Two complete syntheses of (S) -aspartate semi-aldehyde and demonstration that ∆ 2 -tetrahydroisophthalic acid is a non-competitive inhibitor of dihydrodipicolinate synthase

We report, in full, two 3-step syntheses of (S)- aspartate semi-aldehyde, an important synthetic and biosynthetic precursor, from diprotected aspartic acid. The first synthesis proceeds via a thioester, the second via a Weinreb amide. Each route yields pure (S) -aspartate semi-aldehyde in excellent yield. The utility of (S) -aspartate semi-aldehyde prepared in this manner was demonstrated with an inhibition study of dihydrodipicolinate synthase, wherein ∆ 2 - tetrahydroisophthalic acid is shown to be a non-competitive inhibitor with respect to both substrates.


Introduction
The lysine biosynthesis pathway has remained a target for therapeutic agents for many years, although no commercial product has yet been shown to inhibit this pathway. 1,2The enzyme that catalyses the branchpoint of the diaminopimelate pathway to lysine is dihydrodipicolinate synthase (DHDPS).Despite intense scrutiny over many years, no potent inhibitor of this pathway has yet been found.DHDPS catalyses the condensation of (S)-aspartate semi-aldehyde (ASA, 1) and pyruvate (2) to form an unstable heterocycle, formally thought to be dihydrodipicolinate, but now believed to be 4-hydroxytetrahydrodipicolinate (3) (Scheme 1). 2 (S)-ASA is drawn here as the aldehyde, but is actually thought to exist predominantly in the hydrated form. 3,4cheme 1.The condensation of pyruvate and (S)-ASA to form 4-hydroxytetrahydrodipicolinate, catalyzed by DHDPS.
For detailed biochemical analysis of this reaction, a convenient source of pure (S)-ASA is required. 1In particular, for screening of inhibitors of potential therapeutic use, the ASA employed must be free of impurities that may inhibit the enzyme and confuse the results.Enantiomerically pure ASA derivatives are also increasingly important synthetic intermediates, as the aldehyde moiety can be functionalized to yield more complex structures.The potential access to a variety of polyfunctional non-proteinogenic and unnatural amino acids using ASA and its derivatives has already been demonstrated. 2ASA manipulation has also proved to be important in the synthesis of pharmaceuticals, aroma and flavour chemicals, pesticides and herbicides, dyes and pigments. 54][5] There are three methods for synthesizing (S)-ASA that are commonly used for biochemical studies. 3,6,7The later methods 3,7 are derived from the original method of Black and Wright 6 which requires the ozonolysis of (S)-allylglycine.Other methods have also been reported in the literature, which yield either free ASA or diprotected ASA, but they are often complex multi-step procedures and/or are extremely low yielding procedures and have not been widely adopted. 4,8,9n the original synthesis of (S)-ASA, the aldehyde moiety was obtained by oxidative cleavage of the double bond of (S)-allylglycine by ozonolysis. 6The reaction is carried out in a 1 M aqueous hydrochloric acid solution at 0°C, with ozone bubbled through the solution.The reported yield of the desired product is 90-100%, as determined by an enzymatic assay following the conversion of ASA into homoserine by homoserine dehydrogenase. 6However, (S)-ASA produced by this method has been found to have variable purity and no chemical characterization of the product has ever been reported. 3,41][12] However, studies in our laboratory using the DHDPS/DHDPR coupled enzymatic assay 7 have suggested that the ozonolysis product is contaminated by material that has an inhibitory effect on the DHDPS enzyme. 13o address the deficiencies of the Black and Wright method, Tudor and coworkers have investigated the ozonolysis of (S)-N-tert-butoxycarbonyl allyglycine p-methoxybenzyl ester. 3emoval of the protecting groups with TFA affords the hydrate of ASA as a trifluoroacetate salt 4, which can be stored for many months at 0 °C as a stable solid.This method has since been modified so that a Lemieux-Johnson oxidation is used in place of the ozonolysis. 7In our hands, the overall yield of the latter method for the preparation of (S)-ASA was 59%, an increase on previously reported values of 42% and 43%. 3 However, the route is multi-step, involves several purifications, and is particularly tedious for those focusing on biochemical studies of DHDPS.
We report herein, in full detail, two convenient syntheses of (S)-ASA from commercially available α-tert-butyl (S)-N-tert-butoxycarbonyl aspartate (5). 14Literature procedures were employed to activate the aspartic acid side chain, firstly, to a thio-ester 15 and secondly, to a Weinreb amide. 16We also demonstrate the utility of the synthesis in a kinetic analysis of inhibition by ∆ 2 -tetrahydroisophthalic acid.

Results and Discussion
Synthesis of (S)-ASA In the first synthesis, α-tert-butyl (S)-N-tert-butoxycarbonyl aspartate (5) in dichloromethane was reacted with DCC, ethanethiol and DMAP at room temperature.Purification was achieved by flash chromatography yielding the thioester 6 as a clear oil in 94% yield.Reduction of the thioester to the aldehyde 7 was achieved in 84% yield using triethylsilane/10% Pd/C.The method of Tudor and coworkers 3 was used to deprotect the aldehyde 7 by stirring in trifluoroacetic acid in dry dichloromethane, with (S)-ASA being isolated as the hydrated trifluoroacetate salt 4, as a pale yellow solid, in 96% yield.This procedure involves only three steps and was relatively simple, cutting out the need to use osmium tetroxide (not previously mentioned).The overall reaction yield was 75% and the purity of the ASA produced was also of a very high standard, as determined by the DHDPS/DHDPR coupled enzymatic assay (99%).The only drawback of this method is that there are two purification steps required.
In a bid to cut down the number of purification steps required, the procedure of Wernic and coworkers was investigated as an alternative to obtaining (S)-ASA (Scheme 2). 16α-tert-Butyl (S)-N-tert-butoxycarbonyl aspartate (5) was converted to the corresponding Weinreb amide 8 in excellent yield (86%), using N,O-dimethylhydroxylamine hydrochloride, (benzotriazol-1yloxy)tris(dimethylamino) phosphonium hexafluorophosphate (BOP.PF 6 ), and triethylamine.The resulting product 8 was reduced with diisobutyl aluminium hydride at -78 °C to give aldehyde 7 in 95% yield, which required no further purification.The method of Tudor 3 was again used to deprotect the aldehyde 7 to afford (S)-ASA as the hydrated trifluoroacetate salt 4 in 96% yield.This procedure was a much faster and easier method for obtaining (S)-ASA than the oxidation of diprotected (R,S)-allyglycine by osmium tetroxide/periodate or by ozonolysis. 6The overall yield of the reaction from the diprotected aldehyde 5 was also greatly increased (82%) when compared to other literature procedures. 3The sequence has routinely been carried out on a 2 millimole scale.The above procedure is only three steps to the pure aldehyde and only one purification step is required.The purity of the ASA generated was also of a very high standard (99%) as checked by the coupled assay of DHDPS and DHDPR.No evidence of contaminating inhibitory compounds was found.
Inhibition of DHDPS by ∆ 2 -tetrahydroisophthalic acid ∆ 2 -Tetrahydroisophthalic acid is a product mimic of the DHDPS reaction product (Scheme 3).In the literature, ∆ 2 -tetrahydroisophthalic acid has been reported to be a weak inhibitor of DHDPS. 7o date, no detailed kinetic studies have been done to determine the type of inhibition.Inhibition kinetics of ∆ 2 -tetrahydroisophthalic acid with respect to ASA and pyruvate were carried out according to previously described methods, 13 with ∆ 2 -tetrahydroisophthalic concentrations being varied between 1 and 30 mM.The kinetic data were fitted to mathematical models using the Enzfitter computer program that simulated competitive, noncompetitive, uncompetitive and mixed inhibition patterns to determine the best fit and inhibition constant K i .Lineweaver-Burk and Eadie-Hofstee plots were used to show the quality of the generated data.
The inhibition was found to be noncompetitive with respect to both substrates, ASA and pyruvate.The K i for ASA was found to be in the range of (24 -31) mM, while the K i of pyruvate was found to be in the range of (22 -28) mM (Figures 4-7).These data suggest that ∆ 2tetrahydroisophthalic acid does not bind at the active site, which explains the weak inhibition observed.The high quality of the data generated in all cases confirms the utility of (S)-ASA generated by these methods for kinetic study.In conclusion, (S)-ASA has been synthesized from commercially available α-tert-butyl (S)-N-tert-butoxycarbonyl aspartate (5) in three steps via side-chain activation with an overall yield of 82%.This represents a significant advance over previously published routes to (S)-ASA and is the preferred synthetic route to (S)-ASA for future biochemical investigations. 1,13,20The utility of (S)-aspartate semi-aldehyde prepared in this manner was demonstrated with an inhibition study of dihydrodipicolinate synthase, wherein ∆ 2 -tetrahydroisophthalic acid is shown to be a noncompetitive inhibitor with respect to both substrates.

Experimental Section
General Procedures.Starting materials were obtained from Aldrich Chemicals, (Sigma Chemical) Company Ltd (Castle Hill, Australia.)Unless otherwise stated, all synthetic reactions were performed in dry glassware under an atmosphere of oxygen-free nitrogen or argon.All organic extracts were washed with brine and dried over anhydrous magnesium sulfate.After filtration of solutions to remove drying agents, the solvents were removed under reduced pressure. 1H NMR spectra were recorded on either a Varian Unity 300 or Varian Inova 500 spectrometer, operating at 300 and 500 MHz respectively. 13C NMR spectra were obtained on either a Varian Unity 300 or Varian Inova 500 spectrometer, operating at 75 and 126 MHz respectively.For 1 H NMR, all chemical shifts are reported relative to tetramethylsilane (TMS), if run in deuterated chloroform (CDCl 3 ).For samples run in deuterium oxide (D 2 O) the spectra were referenced to the residual protonated solvent at 4.70 ppm, and for samples run in deuterated methanol (CD 3 OD) the spectra were referenced to the residual protonated solvent peak at 3.30 ppm.For 13 C spectra run in deuterated chloroform (CDCl 3 ) the spectra were referenced to the residual protonated solvent at 77.0 ppm, and for samples run in deuterated methanol (CD 3 OD) the spectra was reference to the residual protonated solvent at 49.3 ppm.Melting points were determined using an electrothermal melting point apparatus and are uncorrected.