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TRANSCRIPT
THE FLORIDA STATE UNIVERSITY
COLLEGE OF ARTS AND SCIENCES
SOLID-STATE PHOTOCHEMISTRY OF PROVITAMIN D
By
GOVIND H. KALLUMKAL
A Thesis submitted to theDepartment of Biological Sciences
in partial fulfillment of the requirements for graduation withHonors in the Major
Degree Awarded:Spring, 2017
The members of the Defense Committee approve the thesis of Govind Kallumkal Defended on April 26, 2017 (Signatures on file with Florida State University Honors Program).
Dr. Jack Saltiel Thesis Director
Dr. Mark Kearley Outside Committee Member
Dr. Trisha Terebelski Committee Member
Dr. Jonathan Dennis Committee Member
2
Acknowledgements
My sincerest thank you goes out to Dr. Jonathan Dennis, Dr. Trisha Terebelski, and Dr. Mark Kearley for their guidance, compassion, and instilment of a love for science. Many thanks go out to Dr. Jack Saltiel for taking me into his lab 3 years ago, helping me mature as a researcher while pushing me to think harder and deeper about science. Finally, thanks to Dr. Shipra Gupta for her endless and limitless help through all of my experiments and studies, even when I forgot to turn the scale off. This research was supported by the National Science Foundation through Grant No. CHE-0846636.
3
Solid-State Photochemistry of Provitamin D
Abstract
Of biological importance, vitamin D and its prohormone (provitamin D) are heavily
involved in calcium and iron absorption by the gut. Although photochemistry in the
vitamin D field has been studied widely, studies have been limited to photoreactions
in solution and glassy media. The motivation for this study of the solid-state
photochemistry of provitamin D is the possibility that it may lead to a “green”
method of synthesizing vitamin D and its valuable derivatives while avoiding the use
of photoreaction solvents. Irradiation of provitamin D in the solid state was shown
to give a single product. This product was synthesized, subsequently isolated, and
spectroscopically characterized. It was shown to differ from the other known
primary and secondary photoproducts provitamin, namely previtamin D and its
major photoproducts: lumisterol and tachysterol. Its NMR spectrum was also
compared to those of the acid rearrangement products isotachysterol and
isoprevitamin D.
4
Table of Contents
1 Introduction..............................................................................................................................................7
2 Methods and Materials......................................................................................................................11
2.0 Materials……………………………………………………………………………………………11
2.1 Irradiation of Acetyl Provitamin D and 25-OH Provitamin D.........................12
2.1.1Scaled up Irradiation.......................................................................................12
2.1.2 TLC Experiment ..............................................................................................12
2.1.3 Column Chromatography Separation.....................................................13
2.2 Irradiation of Provitamin D...........................................................................................13
2.2.1 First Separation of Provitamin D and Irradiation Product............13
2.2.2 Second Irradiation of Provitamin D.........................................................13
2.2.3 Fractional Crystallization.............................................................................14
2.2.4 Second Separation of Provitamin D and Irradiation Product......14
2.3 Irradiation of HCl+Provitamin D.................................................................................14
2.4 Irradiation of Base Washed Provitamin D..............................................................14
3 Results and Discussion......................................................................................................................16
3.1 Irradiation of 25-OH Provitamin D............................................................................16
3.2 Irradiation of Acetyl Provitamin D ............................................................................16
3.2.1 Scaled Up Irradiation ....................................................................................17
3.2.2 TLC Experiment.…………………………………………………………………...17
3.2.3 Column Chromatography Separation……………………………………. 17
3.3 Irradiation of Provitamin D.………………………………………………………………..18
3.3.1 First Separation of Provitamin D and Irradiation Product.………20
3.3.2 Second Irradiation of Provitamin D.…………........………………………22
3.3.3 Fractional Crystallization.……………………………………………………..22
3.3.4 Second Separation of Provitamin D and Irradiation Product.…..23
3.4 Strucural Investigation of the Product…………………………………………………28
3.5 Irradiation of HCl+Provitamin D.………………………………………………………...37
3.6 Irradiation of Base Washed Provitamin D……………………………………………..............38
5
3.7 HPLC/GC………………………………………………………………………………………………………38
3.8 Future Work…………………………………………………………………………………………………39
4. References..............................................................................................................................................41
6
1. Introduction
Exposure of the skin to UV light in the sun converts 7-dehydroxycholesterol
to the prohormone vitamin D3 through a sequence of a photochemical ring openings
followed by a thermal 1,7 hydrogen transfer. Vitamin D is essential to human
bodily functions. After being metabolized by the liver and kidney, the compound,
converted to the more active form 1 ,25-dihydroxyvitamin Dα 3, uses nuclear
receptors, commonly reffered to as transcription factors, in order to absorb calcium
from the blood stream, absorb phosphate in the intestine, mobilize calcium in the
bone, and reabsorb the calcium in the kidney. These are for the purpose of making
bones stronger, more pliable, and properly shaped. 1
Vitamin D became a dietary supplement when it was recognized that people
of the northern areas of the earth do not receive enough sunlight. As such, it was
placed in milk, allowing those who do not produce enough vitamin D through
exposure to sunlight; to ingest an artificially synthesized vitamin D. Currently this
“artificial” vitamin D is synthesized through various forms of solution-state
photochemistry. The primary method for this process is photochemical ring opening
of steroidal Δ5,7-dienes. This procedure is based in the photochemical conversion of
3-hydroxy-7-dehydrocholesterol (Pro) to previtamin D3 (quasi-stationary state of
Pre), which is heated in order to form vitamin D3. It has, however, been seen that
there are several compounds that can be synthesized by the irradiation of 1α-
hydroxy-7-dehydrocholesterol. These compounds are referred to as the vitamin D
field.2
7
Lumisterol (Lumi) and tachysterol (Tachy) are other compounds that can be
synthesized through the irradiation of provitamin D (Pro). This is can be performed
using irradiation with appropriately selected wavelengths (e.g., 313 nm for Lumi,
254 nm for Tachy).3 Fluorescence studies of Tachy showed that in EPA (5:5:2 ether,
isopentane, and ethanol by volume) at 77 K Tachy exists as a mixture of three
conformers.3 Those conformers are also formed through the irradiation of Pro at 77
K which indicates that its ring opening leads to 3 conformations of previtamin D
(Pre). The fluorescence studies of the compounds in 77 K EPA showed that pre
photoisomerization in glassy medium follows the one bond twist mechanism
instead of the previously proposed Hula twist.3
It should be noted that the traditional methods for the synthesis of vitamin D
utilize solvents that are considered environmentally unfriendly. Irradiating Pro in
the solid state may alleviate the concerns of those solvent uses. performing the
Scheme 1. Vitamin D field including the various products formed from irradiation of Pro.3,4
HO
RCH3
CH3
Provitamin D (Pro) Previtamin D (Pre)
h
h h
h
h
RCH3
CH3
HOLumisterol (Lumi)
H
RCH3
CH2
HO
Vitamin D
RCH3
Tachysterol (Tachy)
H3C
OH
Vitamin D2:
R = C9H17
21 22
23
24
25
26
27
21 20 22
23
24
25
26
27Vitamin D3:
=
17
17
CH3
CH3R
123
4 5 67
89
10
11
12
13
14 15
1617
18
19
25-Hydroxyvitamin D3:
R = C8H17
R = C8H17
=
=21 20 22
23
24
25
26
27
17
OH
20
8
majority of the reaction in the solid state as opposed to the solution state the
reaction could be considered a “green” chemical reaction. There are several tenets
that would characterize a green reaction. As previously mentioned, the procedure
must minimize the utilization of chemical solvents. Additionally, the minimization of
solvent use makes the experiment safer for those using it and for the environment ],.
Finally, the experiment itself must be efficient, as the majority of it occurs through
irradiation requiring only the use of a lamp.11
Solution
photochemistry in the vitamin D field is relatively well understood, particularly due
to the efforts of Havinga5 and Dauben.6 Saltiel’s work has led to a reinterpretation of
the observations of Havinga4 and coworkers in low T glassy media. The goal of my
Scheme 2. Pathways and conditions for the irradiation of Pro and Pre to tEc and cEc confirmations of Tachy.3
9
study is to examine the photochemistry of Pro in the solid state. As such one could
hope to better understand how crystal structures of Pro and its derivatives can
affect the products formed through irradiation. It is possible for the crystal
environment to control reactivity and possibly open reaction pathways that are not
observed in solution. The recent discovery that in the solid state certain cis,cis-1,4-
diaryl-1,3-butadienes photoisomerize exclusively by a BP process is a relevant
example7. In order to observe the reaction, Pro, the acetate derivative of Pro, and the
25-OH derivative of Pro
were utilized. It was hoped
that the crystal lattice
would preserve the s-cis,s-
cis conformer structure of
the initial Pre photoproduct
such that melting it would
lead directly to pure vitamin D. If the photoreaction were incomplete, a separation
step from unreacted Pro would be required. Unreacted Pro could then be used to
repeat the reaction. The crystal structure of the D2 form of Pro, ergosterol, is a
known monohydrate, Figure 1.9 The anhydrous Pro is particularly difficult to
crystallize8,9. In the monohydrate it is seen that hydrogen bonds control the layered
packing structure of the crystals and water plays a template role in lining the
molecules in a head to head fashion (scheme 4). On the other hand, the 25-OH Pro
utilized in the experiment is anhydrous as it uses its own 3-OH and 25-OH groups to
10
allow for head to tail
stacking without the
incorporation of water
molecules (scheme 2)10.
2. Methods and Materials
2.0 Materials
7-Dehydrocholesterol (Pro) (95%), 25-OH Pro, ethanol (Absolute Anhydrous,
200 Proof), ethyl acetate, hexanes, isopropanol, CDCl3, acetone D6, HCl (37%),
ammonium hydroxide (28%), and tetrahydrofuran (THF), silica gel, aluminum oxide
(activated, neutral, Brockmann I) were obtained from Sigma Aldrich and used as
received. Acetyl Provitamin D was synthesized previously in lab through
esterification of the obtained Pro and methanoic acid. The Rotovap used for
evaporation was manufactured by Büchi. All NMR spectra were obtained using a
Bruker Avance III 500 MHz NMR. All UV spectra were measured using a Varian Cary
300 UV VIS Spectrophotometer. MALDI was conducted on a Bruker Autoflex III
MALDI Time-of-Flight Mass Spectrometer. Irradiation was done with the use of an
Hanovia medium pressure 450-W Hg lamp filtered through Pyrex.
Figure 2. Unit cell of ergosterol monohydrate; viewed along the unique axis, showing the hydrogen bonding.9
11
2.1 Irradiation of Acetyl Provitamin D and 25-OH Provitamin D
Samples of 20 mg acetate of Pro were placed on each of four Pyrex glass
slides labeled 1, 2, 3, and 4. Another slide was made with 20 mg of 25-OH Pro Solid
crystals were weighed and piled onto one irradiation slide. Another slide was then
placed on top and the crystals flattened out. The slides were then taped together
with duct tape and placed at a distance of 10 cm from a 450-W lamp (within a Pyrex
sheath). Ensuing slides are made and irradiated in this manner as well. Sample 1
was irradiated for 5 h, sample 2 for 10 h, sample 3 for 15 h, and sample 4 for 20 h.
The 25-OH slide was irradiated for 10 h. 1H NMR spectra of the first 3 samples in
addition to an un-irradiated sample and that of 25-OH Pro. Progression of the
reaction was observed through 1H NMR through integration of vinyl H peaks at 5.5
and 5.6 as well as those at 6.25 and 6.5. As the reaction progressed the upfield
peaks diminished while those downfield grew.
2.1.1 Scaled Up Irradiation
Five slides with approximately 0.112 g of Acetyl Pro on each slide were made
and irradiated according to the previous parameters for 30 h. 0.412 g of product
mixture was removed from the slides. The NMR spectrum of a crystal sample and
that of the homogenized mixture were taken.
2.1.2 TLC Experiment
In order to assess the ability to separate the product from the starting
compound, the mixture was observed by TLC with a 6% ethyl acetate: 94% hexanes
solution and a silica TLC. The plate was then exposed to iodine.
12
2.1.3 Column Chromatography Separation
A silica column was used for the separation of the product mixture, using a
mobile phase of 1% ethyl acetate: 99% hexane. Once the starting compound had
been fully eluted through the column, ethyl acetate was increased to 2%, then 4%.
An issue that arose in the process was that the solution within the test tubes was
much too dilute for any spot from the product to be visible on the iodized silica
plates. Eventually, after several liters of solvent were eluted through the column, the
chromatography was concluded.
2.2 Irradiation of Provitamin D
Two slides were made with 0.1 g of Pro each. The slides were irradiated for
37 h (with the same parameters as previously) and the progress of the reaction was
determined by 1H NMR spectroscopy reaction was monitored by NMR. This was
done in the same manner as with the acetate, however NMR spectra were taken
after 0, 15, 20, 25, 37, and 41 h of irradiation.
2.2.1 First Separation of Provitamin D and Irradiation Product
The same method described in the previous separation was used in the
separation of Pro and its irradiation product. This time neutral alumina was used
instead of silica and the reaction was then monitored by UV spectrometry. NMR was
performed once the entire product was separated and subsequently concentrated,.
Acetone-d6 was used as the solvent.
2.2.2 Second Irradiation of Provitamin D
Ten slides with 0.1 g on each slide were irradiated in the same manner as
with previous trials.
13
2.2.3 Fractional Crystallization
Two hundred mg of the product mixture was dissolved in ethanol, with the
expectation that the product and Pro would dissolve fractionally allowing for
separation. Another mixture of 0.1 g was dissolved in THF.
2.2.4 Second Separation of Provitamin D and Irradiation Product
Chromatographic separation of the product was attempted by dry loading
250 mg of irradiated Pro crystals on a column of neutral alumina. The solvent
system used was 1:99 ethanol and hexanes. The separation was monitored through
UV spectrometry. Another attempt was made using 0.2 g of product mixture. The
same parameters were once again utilized for the experiment, however extra heed
was paid to prevent the mixing of the product and Pro.
2.3 Irradiation of HCl+Provitamin D
HCl, 10 μL, was added to 0.05 g of Pro. The crystals and HCl were then
ground together for 10 min with mortar and pestle. The crystals were allowed to
dry overnight in air (in the dark). In terms of morphology they were much different.
The crystals were now finer and were slightly darker in color. All of the crystals
were applied to a slide as previously described and irradiated.
2.4 Irradiation of Base Washed Provitamin D
Pro, 0.4 g, was dissolved in ethyl acetate and washed with 1 N ammonium
hydroxide within a separatory flask. Most separations are completed with diethyl
ether as the organic layer, however, a lack of usable diethyl ether, and the solubility
of Pro within ethyl acetate, led to its use as solvent instead. The solubility of ethyl
acetate in water is 8.1% by weight.22 The crystals were then dried using the
14
Rotovap. Irradiation slides were washed in 1 N ammonium hydroxide for 30 min
and then rinsed in water. Base-washed Pro, 0.1 g, was placed between Pyrex slides
and then irradiated in the same manner as previously described.
15
3. Results and Discussion
3.1 Irradiation of 25-OH Provitamin D
1H NMR spectroscopy revealed no reaction after 15 h of irradiation.
3.2 Irradiation of Acetyl Provitamin D
The un-irradiated sample had vinyl H peaks at 5.5, 5.6 while the 3α peak
was seen at δ 4.7 .As the irradiation of the sample progressed, the intensity of those
peaks diminished and new vinyl H peaks appeared as a pair of doublets at 6.25
and 6.5. Based on the intensity of those peaks, conversions to the photoproduct
were 9.6%, 14.9% and16.8 % after 5, 10 and 15 h of irradiation, respectively,
(Figure 3). A new acetate peak was seen at 7.5 and a 3α peak was seen at δ 5.
Figure 3. NMR of Pro-Acetate product mixture NMR after 5, 10, and 15 h of irradiation in CDCl3. Peaks seen between 3.5 and 4 are due to impurities and the presence of ethanol in the NMR sample.
16
3.2.1 Scaled Up Irradiation
Upon 30 h of irradiation conversion was seen to be 45% according to the
NMR integrations of the vinyl hydrogen from pro and the photoproduct. The
product mixture (0.412 g) was then removed from the slides. The NMR spectrum of
the homogenized mixture of slides 1-4 was taken. It was seen that the conversion
percentage was 25% after 30 h, unlike the 45% seen in slide 5 after 30 h (Figure 3).
A possible explanation for this discrepancy is that every time an NMR was taken to
observe the progress of the reaction, some a portion of the irradiated sample was
removed from the irradiation slide. As the reaction progressed, the slide would have
less and less compound, and would layer the slides less thickly. As such, the
radiation would penetrate the compound more than in the non-test slides.
3.2.2 TLC Experiment
Only acetyl-Pro was very visible under 365 nm light, however this is likely
due to a trienol impurity. A more concentrated solution of the product mixture was
spotted on the TLC plate alongside a sample of pure Pro and the analysis was
carried out. The plate was then placed in an iodine chamber. After the plate was
treated with iodine, two spots were observed with good separation. The Rf value of
Pro was 0.315, while that of the product was 0.162.
3.2.3 Column Chromatography Separation
Very little of the product was isolated. Due to the confusion of why so little
product was isolated when approximately 100 mg was theoretically collectable, the
NMR spectrum of the isolated product was measured. The spectrum differed from
those of the starting compound or the photoproduct. Neither the peaks at 5.45 and
17
5.55 (starting compound) nor the peaks at 6.25 and 6.5 (product) were observed.
Instead, two peaks at 7.5 and 7.6 were observed (see figure 4).
Figure 4. NMR of product after separation and rearrangement
Because the NMR spectrum of the mixture was drastically different from and
generally more clean than that of the separated product, it is highly likely that the
separation process may have played a role in the product’s degradation. One
possibility is that the slightly acidic nature of the column caused a rearrangement in
the product.
3.3 Irradiation of Provitamin D
The irradiation of Pro led to the formation of the same peaks seen at 6.25
and 6.5, and the product as such was analogous to that formed in the irradiation of
18
the acetate. After 37 h irradiation, the product reached 25% relative to unreacted
Pro
Figure 5. NMR spectra of Provitamin D after (a)0, (b)15, (c)20, (d)25, (d) 37 h, and (e)homogenized mixture [all in CDCL3].
Irradiation time, h Conversion percentage
0 0 %
15 7.6%
20 10.4%
25 16.5%
37 25%
Homogenized 22.4%
Table 1. Percent conversion of Pro to product after 0, 15, 20, 25, 37, 41 h of irradiation (observed by NMR)
19
0 5 10 15 20 25 30 35 400
5
10
15
20
25
30
f(x) = 0.633638793432608 xR² = 0.989045569101285
Hours Irradiated
Per
cen
t Co
nve
rted
to
Pro
du
ct
Figure 6. Conversion of Pro to Product after 0, 15, 20, 25, and 37 h of irradiation (observed through NMR)
3.3.1 First Separation of Provitamin D and Irradiation Product
In order to mitigate the issues hypothesized from the usage of silica, neutral
alumina was used again. In taking the UV spectra of the separation fractions it was
seen that the spectra of the product was very similar to that of Tachy. The same
peak at 277 nm was observed with shoulders seen at 269 and 281 nm. The Tachy-
like spectrum was not originally evident in the product mixture, however, this was
not a minor product either.
20
Figure 7. UV spectra of product separation fractions (first separation) and combination of separation fractions in CDCl3.
The NMR spectrum was measured following combination and concentration
of product fractions. In order to avoid the issues observed when taking the NMR of
the product in the presence of CDCl3, acetone-d6 was used as the solvent instead. It
was hoped that avoiding CDCl3 and not leaving the product within the solution for a
long period of time could prevent the rearrangement seen in previous separations.
The NMR unfortunately did show the same rearrangement with a large majority of
the product shifting to one with peaks at 7.6 and 7.7. Intriguingly enough, peaks
associated with the product remained. In taking the UV of the product, it now
however failed to look like that of Tachy and what was seen during the separation.
21
The mixture spectrum additionally failed to look like Tachy. The spectrum of the
isolated rearranged product is shown in Figure 7.
Figure 8. NMR spectra of product after separation and possible rearrangement in CDCl3
3.3.2 Second Irradiation of Provitamin D
Due to the failures of previous experimental trials more Pro was irradiated.
Ten slides with 100 mg on each slide were irradiated in the same manner as with
previous trials. After 46 h the conversion was approximately 35%. You keep
mentioning conversions but you do not describe how they were determined.
3.3.3 Fractional Crystallization
Because several previous attempted separations of the product from Pro had
either failed or there was product degradation, other separation techniques were
attempted, one of which being a fractional crystallization. This method relies on the
22
possibility that the product and Pro have differing solubility. Two hundred mg of the
product mixture was dissolved in ethanol, however, no separation was seen as a gel
like product formed after the ethanol was fully evaporated. It was previously seen
that ethanol would leave residues on crystals preventing their subsequent
formation. Another mixture of 100 mg was dissolved in THF, but, instead of crystals,
the same gel like substance formed.
3.3.4 Second Separation of Provitamin D and Irradiation Product
A sample of irradiated crystals, 0.25 g, was dry loaded on a column of neutral
alumina. The solvent system to be used was ethanol to hexanes (1:99). The
separation was monitored through UV spectrometry. However, after completion of
the separation, it was seen through NMR that the product had not been separated
from Pro and the previously mentioned downshifted peaks were visible once again.
Another attempt was made using the last 0.200 g of product mixture that was left.
The same procedure was once again utilized for the experiment, however caution
was employed to prevent mixing of Pro and product fractions. Five separate
fractions were observed, the fifth being the product, this time however it was seen
that the product did not have traces of Pro and was clean enough to be utilized for
further analysis by COSY. The product separated had a mass of 0.02 g. Spectral
characterization of the product can be seen below.
23
240 250 260 270 280 290 300 310 320 330 3400
0.5
1
1.5
2
2.5
Separated Pro
Product mixture after 37 h irradiation
Separated product af-ter NMR
Wavelength (nm)
Ab
sorp
tion
Figure 9. UV Spectra of product mixture, separated Pro, and separated product (after concentration through Rotovap) within ethanol.
240 250 260 270 280 290 300 310 320 330 3400
0.02
0.04
0.06
0.08
0.1
0.12
0.14
Wavelength (nm)
Ab
sorp
tion
Figure 10. UV Spectra of Product Fractions from Column Separation (solvent 99:1 hexanes and ethanol; every 4th test tube was spectrally analyzed).
It was found that the UV spectrum of the product and supposed structure
changed after completion of the separation, as the UV spectra of the various product
fractions differed from the spectrum of the product after concentration by Rotovap.
The spectra of the product as it eluted is in figure 9, while the spectrum of the
24
concentrated product is in figure 8. The change from a structured UV spectrum to an
unstructured one could be explained by possible cis-trans isomerization of isoTachy
and isoPre which will be further studied 3.4. The 1H NMR, 13C NMR, and COSY
analysis of the final product can be found below.
Figure 11. 1H NMR spectra of (a) Pro [in CDCl3], (b) product mixture after 57 h of irradiation [in CDCl3] and (c), the separated product [in CDCl3].
The NMR (Figure 11) clearly shows that the product received from the
separation was the photoproduct. However, additional peaks can be seen in the
separated product, which would mean that impurities were added to the separated
product fraction during chromatography.
25
Figure 12. 13C NMR of (a) Pro [in CDCl3] and (b) separated product [in CDCl3]
26
Figure 13. COSY Spectra of Product in Acetone-d6.
Analysis of the COSY spectrum shows that the vinyl protons are coupled. As
such, in identifying the structure of the product, it can be assumed that the vinyl
hydrogens are on adjacent C atoms.
In order to check the purity of Pro, product mixture, and the product, several
identifiable peaks were integrated and compared (Table 2).
27
Pro C-6 Peak ( 5.55) C-7 Peak ( 5.45) 3α Peak ( 3.6)
1.001 .9985 1
Product Mixture Pro C-6 Peak
( 5.55)
Product C-6 Peak
( 6.5)
3α Peak
(3.6)
1 .3330 1.2765
Product C-6 Peak ( 6.5) C-7 Peak ( 6.25) 3α Peak ( 4)
1.0183 .9925 1.000
Table 2. Integrations of C-6, C-7, and 3α in Pro, the product mixture, and the product. Numbering can be found in scheme 1.
The observation that all of the identifiable peaks in Pro have the same
integration shows that it is pure, particularly considering that each of the observed
peaks has one hydrogen. That the two C-6 peaks when added together equal the
observed 3α peak as seen in the spectrum for the product mixture (Figure 11)
indicates that only one prodcut is forming. Finally, the product integrations once
again show that the product is not a mixture of multiple steroidal compounds.
3.4 Structural Investigation of Product
The identification process for the compound began with the use of the COSY
and UV spectra of the product. Though the identification process for the product is
ongoing, several compounds are being used as starting points for investigation due
to quantifiable similarities with the product. Vitamin D, Pre, Lumi, and Tachy are not
the product for various reasons. Comparison of the NMR’s clearly shows the
differences. Pre12, Tachy13 and vitamin D14, all have more vinyl protons than the
product does (chemical structures can be seen in scheme 1). It can be seen that
28
Lumi is also not the product as the chemical shifts of its NMR differ significantly
from those of the product. It should additionally be noted that none of the known
products have UV spectra that resemble the spectrum of the product15, (note: Tachy
and the rearranged product during elution had similar UV spectra). The chemical
shifts of the vinyl protons can be seen below.
Compound Carbon 6 Hydrogen Shift
Carbon 7 Hydrogen Shift
Additional Vinyl Proton/Carbon #
Product (acetone-d6) 6.50 6.25 ----------
Provitamin (CDCl3) 5.45 5.6 ----------
Previtamin (CDCl3) 5.93 5.66 5.46/C9
Tachysterol (CDCl3) 6.25 6.05 5.36/C19
Lumisterol (CDCl3) 6.26 5.92 5.62/C9
Vitamin D (CDCl3) 6.24 6.03 5.05, 4.82/C19
Table 3. Chemical shifts of hydrogen on carbons 6/7 as well as other vinyl hydrogen in the Product, Pro, Pre, Tachy, Lumi, Vitamin D.
29
Figure 14. Vitamin D Field UV Spectra14
Isotachysterol (isoTachy) is an isomer of Tachy after the migration of a
double bond and has a spectrum quite similar to that of Tachy itself.17 However, the
vinyl protons in its NMR spectrum resemble those in our product. This is due to the
rearrangement of its double bond resulting in an NMR lacking a third vinyl H
present in Tachy16. The product is similar in that it also lacks the same vinyl H peak
associated with C-14.
30
A contradictory factor in stating that the product is isoTachy is due to the fact that
the chemical shifts between the product and isoTachy are not identical. In acetone-
d6 the vinyl peaks associated with isoTachy are at 6.53 and 6.36 compared to those
of the product, which were at 6.50 and 6.25 ppm. In the spectrum for isoTachy
there are allylic hydrogen’s with the chemical shift of 1.75, a similar singlet peak
seen in the product is at 1.47. Additionally, it was seen that the UV spectrum of the
product after the conclusion of the chromatography was not the same as that of
isoTachy.
Isotachysterol Tachysterol
31
Its cis isomer, isoprevitamin D (isoPre) has similar NMR spectrum to that of
the trans isomer albeit with slightly different chemical shifts17, however, in
comparing its UV spectrum to that of our product a very significant match was
observed. The known UV spectra of isoTachy and isoPre are shown in Figure 15.
Figure 15. Reported UV spectra of (5) trans isotachysterol and (11) Isopre.16
An 8 Hz coupling was observed between the doublet splitting of both vinyl
protons of the product, as such it can be inferred that the ethylenic CC bond is most
likely cis and not trans. It is possible for the structure to be a cyclohexene as
coupling of the vinyl hydrogen is 8. MALDI analysis has shown that the product is an
isomer of Pro as the molar masses of both the Pro and product are 384 g/mol.
It should be noted that there was a discernible difference between the UV
spectrum of the product that was subjected to column chromatography and the
32
spectra of the eluted fractions. Those fractions were combined, concentrated, and
the NMR taken. The original photoproduct is proposed to have cis vinyl Hs
considering the aforementioned coupling constants as well as the fact that the initial
UV spectrum of the reaction mixture is similar to a combination of isoPre and Pro.
However, the eluted fractions from the chromatography had a UV spectrum that is
very similar to that isoTachy, while that of the concentrated product is similar to
that of isoPre. It follows that the photoproduct can rearrange during column
chromatography. This information would mean that during the column separation,
the isoPre18 isomerized to trans and went back after the product was eluted out,
though this is highly unlikely. It has been reported that isoTachy can isomerize both
photochemically and thermally. The trans isomer is the more stable.
Scheme 3. Trans-Cis isomerization of isoTachy to isoPre16
Isotachysterol is capable of oxidizing in air without the need for any other
stimulants.17 This may be an explanation as to why earlier separations of the
product from the product mixture ended with the characterized product having
downshifted peaks at 7.8 and 7.9. So far I have failed to propose any structures
that would fit this new NMR spectrum.
33
Formation of isoPre from Pre, the expected photoproduct, would require that
there be a proton transfer agent within the Pro crystals used for irradiation. The
acid catalyzed isomerization of Vitamin D to isotachy has been reported.16 Since Pro
crystallizes as the monohydrate, figure 1, an attractive explanation is that the water
molecules within the lattice act as the proton transfer agent allowing for the
formation of isoTachy or isoPre. This would additionally explain why the 25-OH Pro
did not form the same product (its crystal lattice is formed through head to tail
hydrogen bond interactions of the the two OH groups, figure 2). Further
corroboration of this hypothesis stems from the fact that the conversion of Pro to
product increased by the addition of HCl to Pro. Though isoPre remains similar to
the product, it is unlikely for it to be the product as it would require either acid
catalysis or multiple photons converting Pro to an intermediary state and then to
cis. The irradiation of base washed Pro corroborated this conclusion as the same
product was made as was without base wash.
13C NMR shows that the product is most likely neither isoTachy nor isoPre.
IsoTachy has peaks at 127.1, 124.6, 125.9, 125.4, 131.6, and 149.3 (due to 6
carbons within alkene bonds). IsoPre should also have 6 peaks within the same are.
The product however only has 2 peaks at 131 and 136, this information
additionally would indicate that the product only has two carbons within alkene
double bonds. As such neither isoPre nor isoTachy cannot be the photoproduct.
34
Another potential structure for the product could be toxisterol A. It also lacks
the third vinyl hydrogen and is a known overirradiation product Pro.
This structure, however, is most likely not the structure of the product either.
In comparing the assigned chemical shifts of the product with that of toxisterol A,
there is very little in terms of matching. The vinyl peaks are seen at 6.25 and 6.5 in
the product while those in toxisterol A are at 6.06 and 6.35. Additionally, the
chemical shift of the product’s 3α peak is at 3.9, while that of toxisterol A is at
3.77.21 In this structure it is predicted that the coupling of the vinyl peaks is
approximately 5.6 considering that the double bond is within a cyclopentene ring,
the coupling of the product however is 8.6. In terms of 13C NMR, this structure has
four peaks associated with sp2 carbons, and also cannot be the product considering
it only has two.
35
The structure can be seen below represents a novel and unstudied potential
answer to the unknown structure of the product. Its structure only allows for two
allylic hydrogens thereby strengthening its similarities to the product, while
distancing itself from other compounds such as vitamin D, and Lumi.
Scheme 4. Newly proposed structure and predicted 1H NMR
The proposed structure does have its dissimilarities, one particular one being
that the vinyl protons have the same chemical shift, which is inherently unlike that
of the product. Additionally, it can be seen that the multiplets seen in the predicted
spectra are more up field than seen in the product’s 1H NMR spectra. This does not
necessarily remove this structure being the product, as we currently only have a
36
predicted spectra. It should be noted that the 13C NMR of this structure would have 2
peaks between 100 and 170. This is similar to the 13C of the product. This structure
additionally represents a product of a photochemically allowed process. Excited by
light, the cyclohexadiene opens to form a triene, from which its orbitals form new
bonds associated with that of the product.
Scheme 5. Photochemical formation of novel structure.
3.5 Irradiation of HCl+Provitamin D
It was previously observed by that crystals can have an altered conformation
through the addition of minute amounts of HCl to crystals, and the subsequent
grinding of the crystals to better ensure the incorporation of HCl in the lattice.20 It
was seen that even with the addition of HCl the same product was formed as
previously, however within 33.5 h the conversion was seen to be 60.5%. The NMR of
37
the product mixtures after 33.5 h can be found below. This may be due to HCl, but
could also be due to the alteration I nthe morphology of the Pro crystal after
grinding.
Figure 16. Zoomed in NMR spectra of Pro+HCl product mixture after 33 h of irradiation in CDCl3.
3.6 Irradiation of Base Washed Provitamin D
It was observed that the same product formed in the irradiation of Pro even
after it was washed in base. As such it can be said that the product forming from
irradiation of Pro is not forming through acid catalysis.
3.7 HPLC/GC
HPLC was utilized as a separation technique after the failures seen with the
column separation and the fractional crystallization. The irradiated mixture of
crystals, 10 mg, was dissolved in 4 mL 25:75 mixture of ethanol and hexanes by
volume. The solvent system used in the HPLC was a 1:99 solution of isopropanol
38
and hexanes. The use of isopropanol instead of ethyl acetate allowed UV observation
at lower than 250 nm, the ethyl acetate cutoff. Originally, 3 spots were seen, a spot
at 1 min retention time associated with potential impurities, a spot at 4 min
associated with the product, and a spot at 10 min associated with Pro. This,
however, was replicated several times. The parameters of the experiment were in
turn scaled up to 0.04 g per 4 mL of the same solution. However, ensuing trials
showed a fairly different separation, and products associated with said separation.
Spots appeared at 4 min, 12 min, and 25 min with different associated UV spectra.
The first being the previously mentioned impurity, the second being something
unassociated with the product, and the spot at 25 min being that of Pro. The
following replications of this experiment at various different mixture concentrations
were congruent with the new spots and Rf values and none of the trials replicated
what was originally seen. Due to the relative inefficiency of utilizing HPLC as a mass
separation technique and the inconsistency of the data associated with the HPLC,
the method was in turn considered an inapplicable method for the separation of our
product and Pro.
GC was proven to be an unusable measure of separation as the compounds
being studied were much too large, peaks associated with Pro and the Product were
not seen in various runs of the GC according to different parameters.
3.8 Future Work
The findings from this preliminary study supports the hypothesis that the
product is most likely the novel structure. A crystal structure would clearly identify
the product, and further investigation is therefore necessary.
39
Crystallization of the product has been attempted in the past and a gel-like
substance was formed. The Alabugin group has, however, utilized TADDOL ( , , ', α α α
'-tetraaryl-2,2-disubstituted 1,3-dioxolane-4,5-dimethanol) for co-crystallization α
purposes. TADDOL has previously shown capabilities of co-crystallizing with
hydrogen-bond acceptors. This, in theory,could aid in the crystallization of the
product.
Though COSY analysis has been completed more spectral analyses can be
done. C-14 NMR as well as other 2-D NMR studies will help in the analysis of the
product. In order to properly utilize such techniques a purer sample of the product
will be required. With this in mind, additional Pro is currently being irradiated in
order to receive more product.
Irradiation of the product within solution could also facilitate its
identification. It was previously mentioned that a possible structure for the product
is isoPre. If this is true, then irradiation of the product within solution could cause
an isomerization to isoTachy. By observing the UV spectra and how they change
over time, one should be able to answer the question whether or not the product is
indeed isoPre with an open-ring system.
40
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