1. Synthesis of Templated Polymeric Materials for Plasma LPA Enrichment
By:
Cris Ochoa
Department of Chemistry
Private Investigator: Dr. Robert Strongin

2. Introduction
Ovarian cancer is a deadly disease that around 10 million women in the United States are at risk for, with 21,000 new cases each year
CA125 (cancer-antigen) is the main biomarker used now, but it has a very low detection rate so it is usually discovered only during the late stages and when that occurs, the survival rate is 30%
Previous studies have shown that LPA could be a biomarker that can help identify ovarian cancer in its earliest stage, which would raise the survival rate to 90%

3. Total LPA Concentration (µmol/L)
LPA background
LPA 18:1
LPA 14:0
LPA 20:4
LPA 16:0
LPA 18:0
Lysophosphatidic acid (LPA) is a phospholipid that have been show to stimulate the reproduction of cancer cells and it appears to be in larger numbers in the blood of people with ovarian cancer.
Yield around 53%
Total LPA Concentration (µmol/L)
No Ovarian Cancer
Ovarian Cancer
1.86
11.53
Sedláková, I, J Vávrová, J Tošner, and L Hanousek. "Lysophosphatidic acid (LPA)—a perspective marker in ovarian cancer." Tumour Biology (2011): Web.

4. Objective LPA Analysis Process
LPA Enrichment
From Biological
Sample
Sample
Collection
LPA Analysis
The current way LPA is extracted from other samples is through liquid-liquid extraction of plasma, a method that does not remove other phospholipids surrounding it
The only other study that reported extracting pure LPA was through 2D-TLC, which is much more time consuming than using a MIP
Previous research also included using a diol bonded phase to recover pure LPA, but it had low yield
My role in the project is creating an MIP for LPA that would be allow us to extract the LPA from a sample of plasma, since it would bind mostly to LPA, although it may bind to other phospholipids; however, they can be washed off with solvent
Different functional monomers polymerize to create different templates that bind to LPA. There are two functional monomers that I will be creating that will hopefully be better at binding than any other molecule
Commercially Made
Material
Tailor Made Material
(MIPs)
TLC
LC/MS
HPLC GC
Liquid-Liquid Extraction
Solid Phase Extraction
2D TLC

5. Method Proposed Approach
Strongin’s group has developed a method that uses a reversed phase solid support for the solid phase extraction (SPE) of LPA, but a liquid-liquid extraction is needed beforehand in order to remove impurities
It is proposed that LPA enrichment from biological samples could be carried out by the use of tailor made materials called MIPs (Molecularly Imprinted Materials)
It is anticipated that this will shorten sample process time and be more efficient than liquid-liquid extraction.

6. Molecularly Imprinted Materials
Explain how the red shapes are functional monomers, the template is LPA, and how after it is polymerized the template is washed off and what’s left is a monomer matrix (also called MIP) that binds to anything that resembled the template, which would be any type of LPA.
Picture on the bottom right shows how after the MIP is made what we would do in order to extract LPA from plasma.
Binding

7. Functional Monomers Commonly Used for Molecular Imprinting of Phosphates
1 = 1-(4-vinylbenzyl)-1,4,7,10-tetraazacyclododecane
2 = 1-allyl-1,4,7,10-
tetraazacyclododecane
3 = 1-vinyl imidazole
4 = 4-vinyl pyridine
5 = 2-vinyl pyridine = LPA-Cyclen binding 2 1 6 3 5 4

8. Crosslinking Monomers Used for Molecular Imprinting
7 = EGDMA = DVB
8 = Ethylendimethacrylamide 12 = bis(acrylamide)pyridine
9 = Butanediolmethacrylate (BDMA) 13 = N,O-bismethacryloyl phenylalanine
10 = TRIM

9. Results Functional Monomer Synthesis
This is my overall assignment. Talk about how I repeated the first step with Cyclen as training and learning to use equipment, and how the Boc-protected cyclen was used a lot in the subsequent steps in order to create the two different functional monomers.
Explain the basic concept of what happens between each step mechanistically (first step is protecting groups, seconds is creating different functional monomers and how the Boc protecting groups made it so only one nitrogen was nucleophilic, and third step was washing off the protecting groups so that a cross linking monomer could be introduced so that the monomers can polymerize in a stable and rigid way)
Nothing too in depth, that’s for further slides, just a basic overview.
These will be made so they can be polymerized into a polymer that can specifically bind with LPA.

10. Synthesis of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate (Boc-protected Cyclen) Step 1
The first step of the synthesis was taking cyclen, also known as 1,4,7,10-tetrazacyclododecane, and combining it with Di-tert-butyl dicarbonate in order to add the Boc protecting groups to 3 of the Nitrogens.
Explain each part of this reaction, such as that triethylamine was used to provide a basic environment and to deprotonate 3 of the nitrogens which would then attack the di-tert-butyl dicarbonates.
The CHCl3 was the solvent.
The 75% represents the percent yield that I received upon doing the experiment.
I performed this experiment a total of 4 times, the first 2 were purely training on a small scale (50 mg of cyclen), the third time was on a larger 4x scale and it worked out best, with the 75% yield and it was that time that the NMR shows.
The 4th time I performed the experiment was on a 8x grander scale (400 mg of cyclen) and for some reason the reaction did not go through to completion. The reason why is unknown.
Hwang S.; Cha W.; Meyerhoff M. Polymethacrylates with Covalently Linked Copper(II)-Cyclen Complex for in situGeneration of Nitric Oxide from Nitrosothiols in Blood. Chem. Int. Ed. 2006, 45,

11. 1H NMR Boc-Protected Cyclena a a a d c a a c d c b c b a a a
An NMR is used in order to measure the proton peaks, and the peaks matched with previous research very well
Give brief explanation of NMR spectroscopy and how this was used to determine what I made was the product I wanted (describe shielding and unshielded atoms and integration under the spikes)
CDCl3
EtoAc/Hex c a
EtoAc d b
EtoAc
Hex

12. Synthesis of tri-tert-butyl 1-(4-vinylbenzyl)-1,4,7,10-tetraazacyclododecane-4,7,10-tricarboxylate Step 2
The boc-protected cyclen was added to the 4-Bromo-1-vinyl benzene and refluxed for 72 hours in an inert nitrogen system in order to create this molecule. Butylated Hydroxy Toluene (BHT) was added to prevent the molecule from polymerizing prematurely.
Still currently working on this one.
Explanation of why K2CO3 salt was used and what anh means.
Exp
Reactants ratio
Cylen:RX
Temp
(oC)
Reaction time
(h)
Yield
(%) 1
1:4 90 70 2 96
TBA 3
1:1.5 43

13. 1H NMR Boc-Protected-Benzyl Cyclena a a a c c a a c c c b c c b h a h g a a g f e d a
CDCl3
EtoAc/Hex
EtoAc h g c
Hex f e d b
EtoAc

14. Synthesis of tri-tert-butyl 1-allyl-1,4,7,10-tetraazacyclododecane-4,7,10-tricarboxylate Step 2
Boc-protected cyclen was added to allyl bromide and refluxed for 72 hours under an inert nitrogen system. BHT was once more added to inhibit polymerization.
0.1% of the mass of cyclen – BHT amount
Ran TLC plates after certain amounts of hours to see whether reaction went to completion before 72 hours but it did not appear so.
Hurdle was originally we had 4 equivalents of allyl bromide for each equivalent of the boc protected cyclen, but after 48 hours we saw all the allyl bromide had been used up but the cyclen remained, therefore we added another 4 equivalents of allyl bromide to finish reacting with the cyclen.
Exp
Reactant Ratio
Cyclen:RX
Temp
(oC)
Reaction time
(h)
Yield
(%) 1
1:8 90 70
43.16 2 96
35.34

15. 1H NMR Boc-Protected-Allyl Cyclenf a a e d a a a a
This is after the vacuum filtration, where the solvent was evaporated as much as we could. There are still some solvent peaks but they are much smaller.
CDCl3
EtoAc/Hex a
EtoAc
EtoAc
Hex c b f e d

16. Conclusions Creation of the MIP

17. Future Research
Synthesize deprotected benzyl and allyl monomers.
LPA-Monomer Binding Studies by H NMR.
Synthesis of MIPs.
LPA Screening of MIPs using control samples.
LPA-MIP enrichment from human plasma.

18. Acknowledgements
National Science Foundation
Woman and Infants Hospital of Rhode Island
Brown University
Dr. Robert Strongin, Dr. Martha Sibrian-Vazquez, Jialu Wang, and everyone else in the Strongin lab

19. Questions?
If so, ask away!