Enzymatic synthesis of tryptamine and its halogen derivatives selectively labeled with hydrogen isotopes

Nine isotopomers of tryptamine and its halogen derivatives, labeled with deuterium, tritium in side chain, i.e., [(1R)-2H]-, [(1R)-3H]-, 5-F-[(1R)-2H]-, 5-F-[(1R)-3H]-, 5-Br-[(1R)-2H]-, double labeled [(1R)-2H/3H]-, 5-F-[(1R)-2H/3H]-, and ring labeled [4-2H]-, and [5-2H]-tryptamine, were obtained by enzymatic decarboxylation of l-Trp and its appropriate derivatives in deuteriated or tritiated media, respectively. Intermediates: [5′-2H]-l-Trp used for further decarboxylation was synthesized by enzymatic coupling of [5-2H]-indole with S-methyl-l-cysteine, and [4′-2H]-l-Trp was obtained by isotope exchange 1H/2H of the authentic l-Trp dissolved in heavy water induced by UV-irradiation. Doubly labeled [(1R)-2H/3H]- and 5-F-[(1R)-2H/3H]-tryptamine were obtain by decarboxylation of l-Trp or [5′-F]-l-Trp carried out in 2H3HO incubation medium.


Introduction
Tryptamine, a biogenic amine is also the backbone for a group of compounds known collectively as tryptamines. This group includes many biologically active, natural or synthetic compounds, including neurotransmitters, e.g., serotonin, melatonin [1,2] and psychedelic drugs such as DMT (N,N-dimethyltryptamine) [3,4], and psilocybin. Diversity of tryptamine derivatives due to the large number of substituents in the different positions of the indole ring and the aliphatic chain, therefore, this class of compounds has been widely used in medicinal chemistry for production the psychoactive drugs. The tryptamines have a multiple applications: they regulate many biologically processes, affect the nervous system (serotonin) [5], and are involved in the sleep-wake cycle (melatonin) [2].
In the plants and mammalian the bioamines, products of enzymatic decarboxylation of amino acids play a role of precursors of a wide range of alkaloids and other biologically active compounds [7]. The enzyme aromatic L-ADDC (EC 4.1.1.28), involved in production of tryptamine [8] is also an effective catalyst in the decarboxylation of various aromatic L-amino acids 3,4-dihydroxy-L-phenylalanine (L-DOPA), L-tryptophan, 5 0 -hydroxy-L-tryptophan, phenyl-L-alanine, L-tyrosine, and L-histidine [9]. This pyridoxal phosphate (PLP) containing enzyme is widely distributed in mammalian tissues and is responsible for the production of indolamines (serotonin, melatonin), and catecholamines (dopamine, adrenaline, and noradrenaline) [10]. The enzyme AADC is found in serum of various animals such as guinea pig, rat, monkey, mice and humans. Catecholaminergic and serotonergic cells such as adrenal glands, brain, and other tissues as liver and kidney are rich in this enzyme [11].
From a biological point of view the halogen derivatives of tryptamine are not important for metabolism, nevertheless, in many molecules of biological importance hydrogen can be easy replaced by a halogen, without much changing the activity of these species. The introducing of 18 F, 79 Br, 81 Br, and 131 I in the place of hydrogen atom is widely used for production of radiopharmaceuticals for nuclear medicine and positron emission tomography (PET) [12]. For nuclear medicine it is essential to investigate the metabolism of halogen substituted derivatives of biologically active compounds. Therefore, 18 F-tryptamine as the potential radiopharmaceuticals [13], is a promising agent for monitoring of abnormal brain states occurring in Parkinson and Alzheimer diseases and schizophrenia, should be a subject of such research.
The studies on the inactive compounds allow to determine the involving of halogenated tryptamines in metabolic reactions. The object of our further study was an investigation whether enzyme AADC effectively catalyzes the oxidative deamination of halogenated derivatives of tryptamine presented in Fig. 1. For study some intrinsic details of mechanism of enzymatic oxidative deamination of halogenated tryptamine and its halogenated derivatives we are going to use the kinetic (KIE) and solvent isotope effects (SIE) methods [14][15][16].
In this work we presented the enzymatic methods of decarboxylation of L-Trp and its halogenated derivatives for producing tryptamine and its halogen derivatives specifically labeled with deuterium and tritium needed to KIE and SIE studies. The decarboxylation carried out in deuteriated or tritiated incubation media was used to synthesis of isotopomers labeled in the aliphatic chain, i.e.,

Methods
The 1 H NMR spectra were recorded in 2 H 2 O using tetramethylsilane, TMS, as internal standard on a Varian 500 MHz Unity-plus spectrometer. TLC was used for 3-IAL Fig. 1 The metabolic pathway of L-Trp to 3-indolacetaldehyde, 3-IAL identification of substrates and products in the samples taken in the course of enzymatic reactions. As a developing solvent the water solution of acetonitrile was used (acetonitrile:water; 4:1, v/v). Radioactivity of samples were measured using a liquid scintillation counter Perkin Elmer Tri-Carb 2910TR. 9. The compound 9 was obtained by the same protocol as in case of 8 using as a substrate [5 0 -2 H]-L-Trp synthesized according modified procedures described earlier [18]. The sample of 3 mg (14.6 lmol) of [5 0 -2 H]-L-Trp (100 % 2 H enrichment) was dissolved in 50 mM Tris-HCl buffer pH 5.5 to which 0.6 mg (2.43 lmol) of PLP and 5.2 mg (1 U) L-phenylalanine decarboxylase were added. As a result a 1.8 mg (11.4 lmol) of 9 was obtained (78 % chem. yield) with 100 % of deuterium enrichment.

Results and discussion
Generally, all nine isotopomers of tryptamine and its halogen derivatives were synthesized in course of enzymatic decarboxylation, Fig. 1, of appropriate derivatives of L-tryptophan. For this reaction instead of the enzyme aromatic L-ADDC (EC 4.1.1.28) L-phenylalanine decarboxylase (EC 4.1.1.53) from Streptococcus faecalis [14] (having the similar properties as ADDC) was used. Previous studies [19,20] has strongly documented that enzymatic decarboxylation of a-L-amino acids undergoes together with the replacement of carboxyl group by solvent proton (deuteron/triton) with retention of configuration at the a-carbon (Fig. 3). Therefore, the enzymatic decarboxylation of appropriated derivative of L-Trp carried out in fully deuteriated or tritiated medium produces corresponding tryptamine labeled with deuterium or tritium in configuration (1R). Introduction of one deuterium (or tritium) atom from incubation medium to the newly generated tryptamine and preserving the configuration at the carbon atom in the aposition leads to obtain R-isotopologues of tryptamines [19][20][21][22].
The ]-tryptamine were synthesized in fully deuteriated Tris-DCl buffer, pD 5.9, to which tritiated water was added. In the case when the synthesis was carried out in fully deuteriated incubation medium its pD was corrected to 5.9 value due to higher pK (D 2 O) [23]. In the case of deuteriated media all substrates for decarboxylation reaction were dissolved in almost entirely deuteriated 50 mM Tris-DCl buffer (394 mg, 2.5 mmol) of Tris-HCl were dissolved in 50 mL of D 2 O; and calculated fraction of H ? /D ? ions in this way prepared incubation medium was equal to 0.0005).  Fig. 3 The stereochemistry of the enzymatic decarboxylation of a-amino acids The compound 8, i.e., [4-2 H]-tryptamine, labeled with deuterium in the 4-position of indole ring was synthesized by decarboxylation of [4 0 -2 H]-L-Trp which was obtained by 1 H/ 2 H isotope exchange according the procedure described earlier [24,25]. The exchange was catalyzed with UV light produced by a 250 W mercury lamp. The sample of L-Trp dissolved in 2 H 2 O was irradiated in a sealed and outgassed glass ampoule. The 1 H NMR spectrum showed that deuterium enrichment at the 4 0 position of L-Trp was around 73 %.