Trans -2-Aminocyclohexanols as pH-triggered molecular switches

Cyclohexane-based conformationally controlled ionophores, the emerging new class of molecular switches, provide a new and promising approach to allosteric systems with negative cooperativity. Protonation of trans -2-aminocyclohexanols leads to dramatic conformational changes: due to an intramolecular hydrogen bond, a conformer with equatorial position of ammonio-and hydroxy-groups becomes predominant. Thus, these structures can serve as powerful conformational pH-triggers. The trans -2-aminocyclohexanol moiety has been used for pH-triggered conformational switching of a crown ether and a podand, and their complexes with potassium ion.


Introduction
The development of molecular switches is of great current interest in view of their possible use in many applications, such as drug release, new sensor techniques or information storage and transmission.2][3] Allosteric switches are host compounds containing at least two spatially separated binding sites that are conformationally coupled.When one site is occupied, it changes conformation, and this 'signal', mechanically transmitted by the structure of the molecule, induces a conformational change in the second site, thus increasing (positive cooperativity) or decreasing (negative cooperativity) its affinity to an appropriate guest.Negative cooperativity has been less explored than the positive, though it may be more interesting for applications, such as membrane transport, drug delivery, catalysis, etc. [1][2][3] For example, the presence of a particular effector compound, or a particular pH value could lead to the release or to the uptake of a biologically active substance.

Scheme 1
A promising type of a conformational trigger is provided by trans-2-aminocyclohexanol moiety.0][41][42] Therefore, these structural moieties can be used as conformational counterbalances or locks.
As we suggested in a preliminary publication, 23 another way to control such a conformational equilibrium is an addition of acid to protonate the amino group, and to generate a stronger intramolecular hydrogen bond of O⋅⋅⋅H-N + type, 23,39 e.g. in 3A (Scheme 3). 23This bond would stabilize conformation 3A, thus moving the ester groups away from each other, and decreasing their potential ability to interact with another molecule or ion, for example to form complexes like 1B.

Results and Discussion
To further explore the use of trans-2-aminocyclohexanol moiety as a conformational trigger, we synthesized the model compounds 5-11 (Scheme 4), and evaluated their conformational behaviour in various conditions (Table 1).
ISSN 1424-6376 Page 132 © ARKAT USA, Inc The position of the equilibrium 3A 3B (Scheme 3) was used as an indicator of the changes in intramolecular interactions.The conformer populations (n A , n B ) and the free energy differences between conformers (∆G B-A ) were estimated by 1 H NMR measurements in various solutions (Table 1).The conformer populations were determined using Eliel's equation 43 for signal widths (W = ΣJ HH ) of the cyclohexane protons H 1 , H 2 , H 4 and H 5 , measured as a distance between terminal peaks of a multiplet: W observed = W A n A + W B n B .The signal widths for individual conformers were estimated from measurements for compounds 5-11 and for closely related cyclohexane derivatives with completely biased conformational equilibrium: [16][17][18][19][20]23,44,45 W A = 25.7 Hz (25.0 Hz for 5, 7, 9) and W B = 9 Hz for H OH ; W A = 26.6 Hz (25.5z for 5, 7, 9) and W B = 10 Hz for H NR2 ; and W A = 9 Hz and W B = 30 Hz for H COOR' .The more accurate estimations were usually obtained from the data for H OH (H 5 ) signal.We did not use the averaged chemical shifts for the equilibrium estimations because of their general sensitivity to temperature, to the nature of a solvent, the complex formation, additives, etc.In accordance with the preliminary observations, 20,23 all the studied molecules, except the pyrrolidinyl derivative 7, strongly prefer the conformation 2A (Scheme 2) in nonpolar solvents C 6 D 12 -CCl 4 (1:1) and CDCl 3 .The equilibrium switches to conformation 2B in CD 3 OD.
Apparently, methanol effectively disrupts the intramolecular OH⋅⋅⋅N hydrogen bond that stabilizes 2A.The addition of excess acetic acid causes an opposite switch to conformation A, even in methanol solutions (3A, Scheme 3).Trifloroacetic acid produces a stronger effect.The power of this conformational pH-trigger has been estimated from the measurements for compound 7 as ≥ 12 kJ/mol (Table 1).Hydrogen bonds of both OH⋅⋅⋅N and O⋅⋅⋅H-N + types are known to convert a chair ring into a twist conformation in trans-aminohydroxy steroids 46,47 and some other conformationally locked structures. 42,44This acid-induced twisting of six-membered cycles indicates that the actual power of such triggers may be well above 20 kJ/mol.The latter fact also points out that a relative flexibility of cyclohexane ring sets a natural limit to the effective power of conformational tools (levers, locks, counterbalances) in such systems.If the power applied to both ends of the system exceeds the energy difference between the chair and twist-forms of cyclohexane (23-26 kJ/mol 48 ), then the ring may be screwed (for the relevant discussion see 13,17,27,42,44,49 ).
The macrocycle in 11 and the polyether chains in 10 should be able to complex metal ions, thus providing a second binding site required for modelling of a negative allosteric effect.The necessary geometrical arrangement for such complexation can be achieved only in conformations 10B and 11B.When methanolic solutions of 10 or 11 were saturated with KI, the conformational equilibria were shifted to these B conformations (Table 1, Schemes 5,6) with a relatively small power of approximately 1.5-2 kJ/mol. 23Addition of excess acetic acid to these solutions completely switched the equilibrium back to conformations 10A and 11A.By contrast, the conformational equilibrium for the related non-complexing compound 8 was indifferent to the addition of potassium salt (Table 1).
There is a substantial difference in positions of conformational equilibria for similar structures 5-9 with different NR 2 groups.The preference for conformation A (∆G B-A , in CD 3 OD) decreases in order (Table 1): Et 2 N (2.3 kJ/mol) > piperidyl (0.6) > Me 2 N (-1.5)~ morpholyl (-1.5) > pyrrolidyl (-5.4)This order shows poor correlation with the effective bulkiness of NR 2 groups, i.e. their Avalues.As estimated by simple calculations (PCMODEL molecular mechanics 50 ) for R 2 Ncyclohexanes with no account for solvent effects, they are: Et 2 N (6.7 kJ/mol) > piperidyl (5.1) > pyrrolidyl (4.3) ~ Me 2 N (4.2) ≥ morpholyl (3.6)However, the similar PCMODEL calculations for trans-2-R 2 N-cyclohexanols, which included an intramolecular OH⋅⋅⋅N hydrogen bond, produced the preference for the diequatorial conformation (equivalent to A) that qualitatively parallels the experimental order for compounds 5-9: Et 2 N (17.5 kJ/mol) ≥ piperidyl (17.2) > Me 2 N (15.2) ≥ morpholyl (14.9) > pyrrolidyl (8.5)   Apparently, the geometrical requirements of the intramolecular hydrogen bond play an important role.The formation of hydrogen bond of OH⋅⋅⋅N, or O⋅⋅⋅H-N + type forces NR 2 group to adopt a conformation, which is different from its optimum conformation.In other words, the optimum conformations of different NR 2 groups are not equally suited to the formation of hydrogen bond with the vicinal OH group.The magnitude of this additional strain depends on the structure of NR 2 .A similar observation was made for trans-2-amino-and trans-2dimethylamino-cyclohexanols, 49 where the net gauche-attraction between OH and NR 2 (in C 2 Cl 4 ) was stronger for NH 2 than for the more basic NMe 2 group (3.8 kJ/mol and 2.5 kJ/mol, respectively).
However, if the intramolecular hydrogen bond is not included, and the OH group points away from NR 2 group (which may be the case in methanol solution), the calculated preference for the diequatorial conformation A for trans-2-R 2 N-cyclohexanols still parallels the experimental order for 5-9: Et 2 N (10.3 kJ/mol) > piperidyl (8.9) > morpholyl (8.3) ≥ Me 2 N (7.5) > pyrrolidyl (0.3) Evidently, the steric restrictions imposed by the vicinal oxygen may be sufficient to force the equatorial dialkylamino group into non-optimal position thus affecting the conformational preferences of trans-2-R 2 N-cyclohexanols.

Conclusions
The results of the present study prove that the trans-2-aminocyclohexanol moiety can be used as a conformational pH-trigger for the control of the complex formation by various crown ethers and podands via switching of their preferred conformation.The strong conformational coupling of two different binding sites in compounds like 10 or 11 should allow the development of new heterotropic allosteric systems with high negative cooperativity, which may be especially useful for a selective membrane or drug transport.The variation of NR 2 groups allows a broad tuning of the conformational equilibrium, and thus of the complexing ability of these allosteric ionophores.In addition, the basicity of amino functions could be tuned for a response within a narrow pH range, in which such a switchable system could then liberate or bind drugs or toxic compounds.

Experimental Section
General Procedures. 1 H NMR spectra were recorded on Varian VXR-400 (400 MHz) instrument. 13C NMR spectra were recorded on Varian Mercury-300 (75.5 MHz) instrument.The signals were assigned using COSY, HETCOR and homonuclear spin-spin decoupling techniques.
Exact mass measurements were performed on the JEOL LCMate double-focusing mass spectrometer (Peabody, MA, USA) equipped with atmospheric pressure chemical ionization source at a resolving power of 5000 with polyethyleneglycol as an internal reference.The MS/MS spectra were obtained using the Varian 1200L triple quadrupole mass spectrometer (Walnut Creek, CA, USA) with electrospray source.