A-H-amino acid amides

Scheme 3 Preparation of racemic a-H-amino acid amides by Strecker synthesis.

Figure 8 Resolution of an a-H-amino acid amide by E. coli DH5a/pTrpLAP (A), and P. putida ATCC 12633 (V). Both cells were used in the same cell-to-substrate ratio.

Identically to the enz5miatic resolution process for a-H-amino acid amides by P. putida, we searched for a new biocatalyst for the stereoselective hydrolysis of a,a-disub-stituted amino acid amides (15). Through screening a new biocatalyst Mycobacterium neoaurum ATCC 25795 was obtained that fulfilled the demand for stereoselective hydrolysis [43,44]. 

Analogous to the a-H-amino acid amides, the Strecker synthesis is the most direct way to prepare the disubstituted amino acid amides (15) (Scheme 6). However, in this case the basic hydrolysis of the aminonitrile (14) is hampered by steric interactions and is generally performed in cone. H2SO4 or in HCl-saturated formic acid (both containing 1 eq of water) [45]. 

Enzymatic hydrolysis of the racemic amides by the amino amidase from M, neoaurum affords the (5)-a,a-disubstituted amino acids and the (i )-a,a-disubstituted amino acid amides (15) in almost 100% e.e. at 50% conversion for most a-methyl-substituted compounds E > 200) [43] (see Scheme 7 and Table 7). Only for glycine amides wititi two small substituents the enantioselectivity is decreased for example, for isovaline amide die enantiomeric ratio E = 9 and for (a-Me)allylglycine amide = 40 [44]. Also a-H-amino acid amides are substrates and are hydrolyzed enantioselectively in contrast, however, dipeptides are not hydrolyzed [45]. For all a-methyl-substituted substrates the activity is high. Reactions performed at 5-10 w/w% substrate solutions in water (pH 8, 37 C) with 0.3-1.0 w/w% of freeze-dried biocatalyst are in general completed (i.e., 50% conversion) after 5-48 h. Increasing the size of the small substituent to ethyl, propyl, or allyl dramatically reduces the activity, especially if the large substituent contains no —CHj— spacer at the chiral center. Due to the longer reaction times the enantioselectivity is also reduced [44]. 

In general, the unsubstituted amides of racemic a-H-amino acids (12) are used as substrates that are readily available by Strecker synthesis on aldehydes. The initially formed ami-... 

Thus, a versatile toolbox of (amino)amidases is now available for the resolution of a very broad range of both a-H- and a,a-disubstituted amino acid amides. So far the applicability of this toolbox has been demonstrated for more than 100 natural and synthetic anoino acids. The amino acids can be obtained in enantiomerically pure form for both the D and the l configuration. 

The predominant H-bond in proteins is the bridge between the backbone amide proton of one amino acid and the backbone carbonyl oxygen atom of a second amino acid (see insert to Fig. 9.3). Although scalar couplings across H-bonds to the magnetic isotope 170 are conceivable, the fast relaxation of this quadrupolar nucleus would prevent such observations in... 

When we consider the reverse reactions, the metal-directed hydrolysis of amides, the polarising metal ion may also play a second, and often undesirable, role. In addition to polarising the carbonyl group and activating the carbon atom to nucleophilic attack, the metal may also polarise an amide N-H bond. If we consider the amino acid amide 3.4, the polarisation may be transmitted through the ligand framework to the amide N-H bond. This polarisation may be sufficient to lower the pKa so as to allow deprotonation under the desired reaction conditions (Fig. 3-13). 

Once it was established that pheromone biosynthesis was regulated by a peptide produced in the SEG, the next goal was to identify the peptide. In the purification of any biologically active factor, each purification step requires a sensitive bioassay to measure the active material. In the purification of PBAN, the bioassay consisted of head ligated females that were injected with bioactive fractions. After a 1-3 h period of incubation, the pheromone gland was excised and titers of pheromone determined by gas chromatography (GC). The first PBAN was purified and identified from H. zea (Raina el ah, 1989). Dissection of about 5000 brain-SEG complexes followed by several steps of HPLC purification resulted in a pure peptide that could be sequenced. It was found to be a 33 amino acid peptide with a C-terminal amide (Table 5.1). The peptide was synthesized and was shown to be active in the bioassay in a dose as low as 2 pmol (Raina et al., 1989). In the same year, a PBAN from B. mori was purified and sequenced (Kitamura et al.,... 

Boesten, W.H.J. and Cals, M.J.H. 1987. Process for the enzymatic hydrolysis of d-a-amino-acid amides. US Patent 4 705-52. 

All of the A -Trt amino acid fluorides listed in Table 6 are soluble in diethyl ether, with the exception of Trt-pGlu-F.t The fluorides undergo aminolysis with amino acid esters and amides without loss of configuration, although relatively slowly due to the steric hindrance resulting from the bulky Trt group. For example, the reaction of Trt-pGlu-F with H-His-OMe and Trt-Ile-F with H-Val-OBzl required 30 minutes and 6 hours respectively.t ... 

Ambler, S.J., Dell, C.P., Gilmore, J., Hotten, T.M., Sanchiz Suarez, A., Simmonds, R.G., Timms, G.H., Tupper, D.E., and Urquhart, M.W.J., Novel N-substituted a-amino acid amides as calcium channel modulators, Lilly Industries, Eur. Patent Appl. EP 805147, 1997 Chem. Abstr., 128, 13437, 1997. 

Monomethyl phthalate, BOP, ZnCB, DIPEA, CH3CN, sonication, 16 h, 53-95 % yield. These conditions result in racemization free protection of amino acid amides and esters. PyBOP can also be used as a dehydrating agent. ... 

Figure 18.4. H spectra (region of amide protons) of different amounts of a polystyrene/1 % divinylben-zcne resin (0.27 mmol/g) carrying the tetrapeptide Glu(tBu)-Gly-Lys(Boc)-Leu through a Wang linker. (A) Without presaturation (B) with presaturation of the water signal. The presaturation of the water signal at 3.75 leads to a loss of signal intensity of the second-last amino acid amide proton (8.26 ppm). This phenomenon can be explained by the presence of water in the region of the peptide N terminus.

The basis of the process leading to the enantiomerically pure acid is essentially the same as that for a-H-a-amino acids. However, in this case, a ketone is used as the starting material which undergoes a Strecker reaction, followed by hydrolysis of the resulting aminonitrile to form the racemic a-alkyl-a-amino acid amide. Enzymatic hydrolysis results in the formation of the L-a-alkyl-a-amino acid (Fig. 12.2-2). 

The manufacture of optically active L-a-amino acids from racemic amino acid amides was shown by Mitsubishi Gas Chemical, Japan [117]. In this process different microorganisms were immobilized on polymers made from (meth)acrylic acid esters or urethane acrylates and applied for the stereoselective hydrolysis of racemic amides (Scheme 43). o/L-Leucinamide (rac-136), for example, can be hydrolyzed with Mycoplana bullata cells immobilized on polyethylene glycol dimethacrylate-AT,N -methylenebisacrylamide copolymer at 30 C to produce i-leudne (l-137) over 3,000 h. 

In the above examples, L-a-amino acids induce the natural 13P-chirality. Amines or amino acid derivatives (esters, amides) are much less effective, and a tertiary amino acid, hygrinic acid, was ineffective. For trione 13a, secondary amino acids (e.g., proline) are best for 13b and 13c [R > H, see also the Danishevsky (Scheme 8) and Tsuji (Scheme 10) syntheses below], a primary amino acid (e.g., L-phenylalanine) is preferred. The mechanism of the reaction has not yet been clarified. ... 

---