©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Catalyzed Thermal Isomerization between Previtamin Dand Vitamin Dvia -Cyclodextrin Complexation (*)

Xiao Q. Tian , Michael F. Holick (§)

From the (1) Vitamin D, Skin, and Bone Research Laboratory, Endocrinology Section, Department of Medicine and Department of Physiology, Boston University Medical Center, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To examine the effect of microenviroments on previtamin D vitamin Disomerization, we have conducted kinetic studies of the reaction in an aqueous solution of -cyclodextrin. Our results showed that at 5 °C, the forward ( k) and reverse ( k) rate constants for previtamin D vitamin Disomerization were increased by more than 40 and 600 times, respectively, compared with those in n-hexane ( k, 8.65 10versus 1.76 10s; k, 8.48 10versus 1.40 10s), the fastest rate of this isomerization ever reported at this temperature. Thermodynamic studies revealed that the equilibrium constant of the reaction was significantly reduced by more than 12-fold when compared to that in n-hexane at 5 °C, and the percentage of vitamin Dat equilibrium was increased as the temperature was increased in -cyclodextrin. When complexed with -cyclodextrin, the previtamin D vitamin Disomerization became endothermic ( H= 13.05 kJ mol) in contrast to being exothermic in other media. We propose that thermodynamically unfavorable cZc conformers of previtamin Dare stabilized by -cyclodextrin, and thus the rate of the isomerization is increased. This conformation-controlled process may play an important role in the modulation of previtamin D vitamin Dendocrine system in vivo such as in the sea urchin.


INTRODUCTION

The photobiogenesis of vitamin Din the skin consists of two sequential pericyclic reactions (Fig. 1) (Havinga, 1973; Holick et al., 1980; Moriarty et al., 1980; MacLauglin et al., 1982). The first step involves the ultraviolet-B- (UV-B,() 290-315 nm) induced electrocyclic ring opening of 7-dehydrocholesterol (7-DHC) between Cand Cto form a seco-sterol, previtamin D(preD) (Woodward and Hoffmann, 1965; Havinga, 1973; Esvelt et al., 1978; Jacobs and Havinga, 1979; Holick et al., 1979, 1980). PreDis an obligatory precursor for the biogenesis of vitamin D. Once formed, preDbegins to thermally isomerize to vitamin Dviaan antarafacial [1,7]-sigmatropic hydrogen shift from Cto C(Dauben and Funhoff, 1988a, 1988b; Yamamoto and Borch, 1988; Curtin and Okamura, 1991). The thermal rearrangement of preD vitamin Dis an intramolecular concerted process. Due to the reversibility of this isomerization, vitamin Dand its precursor preDalways coexist and constantly interconvert. This contrasts markedly with all other steroids.

The relevance of preD vitamin Dendocrine system to biological activity was recently implicated in studies (Norman et al., 1993; Dormanen et al., 1994) suggesting the existence of different forms of the 1,25-dihydroxyvitamin D(1,25-(OH)D) receptor: the classic nuclear receptor for 1,25-(OH)Dassociated with genomic activity as well as the uncharacterized membrane receptors for both 1,25-(OH)Dand 1,25-dihydroxyprevitamin D(1, 25-(OH)preD) associated with nongenomic activity. Hobbs et al. (1987) reported the first example that preD vitamin Disomerization could be altered in vivo in the sea urchin Psammechinus miliaris. There were three remarkable features for the reaction in the sea urchin. First and most important, the equilibrium of the reaction is dramatically altered and shifted toward preDat equilibrium (45% in the sea urchin versus 8% in n-hexane at 10 °C). The second striking feature for the reaction in the sea urchin was that the rate of conversion of vitamin D preDwas greatly increased. For example, at 10 °C less than 5% of vitamin Dconverted to previtamin Din n-hexane after 1 month (Tian et al., 1993). In contrast in the sea urchin at the same temperature, it took only about 1-2 days to convert as much as 30-45% of vitamin Dinto preD(Hobbs et al., 1987). Last and most unusual was the percentage of vitamin Dat equilibrium was increased as temperature was increased (72 and 78% at 17.5 and 20 °C, respectively), which is in contrast to all other known reaction systems reported to date which showed a decrease in the amount of vitamin Dwith increasing temperature (Cassis and Weiss, 1982; Yamamoto and Borch, 1985; Tian et al., 1993, 1994).

The shell tissue of sea urchin has been found to have the greatest ability to alter the rate and equilibrium of preD vitamin Disomerization (Hobbs et al., 1987). However, the active agent within the shell has not been identified. It is known that more than 90% of mollusk shell consists of inorganic salts, mainly calcium carbonate, and the remainder are proteins and polysaccharides (Rieke et al., 1992). Both protein component and pure mineral salts fail to catalyze the isomerization, and the effect of saccharides has not been examined (Hobbs et al., 1987).

Cyclodextrins, the naturally occurring, truncated cone-shaped oligosaccharides, have received increasing attention in recent years for their ability to complex a variety of guest molecules including steroids into their hydrophobic cavities in aqueous solution (Saenger, 1984; Liu et al., 1990; Albers and Muller, 1992). These microheteroenvironments have been shown to modify both energetics and dynamics of many chemical reactions (Ueno and Osa, 1991; Pitchumani and Ramamurthy, 1994). Of great importance is their ability to catalyze reactions of a wide variety of guest molecules (Breslow, 1984; Tabushi, 1984; Chen and Pardue, 1993). It is known that -cyclodextrin is capable of forming 2:1 (host/guest) inclusion complexes with vitamin D(Szejtli et al., 1980; Szejtli, 1984; Bogoslovsky et al., 1988). Therefore, we evaluated -cyclodextrin as a possible model to mimic the preD vitamin Dreaction in the sea urchin and investigated the mechanism by which the reaction kinetics was modulated by this constrained medium.


MATERIALS AND METHODS

Chemicals

Crystalline -cyclodextrin, methyl--cyclodextrin (mean degree of substitution, 10.5-14.7), -cyclodextrin, vitamin D(>99%), and 7-DHC (98%) were purchased from Sigma and were used as received without further purification. n-Butanol (>99%) was obtained from Aldrich. High performance liquid chromatography (HPLC) grade n-hexane and 2-propanol were obtained from EM Science (Gibbstown, NJ). PreDwas chemically synthesized by photolysis of 7-DHC solution according to a previous reported method (Tian et al., 1993, 1994). PreDin n-hexane solution was stored in argon-flushed glass ampoules at -70 °C until use, and the purity was checked by HPLC analysis and its UV absorption spectrum.

Preparation of 7-DHCCyclodextrin Complex

The inclusion complex was prepared by a modified method described by Duveneck et al. (1989), i.e. addition of one volume of 1 m M 7-DHC in ethanol to 50 volumes of aqueous -cyclodextrin solution (15 mg/ml). The prepared solutions were stirred at room temperature for at least 4 h and then filtered to remove possible precipitates to obtain a clear solution. The formation of the inclusion complex was verified by the appearance of characteristic UV absorption spectrum of 7-DHC (MacLauglin et al., 1982). Due to 7-DHC's very low water solubility, no UV absorption for 7-DHC could be detected when using pure water as the solvent. The same procedure was used to prepare the inclusion complexes of 7-DHC with -cyclodextrin and methyl--cyclodextrin in aqueous solution.

Photolysis of 7-DHC-Cyclodextrin Complex

Solutions of 7-DHC inclusion complex were placed in argon-flushed quartz tubes and irradiated on ice by UV-B Medical Sunlamps (National Biological Corp., Cleveland, OH) for 1 min (40 mJ cm) (Tian et al., 1993). For kinetic studies, triplicate exposed solutions were incubated at 5, 30, 37, and 50 °C for various durations. Aliquots sampled at each time interval were immediately extracted with a precooled n-butanol/ n-hexane solution (15:85, v/v). The amount of vitamin Dand preDin each sample was quantified by a previously described HPLC method (Tian et al., 1993, 1994).

Kinetic Studies

Due to high excess of -cyclodextrin (host/guest = 660:1) and the virtual insolubility of free 7-DHC in water, it was expected that 7-DHC, preD, and vitamin Dwere completely complexed with -cyclodextrin forming readily water-soluble inclusion complexes (Szejtli et al., 1980; Szejtli, 1984). Therefore, the following reversible thermal isomerization existed in the exposed solutions:

On-line formulae not verified for accuracy

In analogy to the thermal interconversion between free preDand vitamin Din solutions, rate constants ( kand k), equilibrium constant ( K), and thermodynamic activation parameters were calculated by using standard methods for reversible first-order reactions (Tian et al., 1993, 1994). In brief, the rate constants were obtained from the slopes of the plots of ln[( D- D)/( D- D)] versus reaction time t. The equilibrium constants were equal to the ratios of forward rate constants ( k) over reverse rate constants ( k). The standard enthalpy change ( H) for the reaction was calculated from the van't Hoff plot, and activation energy ( E) was obtained from Arrhenius plot. Finally, the activation parameters were calculated from Eyring's equation.


RESULTS

Kinetic Analysis

Incubation of purified preDin -cyclodextrin aqueous solution at 37 °C for 30 min resulted in the conversion of 60% of preDinto vitamin D, in contrast to only 1% of conversion in n-hexane (Fig. 2, A and B). For the reverse reaction, in -cyclodextrin, 19% of vitamin Dwas converted into preDat 37 °C within 30 min, whereas in n-hexane no conversion of vitamin Dinto preDwas detected at the end of 1 h of incubation (Fig. 2, C and D).


Figure 2: HPLC separation and quantification of previtamin D and vitamin D. A, the HPLC profile of thermal isomerization of previtamin Dinto vitamin Din n-hexane at 37 °C. 1% of previtamin Dis converted into vitamin Dat end of 30 min of incubation; B, the HPLC profile of previtamin D vitamin Disomerization in -cyclodextrin at the same temperature. 60% of previtamin Dis converted into vitamin Dduring a period of 30 min of incubation; C, incubation of vitamin Din n-hexane at 37 °C for 1 h. No conversion of vitamin Dto preDwas detected at the end of the incubation; D, whereas incubation of vitamin Din -cyclodextrin solution at 37 °C for 30 min resulted in 19% of vitamin Dbeing converted into preD. Chromatograms were obtained at 254 nm on an Econosphere silica column (250 4.6 mm, 5 µm) with mobile phase containing 0.45% 2-propanol in n-hexane.



The integrated rate equation for the thermal interconversion between preDand vitamin Dinclusion complexes (Eq. 1) was expressed as

 

On-line formulae not verified for accuracy


Figure 3: Comparison of kinetics of previtamin D vitamin D reaction in -cyclodextrin and in n-hexane at 5 °C ( A) and 37 °C ( B). The rate constants of the isomerization ( k) in n-hexane () and in -cyclodextrin ([b<]p) were calculated from the slopes of the straight lines by least-squares analysis. The data presented are means of three determinations.



Effects of Temperature on Rate Constants of PreD Vitamin DInterconversion

The temperature dependence of the rate constant was defined by Arrhenius' equation

 

On-line formulae not verified for accuracy

Effects of Temperature on Equilibrium Constants of PreD Vitamin DInterconversion

The equilibrium constant for preD vitamin Dinterconversion depends strongly on temperature. The effect of temperature on the equilibrium constant K was given by the van't Hoff equation

  

On-line formulae not verified for accuracy

Whereas the reported van't Hoff equation (Tian et al., 1993) for the reaction in n-hexane was

 

On-line formulae not verified for accuracy


Figure 4: Comparison of temperature dependence of equilibrium constants for previtamin D vitamin D isomerization in -cyclodextrin ([b<]p) and in n-hexane (). Effects of temperature on equilibrium constants for the reaction in these two media are opposite, which was determined by the sign of the standard enthalpy changes, i.e. H> 0 or H< 0. For details see ``Results'' and ``Discussion.''



Thermodynamic Analysis

The determined values of standard thermodynamic parameters G, H, and Sfor preD vitamin Dinterconversion in -cyclodextrin solution are given in I together with the reported values for the reaction carried out in n-hexane (Tian et al., 1993). Eyring's Transition-State Theory and the Activation Parameters-According to transition-state theory, the rate of a reaction at any given temperature depends solely on the concentration of the high energy activated complex. Eyring's equation relates the rate constant to quasithermodynamic parameters by the following expressions

 

On-line formulae not verified for accuracy

Effects of Cavity Size and Hydroxyl Groups of Cyclodextrin on Reaction Rate and Equilibrium of PreD Vitamin DIsomerization

Compared to the rate constant in -cyclodextrin at 37 °C, the determined k values in -cyclodextrin ((2.18 ± 0.0035)10s) and in methyl--cyclodextrin ((7.66 ± 0.038) 10s) were decreased by more than 20- and 6-fold, respectively. Whereas the percentage of vitamin Dat equilibrium at 37 °C were increased from 64.2 ± 1.8 in -cyclodextrin to 96.7 ± 0.2 in -cyclodextrin and 96.6 ± 0.2 in methyl--cyclodextrin (Fig. 5).


DISCUSSION

A change in the reaction medium such as polarity, viscosity, etc. , can have a substantial influence on the kinetics of chemical reactions. However, from a chemical point of view, these parameters usually do not have a major impact on an intramolecular concerted process. It has been assumed that the rate and equilibrium of preD vitamin Dinterconversion was only affected by temperature (Hanewald et al., 1961; Schlatmann et al., 1964; Sanders et al., 1969). However, in a biological system, the conventional chemical media have been replaced with diversified physiological environments, such as lipid bilayers, micelles, proteins, nucleic acids, and polysaccharides. In contrast to isotropic solutions, these organized and constrained media have the unusual ability to dramatically modulate the conformational equilibrium of guest molecules that may ultimately lead to catalysis or inhibition by favoring or disfavoring particular conformations. PreDis conformationally flexible and undergoes rotation around C-Csingle carbon bond to create cZc (s- cis,s- cis) and tZc (s- trans,s- cis) conformations (Fig. 1) (Dauben and Funhoff, 1988a, 1988b; Norman et al., 1993). In isotropic solutions cZc conformation is energetically less stable due to steric interactions between Cmethyl group and C/D rings. The cZc conformers are able to undergo alternative reaction pathways. They can either thermally isomerize to vitamin Dor photochemically convert to lumisterol, whereas the tZc conformers are the precursors solely responsible for the photoproduction of tachysterol (Dauben and Funhoff, 1988a, 1988b; Terenetskaya et al., 1992). We hypothesize that the complexation of preDwith -cyclodextrin shifts its conformational equilibrium in favor of formation of cZc conformation, and therefore the rate constant is increased. This hypothesis is supported by the finding that irradiation of 7-DHC-cyclodextrin complex results in marked increase in the formation of lumisterol with a concomitant decrease in the amount of tachysterol compared with the reaction carried out in isotropic solutions.()


Figure 1: Schema for conformation-controlled photolysis of 7-dehydrocholesterol and thermal isomerization between previtamin D and vitamin D. 7-Dehydrocholesterol is first converted by ultraviolet-B irradiation into a seco-steroid, previtamin D. Unlike its precursor, previtamin Dis conformationally mobile, which undergoes rotation around the 5,6 carbon-carbon single bond to create two distinct conformers, i.e. 5,6-s- cis ( cZc) and 5,6-s- trans ( tZc) previtamin D. Photochemically, 5,6-s- cis-previtamin Dis responsible for the formation of lumisterol and 7-dehydrocholesterol, whereas 5,6-s- trans-conformer is the precursor of tachysterol. Previtamin Dis thermally liable, once formed it begins to isomerize to vitamin Dvia5,6-s- cis-conformer by a [1,7]-sigmatropic hydrogen shift. Vitamin D, like its precursor, is also conformationally flexible and undergoes rotation around the 6,7 carbon-carbon single bond. The 6,7-s- cis-conformer of vitamin Dis responsible for the thermal isomerization to previtamin D.



The thermodynamics and kinetics of preD vitamin Dreaction in -cyclodextrin solution medium showed striking similarities to those in the sea urchin. First, the equilibrium of the reaction was greatly shifted to preD, i.e. from 8% in n-hexane to 48% in -cyclodextrin solution at 10 °C, which agrees well with the reported value in the sea urchin (more than 45%) (Hobbs et al., 1987). Second, like the reaction in sea urchin, the rate of the isomerization in -cyclodextrin solution is among the fastest ever known, i.e. more than 40- and 600-fold increases in kand k, respectively (). Third, the percentage of vitamin Dat equilibrium in -cyclodextrin solution is increased as the temperature is raised (Fig. 4), which is determined by the negative slope of the van't Hoff plot ( H> 0) (Equations 4 and 5). And this is in contrast to all other reported reaction media to date (Schlatmann et al., 1964; Cassis and Weiss, 1982; Yamamoto and Borch, 1985; Tian et al., 1993, 1994). Since H= E- E, the mechanism responsible for the positive His that the activation energy for the reverse reaction ( E) is markedly reduced and becomes smaller than E(I), i.e. E- E= H> 0.

Based on Arrhenius' equation (Equation 3) it is evident that the rate constant can be increased either by lowering the activation energy ( E) through the formation of inclusion complex, or by increasing frequency factor ( A) through properly orienting Cand Cof the preDand vitamin Dmolecules, or a combination of both. For the forward reaction, preD vitamin Dboth effects exist and are additive, with Ebeing lowered by 2.5 kJ moland Abeing increased by 17-fold. By this mechanism, the forward rate constant kfor the reaction carried out in -cyclodextrin solution was increased more than 40 times compared to that in n-hexane at 5 °C (Table I). However, the dramatically increased kis a consequence of a significantly lowered Ebeing offset by a smaller A(I). If Awere not decreased, we could have expected that at 5 °C, kfor the vitamin D preDisomerization in -cyclodextrin would be increased by a million fold.

To examine the effects of cavity size of cyclodextrin on the reaction rate of preD vitamin Disomerization, kinetic studies were carried out in -cyclodextrin. We found that when the cavity diameter of cyclodextrin was decreased from 6.2 Å (-cyclodextrin) to 4.9 Å (-cyclodextrin), the rate constant was decreased by more than 20 times. These results indicate that similar to an enzymatic reaction, the size of the hydrophobic cavity had a great influence on the reaction rate. To assess the influence of outer surface hydroxyl groups of cyclodextrin on the reaction rate, comparisons were made between the reactions in -cyclodextrin and in methyl--cyclodextrin. It was found that reaction rate in -cyclodextrin was six times faster than that in methyl--cyclodextrin. Since partial permethylation had no effect on the cavity size and basic conformation of the cyclodextrin (Myles et al., 1994), these data suggest for the first time that the host hydroxyl groups can accelerate the reaction rate of the [1,7]-sigmatropic hydrogen shift between preDand vitamin D. It is interesting to note that the degree of the acceleration of the reaction rate by intermolecular hydroxyl groups is similar to the reported values for intramolecular hydroxyl-directing effects (-facial selectivity) on the reactions involving [1,7]-sigmatropic hydrogen shift (Hoeger et al., 1987, Wu and Okamura, 1990; Curtin and Okamura, 1991). The observation that either changing cavity size or masking host hydroxyl groups resulted in a dramatic increase in the percentage of vitamin Dat equilibrium revealed a novel mechanism by which the equilibrium of preD vitamin Disomerization can be modulated by constrained media. A similar mechanism may exist in vivo by which the preD vitamin Dendocrine system is modulated to meet various physiological requirements.

  
Table: Temperature dependence on the rate constants of preD vitamin Disomerization in an aqueous solution of -cyclodextrin (A) and in n- hexane (B)


  
Table: Temperature dependence on the equilibrium constants of preD vitamin Disomerization in an aqueous solution of -cyclodextrin and in n- hexane


  
Table: Arrhenius rate parameters and thermodynamic values for preD vitamin Disomerization in an aqueous solution of -cyclodextrin (A) and in n- hexane (B)


  
Table: Activation parameters for preD vitamin Disomerization in an aqueous solution of -cyclodextrin (A) and in n- hexane (B)



FOOTNOTES

*
This work was supported in part by Grants RO1-AR-36963 from the National Institutes of Health and 199081769 from the National Aeronautics and Space Administration. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 617-638-4545; Fax: 617-638-8882.

The abbreviations used are: UV-B, ultraviolet-B; 7-DHC, 7-dehydrocholesterol; preD, previtamin D; 1,25-(OH)D, 1,25-dihydroxyvitamin D; 1,25-(OH)preD, 1,25-dihydroxyprevitamin D; HPLC, high performance liquid chromatography.

X. Q. Tian and M. F. Holick, unpublished results.


ACKNOWLEDGEMENTS

We thank David Jackson for his assistance in preparing the graphics.


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