3-(2,5-Cyclohexadienyl)-L-alanine (1,4-Dihydro-L-phenylalanine)
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Its Synthesis and Behaviour in the Phenylalanine Ammonia-Lyase Reaction

Results and Discussion
The chemical synthesis of 2,5-dihydro-L-phenylalanine by Birch reduction has already been reported (scheme 7) [12]. The product was obtained in 43-48% yield after crystallisation but its purity was only 92-95% and it was contaminated by up to 2% L-phenylalanine and 3-5% 3-(1-cyclohexenyl)-L-alanine. In an attempt to obtain 2,5-dihydro-L-phenylalanine in pure form we applied HPLC. The reversed phase separation with a water/methanol system yielded two different fractions. While the second fraction was pure 2,5-dihydro-L-phenylalanine the 1H-NMR-spectrum revealed that the first fraction was not pure L-phenylalanine as first expected, but a mixture thereof with 2,5- and 1,4-dihydro-L-phenylalanine (spectrum 1). This was surprising because it had consistently been reported that the Birch reduction of aromatic compounds bearing an alkyl side chain yields only the product with the side chain at a residual double bond.
BIRCH et al. have performed ab initio calculations about the preferred site of protonation in the radical anion [13-15]. They concluded that the kinetically favoured site of protonation in a substituted (strong pi-donor substituent and CH3) benzene radical anion is the ortho and/or meta position, while the thermodynamically favoured site is the ortho and/or para position. The calculated energy differences for ortho and para protonation were so small that a mixture of both resulting products seemed possible. The second protonation occurs normally para to the first one. A product derived from the first protonation in para position yielding a 1,4-cyclohexadiene with the donor substituent at a reduced ring carbon atom has so far not been reported.
To completely separate and characterise the 1,4-dihydro-L-phenylalanine, a second chromatography was performed. Good separation was obtained by isocratic elution with water. Due to the instability of the dihydro-products with respect to rearomatisation [16,17], the isomers were accompanied by 3-5% rearomatised product after solvent removal.
Samples of the isolated isomers were analysed on a chiral-phase HPLC column to proof that no racemisation took place during Birch reduction and separation. All substances have the L-configuration as deduced from comparison with authentic samples.
To increase the yield of 1,4-dihydro-L-phenylalanine the proton source used in the Birch reduction was changed from tert.-butanol to ethanol. As it can be seen in table 1 the total yield slightly decreased from 95.7 to 87.7 % but the amount of the desired 1,4-dihydro product doubled from 1.5 to 3 %. We explain this as follows: Ethanol is more acidic than tert.-butanol which provides faster protonation of the generated radical anion. Because the protonation is irreversible and the second protonation takes place para to the first one, it can be concluded that 1,4-dihydro-L-phenylalanine is a kinetically controlled product. A further optimisation of the conditions may improve the yield of this minor product.
In kinetic experiments with PAL the two isomers showed different behaviour. As expected, 2,5-dihydro-L-phenylalanine was a substrate for PAL with smaller Km and Vmax values than those for the natural substrate L-phenylalanine (table 2). The Km value was only one third of the value of L-phenylalanine, Vmax was 30 times lower. The smaller Km value corresponds to a higher affinity of the substrate to the active site of PAL. Because the binding pocket is designed to bind the aromatic phenyl ring of L-phenylalanine, it is hydrophobic. An increase in hydrophobicity in this part of the substrate may increase the binding affinity and hence lower the Km value [18]. The smaller reaction velocity can be explained by the steric difference in the ring system. In contrast to the planar phenyl ring the 1,4-cyclohexadienyl ring adopts a boat-like conformation. This deviation from the plane places the MIO group and the double bond of the substrate in an inappropriate steric position, slowing the reaction rate down. Simultaneously, a more tight binding results in a lower dissociation rate of the product, leading to a decreased Vmax value. The weaker acidification of the ß-protons may shift the rate limiting step to the C-H-bond cleavage, also leading to a slower reaction.
1,4-dihydro-L-phenylalanine was no substrate for the enzyme as shown by incubation with PAL. HPLC analysis of withdrawn samples were performed after 2, 4, 6 and 20 hours. During the first two hours the concomitant L-phenylalanine was completely converted into trans-cinnamic acid. No conversion of 1,4-dihydro-L-phenylalanine was observed during 20 hours (graphs a and b). From these data one can conclude that 1,4-dihydro-L-phenylalanine is no substrate of PAL.
It has been known that 2,5-dihydro-L-phenylalanine is an inhibitor of microbial growth [12,19-21]. This inhibitory effect can be overcome by addition of an equimolar amount of L-phenylalanine. According to these data a competitive inhibition of PAL by 2,5-dihydro-L-phenylalanine can be expected. Therefore inhibition kinetics were performed to determine the Ki value of 2,5-dihydro-L-phenylalanine. The results show a competitive inhibition of PAL with a Ki value of 30.6 µM (table 2). 1,4-Dihydro-L-phenylalanine showed also competitive inhibition with a 5.1 times higher Ki value (157 µM) probably due to weaker binding at the active site. Comparing with other known inhibitors the two dihydro-phenylalanines are moderately good inhibitors. They show stronger inhibition than for example open chain unsaturated analogues or the completely hydrogenated analogue, 3-cyclohexyl-L-alanine (table 3) [18].

Title     Abstract      Introduction   Conclusion and Acknowledgements   References