Diketopiperazines, DKP

The cyclic dipeptides or diketopiperazines (DKPs) 6-10 are yet another class of small diffusible signal molecules that has been isolated from the culture supernatants of R aeruginosa, Pseudomonasputida WCS 358 and other Gramnegative bacteria. Their role in cross-talk with AHL-dependent QS has been demonstrated but their physiological function is not known [20,21]. 

Aspartame is relatively unstable in solution, undergoing cyclisation by intramolecular self-aminolysis at pH values in excess of 2.0 [91]. This follows nucleophilic attack of the free base N-terminal amino group on the phenylalanine carboxyl group resulting in the formation of 3-methylenecarboxyl-6-benzyl-2, 5-diketopiperazine (DKP). The DKP further hydrolyses to L-aspartyl-L-phenyl-alanine and to L-phenylalanine-L-aspartate [92]. Grant and co-workers [93] have extensively investigated the solid-state stability of aspartame. At elevated temperatures, dehydration followed by loss of methanol and the resultant cyclisation to DKP were observed. The solid-state reaction mechanism was described as Prout-Tompkins kinetics (via nucleation control mechanism). 

Diketopiperazines (DKPs) are the smallest naturally occurring cyclic peptides. They are folded head-to-tail and conformationally constrained by a six-membered ring with the side chains orientated in a spatially defined manner. The DKP core... 

Studies with tryptophan and tyrosine derivatives and with tryptophan- or tyrosine-containing dipeptides or piperazine-2,5-diones (diketopiperazines, DKP) revealed structural moieties that affect the fluorescence quantum yield of these aromatic amino acids (for a review, see... 

Some peptide derivatives containing, /V-alkyl amino acids, especially Pro or Hyp, have a tendency to undergo cyclization to form piperazine-2,5-diones (diketopiperazines, DKP), or amino acid anhydrides.[149151] Formation of piperazine-2,5-diones is explained by the occurrence of the ds-isomer of the tertiary amide bond (Scheme 28)J19"21 ... 

Aspartame (and its (D,D), (D,L), and (L,D) diastereomers) display double melting points. When recrystallized from aqueous ethanol or water, aspartame melts initially at 190°C, and then solidifies and re-melts at 246-247° C [21]. Other sources give only the 246-7°C value [5]. The explanation for this behavior is that intramolecular self-aminolysis and simultaneous demethanolation of aspartame talks place on initial melting, to yield 3-methylenecarboxyl-6-benzyl-2,5-diketopiperazine (DKP) (C12H14N2O4 = 262.26 g/mole). The latter compound has been reported to melt at 259 1.1° C [22]. 

Peptides have long been recognized as important flavor compounds in processed foods. The taste of peptides per se is veil known in cheeses (1), meat (2), hydrolyzed vegetable protein including soy products T3), cocoa (A, 5) and to a lesser extent in roasted malt (6), corn steep liquor (7) and aged sake (8). Structurally, food peptides can occur as linear protein fragments (1-3, 5), cyclic dimers (diketopiperazines, DKPs)(A, 6-81 and cyclic trimers (7). 

Among the classes of molecules able to form hydrogen-bonding-based tapes, the diketopiperazine (DKP) family now appears to be the most attractive [30]. The molecular packing in this system of crystals is less complex than systems based on CA M or cyclic ureas, because there is only one possible tape. The locations of the 3,6-substituents seems to protect the secondary amide groups from interactions... 

We were able to develop some simple rules in predicting the packing of molecules based on CA M in one dimensional tapes, but not for predicting the three-dimensional packing of these tapes [98,107,145-148]. Conformational polymorphism due to the diphenyl rings, in addition, increased the complexity of the problem in the CAM system. We have found that crystal structures based on diketopiperazines (DKP) are simpler to predict and rationalize than our previous systems, both from observations of existing structures, and from computational methods [30,109]. 

A -protected penultimate amino acid (3) protocol for introduction of the second and third residues when the sequence poses a risk of serious chain loss due to diketopiperazine (DKP) formation (4) stepwise chain elongation using Fmoc protocols to complete on-resin assembly of the desired C-terminal-modified peptide and (5) acidolytic cleavage to release the peptide into solution. 

The sweetener aspartame exists as a hemihydrate (C14H18N2O5 O.5H2O ASH), under ambient conditions. When heated, ASH converted to aspartame anhydrate (ASA), which on further heating decomposed to form a diketopiperazine (DKP) derivative. The XRD patterns of ASH, ASA, and DKP showed pronounced differences (Fig. 12). XRD was used to simultaneously quantify the (i) disappearance of ASH and appearance of ASA in the first reaction and (ii) disappearance of ASA and appearance of DKP in the second reaction. For studying the kinetics of the first reaction, the peaks unique to ASH at 15.9 and 16.4° 20 and the 17.1° 20 peak of ASA were used. For the second reaction, the sum of the integrated intensities of the peaks at 10.2, 11.0, and 11.8° 20 of ASA and the 13.0° 20 peak of DKP were used. While the dehydration of ASH appeared to follow first-order kinetics, the cyclization of ASA was a nucleation-controlled process. Figs. 13 and 14 contain the dehydration and cyclization data at 118 and 180°C respectively. Since the concentrations of the crystalline reactant as well as the product were simultaneously monitored, mass balance calculations of the crystalline phases were possible at each time point. The reaction... 

The formation of diketopiperazines (DKP) is also possible when heating occurs before the ion formation in the mass spectrometric analysis of amino acids. These compounds will further form characteristic ions by the scheme ... 

Another main type of pyrolysis products for peptides includes the diketopiperazines (DKP) and their secondary fragmentation products. The formation of DKP from oligopeptides showed that the generation of DKPs always takes place from neighboring amino acids. The mechanism of DKP formation seems to be the following ... 

This resin (97 R = propyl, = benzyl, R = ethyl -0.10 mmol) was treated with 2 m isobutylamine in DMSO (1 mL, 20 equiv.) for 24 h at 70 °C. The solution was then drained, the resin was washed with CH2CI2 (3 X 10 mL), and the combined filtrate and washings were concentrated in a rotary evaporator. At this stage, only a trace of diketopiperazine (DKP) was detectable, while most of the material remained on the support. The resin was then treated with 95% TFA/5% H2O for 1 h. After filtration, the TFA filtrate was concentrated to afford the desired (crude) product 98 as an oil. 

Scheme 17.3 The formation of diketopiperazine (DKP) not only reduces the total yield but can also lead to increased impurities due to the de-loaded benzyl alcohol s involvement in side reactions.

Table 17.1 Comparison of coupling agents used to suppress the formation of diketopiperazine (DKP).

To begin with, the conformation of the skeleton of cyclic dipeptide, i.e., the diketopiperazine (DKP) ring, will be considered. There are many possible conformations of the DKP ring 8) as shown in Fig. 14. For unsubstituted diketopiperazine, Cyclo-(Gly2) (R = R = H) a planar conformation (A) in crystalline state has been reported 95, 96). In solution, the planar conformation (A) or rapidly-inverting boat conformation (B) (C) may be supposed. This consideration was supported by NMR studies as described below. Monosubstituted diketopiperazines Cyclo-(Gly-X) (R or R = H) can assume two boat conformations, (B) and (C), as well as the planar conformation (A). The C -substituent R or R takes an axial position in (B) and an equatorial or bowsprit position in (C). Kopple and Ohnishi (97) jaroposed the possibility of a twist-boat conformation (D). Rotations around N—C and C —G... 

Diketopiperazine (DKP), a cyclic dipeptide easily formed by the cyclodimerization of amino acid esters. The formation of diketopiperazines is an undesired cycliza-tion reaction by incorporation of the third amino acid by stepwise SPPS. The free amino group of the resin-bound dipeptide can attack the peptide-resin anchorage intramolecularly, resulting in the formation of diketopiperazine [J. R. Spencer etal., Int. J. Pept. Protein Res. 1992, 40, 282]. 

Chromatographic procedures have been developed to separate llsinopril from its principal decomposition product, the diketopiperazine (DKP), a product of intramolecular dehydration. Since there are three optical centers in the molecule, isomers of iisinoprii and its DKP degradate are possible. The sequence of thermal decomposition for iisinoprii to form isomers of DKP is shown in Figure 13. 

Methods for the analysis of lisinopril in dosage forms are summarized in Table VIII. Methods (I), (II) and (III) have been developed to separate lisinopril from its principle degradation product, the SSS diketopiperazine (DKP) and a process impurity APBA (see Section 5.2.1). Method... 

The simplest cyclic peptides are dipeptides (or diketopiperazines), in which the amide linkages must be cis. Crystal structure determinations for a number of cyclic dipeptides have been completed, and the results are summarized in Table I. The diketopiperazine (DKP) ring assumes either a planar, a boat, or... 

In diketopiperazines (DKP-s) both amides are cis, that is the carbonyl oxygen atom and the amide hydrogen are on the same side of the C—N partial double bond. Accordingly, cyclization requires the energy consuming conversion of the more stable transoid to the less stable cisoid form. The energy liberated in the... 

The smallest cyclic peptides built from amino acids are the so-called diketopiperazines (DKP). In recent research, the DKPs and higher functionalized analogs - the thiodiketopiperazines (TDKP) - have become attractive due to their broad biological activity (390). The DKP or TDKP moiety can be found in a great variety of mycotoxins. Both DKPs and TDKPs can, for example, be isolated from Aspergillus, Candida, Chaetomium, Gliocladium, Penicillium, and Verticillium species (7, 390). The most common structural motifs A-D of this class of compounds are depicted in Fig. 10.1. Many of these natural products show C2 symmetry, which means they consist of two identical amino acids (R = R in Fig. 10.1). 

Epipolythiodioxopiperazines (ETPs) belong to a class of secondary metabolite toxins derived from diketopiperazines (DKPs). At least 14 ETPs are known, and most of them are made from a diverse range of filamentous fungi and are characterized by the presence of a disulfide bridge that allows ETPs to cross-link vital proteins via... 

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