Peptide link formation

Outline a design for a combinatorial synthesis for the formation of a combinatorial library of nine compounds with the general formula B using the Furka mix and split method. Outline any essential practical details. Details of the chemistry of peptide link formation are not required it is sufficient to say that it is formed. 

Use has been made of peptide link formation when making artificial molecules like Nylon. One type of Nylon can be made by the condensation of a dicarboxylic acid and a di-amine (Figure 7.3.10). 

Figure 1.4 Schematic of peptide link formation, central virtual bond of polypeptide and protein backbones.

Figure 1.84 Schematic of translation. The mRNA codons are read and converted from nucleoside sequences to protein primary structure by means of cognate aminoacyl-tRNAs. All mRNA codons are translated at a ribosome (prepared from rRNA) that has two cognate aminoacyl-tRNA binding sites P (peptidyl) and A (aminoacyl). All tRNAs are "adaptors" that can bind a particular mRNA codon through their anticodon loop, using Watson-Crick base pairing, and also associate covalently with the appropriate amino acid residue coded for by the corresponding mRNA codon When two cognate aminoacyl-tRNA molecules bind mRNA in P and A sites (a), then both are close enough for peptide link formation to take place with the emergence of a peptide chain (b). As amino acyl tRNA molecules continue to dock sequentially onto mRNA codons (in the direction 5 (c), and amino acid residues continue to be added (W —> C ) (d),...

Furthermore, coupling (peptide link formation) need to be performed under very carefully controlled conditions, otherwise a-carbon racemisation becomes a serious problem, especially during coupling. This is so because, each selected N-terminal protected amino acid... 

Figure 2.1 Peptide link formation. L-a-Amino adds are rich in reactive functional groups. Therefore, chemo-selective peptide Link formation is not possible without protecting groups. A future A/-terminal residue must be a-A/-protected (Pi) (with optional P3 side chain protection) while future C-terminal residue must be a-C protected (P2) (with optional P4 side chain protection). The protected A/-terminal residue must then be activated to allow peptide link formation to take place. Global deprotection reveals a dipeptide product.

Problems in solution phase peptide synthesis have been largely overcome by the development of solid phase peptide synthesis (SPPS). The chemical principles of peptide link formation and peptide synthesis remain the same, but in SPPS the growing peptide chain is anchored to a solid phase resin, thereby easing the iterative process of peptide bond formation, removing the need for crystallisations and purifications after each step of the synthesis, and in some ways simplifying protection/deprotection problems. SPPS earned Bruce Merrifield a Nobel Prize in 1986, and has eased the technical challenges of peptide synthesis to the extent that the process can now be automated. 

Figure 2.2 Modern solid phase peptide synthesis. Process begins with a-N terminal Fmoc deprotection of resin bound C-terminal amino acid residue with piperidine (mechanism illustrated). Peptide link formation follows (typical solvent Al-methylpyrrolidone [NMP]) by carboxyl group activation with dicyclohexylcarbodiimide (DCC) (mechanism illustrated) in presence of hydroxybenzotriazole (HOBt). HOBt probably replaces DCC as an activated leaving group helping to reduce a-racemization during peptide link formation. Other effective coupling agents used in place of DCC/HOBt are HBTU 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate Py-BOP benzotriazole-l-yl-oxy-tns-pyrrolidino-phosphonium hexafluorophosphate. The Process of a-N deprotection, and peptide link formation, continues for as many times as required (n-times), prior to global deprotection and resin removal.

Many medicinally useful peptides have cyclic structures. Cyclization may result if the amino acids at the two termini of a linear peptide link up to form another peptide bond. Alternatively, ring formation can very often be the resnlt of ester or amide linkages that utilize side-chain functionalities (CO2H, NH2, OH) in the constituent amino acids. 

Puromycin, made by the mold Streptomyces al-boniger, is one of the best-understood inhibitory antibiotics. Its structure is very similar to the 3 end of an aminoacyl-tRNA, enabling it to bind to the ribosomal A site and participate in peptide bond formation, producing peptidyl-puromycin (Fig. 27-31). However, because puromycin resembles only the 3 end of the tRNA, it does not engage in translocation and dissociates from the ribosome shortly after it is linked to the carboxyl terminus of the peptide. This prematurely terminates polypeptide synthesis. 

From tables of standard free energies of formation, we find AG,° = -2885 kj-mol-1. Therefore, the maximum nonexpansion work obtainable from 1.00 mol C6H1206(s) is 2.88 X 103 kj. About 17 kj of work must be done to build 1 mol of peptide links (a link between amino acids) in a protein, so the oxidation of 1 mol (180 g) of glucose can be used to build up to about 170 mol of such links. More visually the oxidation of one glucose molecule is needed to build about 170 peptide links. In practice, biosynthesis occurs indirectly, there are energy losses, and only about 10 such links can be built. A typical protein has several hundred peptide links, so several dozen glucose molecules must be sacrificed to build one protein molecule. 

Amino acids and the structure of the polypeptide chain. Polypeptides are composed of L-amino acids covalently linked together in a sequential manner to form linear chains, (a) The generalized structure of the amino acid. The zwitterion form, in which the amino group and the carboxyl group are ionized, is strongly favored. (b) Structures of some of the R groups found for different amino acids, (c) Two amino acids become covalently linked by a peptide bond, and water is lost, (d) Repeated peptide bond formation generates a polypeptide chain, which is the major component of all proteins. 

CD) in comparison with the star shaped analog 43. They also showed that 42 is incorporated easily into phospholipid bilayers. On the other hand, the same group reported the preparation of the coiled coil structure 44 with two amphiphilic 14-residue peptides linked to a bipyridyl template [37], The incorporation of a fluorescent probe, pyrenyl-L-alanine, near the supercoiling region helped them to demonstrate by fluorescence the formation of the proposed structure in water. 

Each molecule (molecular weight 30000) contains one zinc(II) atom, which is (approximately) tetrahedrally coordinated to two N atoms and one O atom from amino-acid residues plus a water molecule. The structures of both the enzyme and some enzyme-substrate complexes have been carefully studied and the detailed mechanism of the hydrolysis is now quite well understood. Without going into details, a crucial factor appears to be the pronounced distortion from regular tetrahedral coordination about the Zn(II), apparently imposed by the conformational requirements of the polypeptide chain. The conflict of interest between the needs of the Zn(II) atom - which, when four-coordinate, always assumes tetrahedral coordination - and the ligands induces an entatic state, a condition of strain and tension which enhances the reactivity at the active site. The Zn atom binds the substrate peptide via the O atom of the —CONH— peptide link, and the entatic state of the free enzyme facilitates formation of the enzyme-substrate complex. 

If individually positioned residues are in close contact, the program evaluates the possibility of peptide-bond formation and thus links single residues to a peptide. Furthermore, ligand-independent extension (i.e. residues without direct contact to vectors) of the pseudoreceptor can be used in order to complete the peptidic receptor site (e.g. entirely closed shell around the ligand molecules). 

Preliminary results show that pentaco-ordinate phosphoranes are practical reagents for the formation of the peptide link. Thus, application of the phosphorane (3b) (7) in the stringent Izumiya test (14)... 

Peptide bond formation the second step, peptide bond formation, is catalyzed by peptidyl transferase, part of the large ribosomal subunit. In this reaction the carboxyl end of the amino acid bound to the tRNA in the P site is uncoupled from the tRNA and becomes joined by a peptide bond to the amino group of the amino acid linked to the tRNA in the A site (Fig. 5). A protein with peptidyl transferase activity has never been isolated. The reason is now clear in E. coli at least, the peptidyl transferase activity is associated with part of the 23S rRNA in the large ribosomal subunit. In other words, peptidyl transferase is a ribozyme, a catalytic activity that resides in an RNA molecule (see also Topic G9). 

The phenanthridine alkaloid lycorine (narcissine, galanthidine) (MD—Phe G5N C6) has a widespread occurrence and inhibits protein synthesis. Like lycorine, the structurally similar alkaloids dihydrolycorinine, haemanthamine, narciclasine, pretazettine and pseudolycorine also inhibit protein synthesis at the level of peptide bond formation. Galanthamine (lycorimine) (Phe C6N C40 C6 ), from daffodil bulbs but also of widespread occurrence, is both a nACh-R allosteric modulator and an inhibitor of AChE. Galanthamine is clinically employed in the treatment of Alzheimer s disease (dementia linked to deficiency in acetylcholine-mediated signalling in the central nervous system). 

Biotin is bound covalently to enzymes by a peptide link to the e-amino group of a lysine residue, forming biotinyl-e-amino-lysine or biocytin (see Figure 11.1). This postsynthetic modification is cattdyzed by holocarboxylase synthetase with the intermediate formation of biotinyl-5 -AMP. In bacteria, this intermediate tdso acts as a potent repressor of till four enzymes of biotin synthesis. 

To produce a carboxyamide peptide The peptide will be linked to the modified Rink linker via an amide bond. The attachment of the first residue can be carried out under conditions for peptide bond formation (e.g., with TBTU) by using the activation procedures described in Subheading 3.3.2,2, (Methods A-E) of this chapter. Do not forget to deprotect the linker before coupling of the amino acid. 

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