon stability. Instead, stability increases markedly with the branching of the alkyl substituent, on the two nitrogen atoms. Thus, diethylcarbodiimide polymerizes in a few days, while dicyclohexylcarbodiimide is stable for months. It is the latter reagent that has found the most use in protein synthesis.  在使用碳二亚胺来合成酰胺时,必须考虑 “R” 基的选择。脂 肪族和芳香族的碳二亚胺的稳定性取决于取代基。二环己基 碳二亚胺稳定性好,常用于蛋白质的合成。

168  Dicyclohexylcarbodimide 二环己基碳二亚 胺 (DCC) may then be used for the formation of peptide bonds:  不含羟基的有机溶剂(质子惰性的)  第二分子氨基酸与酐中间体反应 DCU 二环己基脲

169  Under the mild reaction conditions (0 ℃ ), the second amino acid (carboxyl protected) will not react with DCC, but only with the anhydride-like intermediate. The DCU formed is insoluble in most organic solvents so that most often separation from the product merely involves filtrations 过滤. However, it occasionally may be difficult to remove the last trace of DCU by filtration. As such, the use of carbodimides that are soluble in aqueous solvent may be necessary. One example is 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride. 1- 乙基 - 3 -( 3 -二甲基氨丙基)-碳二亚胺盐酸 The urea by- product formed is also water soluble.

170  So that after reaction in an organic solvent, it is possible to remove any unreacted carbodimide and the urea simply by washing with water. It should be noted that significant racemization, via azlactone formation of the anhydride-like intermediate, can occur during the synthesis of a large peptide (protein) molecule. However, for a smaller polypeptide, this is minimal.  合成大的肽(蛋白质)分子时会有明显的外 消旋化作用,但合成较小的多肽时外消旋化 很小。

171  (2)Woodward’s Reagent 伍德瓦尔德试剂  This reagent is a dehydrating agent which has been used much less for peptide bond formation than the carbodimides.  该试剂也用作合成肽的脱水剂,但不如碳二 亚胺应用广泛。

172  Reaction first occurs with a carboxylate function after base catalyzed decomposition of the isoxazolium salt:  The presence of base in the reaction media is unfavorable, and a variety unweanted reactions, including significant racemization, can occur.  反应首先是异 口恶 唑盐在碱作用下分解,生成活泼的类似烯 酮的化合物,再与羧酸盐反应,生成类酸酐中间体。  其次是该类酸酐中间体与另一分子氨基酸反应生成肽键。  优点:方法简便,产率高,副产物为水溶性易于除去。  缺点:易外消旋化。


174  (3)EEDQ (Belleau’s 试剂)  2-Ethoxyethoxycarbonyl-1,2- dihydroquinoline ( 2- 乙氧基乙氧羰基- 1 , 2 -二氢喹啉) is also a coupling agent that may be used for peptide bond synthesis

175  EEDQ 的制备:  EEDQ is readily synthesized from quinoline and ethyl chloroformate:  由喹啉与氯甲酸乙酯反应制备 EEDQ 。

176  Under mild (room temperature) reaction, EEDQ will not react with amines. In the presence of a hydroxylic solvent, alcoholysis of the carboethoxy function will not occur, but instead the ethoxy function will be exchanged, if a trace of Lewis acid (i.e., BF3) is present. The ethoxy function is good leaving group which may be introduced to do so upon protonation. The driving force is the opportunity to gain aromaticity.  在温和(室温)的反应条件下, EEDQ 不与胺反应。在羟基 溶剂存在下,乙氧羰基不发生醇解。如果有微量的路易斯酸 存在,乙氧基将被交换。乙氧基是一个好的离去基,它可因 质子化而离去,从而使体系获得芳香性。


178  The carboxylate function will readily add to the cation, after which mixed anhydride formation will occur. This will in turn react with the amino function of a second amino acid to give peptide bond formation. Further, the mixed anhydride so formed does not accumulate in solution (its formation is rate limiting ) but instead suffers immediate nucleophilic attack by the amine. Azlactone 吖内酯 formation does not have a chance to occur, and so no significant racemization is observed during polypeptide synthesis.  乙氧基离去后形成的阳离子易于和羧酸盐反应而形成混酐, 接着又与第二分子氨基酸反应形成肽键。由于形成的混酐立 即与胺反应,形成吖内酯的几率小,因而不易产生消旋化。

179  合成二肽:  The special reactivity of EEDQ toward carboxylate functions can be understood by noting that displacement of the ethoxy function by any other nucleophile (i.e.,alcohol or thiol exchange reaction) leads to an analogue of the starting material. However, displacement by the carboxylae proceeds through a six-membered transition sate, and leads to the formation of a stable 10 π- electron aromatic structure.

180 不积累,无吖内酯形成

181  (4)Leuch’s Anhydride Leuch 酸酐  用光气作偶联剂  Reaction of an unprotected amino acid with phosgene provides a simple method of peptide bond formation. The reaction begins with N- acylation of the amino acid, followed by cyclization to give a reactive anhydride-like intermediate:  氨基酸首先 N -酰化,环化形成类酸酐中间体,然 后与另一分子氨基酸反应,脱去 CO 2 后得到二肽。

182 Leuch 酸酐  氨基甲酸

183  The anhydride formed will only undergo nucleophilic attack at one of the two carbonyl functions, as the untouched carbonyl is both less electrophilic (flanked by a nitrogen and an oxygen atom) and a better leaving group. The carbamate formed can be readily decomposed to the dipeptide and carbon dioxide. The phosgene act as a coupling agent since once the anhydride is formed, it undergoes displacement by a second amino acid. The anhydride is not isolated.

184  Further, the carbamate is not readily isolated, and the reaction is difficult to control. It is therefore possible for the dipeptide product to be N-acylated, undergo anhydride formation and then be attacked by a third amino acid, etc. the Leuch’s anhydride methodology is best suited for homopolymer 均聚物 synthesis. However, by careful control of reaction conditions (0 ℃,pH=12) it is possible to stop the reaction at the stage of the carbamate 氨基甲酸 and sequentially add the next monomeric unit, and then allow the reaction to proceed by freeing the amino function. By so doing, large polypeptide chains of desired sequence have been synthesized.  必须严格控制反应条件( 0 ℃,pH=12).

185  (5)Solid Phase Synthesis 固相合成 (Merrifield’s Method)  This technique combines the above methodology (amino and carboxyl protecting group, coupling agents, etc.) but greatly reduces the processing of intermediates or “workup” associated with these techniques. The necessity to obtain the product of each coupling reaction in pure crystalline form is eliminated, and excess reagents and by-products are simply removed by filtration and washing with appropriate solvent systems. This amounts to a lot of work if the synthesis of a large polypeptide molecule is to be considered. This is accomplished by the use of an insoluble polystyrene matrix.  技术方法同上。  减少中间的分离步骤。  使用不溶性的聚苯乙烯基质。

186  Step 1: 在聚苯乙烯上引入氯甲基  A reactive center is introduced into the polymer support by cholromethylation 氯甲基 化. The benzyl cholride linkage formed has already been noted to be very susceptible to SN2 displacements and so it is easily replaced by the carboxyl function, which can in turn be removed (by acidolysis ) at a later date.


188  Step 2: 接上氨基酸  Note that had the amino function been used to displace the cholrine, then removal of the amino acid from the resin 树脂 later would be impossible.


190  Step 3: 去保护基  Once the amino acid is covalently linked to the resin, the tert-BOC function may be removed by treatment with acid which will not affect the benzyl linkage to the support.


192  Step 4: 与另一分子氨基酸反应  The freed amino function may now react with a second (tert-BOC protected) amino acid to form the peptide linkage. the tert- BOC group removed and the bound dipeptide may react with a third amino acid, etc.


194  Step 5: 去保护基;重复上述反应,形成多肽  The tert-BOC group again removed and the bound dipeptide may react with a third amino acid, etc.


196  Step 6: 将多肽与树脂分离  Again, workup after each reaction consists of simply filtering the bound product and washing away unreacted reagents and unwanted side products. After the peptide of specified sequence has been synthesized, it is removed from the resin by treatment with HBr or HF in trifluoroacetic acid.

197  Any coupling method may be used, but that which has found the most use to date has been DCC.  As the workup of each coupling reaction is so simple, it has been possible to automate the procedure, thus allowing for an even more rapid polypeptide synthesis. As such, the first chemical synthesis of an enzyme (bovine pancreatic ribonuclease, 124 amino acid residues) was accomplished by this method.

198  总结:  蛋白质的化学合成

199 合成步骤 ( 1 )接保护基 PG-NH-CH 2 -COOH + NH 2 -CH 2 -COO-PG ( 2 )羧基活化 PG-NH-CH 2 -CO - ( 3 )肽键形成 PG-NH-CH 2 -CONH-CH-COO-PG ( 4 )去保护基 NH 2 -CH 2 -CONH-CH-COOH R PG

200  1 氨基的保护基:  叔丁氧羰基;  苄氧羰基;  肽酰基;  对甲苯磺酰基;  三苯甲基;  甲酰基;  三氟乙酰基

201  2 羧基的保护基  (1) 形成酯:  甲酯,乙酯;  苄酯;  叔丁酯  (2) 2-( 三甲硅基)乙基  侧链的保护

202  3 肽键的形成  (1) 羧基活化法:  酰氯 ;  酸酐 ;  叠氮化物  (2) 原位活化法:  偶联剂 DCC (二环己基碳二亚胺);  伍德瓦尔德试剂;  EEDQ ;  Leuch 酸酐;  固相合成

203  习题课:  以赖氨酸和甘氨酸为原料用化学方法合成赖 氨酰甘氨酸二肽。

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