The chemo- enzymatic synthesis of labeled l-amino acids and some of their derivatives

This review compiles the combined chemical and enzymatic synthesis of aromatic l-amino acids (l-phenylalanine, l-tyrosine, l-DOPA, l-tryptophan, and their derivatives and precursors) specifically labeled with carbon and hydrogen isotopes, which were elaborated in our research group by the past 20 years. These compounds could be then employed to characterize the mechanisms of enzymatic reactions via kinetic and solvent isotope effects methods.


Introduction
This review deals with combined chemical and enzymatic synthesis of aromatic L-amino acids and bioamines labeled specifically with carbon and hydrogen isotopes. These compounds play an essential role in biochemical processes of life. Therefore, in the past the majority of very laborious syntheses have been carried out to provide these biologically active compounds, which were used as analytical, diagnostic, or therapeutic agents. However, the main impact on searches for new improved methods of synthesis comes from nuclear medicine, biochemistry, and pharmacy. Information on these methods are scattered, although a large knowledge may be taken starting from the large monograph published quite a long time ago [1], or from subsequently issued book [2][3][4]. In response to the growing demands for the labeled compounds, recently enzymatic methods were introduced, leading to the formation of needed biologically active products. However, there are no literature reviews devoted only to the synthesis of labeled compounds of particular relevance to the field of life science. Our research group investigates the mechanisms of reactions catalyzed by enzymes. We employ isotopic techniques, particularly kinetic isotope effect (KIE) and solvent isotope effect (SIE) methods [5,6], which require the use of selectively labeled compounds. For the abovementioned purposes the combined chemical and enzymatic synthesis of isotopomers of L-aromatic amino acids, its precursors, and derived bioamines, selectively labeled with isotopes of carbon and hydrogen were elaborated. In this paper we review previously published methods of synthesis of isotopomers of L-phenylalanine, L-tyrosine, L-DOPA, Ltryptophan, their derivatives, and precursors, all of which are specifically labeled with isotopes of hydrogen and carbon.
The metabolism of L-Phe is also connected with one of the human genetic disease-phenylketonuria (PKU), which is accompanied by elevated levels of L-Phe (1) metabolites such as phenylpyruvate and phenyllactate in body fluids. The knowledge about the mechanism of enzymatic conversion of L-Phe (1) into phenylpyruvic acid, PPA (3) is essential for proper therapy of PKU patients. One of the metabolic paths of conversion of (1) into (3) is reversible, oxidative deamination catalyzed by enzyme L-phenylalanine dehydrogenase (PheDH, EC 1.4.1.20) [13, 14] (Fig. 2).
The above two multistep reactions involve several intermediates, and therefore it is important to determine the structure of active complexes formed in the rate determining step. The number of arising questions can be minimized by determining kinetic isotope effects, KIE, of carbon 14, deuterium and tritium, as well as, the deuterium solvent isotope effects, SIE. Aforementioned studies require the use of the optically active forms of (1) specifically labeled with deuterium or tritium in desired (3R) and (3S) positions. The introduction of label in these specific positions only by chemical methods is a very tedious, time consuming, and sometimes even impossible, therefore, the combined chemical and enzymatic approaches were used.
For the preparation of labeled enantiomers of phenylalanine, the experimental procedures described in the literature resulted in multilabeled products or those labeled specifically with deuterium in irrelevant positions [15][16][17][18][19]. Also in the earlier reported studies on the synthesis of stereoisomers of [3-2 H]-and [3-3 H]-Phe the desired products were obtained as a result of tedious, multi step chemical synthesis [20][21][22][23][24]. Furthermore, often the enzymatic approach was applied to separate L-and D-isomers as the last step.
For synthesis of specifically labeled isotopomer, [(3S)-3 H]-L-Phe (1a) properties of the enzyme PAL were used. This enzyme, under proper conditions, catalyzes addition of ammonia to (E)-cinnamic acid (2) resulting in formation of L-Phe (1) [7]. The synthesis of (1a) was performed according to Fig. 3. Addition of ammonia to cinnamic acid, catalyzed by PAL, was carried out in the buffer containing tritiated water, HTO, leading to formation of (1a) [25,26].
The same approach was taken to obtain deuterium labeled [(3S)-2 H]-L-Phe (1b). In this case, addition of ammonia was carried out in fully deuterated phosphate buffer.
The characteristic of isotopomers of L-Phe (1) are collected in Table 1.
Synthetic route of [2 0 ,6 0 -3 H 2 ]-L-Tyr (14i) which consists of a combination of chemical and enzymatic methods [60] is shown in Fig. 14 (15) and tritiated water. The literature data [56,57,61] show that phenol can be catalytically exchanged with deuterated or tritiated water selectively in the o-and p-positions or per labeled. By the reverse acid catalyzed exchange of uniformly tritiated In the literature there are reports of preparation of isotopomers of L-Tyr labeled with stable and radioactive carbon isotopes using classical chemical methods. Doubly labeled stereoisomers, i.e., threo-and erythro-[1-13 C, 2,3-2 H 2 ]-L-Tyr, used for subsequent spectroscopic studies, were afforded in the multistep chemical synthesis [19]. Similarly, the pure chemical approach was applied for synthesis o [2-11 C]-L-Tyr [62]. The demand for compounds labeled with short-lived 11 C that are used as a diagnostic in nuclear medicine (i.e., positron emission tomography, PET) has prompted the efforts to synthesize amino acids labeled with this nuclide. Using 11 CO 2 as a source of label and applying the combined chemo-and multienzymatic methods the following isotopomers labeled in side chain were obtained: [1-11 C]-L-Tyr [63], [2-11 C]-L-Tyr [64] and [3-11 C]-L-Tyr [43].  (Fig. 15). In the next step, labeled L-Phe was oxidized to L-Tyr using an enzyme phenylalanine 4 0 -monooxygenase from rat liver [52,65]. The hydroxylation of L-Phe to L-Tyr was carried out in the presence of a cofactor and the enzyme catalase (EC 1.11.1.6) that protects L-Tyr from hydrogen peroxide formed during incubation. The general route for the synthesis of labeled L-Tyr is shown in Fig. 15.
The characteristic of L-Tyr (14) isotopomers are collected in Table 2.  17), plays a significant role in many metabolic processes [69]. It is a precursor of biogenic amine-dopamine, DA, (25)-an important neurotransmitter in the nervous system of mammals. DA is formed in the brain as a result of decarboxylation of L-DOPA catalyzed by enzyme aromatic Lamino acid decarboxylase (EC 4.1.1.28) [70,71] (Fig. 17).
The mechanism of decarboxylation is not clear up to now, so for KIE and SIE studies specifically labeled isotopomers of L-DOPA are needed.
The original literature data concerning the synthesis of DL-DOPA specifically labeled with deuterium and tritium in different positions of ring and side chain are dated [72,73] and yielded products useless for biological studies.
The ring deuteration of L-DOPA (17) was carried out using acid catalyzed isotope exchange method at elevated temperature [78] (Fig. 20). No significant change of proton NMR signal integrations, corresponding to methylene and methine groups of the side chain, have been noticed in the course of experiments. The incorporation of deuterium takes place only into the aromatic ring of L-DOPA (17) yielding [2 0 ,5 0 ,6 0 -2 H 3 ]-L-DOPA (17f). Also the rates of H/D exchange are practically the same for the protons in 2 0 , 5 0 , and 6 0 ring positions. Tritiation of (17) carried out in the same conditions using HTO as a source of 3 H-label leads to [2 0 ,5 0 ,6 0 -3 H 3 ]-L-DOPA (17 g).
L-DOPA labeled with 14 C in carboxyl group, needed as internal radiometric standard, was synthesized [79] from [1-14 C]-L-Tyr (14j) according to Fig. 18. The literature data concerning the chemical and combined chemoenzymatic synthesis of L-DOPA bearing 11 C-label are very tedious and were designed to obtain the products for PET diagnosis. Chemical [80,81] and chemo-enzymatic [63] routes are applied for synthesis of reports on the synthesis of uniformly ring labeled [U-14 C]-L-DOPA using [U-14 C]-phenol as a substrate [83].
The characteristic of L-DOPA (17) isotopomers are collected in Table 3.
The suggested mechanism of decomposition of L-Trp postulates proton transfer from the side chain to the C-3 carbon atom of the indole ring. This hypothesis should be verified by measuring the KIE for deuterium, tritium and carbon-14, as well as, the deuterium solvent isotope effects, SIE. For such kind of studies there is a need for isotopomers of L-Trp and 5 0 -OH-L-Trp specifically labeled with deuterium and tritium at the a-carbon position. Unfortunately, while the literature provides several synthetic methods leading to preparation of different isotopomers of tryptophan and its hydroxyl derivative labeled with deuterium and tritium specifically or nonspecifically, these reports are of little value for this purpose.  [89]. Four isotopomers of L-Trp labeled with deuterium specifically in indole ring have been obtained by coupling labeled indoles with L-serine catalyzed by extracts of E. coli cells containing enzyme tryptophan synthetase [90]. Also, the various isotopomers of tryptophan labeled with deuterium and tritium at the 2and 3-positions of side chain were synthesized by chemical methods [91][92][93].  [95]. In addition, the isotopomers doubly labeled with deuterium and 13 C were prepared [19,96] for spectroscopic studies.

Synthesis of dopamine labeled with hydrogen isotopes
The biogenic amine, dopamine, DA, (25) plays an important role in many physiological functions as a neurotransmitter in the nervous system of mammals [112,113]. DA (25) is also involved as a precursor in the synthetic enzymatic route of the other catecholamines as noradrenaline (34) and adrenaline (35) [114,115].
The mechanism of b-hydroxylation of DA, leading to formation of noradrenaline, catalyzed by the enzyme dopamine b-hydroxylase (EC 1.14.17.1) (Fig. 27) are not completely clear up to now.
The literature data on the synthesis of labeled DA is very old and scarce. Dideutero [2-2 H 2 ]-DA was obtained by reduction of 3,4-dimethoxyphenylacetonitrile with LiAlD 4 as [1-2 H 2 ]-DA was prepared from homoveratric acid by incorporation of deuterium into the side chain with exchange procedure [116]. The different isotopomers of DA tritiated in the 2-and 3-positions were obtained from (dihydroksyphenyl)ethyl alcohols as the result of three step chemical procedures [117]. Also, the very old data reports on chemo-enzymatic preparation of DA labeled with  [120]. Therefore, to study processes in Fig. 27 using KIE and SIE methods, a new simpler synthesis of deuterium or tritium labeled isotopomers of DA was elaborated.
Previous studies have shown that enzymatic decarboxylation of L-amino acids occurs with retention of configuration at the a-carbon [123,124]. This fact has been used to obtain two (1S)-isotopomers of (25) labeled with deuterium or tritium by enzymatic decarboxylation of specifically labeled isotopomers of L-DOPA (17) i.e.,  [77] (Fig. 29). For these reactions enzyme tyrosine decarboxylase (EC 4.1.1.25) was used.

Synthesis of tyramine labeled with hydrogen isotopes
Tyramine, TA (36), a biogenic amine, plays an important role in many metabolic processes. It is one of the trace amines in the central nervous system in humans [125,126]. TA may also be a substrate for enzymatic hydroxylation to another important neurotransmitter such as DA (25), catalyzed by enzyme tyrosinase (EC 1.14.18.1), Fig. 32.
Some isotopomers of TA labeled with deuterium, tritium and 14 C have been obtained during the study on the stereochemistry of enzymatic elimination of ammonia [127] and decarboxylation of L-Tyr [51]. Unfortunately, these chemical multistep syntheses are very labor intensive. For our purposes, to better understand the reaction of hydroxylation, specifically labeled isotopomers of (36), needed for KIE and SIE studies, were synthesized.
The characteristic of TA (36) isotopomers are collected in Table 6.

Synthesis of histamine labeled with hydrogen isotopes
The biogenic amine histamine, HA (37) plays an important role in various physiological function as a key mediator of cell growth, gastric secretion, acute allergic inflammation, and neurotransmitter for blood pressure [128][129][130]. In humans and experimental animals HA is mainly metabolized on the two pathways, Fig. 36 [131][132][133]. In humans about three quarters of HA is methylated to N s -methylhistamine, sMeHA (38) by enzyme N-methyltransferase (EC 2.1.1.8), and subsequently this intermediate is oxidized to N s -methylimidasole acetalaldehyde (39) by enzyme diamine oxidase (DAO, EC 1.4.3.6). The remaining quarter of HA, however, is indirectly biotransferred into imidasole acetalaldehyde (40) by DAO. (According to the recommendation of IUPAC [134], the nitrogen atoms of the imidasole ring are denoted by p and s, carbon atoms in the side chain as a, and b and ring carbon atoms as 2, 4, 5).
Despite of many studies the mechanism of the removal of excess of HA (37) from human body is not completely understood. Therefore, we planned experiments to investigate some details of methylation and oxidation reactions presented in Fig. 36, by applying the KIE and SIE methods. For this kind of study the isotopomers of HA and N-methyl-HAs specifically labeled with deuterium and tritium are needed. In the literature there is description of the synthesis of sMeHA and pMeHA tritiated selectively in the methyl group [135]. The product obtained consists of two (s and p) isomers, which separation was unsuccessful. Also, the preparation of tritiated (N s -C[ 3 H 3 ])-HA from [ 3 H]CH 3 I by chemical method is described [136], as well as the synthesis of deuterated (N s -C[ 2 H 3 ])-HA [137].  were obtained in the same manner as in Fig. 37 by enzymatic decarboxylation of N p -methyl-L-histidine [138].
The characteristic of HA (37) isotopomers are collected in Table 7.

Synthesis of phenylpyruvic acid labeled with hydrogen and carbon isotopes
Phenylpyruvic acid, PPA (3) is a product of oxidative deamination reaction of L-Phe (1) presented in Fig. 2. In the course of this reaction some tautomerization of PPA takes place, and in the process the stereospecific abstraction of proton from 3-position of PPA is involved [141]. The numerical values of isotope effects allowed us to elucidate the intrinsic details of this mechanism. This kind of studies require the use of isotopomers of PPA labeled with deuterium and tritium in 3 position, and also the 14 C-labeled isotopomer of PPA used as internal radiometric standard in the course of KIE assays. In the literature there are a few papers that describe the synthesis of deuterium-, [141] tritium-, [142] and 14 C-labeled [143] isotopomers of PPA. Most of them yielding isotopomers bearing the label in position not useful for study of mechanism of reaction presented in Fig. 2  Isotopomer [1-14 C]-PPA (3d) was synthesized as above using [1-14 C]-L-Phe (1 h) as a substrate [144].
The characteristic of PPA (3) isotopomers are collected in Table 8.
Synthesis of halogen derivatives of L-Phe, L-Tyr and L-Trp labeled with hydrogen isotopes Halogenated derivatives of L-Phe (1), L-Tyr (14) and L-Trp (29), labeled with short-lived radioisotopes i.e., 18 F, 123 I, 125 I or 76 Br are recently applied in nuclear medicine for diagnosis of many types of tumours and neurodegenerative diseases using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).   (45), catalyzed by PAL, was carried out in deuterated or tritiated buffer solutions [150,151].
The characteristic of halogenated derivatives of L-Tyr (50,51,52) are collected in Table 10.
We have also developed the method for synthesis of halogenated derivatives of L-Trp (29), selectively labeled with hydrogen isotopes at the a-position of the side chain i.e.,  [150,151] and carried out in deuterated or tritiated incubation medium, according to Fig. 43. In all cases 2-mercaptoethanol was used to prevent the growth of bacteria and fungi during incubation.
The characteristic of halogenated derivatives of L-Trp (53,54,55) are collected in Table 11.

Conclusions
Taking into account the advantages of enzymatic synthetic methods, it is foreseeable that this type of reactions will gain a stronger presence in preparation of biologically active labeled compounds. While introduction of isotopic carbon atom to the backbone of a molecule may create some synthesis challenges, in majority of cases however, enzymatic syntheses are still easier to carry out than classic multistep organic syntheses. Furthermore this issue is greatly minimized when dealing with the substitution of the stable atom for radioactive one bonded to backbone of molecule (either isotopes of hydrogen and halogens) or addition of functional group bearing isotopic (for instance 11 C-13 C-or 14