Prolyl-proline

Preparative Methods reduction of A(-[A((-benzyloxycarbo-nyl)-prolyl]proline methyl ester with Lithium Aluminum Hydride affords the title reagent in 81% yield. 

All diamino alcohols (2a-g) were derived from N-Z-(S)-proline via the intermediate N-[(N-benzyloxycarbonyl)prolyl]proline methyl ester (4). The diamino alcohol 2a WHS obtained by the reduction of 4 with LiAlH, and 2b-e were obtained by the reaction of 4 with the corresponding Grignard reagents. Diamino alcohols 2f-g and triamino alcohol 5 were obtained by the following sequence of reactions ... 

Kemp, D.S. and Curran, T.P. (1988) (2S,5S,8S.llS)-l-acetyl-1.4-diaza-3-keto-5-carboxy-10-thia-tricyclo-[2.8.0 ]-tridecane, 1, synthesis of prolyl-proline-derived, peptide-functionalized templates for a-helix formation. Tet. Lett. 29 4931-4934. 

Further difficulties must be expected if the amine component contributes to an already existing steric effect. For instance, in the attempted preparation of prolyl-proline via a hydroxysuccinimide ester, attack on the activated ester carbonyl is accompanied by an attack on one of the succinimide carbonyl groups ... 

The synthesis described met some difficulties. D-Valyl-L-prolyl resin was found to undergo intramolecular aminoiysis during the coupling step with DCC. 70< o of the dipeptide was cleaved from the polymer, and the diketopiperazine of D-valyl-L-proline was excreted into solution. The reaction was catalyzed by small amounts of acetic acid and inhibited by a higher concentration (protonation of amine). This side-reaction can be suppressed by adding the DCC prior to the carboxyl component. In this way, the carboxyl component is "consumed immediately to form the DCC adduct and cannot catalyze the cyclization. 

Other interesting examples of proteases that exhibit promiscuous behavior are proline dipeptidase from Alteromonas sp. JD6.5, whose original activity is to cleave a dipeptide bond with a prolyl residue at the carboxy terminus [121, 122] and aminopeptidase P (AMPP) from E. coli, which is a prohne-specific peptidase that catalyzes the hydrolysis of N-terminal peptide bonds containing a proline residue [123, 124]. Both enzymes exhibit phosphotriesterase activity. This means that they are capable of catalyzing the reaction that does not exist in nature. It is of particular importance, since they can hydrolyze unnatural substrates - triesters of phosphoric acid and diesters of phosphonic acids - such as organophosphorus pesticides or organophosphoms warfare agents (Scheme 5.25) [125]. 

Very few post-translational modifications have been found on tropoelastin. However, hydroxylation of 25% of the proline residues is observed [10]. The enzymatic modification of proline to hydroxyproline (Hyp) is performed by prolyl hydroxylase [11]. The purpose of this hydroxylation remains unclear and it is even proposed that Hyps in tropoelastin are a by-product of collagen hydroxylation as this occurs in the same cellular compartment [8]. 

Figure 28-11. The prolyl hydroxylase reaction. The substrate is a proline-rich peptide. During the course of the reaction, molecular oxygen is incorporated into both succinate and proline. Lysyl hydroxylase catalyzes an analogous reaction.

The a-ketoacid-dependent enzymes are distinguished from other non-haem iron enzymes by their absolute requirement for an a-ketoacid cofactor as well as Fe(II) and O2 for activity. They catalyse two types of reaction (Table 2.3), hydroxyla-tion and oxidation. In both, the a-ketoglutarate is decarboxylated and one oxygen atom introduced into the succinate formed in the hydroxylases, the other oxygen atom is introduced into the substrate, while in the oxidases it is found in water, together with the cyclized product. In general these enzymes require one equivalent of Fe(II) an a-ketoacid, usually a-ketoglutarate and ascorbate. Examples of these enzymes include proline 4-hydroxylase, prolyl and lysyl hydroxylase, which... 

The formation of 0-seryl or 0-prolyl esters (Figure 1) of certain N-hydroxy arylamines has been inferred from the observations that highly reactive intermediates can be generated in vitro by incubation with ATP, serine or proline, and the corresponding aminoacyl tRNA synthetases (11,12,119). For example, activation of N-hydroxy-4-aminoquinoline-l-oxide (119,120), N-hydroxy-4-aminoazobenzene (11) and N-hydroxy-Trp-P-2 (121) to nucleic acid-bound products was demonstrated using seryl-tRNA synthetase from yeast or rat ascites hepatoma cells. More recently, hepatic cytosolic prolyl-, but not seryl-, tRNA synthetase was shown to activate N-hydroxy-Trp-P-2 (12) however, no activation was detectable for the N-hydroxy metabolites of AF, 3,2 -dimethyl-4-aminobiphenyl, or N -acetylbenzidine (122). 

Once it is part of a cyclic dipeptide, the prolyl residue becomes susceptible to enantiomerization by base (see Section 7.22). The implication of the tendency of dipeptide esters to form piperazine-2,5-diones is that their amino groups cannot be left unprotonated for any length of time. The problem arises during neutralization after acidolysis of a Boc-dipeptide ester and after removal of an Fmoc group from an Fmoc-dipeptide ester by piperidine or other secondary amine. The problem is so severe with proline that a synthesis involving deprotection of Fmoc-Lys(Z)-Pro-OBzl produced only the cyclic dipeptide and no linear tripeptide. The problem surfaces in solid-phase synthesis after incorporation of the second residue of a chain that is bound to the support by a benzyl-ester type linkage. There is also the added difficulty that hydroxymethyl groups are liberated, and they can be the source of other side reactions. 

Another important 2-OG dependent oxygenase in mammals is prolyl-4 hydroxylase, which catalyzes the hydroxylation of the proline residue in collagen (Scheme 5). This reaction is essential for the structure of the collagen triple helices (9,34 6). An overproduction of collagen is related to fibrotic diseases such as rheumatic arthritis. Thus collagen prolyl-4 hydroxylase is a target for therapeutics (34,36). 

Prolyl 4-hydroxylation is the most abundant posttranslational modification of collagens. 4-Hydroxylation of proline residues increases the stability of the triple helix and is a key element in the folding of the collagen triple helix. " In vertebrates, almost all the Yaa position prolines of the Gly-Xaa-Yaa repeat are modified to 4(I( )-hydroxylproline by the enzyme P4H (EC 1.14.11.2), a member of Fe(II)- and 2-oxoglutarate-dependent dioxygenases. This enzyme is an 0 2/ b2-type heterotetramer in which the / subunit is PDI (EC 5.3.4.1), which is a ubiquitous disulfide bond catalyst. The P4H a subunit needs the 13 subunit for solubility however, the 13 subunit, PDI, is soluble by itself and is present in excess in the ER. Three isoforms of the a subunit have been identified and shown to combine with PDI to form [a(I)]2/ 2) [< (II)]2/32> or [a(III)]2/32 tetramers, called the type... 

Disulfide bond formation within the individual propeptides precedes folding and trimers are then formed by association of the C-terminal propeptides." Disulfide bonds between the chains are then formed and this formation is most likely catalyzed by PDI." As triple helix formation proceeds, the rate-limiting step in this process is the cis—trans isomerization of peptidyl-Pro bonds. This process can be catalyzed by peptidyl-prolyl cis—trans isomerases (cyclophilins and FKBPs). This activity is required to convert the proline residues to the trans form required for triple helix formation." " " ... 

Selected prolines and lysines are hydroxylated by prolyl and lysyl hydroxylases. These enzymes, located in the RER, require ascorbate (vitamin C), deficiency of which produces scurvy. 

So what does this magical molecule do Actually, it does two things, one rather more crystalline clear than the other. The crystal clear thing that ascorbic acid does is act as coenzyme for an enzyme known as prolyl hydroxylase. This enzyme catalyzes the conversion of the amino acid proline to hydroxyproline, a major, if exotic, amino acid in the structural protein collagen ... 

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