From the
To examine the effect of microenviroments on previtamin D
The photobiogenesis of vitamin D
The relevance of preD
The
shell tissue of sea urchin has been found to have the greatest ability
to alter the rate and equilibrium of preD
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
On-line formulae not verified for accuracy
In analogy to the thermal interconversion between free
preD
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
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
On-line formulae not verified for accuracy
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
Based on Arrhenius' equation (Equation 3) it is
evident that the rate constant can be increased either by lowering the
activation energy ( E
To examine the effects of cavity size of cyclodextrin
on the reaction rate of preD
We thank David Jackson for his assistance in preparing
the graphics.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
vitamin D
isomerization, 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 D
isomerization were increased by more than 40 and 600 times,
respectively, compared with those in n-hexane
( k
, 8.65
10
versus 1.76
10
s
;
k
, 8.48
10
versus 1.40
10
s
), 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
D
at equilibrium was increased as the temperature was
increased in
-cyclodextrin. When complexed with
-cyclodextrin, the previtamin D
vitamin
D
isomerization became endothermic
(
H
= 13.05 kJ
mol
) in contrast to being exothermic in other media.
We propose that thermodynamically unfavorable cZc conformers
of previtamin D
are 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 D
endocrine system in vivo such as in the sea urchin.
in 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
C
and C
to 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). PreD
is an
obligatory precursor for the biogenesis of vitamin D
. Once
formed, preD
begins to thermally isomerize to vitamin
D
viaan antarafacial
[1,7]-sigmatropic hydrogen shift from C
to
C
(Dauben and Funhoff, 1988a, 1988b; Yamamoto and Borch,
1988; Curtin and Okamura, 1991). The thermal rearrangement of
preD
vitamin D
is an intramolecular
concerted process. Due to the reversibility of this isomerization,
vitamin D
and its precursor preD
always coexist
and constantly interconvert. This contrasts markedly with all other
steroids.
vitamin D
endocrine 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)
D
associated with
genomic activity as well as the uncharacterized membrane receptors for
both 1,25-(OH)
D
and 1,25-dihydroxyprevitamin
D
(1, 25-(OH)
preD
) associated with
nongenomic activity. Hobbs et al. (1987) reported the first
example that preD
vitamin D
isomerization 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
preD
at 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
preD
was greatly increased.
For example, at 10 °C less than 5% of vitamin D
converted to previtamin D
in 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 D
into preD
(Hobbs et al., 1987). Last and most unusual was the
percentage of vitamin D
at 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
D
with increasing temperature (Cassis and Weiss, 1982;
Yamamoto and Borch, 1985; Tian et al., 1993, 1994).
vitamin
D
isomerization (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).
-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 D
reaction in the sea urchin and investigated the mechanism by
which the reaction kinetics was modulated by this constrained medium.
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). PreD
was chemically synthesized by
photolysis of 7-DHC solution according to a previous reported method
(Tian et al., 1993, 1994). PreD
in
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-DHC
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 Complex
-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
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-Cyclodextrin
Complex
) (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 D
and preD
in 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 D
were 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:
and vitamin D
in solutions, rate constants
( k
and 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.
Kinetic Analysis
Incubation of purified
preDin
-cyclodextrin aqueous solution at 37 °C
for 30 min resulted in the conversion of 60% of preD
into
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 D
was
converted into preD
at 37 °C within 30 min, whereas in
n-hexane no conversion of vitamin D
into
preD
was 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 D
into
vitamin D
in n-hexane at 37 °C. 1% of
previtamin D
is converted into vitamin D
at end
of 30 min of incubation; B, the HPLC profile of previtamin
D
vitamin D
isomerization in
-cyclodextrin at the same temperature. 60% of previtamin D
is converted into vitamin D
during a period of 30 min
of incubation; C, incubation of vitamin D
in
n-hexane at 37 °C for 1 h. No conversion of vitamin
D
to preD
was detected at the end of the
incubation; D, whereas incubation of vitamin D
in
-cyclodextrin solution at 37 °C for 30 min resulted in 19% of
vitamin D
being 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 D
inclusion complexes (Eq. 1) was expressed as
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
The
temperature dependence of the rate constant was defined by
Arrhenius' equation
Vitamin D
Interconversion
Effects of Temperature on Equilibrium Constants of
PreD
The equilibrium constant for preD Vitamin D
Interconversion
vitamin D
interconversion depends strongly
on temperature. The effect of temperature on the equilibrium constant K
was given by the van't Hoff equation
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
S
for preD
vitamin D
interconversion 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
Effects of Cavity Size and Hydroxyl Groups of
Cyclodextrin on Reaction Rate and Equilibrium of PreD
Compared to
the rate constant in Vitamin D
Isomerization
-cyclodextrin at 37 °C, the determined
k values in
-cyclodextrin ((2.18 ±
0.0035)
10
s
) and in
methyl-
-cyclodextrin ((7.66 ± 0.038)
10
s
) were decreased by more than
20- and 6-fold, respectively. Whereas the percentage of vitamin D
at 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).
vitamin D
interconversion 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. PreD
is
conformationally flexible and undergoes rotation around
C
-C
single 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 C
methyl group and C/D rings. The cZc
conformers are able to undergo alternative reaction pathways. They can
either thermally isomerize to vitamin D
or 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 preD
with
-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 D
is 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 D
is responsible for the formation of lumisterol and
7-dehydrocholesterol, whereas 5,6-s- trans-conformer is the
precursor of tachysterol. Previtamin D
is thermally liable,
once formed it begins to isomerize to vitamin D
via5,6-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 D
is responsible
for the thermal isomerization to previtamin
D
.
The thermodynamics and kinetics
of preD vitamin D
reaction 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 k
and k
,
respectively (). Third, the percentage of vitamin D
at 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
H
is that the activation
energy for the reverse reaction
( E
) is markedly reduced and
becomes smaller than E
(I), i.e. E
-
E
=
H
> 0.
) through the
formation of inclusion complex, or by increasing frequency factor
( A) through properly orienting C
and C
of the preD
and vitamin D
molecules, or a
combination of both. For the forward reaction, preD
vitamin D
both effects exist and are additive, with
E
being lowered by 2.5 kJ
mol
and A
being increased by
17-fold. By this mechanism, the forward rate constant k
for 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 k
is a consequence of a significantly lowered
E
being offset by a smaller
A
(I). If A
were
not decreased, we could have expected that at 5 °C,
k
for the vitamin D
preD
isomerization in
-cyclodextrin would be increased by a
million fold.
vitamin D
isomerization, 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 preD
and
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 D
at equilibrium revealed a novel mechanism by which the
equilibrium of preD
vitamin D
isomerization can be modulated by constrained media. A similar
mechanism may exist in vivo by which the preD
vitamin D
endocrine system is modulated to
meet various physiological requirements.
Table:
Temperature dependence on the rate constants of
preD vitamin D
isomerization in an
aqueous solution of
-cyclodextrin (A) and in n- hexane
(B)
Table:
Temperature dependence on the
equilibrium constants of preD vitamin D
isomerization in an aqueous solution of
-cyclodextrin and in
n- hexane
Table:
Arrhenius rate parameters and thermodynamic
values for preD vitamin D
isomerization in an aqueous solution of
-cyclodextrin (A)
and in n- hexane (B)
Table:
Activation parameters for preD vitamin D
isomerization in an aqueous
solution of
-cyclodextrin (A) and in n- hexane (B)
, previtamin D
;
1,25-(OH)
D
, 1,25-dihydroxyvitamin
D
; 1,25-(OH)
preD
,
1,25-dihydroxyprevitamin D
; HPLC, high performance liquid
chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.