Peptide production

These methodologies have been reviewed (22). In both methods, synthesis involves assembly of protected peptide chains, deprotection, purification, and characterization. However, the soHd-phase method, pioneered by Merrifield, dominates the field of peptide chemistry (23). In SPPS, the C-terminal amino acid of the desired peptide is attached to a polymeric soHd support. The addition of amino acids (qv) requires a number of relatively simple steps that are easily automated. Therefore, SPPS contains a number of advantages compared to the solution approach, including fewer solubiUty problems, use of less specialized chemistry, potential for automation, and requirement of relatively less skilled operators (22). Additionally, intermediates are not isolated and purified, and therefore the steps can be carried out more rapidly. Moreover, the SPPS method has been shown to proceed without racemization, whereas in fragment synthesis there is always a potential for racemization. Solution synthesis provides peptides of relatively higher purity however, the addition of hplc methodologies allows for pure peptide products from SPPS as well. 

Fig. 1. Schematic drawing of precursors for selected brain oligopeptides. Shaded areas represent the location of sequences of active peptide products which are normally cleaved by trypsin-like enzymes acting on double-basic amino acid residues. Precursors are not necessarily drawn to scale, (a) CRF precursor (b) proopiomelanocortin (POMC) (c) P-protachykinin (d) proenkephalin A (e) CGRP precursor (f) preprodynorphin, ie, preproenkephalin B. Terms are...

Inhibitors as well as substrates bind in this crevice between the domains. From the numerous studies of different inhibitors bound to serine pro-teinases we have chosen as an illustration the binding of a small peptide inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH to a bacterial chymotrypsin (Figure 11.9). The enzyme-peptide complex was formed by adding a large excess of the substrate Ac-Pro-Ala-Pro-Tyr-CO-NHz to crystals of the enzyme. The enzyme molecules within the crystals catalyze cleavage of the terminal amide group to produce the products Ac-Pro-Ala-Pro-Tyr-COOH and NHs. The ammonium ions diffuse away, but the peptide product remains bound as an inhibitor to the active site of the enzyme. 

Breakdown of the amide dihydrate occurs by a mechanism similar to its formation. The ionized aspartate carboxyl (Asp in Figure 16.27) acts as a general base to accept a proton from one of the hydroxyl groups of the amide dihydrate, while the protonated carboxyl of the other asparate (Asp in this case) simultaneously acts as a general acid to donate a proton to the nitrogen atom of one of the departing peptide products. 

These procedures illustrate the use of N-ethyl-5-phenylisoxazolium-3 -sulfonate as a reagent for peptide synthesis.2-3 Procedure A is recommended for peptides that are not soluble in either organic solvents or in water. Procedure B illustrates the formation of a peptide that is soluble both in organic solvents and in water. Por peptides that are soluble in organic solvents and insoluble in water, the submitters recommend the use of Procedure B, except that the peptide product may be recovered directly from its solution in ethyl acetate after this organic solution has been washed successively with aqueous 5% sodium bicarbonate, water, aqueous 1 M hydrochloric acid, and water. Table I summarizes the preparation of various peptides by these procedures. Some more complex examples from other laboratories are listed elsewhere.2b... 

Characterization of peptide substrates led to the observation that the N-terminal peptide product was able to inhibit the enzyme (Llinas-Brunet et al. 1998 Steinkuhler... 

The third prohormone from which opioid peptides are derived is pro-opiomelanocortin, which yields a number of nonopioid and opioid peptide products (O Donohue and Dorsa 1982). Of these products, beta-endorphin, an untriakontapeptide isolated from camel pituitary gland by Li and Chung (1976)) is thought to interact primarily with mu and delta receptors. 

Bihel, F. Quelever, G. Lelouard, H. Petit, A. Alves de Costa, C. Pourqie, O. Checler, F. Thellend, A. Pierre, P Kraus, J.-L. Synthesis of new 3-alkoxy-7-amino-4-chloro-isocoumarin derivatives as new beta-amyloid peptide production inhibitors and their activities on various classes of protease. Bioorg. Med. Chem. 2003, 11, 3141-3152. 

Calcitonin and other peptide products of the calcitonin gene are known to be el-... 

Figure 12.3 M ALDI analysis of peptides from Bacillus subtilis sp. 168 vegetative cells prepared in situ.87 (a) Survey spectrum of peptide products. Protein assignments are listed in the figure, (b) Spectrum of product ions of unimolecular decomposition of the peptide with m z 2606.

FIGURE 1 8-5 Tissue-specific processing of the pro-opiomelanocortin (POMC) precursor yields a wide array of bioactive peptide products. Processing of the POMC precursor varies in various tissues. In anterior pituitary, adrenocorticotropic hormone (ACTH (1-39)) and P-1 ipo tropin (P-LPH) are the primary products of post-translational processing. Arcuate neurons produce the potent opiate P-endorphin (P-endo (1-31)) as well as ACTIK1 -13) NIT,. Intermediate pituitary produces a-melanocyte-stimulating hormone (aMSH), acetylated P endof 1 31) and P-endo(l-27). NTS, nucleus tractus solitarius. 

Melanotropes and CNS neurons making POMC express both PCI and PC2 and, thus, the smaller peptide products... 

Similarly, five closely related melanocortin receptors that respond to various peptides derived from the POMC precursor have been identified (Fig. 18-7) [24]. As expected, the receptor on adrenal cortical cells responds best to ACTH, which normally stimulates adrenal steroidogenesis, and the receptor on melano cytes responds best to aMSH, which causes skin darkening. However, the pattern of melanocortin receptor expression in the brain is not simply explained by the known patterns of peptide expression in the brain or by the known effects of POMC-derived peptides when applied to various brain regions. With this number of peptide receptors, it is obvious that production of final peptide products must be precisely controlled and that different biosynthetic processing pathways can dramatically affect the biological activity observed (Figs 18-5,18-7). 

Edbauer, D., Winkler, E., Haass, C. and Steiner, H. Presenilin and nicastrin regulate each other and determine amyloid beta- peptide production via complex formation. Proc. Natl Acad. Sci. USA 99 8666-8671, 2002. 

Co(III)-chelated amino acid ester reactant and/or peptide product (Scheme 1). This basic difficulty was quickly pointed out (5), and has subsequently been examined and commented upon by others (6, 7). Such criticisms are well-founded since epimerization (or racemization) is a common problem, at least to some degree, in all chemical methods of synthesis where acyl-activation is employed. As a result, metal-activation methods have received little attention. However, since 1981 we have refined the Co(III) method such that very fast, clean, couplings can now be carried out using A-[Co(en)2((S)-AAOMe)]3+ reagents, which involve minimal (<2%) epimerization/racemization provided experimental conditions are strictly adhered to. 

Additives increase efficiency in carbodiimide-mediated reactions by preventing intermediates from undergoing side reactions and by transforming them into activated esters that become the precursors of the peptide products. A-hydroxysuccinimide and 4-hydroxy-3-oxo-3,4-dihydrobenzotriazine are the best nucleophiles or acceptors of activated species. They trap oxazolones before they have time to isomerize. The popularity of HONSu as an additive has diminished considerably during the... 

The two hydrolytic reactions of /V-(phenylacetyl)glycyl-D-valine (6.47) yield Gly-D-Val (6.48) and D-Val (6.49), respectively, as peptide products. The pH-rate profiles of the reactions at 37° (Table 6.4) were practically iden-... 

In neutral and alkaline solutions, the iso-Asp/Asp-peptide products were always formed in a ca. 3 1 ratio. In the Val-Xaa-Asn-Ser-Val series, the nature of the N-flanking residue had little effect on the rate of deamidation, regardless of size. However, the actual effect may have been partly masked by cleavage of the Asn-Ser bond (Sect. 6.3.2A), which proceeds at a rate ca. 10% that of deamidation. In the Val-Ser-Asn-Xaa-Val series, the nature of the C-flanking residue did have a major influence. As expected, the most unstable peptide was Val-Ser-Asn-Gly-Val (tll2 ca. 6 h at pH 7.3 and 60°). A second group of peptides had t1/2 values in the range of 25-75 h, which increased in the order Xaa=His[Pg.325]

The protein kinase mechanism uses aspartate as a general base, weakening the hydrogen—oxygen bond of the hydroxyl-containing residues. This allows the hydroxyl nucleophile of the substrate to attack the 7-phosphate of ATP, producing a phosphorylated peptide product and ADP (Scheme 2). 

Structural characterization of NRPSs has yielded significant insight into the enzymology of these complex biosynthetic machines and has provided a framework for engineering these systems toward novel function. As summarized in this section, X-ray crystal and NMR structures have been determined for both individual NRPS domains and multidomain constructs. Overall, these studies support a monomeric structure for NRPS assembly line units where significant domain motion is necessary to allow participation of the various active sites in the chemistry leading to peptide products. 

Wong, G.T., Manfra, D., Poulet, F.M., et al. (2004) Chronic treatment with the y-secretase inhibitor LY-411,575 inhibits P-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation. J. Biol. Chem., 279, 12876-12882. 

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