Synthesis of Protein Molecular Probes

1
Synthesis of Protein Molecular Probes

• by
• SHILO ANNIS
• and
• AMBER HAYER

2
Scope of Research

• Oxidative stress has been implicated in numerous
  degenerative diseases of aging, including heart
  disease
• Proteins are modified in-vivo by products of
  lipid peroxidation, such as 4-hydroxynonenal
  (HNE). The HNE-protein adducts are isolated from
  the mitochondria of young and old rats to
  evaluate the occurrence and impact of oxidative
  stress on heart muscle tissue.

3
Scope of Research

• Primary synthetic target biotin derivative
  coupled to an acid liable benzyl ester and a
  carbonyl reactive functional group

Slide courtesy of Professor Prudente, University
of Southern Maine
4
Our Role in Professor Prudentes Research

• Professor Prudente has a long way to go

5
Assignments

• Ambers Protecting Group
• tert-butyl dicarbonate (TBOC)
• Shilos Protecting Group
• Fluorenylmethoxycarbonyl (FMOC)

6
Ambers Procedures

• Chemical Reaction
• Extraction
• Evaporation for Crude Product
• Thin Layer Chromatography (TLC)
• Recrystallization
• High Pressure Liquid Chromatography (HPLC)
• Nuclear Magnetic Resonance (NMR)

7
Ambers ProcedureDay 1Protected
O-(Carboxymethyl) Hydroxylamine Hemi
hydrochloride with Ditertbutyl Dicarbonate
(T-Boc) using Biochemistry literature as a
reference
1 ½ HCL

O-(Carboxymethyl) hydroxylamine hemi
hydrochloride MW109g Added to triethylamine
Et3N and dissolved in water
ADD Ditertbutyl dicarbonate MW218.3 (t-boc
protecting group) in dioxane

T-BOC PROTECTED PRODUCT
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EXTRACTIONSDay 2

• Extractions are used to separate a compound from
  a mixture.
• Performed extraction (3x) with Ethyl Acetate
  CH3CH2OC(O)CH3 This gets rid of the un-reacted
  ingredients.

9
THIN LAYER CHROMATOGRAPY (TLC)Day 3

• Quick technique to used to determine how many
  components are in a mixture.
• Used plastic plates coated with silica gel.
• Looks like I might have one substance, possibly
  some impurities.

Pictures from http//inst.sfcc.edu/chemscape/cato
fp/chromato/tlc/tlc.htm
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RECRYSTALLIZATIONDay 3

• A method used to remove the desired product from
  impurities.
• The desired product crystallizes out of solution
  and the impurities remain in the solution.
• Boiled Ethyl Acetate and added to crude product
  until it was all dissolved. Covered and put in
  the freezer.

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Removing crystallized productDay 4

• Now have crystallized product in solution.
• Added hexanes to pull out any more pure product
  from the solution.
• Used filter and vacuum to remove and dry the
  crystals from the solution.
• Dissolved a small amount of product in 11 ratio
  of H2OCH3CN to use for HPLC.

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High Performance Liquid Chromatography (HPLC)

• HPLC is a method of analysis that separates
  compounds in a mixture.
• Each compound will have its own characteristic
  peak on the graph.
• Process was set to take 41 minutes.

Pictures from http//www.pharm.uky.edu/ASRG/HPLC/
hplcmytry.html
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HPLC- RUN 1
Thought the tallest spike would be my product and
other spikes were impurities.
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HPLC- RUN 2
Tall spike from previous run didnt show up-
Results are inconclusive.
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NUCLEAR MAGNETICRESONANCE SPECTROSCOPY(NMR)Day 5

• NMR is an analytical tool used in the
  identification process of a compound.
• Measures protons in a mixture and the grouping of
  these protons.

Picture from http//en.wikipedia.org/wiki/NMR_spe
ctroscopy
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Ambers NMR Graph
CH3 C-CH3 CH3

H-H
NH
OH
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RESULTS

• Ambers product was pure and ready to be used in
  the continuing experiment.

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What have I learned?

• Different Purification techniques
• -Extractions
• -Filtering/Vacuuming
• -Recrystallization
• Different Identification techniques
• -Thin Layer Chromatography (TLC)
• -High Performance Liquid Chromatography
  (HPLC)
• -Nuclear Magnetic Resonance Spectroscopy
  (NMR)

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Shilos Procedures

• Chemical Reaction
• Extraction
• Evaporation for Crude Product
• TLC
• Flash Column Chromatography
• NMR

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Shilos ProcedureDay 1
Protected O-(Carboxymethyl) Hydroxylamine Hemi
hydrochloride with Fluorenylmethoxycarbonyl
(FMOC) using Biochemistry literature as a
reference
O-(Carboxymethyl) hydroxylamine hemi
hydrochloride MW109g Added to triethylamine
Et3N and dissolved in water
ADD Fluorenylmethoxycarbonyl MW258.7 (FMOC
protecting group) in dioxane
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EXTRACTIONSDay 2

• Performed extraction (3x) with Ethyl Acetate
  CH3CH2OC(O)CH3 This got rid of the un-reacted
  ingredients.
• Keeping track of our stuff is important here.
• It helps if you know the relative density of each
  substance

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TLCDay 3

• Used plastic plates coated with silica gel.
• First observed the plates under the UV lamp and
  traced the substances spots
• Then used a hot plate to dry the plates after
  they were stained

Pictures from http//inst.sfcc.edu/chemscape/cato
fp/chromato/tlc/tlc.htm
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Flash Column ChromatographyDay 4

• Made my sample into solution using ethyl acetate
  and methanol
• Under pressure collected 29 fractions of the
  sample

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Flash Column ChromatographyDay 4

• Using the 29 fractions it was determined where
  our stuff was by matching the crude product
  with the most similar TLC pattern
• Determined that fractions 13-22 were to be kept
  for further analysis

Fractions 10 11 12 13
13 16 19 22
Crude Sample
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NUCLEAR MAGNETICRESONANCE SPECTROSCOPY(NMR)Day 5
Still have a lot of impurities
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What Have I Learned?

• Exposure to Literature
• Scaling the Reaction
• Extraction
• TLC
• Flash Column Chromatography
• NMR

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What Have I Learned?

• Exposure to Literature

This is where we began
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What Have I Learned?

• Scaling the Reaction
• HO2CCH2ONH2 (7.0 g, 64mmol) ½ HCL
• Et3N (8.1g, 80mmol)
• Protecting Group (FMOC) (15.3g 70mmol)

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What Have I Learned?

• Extraction
• Very important to keep track of your stuff
• You just may find it somewhere else

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What Have I Learned?

• TLC
• A very good refresher of the lessons in CHY 114
• Understanding how the plate will separate
  different substances based on their solubility
• The trial and error of finding the right solvent
• Knowing what should be seen under UV light

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What Have I Learned?

• Flash Column Chromatography
• The importance of packing it very well
• Finding the perfect rate (2/minute)
• Collecting fractions

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What Have I Learned?

• NMR
• How to operate the NMR
• Roughly how to read or interpret a NMR printout

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What Have I Learned?

• A very well constructed lab course explains
  volumes
• Critical thinking and analysis
• Techniques and when to use them