L-Norleucine

One example of a naturally occurring diazirine, duazomycin A (137 Scheme 11.20), has been reported, isolated in 1985 from a Streptomyces species during a screen for herbicidal compounds [196], It was fotind to inhibit de novo starch synthesis and it was suggested that this is due to direct inhibition of protein synthesis. Duazomycin A is structurally related to 6-diazo-5-oxo-L-norleucine (138), also reported as a natural product from Streptomyces [197], which acts as a glutamine antagonist and inhibits purine biosynthesis [198],... 

Fig. 34.—Fixation of o-Norleucine (Sweet) and L-Norleucine (Bitter) in Rectangular Coordinates in Allowed and Forbidden Positions. ...

An alternative to extraction crystallization is used to obtain a desired enantiomer after asymmetric hydrolysis by Evonik Industries. In such a way, L-amino acids for infusion solutions or as intermediates for pharmaceuticals are prepared [35,36]. For example, non-proteinogenic amino acids like L-norvaline or L-norleucine are possible products. The racemic A-acteyl-amino acid is converted by acylase 1 from Aspergillus oryzae to yield the enantiopure L-amino acid, acetic acid and the unconverted substrate (Figure 4.7). The product recovery is achieved by crystallization, benefiting from the low solubility of the product. The product mixture is filtrated by an ultrafiltration membrane and the unconverted acetyl-amino acid is reracemized in a subsequent step. The product yield is 80% and the enantiomeric excess 99.5%. 

Nitrogen can be transferred from glutamine into many other substrates.102 Several antibiotic analogs of glutamine have been useful in studying these processes. Examples are the streptomyces antibiotics L-azaserine and 6-diazo-5-oxo-L-norleucine (DON). 

The oxidation of L-norleucine, L-leucine, L-isoleucine, and r.-r-leucine with permanganate in strong acid medium showed an autocatalysis by Mn(II), except in the case of L-leucine. For the autocatalytic activity to initiate, a certain concentration of Mn(II) is required. Moreover, the autocatalytic phenomenon vanishes in concentrations of sulfuric acid that are greater than 4.3 mol dm-3. The oxidation showed a good correlation in a biparametric equation, with p = -4.57 and Sc = 2.23 at 318 K.43... 

Chemical Name L-Norleucine, 6-diazo-N-(6-diazo-N-L-y-glutamyl-5-oxo-L-norleucyl)-5-oxo-... 

A short-chain L-amino acid, e.g. L-alanine (sweet) can occupy the positions p (+) p (-) a (+x, -y, -z), whereas L-norleucine (bitter) in stretched conformation cannot do so because of forbidden positions (Fig.10), altough it can occupy p (+) a (+x, -y, +z) after 60° rotation about the y-axis (Fig.10). This rotation about the y-axis is only one possibility, for with monopolar occupation, where fixation of the origin is no longer meaningful, rotation about other axes is also possible, so that this situation must be generally denoted as p (+) a (+x, -y, z). 

Figure 10. Fixation of sweet and bitter compounds in rectangular coordinates— D-norleucine and L-norleucine in allowed and forbidden positions...

The present study deals first with the papain-catalyzed attachment of lipophilic L-norleucine n-dodecyl ester to a hydrophilic protein. To determine more clearly the mode of reaction, well-defined asi-casein was selected as the protein for this purpose. 

Primary Mode of Reaction and Consequences (9). Bovine a8i-casein (Variant B) was prepared from fresh milk (12) and immediately succinylated to increase water dispersibility (13). This succinylated asi-casein preparation, molecular weight of ca. 25,000 daltons, was used as the hydrophilic protein substrate. The compound L-norleucine 1-13C-dodecyl ester, prepared from K13CN and 1-bromoundecane through four steps (14,15,16), was used as the lipophilic nucleophile. C-13 NMR measurements showed that this sample gave only one signal at a distance of 65.2 ppm from the signal of tetramethylsilane (TMS). 

The enzymatic process was carried out under the following conditions medium, 20% (v/v) acetone in 1M carbonate (pH 9) containing lOmAf 2-mercaptoethanol concentration of succinylated < si-casein in medium, 20% (w/w) concentration of L-norleucine l-13C-dodecyl ester in medium, 0.25M concentration of papain (recrystallized) in medium, 0.02% (w/w) incubation temperature, 37°C and incubation time up to 60 min. For further details refer to the published papers (7,8). 

Another experiment was carried out with the 5-min, 15-min, and 30-min incubation mixtures in order to determine the correlation between their content of the 20,000-dalton product and the emulsifying activity. The result was that the emulsifying activity increased in accordance with the formation of this polypeptide (see Table II). No increase in the emulsifying activity results when succinylated asi-casein is incubated with papain in the absence of L-norleucine n-dodecyl ester. 

All of the data from Table II indicate that, under the unconventional conditions intentionally set in the present study, papain can catalyze the aminolysis of the ES intermediate that probably occurs from succinylated asi-casein (substrate) and papain (enzyme) by L-norleucine n-dodecyl ester (nucleophile), with formation of a surface-active 20,000-dalton product to which this lipophilic nucleophile is attached covalently as illustrated in Figure 1. The observed amphiphilic function of the 20,000-dalton product is probably a result of the formation of a localized hydro-... 

Table I. Properties of a 20,000-Dalton Polypeptide Formed from Succinylated asi-Casein by Treatment with Papain in the Presence of L-Norleucine l-13C-Dodecyl Ester...

An instructive experiment is to look at three polymers with rather similar side chains poly(L-leucine), poly(L-norleucine), and poly(L-methionine) to compare their surface properties. Finally as an example of extreme side chain flexibility we consider poly(y-n-decyl-L-glutamate), which while quite unlike any protein (more like fat bacon), its surface properties are of some interest and perhaps help us to understand the properties of more protein-like molecules. 

Poly ( L-leucine), Poly ( L-norleucine), and Poly ( L-methionine). 

Structure of the Collapsed Monolayers. IR spectra of specimens prepared from air dried collapsed monolayers were typical of specimens in the a-helical conformation with no indication of any p conformation. Electron diffraction patterns gave a similar result. The patterns for poly-(L-leucine) and poly(L-norleucine) are similar to poly(L-norvaline) (12) with low crystallinity. A strong equatorial reflection at 10.94 0.10 A is observed in poly(L-leucine). If we assume as previously (5) that this is the 100 reflection from a hexagonal cell, the calculated area per residue in the monolayer is 17.3 A, assuming the molecular separation is the same as in the collapsed film. This figure is in agreement with the observed area of 16 A in view of the difficulties encountered in spreading the monolayer. 

Contact Angle Measurements. The angle of contact of 93° for poly(L-norleucine) has been reported previously. Problems of preparing uniform adherent films of poly(L-methionine) and hysteresis effects combined to make measurements of 0 very imprecise it is estimated at 55° 10°. No measurements at all were possible with poly(L-leucine) for similar reasons. 

Discussion. Considering first the pair of polymers poly (L-norleucine) and poly(L-leucine), the marked differences in the pressure-area isotherms (Figure 3) contrast with the closeness in constitution. The results are good support for the view that the more flexible side chain of poly(L-norleucine) will cause the energy of interaction of adjacent molecules to be less sensitive to their relative directions, compared with poly(L-leucine), so that the plateau is flatter. 

From Equations 1 and 2 the calculated values of jpi for poly(L-methionine) and poly (L-norleucine) are 8.5 erg cm (taking W = 14 dyne cm ) and 28. erg cm (5), respectively. The relative magnitudes of these figures may be compared with measurements of the hydropho-bicity of methionine and leucine side chains (relative to glycine) from solubility measurements by Nozaki and Tanford (29) who find energies... 

The value obtained for for poly (L-methionine), 50 erg cm, is significantly higher than the corresponding value of 34 erg cm for poly(L-norleucine). Since for a sufficiently hydrophobic polymer is closely related to the work of cohesion, this would contribute to the markedly higher crystallinity of poly (L-methionine). 

Polymers of the naturally occurring amino acids alanine, leucine, and methionine all show interactions which depend on the relative directions of the backbones. In contrast, poly(L-norleucine) shows less specific interactions clearly for the hydrophobic regions of proteins to function in a precise manner the natural amino acids are most suitable. 

Two other residues which have been modified by affinity labels containing diazo linkages are sulfhydryl groups and histidine. Hartmann (1963) has shown that 6-diazo-5-oxo-L-norleucine (XXII),... 

---