Synthesis of a Cyanine Dye and DNA Detection

1
Synthesis of a Cyanine Dye and DNA Detection
Part I
2
Cyanine Dyes

• Definition
• Any of a class of dyes containing a -CH group
  linking two heterocyclic rings containing
  nitrogen.

Heterocycles
Odd number of Carbon Bridge
3
Cyanine Dyes

• The chromophores contain two nitrogen centers
  linked by a conjugated system of an odd number of
  carbon atoms.
• The positive charge can be delocalized over both
  the nitrogen atoms.

4
Cyanine Dyes

• The first cyanine dye was synthesized as early as
  1856 by Williams and because of its blue colour
  it was named cyanine.
• In 1873 Vogel found that cyanine could be used to
  increase the intensity of colors in photographic
  plates.

5
Cyanine Dyes

• Cyanine dyes can be divided into two groups.
• Symmetrical dyes
• Asymmetrical dyes.

6
Cyanine Dyes
7
Cyanine Dyes

• Intercalation
• Bind to DNA by inserting and stacking between the
  base pairs of the duplex
• Intercalation is a non covalent interaction in
  which the molecule is held perpendicular to the
  helix axis
• This causes the DNA duplex to elongate

8
Cyanine Dyes

• Intercalating asymmetric cyanine dyes
• The use of asymmetrical cyanine dyes in DNA
  research started in the late 20th century
• Found to be fluorescent when they intercalate
  into DNA.

9
Cyanine Dyes

• Advantages
• Intercalating cyanine dyes, 1000 fold increase in
  fluorescence.
• Disadvantages
• Sequence selectivity, which can lead to
  inhomogeneous binding to DNA
• Fluorescent both in ss DNA and ds DNA.

10
Cyanine Dyes

• Groove binding
• Proteins bind primarily through major groove
  interactions and small molecules usually prefer
  minor groove binding.
• Minor groove binding molecules are often crescent
  shaped, cationic ligands.
• Most of the minor groove binders interact with
  DNA by forming hydrogen bonds to the base pairs,
  which stabilize the complex.

11
Minor Groove Binding Dyes

• Minor Groove Binding Dyes.
• Groove binders can be sequence specific.
• Lower affinity for ssDNA

12
Cyanine Dyes

• To create a dye that combines the features of
  minor groove binding ligands and the photo
  physical properties of intercalating cyanine
  dyes, minor groove binding asymmetrical cyanine
  dyes has been developed.
• The first one that was designed is called BEBO.

13
Todays Task

• Synthesis an asymmetric cyanine dye.
• Its a three step reaction

14
Step 1 Procedure

• Take 1 mL of 2-methylbenzo 1, 3 thiazole in a
  25 mL of RB flask and mix it with the
  followings.
• 1. 3 mL of methyl Iodide.
• 2. 3 mL of Dimethyl Formamide.
• Set up a refluxion apparatus with cold water
  circulation and do the refluxion for 1 hour.
• Methyl Iodide has low boiling point do not
  forget to circulate cold water in the condenser.

15
Step 1 Procedure

• Cool the reaction mixture to room temperature.
• Add 7 mL of Diethyl ether and swirl the flask.
• Filter the solid using a frit funnel.
• Wash the product with 4 mL of Diethyl ether and
  dry the product in air.
• Through the ether waste into the waste container.
• Weigh the product and Calculate the of yield.

16
Step 1 Chemical Equation
We are increasing the acidity of the methyl group
by methylation on Nitrogen
17
Step 2 Procedure

• Take 0.47 gram of the 1, 2 dimethylbenzo 1, 3
  thiazolium iodide and grind it with
• 1. 0.2 gram of 2, 6 dimethyl 4 pyrone.
• 2. 1 drop of 70 perchloric acid.
• Transfer into a long test tube.
• Carefully heat the mixture using Bunsen Burner.
• Stop heating once the mixture become liquid.

18
Step 2 Procedure

• Cool the reaction mixture add 10 mL of hot water
  and thoroughly mix the contents using spatula or
  a glass rod.
• Filter the brown precipitate using a frit funnel.
• Wash the solid with 10 mL of hot water and dry on
  air.
• Weigh the product and calculate the yield.

19
Step 2 Chemical Equation
20
Step 3 Procedure

• Place all the solid which you obtained in step II
  (CY 39) in a 25 mL RB flask.
• Add 1.3 molar equivalent of 6 aminohexanoic
  acid.
• Add 7 mL of dimethyl formamide per 1mmol of CY39.
• Do not use more dimethyl formamide, because it
  has high boiling point and its very difficult to
  remove.

21
Step 3 Procedure

• Set up a refluxion apparatus with cold water
  circulation and do the refluxion for 10 minutes.
• Evaporate all the dimethyl formamide and
  crystallize the brown red dye from water.
• Weigh the product and calculate the yield.

22
Step 3 Chemical Equation
CY 39
D 7
23
Notes

• Work 4 students together.
• Always wear your safety glass.
• Perchlorates are explosive in nature, so handle
  with great care.
• Do not use I repeat Do not use or store diethyl
  ether when you do second step.
• Give me your lab note book for final Grading
  before you go home.