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1 1264 Chapter 26 mino cids, Peptides, Proteins, and ucleic cids 11. Merrifield Solid-Phase Synthesis (Section 26-7) ClC 2 C 2 C 3, SnCl 4 P C 3 C 2 P = polystyrene PC 2 Cl R 1. (C 3 ) 3 CCCC R 2., 2 PC 2 CC 2 R 1. (C 3 ) 3 CCCC, DCC 2., 2 R R PC 2 CCCC 2 F PC 2 F R R 3 CCCC Important Concepts 1. Polypeptides are poly(amino acids) linked by amide bonds. Most natural polypeptides are made from only 19 different l-amino acids and glycine, all of which have common names and threeand one-letter abbreviations. 2. mino acids are amphoteric; they can be protonated and deprotonated. 3. Enantiomerically pure amino acids can be made by classical fractional crystallization of diastereomeric derivatives or by enantioselective reactions of appropriate achiral precursors. 4. The structures of polypeptides are varied; they can be linear, cyclic, disulfide bridged, pleated sheet, a-helical or superhelical, or disordered, depending on size, composition, hydrogen bonding, and electrostatic and London forces. 5. mino and nucleic acids are separated mainly by virtue of their size- or charge-based differences in ability to bind to solid supports. 6. Polypeptide sequencing entails a combination of selective chain cleavage and amino acid analysis of the resulting shorter polypeptide fragments. 7. Polypeptide synthesis requires end-protected amino acids that are coupled by dicyclohexylcarbodiimide. The product can be selectively deprotected at either end to allow for further extension of the chain. The use of solid supports, as in the Merrifield synthesis, can be automated. 8. The proteins myoglobin and hemoglobin are polypeptides in which the amino acid chain envelops the active site, heme. The heme contains an iron atom that reversibly binds oxygen, allowing for oxygen uptake, transport, and delivery. 9. The nucleic acids are biological polymers made of phosphate-linked base-bearing sugars. nly four different bases and one sugar are used for D and R, respectively. ecause the base pairs adenine thymine (uracil for R) and guanine cytosine are held by particularly favorable hydrogen bonding, a nucleic acid can adopt a dimeric helical structure containing complementary base sequences. In D, this arrangement unwinds and functions as a template during D replication and R synthesis. In protein synthesis, each amino acid is specified by a set of three consecutive R bases, called a codon. Thus, the base sequence (genetic code) in a strand of R translates into a specific amino acid sequence in a protein. 10. D sequencing relies on restriction enzymes, radioactive labeling, and specific chemical cleavage reactions to small fragments, analyzed by electrophoresis. 11. D synthesis employs silica as a support on which the growing oligonucleotide sequence is built up with the help of base, alcohol, and phosphite phosphate protecting groups. 12. The polymerase chain reaction (PCR) makes multiple copies of D. Problems 30. Draw stereochemically correct structural formulas for isoleucine and threonine (Table 26-1). What is a systematic name for threonine? 31. The abbreviation allo means diastereomer in amino acid terms. Draw allo-l-isoleucine, and give it a systematic name. 32. Draw the structure that each of the following amino acids would have in aqueous solution at the indicated p values. (a) lanine at p 5 1, 7, and 12; (b) serine at p 5 1, 7, and 12; (c) lysine

2 } } Problems Chapter at p 5 1, 7, 9.5, and 12; (d) histidine at p 5 1, 5, 7, and 12; (e) cysteine at p 5 1, 7, 9, and 12; (f) aspartic acid at p 5 1, 3, 7, and 12; (g) arginine at p 5 1, 7, 12, and 14; (h) tyrosine at p 5 1, 7, 9.5, and Group the amino acids in Problem 32 according to whether they are (a) positively charged, (b) neutral, or (c) negatively charged at p Show how the pi values for the amino acids in Problem 32 (see Table 26-1) are derived. For each amino acid that possesses more than two pk a values, give the reason for your choice when calculating pi. 35. Show how ell-volhard-zelinsky bromination followed by amination can be used to synthesize each of the following amino acids in racemic form: (a) Gly; (b) Phe; (c) la. 36. Show how the use of a Strecker synthesis can give each of the following amino acids in racemic form: (a) Gly; (b) Ile; (c) la. 37. What amino acid would result from carrying out the following synthetic sequence on C(C 2 C 2 C 3 ) 2? 1. C 3 C 2 2 a 1, C 3 C 2 ; 2. r(c 2 ) 4 CC 3 ; 3. 1, 2, D. 38. Using either one of the methods in Section 26-2 or a route of your own devising, propose a reasonable synthesis of each of the following amino acids in racemic form. (a) Val; (b) Leu; (c) Pro; (d) Thr; (e) Lys. 39. (a) Illustrate the Strecker synthesis of phenylalanine. Is the product chiral? Does it exhibit optical activity? (b) It has been found that replacement of 3 by an optically active amine in the Strecker synthesis of phenylalanine leads to an excess of one enantiomer of the product. ssign the R or S configuration to each stereocenter in the following structures, and explain why the use of a chiral amine causes preferential formation of one stereoisomer of the final product. C 2 C 2 C, C 3 C } E 2 ) 3 C C 2 C D C C [ Mainly [ } 1., , Pd C C 2 C 3 [ Mainly C 40. The antibacterial agent in garlic, allicin (Chemical ighlight 9-4, Problem 73 in Chapter 9), is synthesized from the unusual amino acid alliin by the action of the enzyme allinase. ecause allinase is an extracellular enzyme, this process takes place only when garlic cells are crushed. Propose a reasonable synthesis for the amino acid alliin. (int: egin by designing a synthesis of an amino acid from Table 26-1 that is structurally related to alliin.) 3 2 C PCC 2 SC 2 CC lliin 41. Devise a procedure for separating a mixture of the four stereoisomers of isoleucine into its four components: (1)-isoleucine, (2)-isoleucine, (1)-alloisoleucine, and (2)-alloisoleucine (Problem 31). (ote: lloisoleucine is much more soluble in 80% ethanol at all temperatures than is isoleucine.)

3 1266 Chapter 26 mino cids, Peptides, Proteins, and ucleic cids 42. Identify each of the following structures as a dipeptide, tripeptide, and so forth, and point out all the peptide bonds. (C 3 ) 2 C C 3 SC 2 C C 2 C 2 (a) 3 CCC C C C (b) 3 CCC C C 2 C 3 C C 2 (C 2 ) 3 3 (c) 3 CCC C C C C C C 2 C 2 C 2 C(C 3 ) 2 (d) 3 CCC 2 C C 2 CC C C C 43. Using the three-letter abbreviations for amino acids, write the peptide structures in Problem 42 in short notation. 44. Indicate which of the amino acids in Problem 32 and the peptides in Problem 42 would migrate in an electrophoresis apparatus at p 5 7 (a) toward the anode or (b) toward the cathode. 45. Silk is composed of b sheets whose polypeptide chains consist of the repeating sequence Gly- Ser-Gly-la-Gly-la. What characteristics of amino acid side chains appear to favor the b-sheet configuration? Do the illustrations of b-sheet structures (Figure 26-3) suggest an explanation for this preference? 46. Identify as many stretches of a helix as you can in the structure of myoglobin (Figure 26-8C). Prolines are located in myoglobin at positions 37, 88, 100, and 120. ow does each of these prolines affect the tertiary structure of the molecule? 47. f the 153 amino acids in myoglobin, 78 contain polar side chains (i.e., rg, sn, sp, Gln, Glu, is, Lys, Ser, Thr, Trp, and Tyr). When myoglobin adopts its natural folded conformation, 76 of these 78 polar side chains (all but those of two histidines) project outward from its surface. Meanwhile, in addition to the two histidines, the interior of myoglobin contains only Gly, Val, Leu, la, Ile, Phe, Pro, and Met. Explain. 48. Explain the following three observations. (a) Silk, like most polypeptides with sheet structures, is water insoluble. (b) Globular proteins such as myoglobin generally dissolve readily in water. (c) Disruption of the tertiary structure of a globular protein (denaturation) leads to precipitation from aqueous solution. 49. In your own words, outline the procedure that might have been followed by the researchers who determined which amino acids were present in vasopressin (Exercise 26-10). 50. Write the products of a single Edman degradation of the peptides in Problem What would be the outcome of reaction of gramicidin S with phenyl isothiocyanate (Edman degradation)? (int: With which functional group does this substance react?) 52. The polypeptide bradykinin is a tissue hormone that can function as a potent pain-producing agent. y means of a single treatment with the Edman reagent, the -terminal amino acid in bradykinin is identified as rg. Incomplete acid hydrolysis of the intact polypeptide causes random cleavage of many bradykinin molecules into an assortment of peptide fragments that includes rg-pro-pro- Gly, Phe-rg, Ser-Pro-Phe, and Gly-Phe-Ser. Complete hydrolysis followed by amino acid analysis indicates a ratio of 3 Pro, 2 Phe, 2 rg, and one each of Gly and Ser. Deduce the amino acid sequence of bradykinin. 53. The amino acid sequence of met-enkephalin, a brain peptide with powerful opiatelike biological activity, is Tyr-Gly-Gly-Phe-Met. What would be the products of step-by-step Edman degradation of met-enkephalin?

4 Problems Chapter The peptide shown in Problem 42(d) is leu-enkephalin, a relative of met-enkephalin with similar properties. ow would the results of Edman degradation of leu-enkephalin differ from those of met-enkephalin? 54. Secreted by the pituitary gland, corticotropin is a hormone that stimulates the adrenal cortex. Determine its primary structure from the following information. (i) ydrolysis by chymotrypsin produces six peptides: rg-trp, Ser-Tyr, Pro-Leu-Glu-Phe, Ser-Met-Glu-is-Phe, Pro-sp-la- Gly-Glu-sp-Gln-Ser-la-Glu-la-Phe, and Gly-Lys-Pro-Val-Gly-Lys-Lys-rg-rg-Pro-Val- Lys-Val-Tyr. (ii) ydrolysis by trypsin produces free lysine, free arginine, and the following five peptides: Trp-Gly-Lys, Pro-Val-Lys, Pro-Val-Gly-Lys, Ser-Tyr-Ser-Met-Glu-is-Phe-rg, and Val-Tyr-Pro-sp-la-Gly-Glu-sp-Gln-Ser-la-Glu-la-Phe-Pro-Leu-Glu-Phe. 55. Propose a synthesis of leu-enkephalin [see Problem 42(d)] from the component amino acids. 56. The following molecule is thyrotropin-releasing hormone (TR). It is secreted by the hypothalamus, causing the release of thyrotropin from the pituitary gland, which, in turn, stimulates the thyroid gland. The thyroid produces hormones, such as thyroxine, that control metabolism in general. C 2 CCCCCC 2 Pyroglutamic acid istidine Prolinamide The initial isolation of TR required the processing of 4 tons of hypothalamic tissue, from which 1 mg of the hormone was obtained. eedless to say, it is a bit more convenient to synthesize TR in the laboratory than to extract it from natural sources. Devise a synthesis of TR from Glu, is, and Pro. ote that pyroglutamic acid is the lactam of Glu and may be readily obtained by heating Glu to 1408C. 57. (a) The structures illustrated for the four D bases (Section 26-9) represent only the most stable tautomers. Draw one or more alternative tautomers for each of these heterocycles (review tautomerism, Sections 13-7 and 18-2). (b) In certain cases, the presence of a small amount of one of these less stable tautomers can lead to an error in D replication or mr synthesis due to faulty base pairing. ne example is the imine tautomer of adenine, which pairs with cytosine instead of thymine. Draw a possible structure for this hydrogen-bonded base pair (see Figure 26-11). (c) Using Table 26-3, derive a possible nucleic acid sequence for an mr that would code for the five amino acids in met-enkephalin (see Problem 53). If the mispairing described in (b) were at the first possible position in the synthesis of this mr sequence, what would be the consequence in the amino acid sequence of the peptide? (Ignore the initiation codon.) 58. Factor VIII is one of the proteins participating in the formation of blood clots. defect in the gene whose D sequence codes for Factor VIII is responsible for classic hemophilia. Factor VIII contains 2332 amino acids. ow many nucleotides are needed to code for its synthesis? 59. In addition to the conventional 20 amino acids in Tables 26-1 and 26-3, two others, selenocysteine (Sec) and pyrrolysine (Pyl), are incorporated into proteins using the nucleic acid based cell machinery described in Section The three-base codes for Sec and Pyl are UG and UG, respectively. These codes normally serve to terminate protein synthesis. owever, if they are preceded by certain specific base sequences, they instead cause incorporation of these unusual amino acids and continued growth of the peptide chain. Pyl is rare, occurring only in some ancient bacteria. Sec, containing the side chain C 2 Se, is widespread; in fact, it occurs in at least two dozen human proteins that rely on the reactivity of the essential trace element selenium for their function. The pk a for the Se group in Sec is 5.2. (For comparison, the pk a for the S in Cys is 8.2.) (a) Draw the structure of Sec at p 7; compare it with that of Cys at the same p. (b) Determine pi for Sec. (c) Given the relationship between sulfur and selenium in the periodic table, and the pk a differences between S and Se, how do you expect the chemical reactivities of Sec and Cys to compare? 60. droxyproline (yp), like many other amino acids that are not officially classified as essential, is nonetheless a very necessary biological substance. It constitutes about 14% of the amino acid content of the protein collagen. Collagen is the main constituent of skin and connective tissue. It is also present, together with inorganic substances, in nails, bones, and teeth. (a) The systematic name for hydroxyproline is (2S,4R)-4-hydroxyazacyclopentane-2-carboxylic

5 1268 Chapter 26 mino cids, Peptides, Proteins, and ucleic cids acid. Draw a stereochemically correct structural formula for this amino acid. (b) yp is synthesized in the body in peptide-bound form from peptide-bound proline and 2, in an enzymecatalyzed process that requires vitamin C. In the absence of vitamin C, only a defective, yp-deficient collagen can be produced. Vitamin C deficiency causes scurvy, a condition characterized by bleeding of the skin and swollen, bleeding gums. In the following reaction sequence, an efficient laboratory synthesis of hydroxyproline, fill in the necessary reagents (i) and (ii), and formulate detailed mechanisms for the steps marked with an asterisk. ClC 2 i C 2 a C(CC 2C 3) 2* CC 2 C 3 C 2 S 2Cl 2 Cl CC ii* C 2 C 3 2 hydroxyproline (c) Gelatin, which is partly hydrolyzed collagen, is rich in hydroxyproline and, as a result, is often touted as a remedy for split or brittle nails. Like most proteins, however, gelatin is almost completely broken down into individual amino acids in the stomach and small intestine before absorption. Is the free hydroxyproline thus introduced into the bloodstream of any use to the body in the synthesis of collagen? (int: Does Table 26-3 list a three-base code for hydroxyproline?) 61. The biosynthesis of oligosaccharides (Chapter 24) employs the chemical constituents of proteins and nucleic acids as well as carbohydrates. In the example shown, a disaccharide linkage is created between a molecule of galactose and a molecule of -acetylgalactosamine. The galactose (the donor sugar) is carried into the process as a uridine diphospate ester, and the acceptor galactosamine is held in place by a glycoside linkage to the hydroxyl group of a serine residue in a protein. The enzyme galactosyl transferase specifically forms a disaccharide linkage between C1 of the donor and C3 of the acceptor: 6 C P P 3 2 6C C C 2 C 3 2 C Serine Uridine diphosphate galactose C 3 -cetylgalactosamine protein C 2 C 2 C C 3 C C 2 C P P Galactose 1 3 -acetylgalactosamine protein Uridine diphosphate (UDP)

6 Problems Chapter What type of basic mechanistic process does this reaction resemble? Discuss the roles played by the various participants of the reaction. 62. Sickle-cell anemia is an often fatal genetic condition caused by a single error in the D gene that codes for the b chain of hemoglobin. The correct nucleic acid sequence (read from the mr template) begins with UGGUGCCCUGCUCCUGGGGG..., and so forth. (a) Translate this into the corresponding amino acid sequence of the protein. (b) The mutation that gives rise to the sickle-cell condition is replacement of the boldface in the preceding sequence by U. What is the consequence of this error in the corresponding amino acid sequence? (c) This amino acid substitution alters the properties of the hemoglobin molecule in particular, its polarity and its shape. Suggest reasons for both these effects. (Refer to Table 26-1 for amino acid structures and to Figure 26-8C for the structure of myoglobin, which is similar to that of hemoglobin. ote the location of the amino acid substitution in the tertiary structure of the protein.) Team Problem 63. mino acids can be used as enantiomerically pure starting materials in organic synthesis. Scheme I depicts the first steps in the synthesis of a reagent employed in the preparation of enantiomerically pure b-amino acids, such as that occurring in the side chain of taxol (Section 4-7). Scheme II features an ester of the same amino acid for the preparation of an unusual heterocyclic dipeptide used in the study of polypeptide conformations. Scheme I: Synthesis of an enantiomerically pure reagent 2 2 * C 2 K Potassium salt of asparagine C K Six-membered nitrogen heterocyle 1. ac3, Cl 2., 2 C3 C Consider the following questions: 1. There are two diastereomers of formed in a 90 : 10 ratio. The major isomer is the one that can attain the most stable chair conformation. re the two substituents on the ring cis or trans to each other? Label their positions as equatorial or axial. 2. Which nitrogen is nucleophilic and yields the carbamic ester (Section 20-6)? Scheme II: Synthesis of an unusual heterocyclic dipeptide 2 2 * 1,1-Dimethylethyl (tert-butyl) ester of asparagine C C (cyclic compound) Fmoc amino acid chloride D eterocyclic dipeptide Fluorenylmethyloxycarbonyl(Fmoc) amino acid chloride R & Cl The Fmoc protecting group (highlighted in the box) is used instead of Cbz or oc, with which you are familiar, because the amino acid chloride is necessary to make the new amide bond. either the Cbz nor the oc group is stable under these conditions. Consider the following questions: 1. What functional group is generated in C? 2. Where is the peptide bond in D? Circle it. Reconvene to discuss the answers to the questions posed in each scheme and the structures you proposed for D.

7 1270 Chapter 26 mino cids, Peptides, Proteins, and ucleic cids C 2 C 3 Preprofessional Problems 64. Structure (shown in the margin) is that of a naturally occurring a-amino acid. Select its name from the following list. (a) Glycine; (b) alanine; (c) tyrosine; (d) cysteine. 65. The primary structure of a protein refers to: (a) cross-links with disulfide bonds; (b) presence of an a helix; (c) the a-amino acid sequence in the polypeptide chain; (d) the orientation of the side chains in three-dimensional space. 66. Which one of the following five structures is a zwitterion? (a) 2 CC 2 C 2 (b) 2 2 2CC2C2C2 (c) 3 1 C 2 C 2 2 l (d) C 3 (C 2 ) 16 C 2 2 K 1 (e) C i šð i šð l C 67. When an a-amino acid is dissolved in water and the p of the solution adjusted to 12, which of the following species is predominant? (a) RCC (b) RCC (c) RCC (d) RCC ow many stereocenters are present in the small, naturally occurring protein glycylalanylalanine? (a) Zero; (b) one; (c) two; (d) three.