Synthesis of Nitroxide Spin Label

1
Synthesis of Nitroxide Spin Label Catherine
Campos Dr. Hirsh Summer 2005
2

• Goals
• To follow a synthesis to produce the nitroxide
  spin label, 4-ethynyl-2,2,6,6-tetramethyl-3,4-dehy
  dropiperidine-1-oxyl nitroxide (B) from TEMPONE
  (A).
• To attach the nitroxide radical to a
  deoxyuridine phosphoramidite.
• To synthesize single stranded DNA, A19mer,
  incorporating nitroxide-labeled dU.

Overall Reaction
B
A
3

• Part One of Synthesis
• In part one of the synthesis, the starting
  material, TEMPONE, is converted to the product
  shown below via the following nucleophilic acyl
  addition reaction

LiBr
4

• Tracking Reaction Progress
• Column chromatography was used to purify the
  product, and TLC was used to determine which
  fractions collected from the column contained
  product.
• It was discovered that the compound was not UV
  reactive, so a KMnO4 dip was used to develop the
  TLC plates.
• It was determined that the spot with the higher
  Rf value was the starting material (a carbonyl)
  and the spot with lower Rf value was the product
  (an alcohol).

5

• Problems
• Poor separation (fractions had more than one
  spot)
• Low yield (exposure to water)
• Solutions
• Corresponded with author of synthesis about ways
  to eliminate exposure of reagents to water (use
  glove box).
• Did a TLC analysis of starting material and
  fraction from column.
• Decided to use a less polar solvent when
  performing the column chromatography to get
  better separation.

• Results of TLC Analysis
• Higher Rf value product
• Lower Rf value starting material

6

• Results of Previous Solutions
• When the lithium bromide was added in the glove
  box, and the polarity of the solvent was
  decreased, the product was obtained in a 35
  yield, which was enough to progress to the second
  part of the synthesis.
• The diffuse reflectance apparatus of the IR
  spectrometer was used to characterize the product
  and the spectrum shown below was obtained.

7

• Part Two of Synthesis
• In the second part of the synthesis, dehydration
  occurs by the following mechanism to yield the
  product shown below.

8

• More Problems
• Poor yield
• Product began to crystallize when it was supposed
  to be an oil
• There was a small OH peak present (possible
  wetness)
• Analysis of Problems
• The poor yield may be a cause of not allowing the
  reagents to react for a long enough period of
  time.
• IR spectra were taken of both the oil and the
  crystalline product (Both had very similar
  spectra.).

Oil
Crystals
9

• 1H NMR Analysis of Products
• The synthesis says to use phenylhydrazine to
  reduce the nitroxide radical to the corresponding
  hydroxylamine, but it does not state the quantity
  or concentration of phenylhydrazine needed.
• Found another paper by the same author that says
  to use 1.5 molar equivalents of phenylhydrazine.
  Once again, the concentration was not specified
  and the 1H NMR spectrum obtained did not reveal
  much about the protons in question.
• An article on reducing nitroxide radicals was
  found in the Journal of Organic Chemistry which
  called for using 0.5 molar equivalents of 0.03 M
  phenylhydrazine. This procedure produced much
  better results.

TIPS
TIPS
0.5 Molar Equivalents
1.5 Molar Equivalents
CH3 Protons
CH3 Protons
CH2 Protons
CH2 Protons
10

• Future Plans
• To move onto part 3 of the synthesis (See
  mechanism below)

• To attach the radical generated in part 3 to the
  deoxyuridine phosphoramidite (above right) on a
  DNA synthesizer column, using a palladium
  catalyst.
• To synthesize stranded DNA, A19mer with the
  attached nitroxide spin label.
• To observe the interactions between the
  nitroxide spin label on one half of the DNA
  Duplex and the EDTA ligand on the other half.

11

• Acknowledgements
• Dr.Donald Hirsh
• Merck SURF 2005
• Dr. Lynn Bradley
• Dr. Georgia Arvinitis
• Dr. Peter Gannett
• Alyza Szajna
• The TCNJ Chemistry Department
• Brad Hutnik
• Dr. Bradleys Research Students
• The Stockroom Angels (Pam, Christina, Emily and
  Smita)
• Joyce Gaiser
• 1.