Stereocontrolled synthesis of deuterated phenylalanine derivatives through manipulation of an N -phthaloyl protecting group for the recall of stereochemistry. Application in the study of phenylalanine ammonia lyase

The enantiomers of [2-2 H 1 ]phenylalanine and all four stereoisomers of [2,3-2 H 2 ]phenylalanine have been prepared from ( S )-phenylalanine through the introduction of a chiral centre onto an N - phthaloyl protecting group for the recall of stereochemistry. Studies of the interaction of these labelled phenylalanines with ( S )-phenylalanine ammonia lyase show that both the C-2 and C-3 hydrogens of the product trans -cinnamate undergo exchange with solvent in the presence of the enzyme. The mechanistic implications of this observation are discussed.


Introduction
(S)-Phenylalanine ammonia lyase (PAL) is a plant enzyme which catalyses the elimination of ammonia and a proton from (S)-phenylalanine 1 (Scheme 1), to give trans-cinnamic acid 2 as required for the biosynthesis of lignins, flavanoids and coumarins. 1,2The catalysis by PAL has been studied extensively and was thought to involve activation of the substrate through addition of its amino group to a dehydroalanine prosthetic group at the enzyme active site. 3However, recent work indicates that the dehydroalanine is probably incorporated in a methylidene imidazolone 3, and that electrophilic attack of this residue at the ortho-position of the substrate's aromatic ring is more likely to be the mechanism of substrate activation. 4cheme 2 (S)-Phenylalanine 1 was treated with phthalic anhydride, and then with acidified methanol, to give the phenylalanine derivative 9. 12 The reduction and solvolysis of this compound was carried out using the procedure of Speckamp et al. 13 Accordingly, treatment with sodium borohydride in methanol at -10 °C for 15 min, followed by acidification and stirring at room temperature for 16 h, afforded a ca.1:1 mixture of the methoxy amides 10 and 11.These diastereomers were separated by chromatography on silica and obtained in yields of 36 and 31%.The individual It was not necessary to assign the stereochemistry of the methoxy amides 10 and 11 at the 3position of the isoindoline moiety in order to exploit the new chiral centre of these compounds, to distinguish and separate the stereoisomers of the deuterated derivatives 12, 14, 15 and 17 and assign their stereochemistry at the amino acid α-position.While the deuteration of 10 occurs with epimerisation at the α-carbon, the products 12 and 15 are separable diastereomers, with the one 12 having similar physical and spectral properties to those of the starting material 10, and therefore possessing α-(S)-stereochemistry.It follows that the other product 15 has α-(R)stereochemistry and similar physical and spectral properties to those of the diastereomer 11 of the precursor 10, since 15 is the deuterated enantiomer of 10.A similar rationale applies for the reaction of 11.Removal of the protecting groups from the deuterated methoxy amides 12, 14, 15 and 17 occurs with retention of configuration at the α-carbon, so the pairs of diastereomers 12 and 14, and 15 and 17, afford 13 and 16, respectively.
Using a similar approach it was possible to prepare each of the four stereoisomers of α,βdideuterated phenylalanine 25, 28, 33 and 36 in a stereocontrolled manner, beginning with the (S)-phenylalanine derivative 9 (Scheme 4).Previously we have reported the use of the Nphthaloyl protecting group for side chain halogenation of amino acid derivatives without loss of stereochemical integrity at the α-carbon. 14Accordingly, the phenylalanine derivative 9 reacted with N-bromosuccinimide to give the bromides 18 and 19, which underwent deuterolysis with retention of configuration on treatment with deuterium over palladium on carbon, 6 to give 20 and 21, respectively.When the (2S,3S)-phenylalanine derivative 20 was elaborated, as shown in Scheme 3 for the non-deuterated analogue 9, 25 and 28 were obtained, while a similar series of reactions carried out using 21 as the starting material gave 33 and 36 (Scheme 5).The deuterides 25, 28, 33 and 36 were obtained as single enantiomers (>95% ee).They were diastereomerically pure within the limits of detection using 1 H NMR spectroscopy.Their mass spectra showed that they were ca.95% dideuterated.
The synthesis of the deuterated phenylalanines 16, 28 and 36 involves overall inversion of stereochemistry at the α-carbon.The methodology is general, as shown through the conversion of the (S)-isomers of alanine 38a, valine 38b and leucine 38c to the corresponding (R)enantiomers 44a-c, as illustrated in Scheme 6.
With the deuterated phenylalanines 13, 16, 25, 28, 33 and 36 in hand, their reactions with PAL in 0.04 mol L -1 sodium borate buffer (pH 8.7) at 30 C were investigated.The samples of trans-cinnamic acid 2 isolated from these reactions contained various amounts of deuterium, decreasing as either the enzyme-substrate ratio or the incubation time increased, to the extent that unlabelled cinnamate 2 was obtained if sufficient enzyme and long incubation times were employed.When (S)-phenylalanine 1 was used as the substrate and the reaction was carried out in deuterium oxide, the initial product was unlabelled cinnamate 2, but after further incubation extensive deuterium incorporation occurred at both the α-and β-positions.No deuterium incorporation was observed in the absence of enzyme.These exchange processes do not alter the principle conclusions drawn from work with the β-deuterated phenylalanines 4a,b and 5a,b, that were based on the extent of deuterium retention in samples of the cinnamate 2 formed initially.It is still reasonable to conclude that (S)-phenylalanine 1 reacts via loss of the pro-S hydrogen, while (R)-phenylalanine reacts mainly by loss of the pro-R hydrogen.However, the exchange of the C-2 and C-3 hydrogens of the cinnamate 2 with deuterated solvent, and vice versa, in the presence of the enzyme does make it impractical to use the deuterated phenylalanines 13, 16, 25, 28, 33 and 36 to examine minor reaction pathways or study subtle isotope effects.As to how the exchange processes occur, it seems likely that the cinnamate 2 bound in the active site of PAL can undergo reversible nucleophilic addition at either the αor β-position.Reaction at the βposition is the normal pathway for conjugate addition, reflecting the polarisation of the cinnamate 2. The alternative pathway is probably facilitated by PAL through reaction of the methylidene imidazolone 3 with the aromatic ring of the cinnamate 2 (Figure 1).
# The stereochemistry at this position has been assigned arbitrarily and may be the reverse, University.HPLC was performed using a Waters µ-Porasil silica column (5 µm silica, 19 • 300 mm), eluting with hexanes-ethyl acetate (5:1).GC was performed using a Chirasil-Val capillary column (0.3 mm • 25 m) and argon as the carrier gas with a flow rate of 0.5 mL min -1 .Materials.(S)-Phenylalanine 1, (S)-alanine 38a, (S)-valine 38b and (S)-leucine 38c and the corresponding (R)-enantiomers and racemic materials were purchased from Sigma Chem.Co. and used to prepare the esters 9 and 39a-c using standard methods. 12Reaction of the ester 9 with Nbromosuccinimide to give the bromides 18 and 19, and their conversion to the corresponding deuterides 20 and 21, was carried out as previously reported. 6,14PAL (grade I from Rhodotorula glutinis) was obtained as a solution in 60% glycerol, 3 • 10 -3 mol L -1 tris-hydrochloric acid, pH 7.5, with an activity of ca. 3 • 10 3 units L -1 .

General procedure for reduction and solvolysis of the phthalimides 9, 20, 21 and 39a-c
Sodium borohydride (190 mg, 5 mmol) was added slowly to a solution of the phthalimide (4.5 mmol) in dry methanol (50 mL), maintained at -10 °C.The mixture was stirred at that temperature for 15 min, then it was acidified through the cautious addition of thionyl chloride (1.0 g, 8.5 mmol).The resultant solution was allowed to warm to room temperature, then it was stirred for 16 h at room temperature, before it was poured into dilute aqueous ammonium chloride (50 mL).This solution was extracted with dichloromethane and the extract was dried and concentrated under reduced pressure.Analysis of the residual oil using 1 H NMR spectroscopy and HPLC showed that the corresponding α-methoxy amides were produced in a ca.1:1 ratio.They were separated through chromatography on silica, eluting with hexanes-ethyl acetate.

General procedure for epimerisation of the a-methoxy amides 40a-c and 41a-c
A solution prepared from sodium (48 mg, 2 mmol) and methanol (1 mL) was added to a solution of the αmethoxy amide (1 mmol) in methanol (20 mL), and the mixture was heated at reflux for 4 h, then it was poured cautiously into dilute aqueous hydrochloric acid (50 mL).The resultant solution was extracted with dichloromethane and the extract was dried and concentrated under reduced pressure.Analysis of the residual oil using 1 H NMR spectroscopy and HPLC showed that the starting material and the corresponding isomeric α-methoxy amide were present in a ca.1:1 ratio.They were separated through chromatography on silica, eluting with hexanes-ethyl acetate.The α-methoxy amides 42a-c and 43a-c prepared in this manner were isolated in yields ranging from 32-43%, and had properties comparable with those of their corresponding enantiomers 41a-c and 40a-c.General procedure for deprotection of the amino acid derivatives 12, 14, 15, 17, 24 26, 27, 29, 32, 34, 35, 37, 42a-c and 43a-c.A solution of the amino acid derivative (0.5 mmol) in a 2:1 mixture of 6N hydrochloric acid and acetic acid (20 mL) was heated at reflux for 5 h and stirred at room temperature for 16 h, before being concentrated under reduced pressure.Water was added to the residue and the mixture was filtered.The filtrate was concentrated under reduced pressure and this residue was dissolved in a mixture of ethanol (10 mL), aniline (0.7 mL) and dichloromethane (10 mL).The solution was allowed to stand at 4 C for 24 h, and the crystals which formed were separated by filtration and washed with dichloromethane, to give the corresponding free amino acid.
The amino acids 13, 16, 25, 28, 33, 36 and 44a-c prepared in this manner from 12 and 14, 15 and 17, 24 and 26, 27 and 29, 32 and 34, 35 and 37, and 42a-c and 43a-c, respectively, were isolated in yields ranging from 78-91%.The samples of (R)-alanine (44a), (R)-valine (44b) and (R)-leucine (44c) were identical with authentic specimens.The samples of the labelled phenylalanine derivatives 13, 16, 25, 28, 33 and 36 had properties comparable with those of unlabelled (S)-phenylalanine 1 and the (R)-enantiomer.Each of the amino acids 13, 16, 25, 28, 33, 36 and 44a-c was shown to be a single enantiomer (>95% ee) using the following procedure.Treatment of a small sample (ca. 1 mg) with acetic anhydride (5 mg) and triethylamine (10 mg) in water (1 mL) for 2 h at room temperature, followed by acidification and extraction with ethyl acetate, gave the corresponding acetamide.This was added to methanol (1 mL) which had been pretreated with thionyl chloride (10 mg), and the mixture was stirred at room temperature for 2 h, then concentrated under reduced pressure, to give the crude N-acetylated amino acid methyl ester, which was analysed by GC and compared with racemic material.The dideuterides 25, 28, 33 and 36 were diastereochemically pure, within the limits of detection using 1 H NMR spectroscopy (>95%).