Computer-assisted automated synthesis. III. Synthesis of substituted N-(carboxyalkyl) amino-acid tert-butyl ester derivatives

A versatile automated synthesis apparatus, equipped with a chemical artificial intelligence, was developed to prepare and isolate a wide variety of compounds. The apparatus was to the synthesis of substituted N-(carboxyalkyl)amino-acids. The apparatus [1,2] is composed of units for performing various tasks,for example reagent supply, reaction, purification and separation, each linked to a control system. All synthetic processes, including washing and drying of the apparatus after each synthetic run, were automatically performed from the mixing of the reactants to the isolation of the products as powders or crystals. The reaction of an amino-acid tertbutyl ester acetic acid salt with a 2-keto acid sodium salt produces an unstable intermediate, Schiff base, which is reduced with sodum cyanoborohydride to give a substituted N-(carboxyalkyl)aminoacid tert-butyl ester sodium salt. The equilibrium and the consecutive reactions were controlled by adding sodium cyanoborohydride using the artificial intelligence software, which contained novel kinetic equations [3] and substituent effects [4]. Substitued N-(carboxyalkyl)amino-acid tert-butyl esters, 90 derivatives, were automatically synthesized using the computerassisted automated synthesis apparatus. The syntheses were performed unattended 24 hours a day, except for supplying the raw materials, reagents and solvents. The apparatus is extremely valuable for synthesizing many derivatives of a particular compound. The configurations of the products were determined by circular dichroism measurements.


Introduction
In general, organic syntheses are time-consuming; involving the preliminary estimation of the reaction conditions, mixing of the raw materials and reagents, control of the reaction conditions, concentration, extraction, purification and finally isolation of the desired products. Although some research on laboratory automation, especially for synthesis, has been attempted [5][6][7][8], an automatic apparatus for the estimation of the optimum reaction conditions, as well as control of all the synthetic processes, has remained an important objective. The authors recently reported a reaction-control methods based on the novel kinetic equations and substituent effect, which made up the computer software for the automated apparatus, and the design and construction of fully automated synthesis apparatus [1]. This paper deals with an application of the apparatus for the synthesis of substituted N-(carboxyalkyl)aminoacids--known as 'unusual amino acids' [9]. Several of these unusual amino-acids have been found to occur in nature; for example strombine, an active fish attractant extracted from Strombus gigas [10], and N-(carboxymethyl)-L-serine which has been isolated from asparagus shoots 11 ]. However, the only derivatives ofN-(carboxyalkyl)amoni-acids which have been synthesized, are a series of N-(carboxymethyl)amino-acids [12].
The reaction ofamino-acid tert-butyl ester acetic acid salt (1) with keto acid (2) in methanol gives an unstable intermediate. Schiff base, which is then reduced with sodium cyanoborohydride to afford the N-(carboxyalkyl)amino-acid tert-butyl ester (3). The products obtained from natural L-amino acids, all of which have an S-configuration on the chiral centre, except for cysteine and methionine, were generally a mixture of diastereoisomers [(S,R)and (S,S)-isomers]. As the diastereoisomers were separated, their configurations were also determined by means of circular dichroism (CD) mesurement.

Results and discussion
Procedure for the automated synthesis The automated synthesis apparatus used is shown in figure 1. All synthetic processes, including washing and drying of the. apparatus after a synthetic run, were automatically performed; from the mixing of reactants to R-CH-NH2HOAc + O=C-R' kl CO2Bu CO2Na (1) the isolation of the products as powders. The prediction of the optimum reaction conditions and the reaction control in real time, are accomplished using the novel kinetic equations and substituent effect which make up the computer software, as previously reported [4].
As a typical automated synthesis using the apparatus, the reaction of L-leucine tert-butyl ester acetic acid salt (4) with 2-ketoisocaproic acid sodium salt (5)  2"9 28"5 2-0 52"4 "4 89"3 0-9 145"2 0-6 155"2 Finish the reaction was stopped after 155 min, and the resulting mixture was transferred to the concentration flask. After evaporation to dryness under reduced pressure, 10 ml of aqueous sodium hydroxide was added to the residue with stirring. The solution was then injected into the purification unit. The high performance liquid chromatographic (HPLC) chart, which was displayed on a CRT monitor during the purification stage, is presented in figure 3.
The peak at the retention time (27"2 min) was not clearly identified but it was attributed to decomposition products of the sodium cyanoborohydride and the keto acid. The two peaks having retention times of 52-3 and 66"3 min were identical to those of the diastereoisomers of N-(3methyl-1-sodioxycarbonyl) butyl-L-leucince tert-butyl ester (6). The analytical yield calculated from the area of these two peaks was almost quantitative on the basis of       the calculated yield presuming consumption of reducing agent. The fractions collected at these two peaks were transferred to the freeze-drying unit, but the fraction eluting from 54"8 to 58"0 min was not collected. Thus the combined synthetic yield of both isomers of 6 was 72"5%.
A variety of substituted N-(carboxyalkyl)amino-acid tritbutyl ester derivatives (90 compounds) were automatically synthesized in a similar manner to that described above, and the yields with the elemental analysis are presented in table 2. The 1H NMR data are shown in tables 3 to 11. Since no significant influence from the amino-acid substituents was found, the addition rate of sodium cyanoborohydride was computed using the substituent constants of the 2-keto acids. However, the forward rate constants for glyoxylic acid and pyruvic acid were sufficiently large, in these case, for the reducing agent to be added to the reaction mixture in an uncontrolled process.

Configurational assignment of the products
The natural L-amino-acids, except for those containing sulphur (cysteine and methionine), have S-configuration on the chiral centre. Thus the reaction ofan L-amino-acid tert-butyl ester with a 2-keto acid generally gives two diastereoisomers, [S,R]and [S,S]-isomers. All of the diastereoisomers derived from the 2-keto acids, except those from glyoxylic acid, pyruvic acid and 2-keto dicarboxylic acid, were separated from each other in the purification unit of the synthesis apparatus. Subsequently, the configurations of the newly formed chiral centres were determined. As a typical example, each diastereoisomer of 6 was derivatized into its N-(carboxy-3-methyl)-butyl-L-leucine disodium salt (7) having symmetrical structure. The first eluted peak (6-a) of 6, converted into the corresponding disodium salt (7-a) by deblocking with trifluoroacetic acid and purified on a Sephadex LH-20 column, gave the meso-compound having no [0]D value, whereas the disodium salt (7-b) derived from the second eluted peak (6-b) showed a positive [0]D value (+39-2). Thus the configuration of the newly formed chiral centre for 6-a was determined as R, and that of 6-b as S.  In the H NMR spectra of substituted N-(carboxyalkyl)amino-acid tert-butyl ester, the chemical shift of the tertbutyl ester group (1"48-1-50 ppm) in the A-isomers was found to be generally at lower field (ca. 0"01 ppm) than in the B-isomers. The chemical shifts of the methine protons on the two chiral centres of the A-isomers (around 2-9-3"6 and 2-7-3"2 ppm) were observed at higher field (0-05-0-1 ppm) than those of the B-isomers.

Conclusion
The reaction control equations incorporated in a computer program have been applied to the synthesis of 192 substituted N-(carboxyalkyl)amino-acids, and all synthetic processes were automatically performed from the mixing of reactants to the isolation of the products as powders. The automated apparatus was run continuously for 24 hours, giving an average rate of synthesis of three compounds per day. The apparatus extremely valuable for synthesizing many derivatives of a particular compound. Even if the chemical yields are low under the optimum conditions. It is still possible to obtain a sufficient amount of the desired product by repeating the reaction. Moreover, it is possible to greatly reduce the labour-intensive nature of the many syntheses which are a necessary part of pharmaceutical research.