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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