2.2.1 Tryptophan, serotonin

The facilitated transport of zwitterionic, lipophilic amino acids, dipeptides tripeptides, serotonin, dopamine and the nucleosides adenosine, guanosine, thymine, and cytosine was investigated across CHCl₃ BLM and found effective to various extents with different {M-^tBu₄-salphen-18-cr-6} complexes. In this paper, we report on our transport studies with tryptophan and serotonin. The cell shown in Fig. 6 was used.

The Ni²⁺ complex, I, and its derivatives, the corresponding Cu²⁺ complex and the benzo-18-C-6 crown ether, that was investigated as a control, were equally ineffective in the transport of tryptophan [15]. In contrast, the Mn³⁺ complex, II, readily transported tryptophan (Fig. 7) by a process driven by the tryptophan concentration gradient [12,14]. The concentrations of tryptophan in the receiving and source compartments asymptotically reach equal values as the transport progresses. Nearly as effective in transport was the neutral {Fe³⁺-^tBu₄-salphen-3n-cr-n}₂O, n = 6, complex (Fig. 8).

We have attributed the poor transport performance of I and of the Cu²⁺ analog to the stability of the square planar Ni²⁺ and Cu²⁺ ions and their inability to bind axial ligands.

The importance of the unencumbered crown ether unit in II becomes apparent when the transport properties of II are compared to those of the {[Mn-^tBu₄-salphen]₂-[18-cr-6]}²⁺·2 I^– carrier with two Mn-^tBu₄-salphen units surrounding the 18-cr-6 unit (Fig. 9). The inferior performance of the latter may be due to the fact that in this carrier the hydrophilic part of the molecule is flanked by two very hydrophobic units and is not free to assist in retrieving the tryptophan molecule from the water solution. It is possible that the observed difference in transport rates is due to the presence of only two aliphatic ether oxygen donors in the {[Mn³⁺-^tBu₄-salphen-X}₂{[18-cr-6] carrier and the consequent weaker interactions possible with the dibenzo-crown unit. This possibility can be tested using derivatives of II that also contain two aliphatic ether donors.

Various modes of interaction can be envisioned between the carrier and the zwitterionic form of tryptophan. They may involve such interactions as: (a) carboxylate coordination to the Mn³⁺ center, (b) H-bonding of the -NH₃⁺ to the crown ether cavity of the carrier, (c) carboxylate coordination to an alkali metal bound in the crown ether cavity, and (d) H-bonding interactions between the indole NH proton and a hydrogen bond accepting site within the carrier. There is no apparent difference in the tryptophan transport rate when the 18-cr-6 unit in II is replaced by a 24-cr-8 unit.

The bathochromic shifts of the 470-nm band, caused by axial ligand coordination to the Mn(III) center, in derivatives of II with various counterions, indicate that this electronic absorption is very likely due to a M-ligand charge transfer, MLCT, process. The relative substrate transport rates decrease as the bathochromic shift of the MLCT band (at 470 nm in the BPh₄^– anion) increases. The differences in the bathochromic shift of the CT absorption reflect differences in the magnitude of Mn(III)-axial-ligand interactions. Generally, the transport data indicate [12] that the weakly coordinating anions give carriers that transport amino acids more effectively in the order: B(Ph)₄^– > I^– = Piv^– > Cl^– > Ac^–.

Transport studies were conducted to determine whether the ditopic receptors are more efficient transporters than an equimolar mixture of two similar monotopic half-receptors. The transport properties of a 1:1 mixture of the Mn-veratrol complex (Fig. 10) and benzo-18-crown-6 were compared to those of {Mn³⁺-^tBu₄-salphen-18-cr-6}⁺ X^– or {Mn³⁺-^tBu₄-salphen veratrol}⁺ X^–. The results show [1] that, in an equimolar mixture, the two carrier components function synergistically. The rate of transport observed with II however was still about 30% faster and indicates moderate ditopic cooperativity. The same experiments carried out with serotonin show greater ditopic cooperativity and can be attributed to a better dimensional matching between the donor–acceptor pairs within the serotonin-II complex. In II the acidic and basic sites are closely spaced such that that the simultaneous binding of the tryptophan zwitterion by both the carboxylate group (to the Mn³⁺) and the ammonium group (to the crown ether) is not possible unless the crown ether unit bends severely (to a right angle or more) towards the salphen unit. The ditopic cooperativity shown in the transport of substrates has been reported previously [17] for the crown-ether-functionalized boronic acids in the facilitated transport of catecholamines. The synergism displayed by the mixture of the two separate carrier components of II in the transport of tryptophan has been reported previously [17], for equimolar mixtures of arylboronic acids and crown ethers, in the transport of L-phenylalanine through CHCl₃.

Molecular mechanics calculations [12] on the tryptophan-II complex (Fig. 11) support this contention and show that when tryptophan is placed in the pocket of the carrier that contains a water molecule axially coordinated to the Mn(III) center, the relative energy of the guest-host complex decreases. In the energy minimized structure, in addition to RNH₃⁺ hydrogen bonding with the crown ether appendix, short hydrogen bonds are found between the amino acid carboxylate group and the Mn(III)-bound water molecule (Fig. 11A). These hydrogen bonds (N–H···O, 2.69 Å; C···H–O–H, 2.51 Å) are to be contrasted with those found in the energy-minimized structure without the Mn(III)-bound water molecule (Fig. 11B). In the latter case, the only hydrogen bonding interaction is between the RNH₃⁺ unit and the crown ether (N–H···O, 2.69Å; C–O···Mn, 3.79 Å).

The same type of calculation was carried out with serotonin. With a water molecule axially bound to the Mn(III) ion in II, the energy-minimized structure (Fig. 12A) shows the relative energy of the serotonin-II complex much higher than that of the complex without an axially bound water molecule (Fig. 12B). The lower energy of the latter is not unexpected considering the close distance matching of the two donor-acceptor pairs [12].

Thus far the transport data suggest that the initial carrier-amino acid contact is the rate-determining step and occurs between the crown ether unit of the carrier and the –NH₃⁺ unit of the zwitterion. This suggestion also is supported by the observed inhibition of transport in the presence of the crown-blocking K⁺ ion. The slow transport rate by the benzo-18-crown-6 molecule shows that a crown ether interaction with the amino acid alone is not sufficient for effective transport, although it should be recognized that the crown ether is less hydrophobic than the ^tBu-salphen-crown ether receptor.

For the facilitated transport of a given solute through a membrane (Fig. 13), the flux [18], J_solute, is defined as [19]:

From the apparent Michaelis constants (K_m) of 25 mM and 245 mM, carrier-permeant extraction constants (K_ex = 1/ K_m) of 40 M^–1 and 4 M^–1 were determined, respectively, for the tryptophan and serotonin carrier complexes. Theoretically, the transport of serotonin as the hydrochloride derivative should be more appropriately treated, as a symport-transport case [19]. The successful application of the same Michaelis–Menten kinetics treatment for serotonin HCl, as that used for tryptophan, however suggests that the HCl derivative of serotonin in CHCl₃ behaves as a tight ion pair rather than as an electrolyte.

Rate studies as a function of temperature and Arrhenius plots were used to determine activation energies and activation parameters for the process of transport of tryptophan and serotonin [12].

The ΔEs of activation of 20 and 16 kcal mol^–1 indicate diffusion-controlled processes [19] through the Nernst layer at the CHCl₃–H₂O interphase.

The transport of serotonin·HCl by II⁺–I^– proceeds at a respectable rate, but which is still half of that of tryptophan. The transport of dopamine (in a buffered solution at pH 7.2) by the same carrier is slower, but still much better than the transport observed with the benzo-18-cr-6 carrier) [1]. The difference in transport rates between serotonin and dopamine is probably due to differences in hydrophobicity between these two molecules. The distance between the electron-pair donor hydroxyl groups and the hydrogen-bond donor amino groups in both are very similar, and for both, {Mn³⁺-^tBu₄-salphen-18-cr-6}⁺, II, can serve as a ditopic receptor. The flux [20] of the previously reported [19] transport of serotonin by the boronic-acid-functionalized crown-ethers is nearly 100 times smaller than that determined for II.

For the transport of tryptophan, through a CHCl₃ BLM, the dependence of transport rate on the stirring rate of the BLM (Fig. 15) unequivocally indicates a diffusion-controlled process [20]. An increase in the stirring rate is expected to reduce the thickness of the Nernst layer. With a hindered carrier (Fig. 9B), difficulty in rapidly penetrating the interphase may introduce a slow kinetic step that may in fact become the rate-determining step.

The lipophilicity of the amino acids is correlated to their solubility in water [2,3,21]. The transport rates of the three lipophilic amino acids, tryptophan, leucine and phenyl alanine (Table 1) roughly follow their insolubility in water and indicate that π interactions, possible with tryptophan but not with leucine, are not very important in the transport process. The faster transport rate observed with the less hydrophilic tryptophan again suggests that, for II, the pickup of the amino acid by the carrier (rather than the release) is the rate-determining step in the transport process. A comparison of the fluxes for the transport of these amino acids by different carriers across a CHCl₃ BLM is shown in Table 1 (flux is defined as: [mmol substrate transported] [mmol carrier]^–1 s^–1 cm^–2.