Evidence for Catechol Ring-Induced Conformational Restriction in

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Evidence for Catechol Ring- Induced Conformational Restriction in Neurotransmitters Haruhiko Mitsuda,† Mitsuhiko Miyazaki,† Iben B. Nielsen,‡ Pierre C-arc-abal,‡ Claude Dedonder,‡ Christophe Jouvet,‡ Shun-ichi Ishiuchi,† and Masaaki Fujii*,† †

Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan, eculaire du CNRS and CLUPS, Universit e Paris-Sud 11, 91405 Orsay cedex, France and ‡Laboratoire de Photophysique Mol

ABSTRACT In the neurotransmission process, a specific neurotransmitter binds to a specific receptor in a “key and lock” recognition process. Neurotransmitters, such as the catecholamines, are flexible molecules that can change their shape easily in principle. However, conformations of both the “key” and “lock” must be quite limited to achieve high selectivity. We have investigated the conformational diversity of catecholamines and related molecules by laser spectroscopy. Molecules of the tyrosine family, which contain a phenolic aromatic chromophore, exist as several conformers. In contrast, a single conformer is observed in dopa and other catecholamines, which contain two hydroxyl groups on the benzene moiety (catechol). This demonstrates that the presence of a catechol ring restricts significantly the number of stable conformations. SECTION Molecular Structure, Quantum Chemistry, General Theory

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n the neurotransmission process, the neural signal is transmitted by specific molecules known as neurotransmitters from one neuronal cell to the next. A specific neurotransmitter emitted from a presynaptic cell binds to a specific receptor on a postsynaptic cell. This molecular recognition process is often likened to a “key and lock”. For catecholamines, which are typical neurotransmitters, the corresponding receptor consists of a single-strand protein of about 400 residues, known as G protein coupling receptor, GPCR. GPCR captures the catecholamine in its binding site by making a number of hydrogen bonds. In the binding site, serine and aspartic acid are believed to bind the catecholamine.1,2 For this kind of molecular recognition process, the robust binding by multisite hydrogen bonds plays a crucial role. Since neurotransmitters are flexible molecules, they can change their conformation easily, but the binding sites in neurotransmitters must be located at favorable positions to bind to the receptor. Thus, this particular conformation must be energetically advantageous. From this point of view, the stability of each conformer is important. If a specific conformation is much more stable than the other conformations, this single conformation will be favored, and thus, the receptor can easily recognize the corresponding molecule and only a few selected molecules can be recognized. So far, the hydrogen bond sites are emphasized in terms of bond making with specific amino acid residues in the receptor protein. Of course, this is their most important function, but we demonstrate in this paper that collective hydrogen bonding has another function, which is to induce restriction of the conformation of the neurotransmitter molecule. In this Letter, it is shown that catecholic OHs restrict the conformational landscape very significantly relative to phenolic OH.

r 2010 American Chemical Society

Our measurements were performed by applying laser spectroscopy to gaseous neurotransmitters and related molecules at ultracold temperature achieved by laser desorption and supersonic jet techniques, which allow the investigation of gaseous biological molecules.3-12 Supersonic jet cooling can be seen as a method to freeze molecules very rapidly, so that the different conformations collapse to nearby minima on the potential energy surface and all of the conformers separate as isomers.13-17 The number of isomers (conformers at room temperature) detected can be seen as a snapshot of all of the configurations that the system is exploring at room temperature. These conformers have different electronic transitions, which are broadened and overlap under roomtemperature conditions. Jet cooling reduces the broadening drastically, so that the different transitions can be discriminated. The conformation in a supersonic jet is completely solvent-free so that it can be related to the conformation in hydrophobic circumstances, such as in a membrane or a receptor. Resonance-enhanced multiphoton ionization (REMPI) spectroscopy was employed to measure the electronic spectra. A tunable ultraviolet (UV) laser was applied perpendicular to the supersonic jet of catecholamine molecules, and its wavelength was scanned. If the energy of this laser is in resonance with an electronic transition of the molecule, efficient ionization due to resonant enhancement of the twophoton ionization process takes place. The ion signal was

Received Date: February 10, 2010 Accepted Date: March 10, 2010 Published on Web Date: March 17, 2010

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DOI: 10.1021/jz100186h |J. Phys. Chem. Lett. 2010, 1, 1130–1133

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Figure 1. Biosynthesis pathway of the catecholamines.

monitored as a function of the laser wavelength. The obtained REMPI spectrum corresponds to the UV absorption spectrum of a specific molecule in ultracold isolated conditions. In the REMPI spectrum, the electronic transitions of conformers that coexist in the supersonic jet are observed. To determine how many species contribute to the REMPI spectrum, UV-UV hole burning spectroscopy was applied.18,19 First, the sample was irradiated with a strong UV laser, the burn laser, whose wavelength was scanned. After an appropriate delay time, a second UV laser, the probe laser, tuned to a specific band observed in the REMPI spectrum, was irradiated, and the REMPI signal due to this probe laser was monitored. The REMPI signal is proportional to the ground-state population of the specific conformer selected by the energy of the probe laser. If the frequency of the burn laser is resonant with an electronic transition of this monitored conformer, the REMPI signal due to the probe laser decreases because of the population loss by the burn laser. Thus, a conformer-selective depletion spectrum (hole-burning (HB) spectrum) can be obtained. Since different conformers have different electronic transitions, we can count the number of stable conformers as the number of different HB spectra. Catecholamines are biologically synthesized from tyrosine or phenylalanine. Figure 1 shows the biosynthesis pathway of the catecholamines. Dopa is synthesized from tyrosine by tyrosine hydroxylase, which introduces a second OH group in the ortho position with respect to the phenolic OH of tyrosine and the meta position with respect to the peptidic chain. Dopa, which is well-known as an antiparkinsonism drug, was not regarded as a neurotransmitter in the past, but recently, it was reported that dopa itself also acts as a neurotransmitter.20 Dopa is further converted to dopamine by dopadecarboxylase. Dopamine is converted to noradrenaline by dopamine β-hydroxylase, and noradrenaline is converted to adrenaline by phenylethanolamine N-methyl transferase. Conformational studies of noradrenaline and adrenaline in the gas phase have already been reported. A single conformer is reported for noradrenaline, and two conformers are observed for adrenaline.21,22 These small numbers of conformations are worthy of remark because tyrosine, which is the starting material in the biosynthesis pathway, has eight conformers.23 Since a small number of conformers are observed for noradrenaline and adrenaline, despite their flexibility, these conformers must be especially stable. The stability of the specific conformation of these catecholamines, which have ethanolamine chains, has been explained by hydrogen bonding between the OH group attached to the β carbon atom and the amino group.21,22 If such an interaction within the chain is indeed responsible for the conformational restriction, many conformations should be observed for dopa, which has


Figure 2. REMPI and HB spectra of dopa. 0° represents the transition from the zero-vibrational level of the S0 state to that of S1. Progressions of ν1, ν2, and ν3 correspond to 33 cm-1, ring-chain torsion; 39 cm-1, COOH bending; and 73 cm-1, CR-Cβ torsion, respectively.

the same chain as tyrosine, for which eight conformers are observed. To confirm it, we have measured the number of conformers of dopa using REMPI and HB spectra. Figure 2 shows REMPI and HB spectra of dopa in the region of the S1 r S0 transition. The HB spectrum is the mirror image of the REMPI spectrum, which means that dopa has a single conformation. This result is surprising because the number of stable conformers is dramatically different for dopa and tyrosine, despite the same amino acid chain. The difference between tyrosine and dopa is that tyrosine has a phenol ring and dopa has a catechol ring. This result suggests that the hydrogen bonding interaction within the chain is not important, and the aromatic chromophore, catechol or phenol, is a key factor for conformational stability. If this hypothesis is right, a lot of conformers should be observed for molecules derived from noradrenaline and adrenaline in which a catechol is replaced by a phenol chromophore. Thus, we have investigated synephrine, which has the same ethanolamine chain as adrenaline but a phenolic ring rather than a catecholic one. Figure 3 shows the REMPI and HB spectra of synephrine in the region of the S1 r S0 transitions. Six different HB spectra were measured by probing bands labeled A-F in the REMPI spectrum. As can be seen in the figure, all of the HB spectra present different spectral structures. Therefore, we can conclude that synephrine has at least six stable conformers. The results obtained for dopa and synephrine show that the structure of the aromatic ring also contributes to the conformational flexibility in this series and that the flexibility of molecules that have a catechol ring is significantly restricted. Two hypotheses can be proposed: (i) The conformational restriction is linked to the meta position of the OH group because it is closer to the chain. (ii) The catechol chromophore makes the difference, that is, the two OH groups in adjacent positions are necessary for the conformational stabilization.

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One should notice that, for “in vivo” neurotransmitters, the amino group is protonated, and it will be then of considerable interest to study similar cold protonated molecules to see if the effect evidenced in this paper still persists. The conformation of protonated species as well as other molecules related to catechol amines should be studied24 and will be reported in the near future.

EXPERIMENTAL METHODS For the laser desorption, we used a graphite disk whose lateral face was coated by a mixture of the sample and graphite powder (1:2 by weight). The sample disk was placed just under a pulsed valve (Parker: General Valve), and its lateral face was irradiated by the desorption laser (1064 nm of Nd3þ:YAG laser at 20 Hz). Desorbed molecules were blown away with the supersonic flow of Ar gas expanded thorough the pulsed valve and were cooled down by a collisional cooling process. Through a skimmer whose diameter was 2 mm, a supersonic jet containing the sample molecules was trimmed away to a molecular beam, which was irradiated UV lasers. The produced cation was detected by a dynode convertor detector through a time-of-flight mass spectrometer. The signal was amplified by a preamplifier (NF: BX-31A) and was measured by a digital oscilloscope (Agilent: DSO6050). The acquired wave was transferred to a PC, and the area of the signal peak was continuously averaged and recorded as a function of the wavelength of the UV laser. The UV laser beams for measurement of REMPI and HB spectra were obtained by second harmonic generation in nonlinear crystals (Inrad: KDP with AUTO TRACKER III) of the output of dye lasers (Lumonics: HD-500) excited by the third harmonic of Nd3þ:YAG lasers (Spectra Physics: GCR170, INDI). The YAG lasers were fired synchronously with the pulsed valve.

Figure 3. REMPI and HB spectra of synephrine. Each HB spectra, A-F, was measured by fixing the wavelength of the probe laser to the A-F bands observed in the REMPI spectrum.

Figure 4. REMPI and HB spectra of meta-tyrosine. Each HB spectra, A-N, was measured by fixing the wavelength of the probe laser to the A-N bands observed in the REMPI spectrum.

AUTHOR INFORMATION

To test the first hypothesis, that is, the effect of a OH group at the meta position, the number of stable conformers of meta-tyrosine was measured, meta-tyrosine being an isomer of tyrosine with the OH group in the meta position and the same amino acid chain as tyrosine and dopa. Figure 4 shows the REMPI and HB spectra of meta-tyrosine. The REMPI spectrum shows a complicated band pattern. HB spectra were measured by probing bands A-N of the REMPI spectrum, and they show that meta-tyrosine has at least 14 conformers. This result clearly demonstrates that the presence of the hydroxyl group in the meta position imparts no restriction on the number of conformations, but on the contrary, actually increases the number of conformers. The structural restriction cannot be achieved only by a single OH group in either the meta or para position, implying that the two OH groups probably have a cooperative effect on the chain conformation. It is very intriguing that a catechol can restrict the flexibility of the chain. It was previously believed that the role of the catechol group was to anchor the molecule to the receptor with hydrogen bonds. Our results reveal that a second function of the catechol ring is to lock the molecule into a specific conformation which would help efficient molecular recognition by a corresponding receptor.


Corresponding Author: *To whom correspondence should be addressed. E-mail: mfujii@ res.titech.ac.jp. Phone and Fax: þ81-45-924-5250.

ACKNOWLEDGMENT The authors thank Professor J. D. Woodward for stimulating dicussions and helpful comments on the preparation of manuscript. This study was supported in part by a Grant-in-Aid for Scientific Research KAKENHI in the priority area (477) “Molecular Science for Supra Functional Systems” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan and the France-Japan Collaboration Program (SAKURA) from EGIDE, France, and the Japan Society for Promotion of Science.

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Evidence for Catechol Ring-Induced Conformational Restriction in

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