Biomedical Engineering Reference
In-Depth Information
Table 9.6. Protocol for Chemical Synthesis of PDTC
Reactions
1.
Dipicolinic acid (pyridine-2,6-dicarboxylic acid) + thionyl chloride-
!
pyridine-2,6-dicarboxylic acid
chloride
2.
Acid chloride + hydrogen sulfide (in pyridine) ! pyridine-2,6-dithiocarboxylate (pyridine salt)
Ingredients
Chemical
FW
Mass (g)
Mol
Density
Vol (mL)
Use
Dipicolinic
acid
167.1
5
0.03
Use
Thionyl
chloride
119
71.2
0.598 (10
as much)
1.631
43.7
Expect
Acid chloride
204.1
61.123
0.03
Expect
PDTC salt
278
8.34
0.03
Expect
PDTC salt
199.1
5.973
0.03
Procedure:
1. Clean all glassware with soap, deionized water, and acetone. Weigh flasks. Mark flasks for easy
solvent addition (45 mL and 75 mL for the 100 mL round bottomed flask with a stir bar; 300 mL for the
three necked 500 mL round bottomed flask). Bake glassware overnight at 150
C. List of glassware
to be baked: 100 mL round bottomed flasks (one with stir bar), 50 mL round bottomed flasks, 50 mL
pipets, a reflux tube, distillation apparatus, three necked flask with a stir bar, a 125 mL addition
funnel. Get dry ice and store it at 78
C freezer. Weigh 5 g dipicolinic acid into a 100 mL round
bottomed flask. Be sure that there are no chunks. Add 45 mL thionyl chloride into flask. Connect
flask, tubes for water circulation, and gas lines to reflux tube. Repeat vacuuming and purging with
nitrogen into system. Emerge part of the flask into an oil bath with a paper clip. Under nitrogen, heat
and stir until all dipicolinic acid dissolves.
2. Distillation: Connect the reaction flask to a distillation setup, which contains water circulation and is
under nitrogen. Thionyl chloride is distilled into another round-bottomed flask. Thionyl chloride is
very acidic. To dispose of it, neutralize it with base gradually while stirring. The addition of base
causes boiling.
3. Remove residual thionyl chloride with a pump, operated overnight. There should be a clean trap in
isopropanol/dry ice bath for the pump. Acid chloride can be stored at 78
C.
4. Saturate 300 mL pyridine with H
2
S in the 500 mL three necked round bottomed flask. All three necks
are sealed with rubber septa. Hydrogen sulfide gas is bubbled into pyridine with a long needle.
Another neck is connected to a nitrogen line (through a needle) that is connected to a bubbler.
Dissolve acid chloride in 75 mL acetone (water free). Add acid chloride in acetone into hydrogen
sulfide saturated pyridine drop by drop with an addition funnel. This funnel is connected to nitrogen
line with a bubbler. Mixture should turn orange.
5.
Filter out precipitate (acid chloride). Rotoevaporate off pyridine. Crystallize product (pyridine salt of
PDTC) with chloroform using a heat gun. If crystals do not form, place flask into refrigerator. Filter to
separate crystals and wash crystals with ice cold ethyl acetate. Dry crystals with an aspirator.
6.
To make the free acid of PDTC, dissolve 500 mg pyridine salt in 100 mL water and bring pH to 1.5
with H
2
SO
4
. At once, extract five times with 20 mL methylene chloride. Rotoevaporate off solvent.
since other levels of regulation (e.g., translational, post-translational) may be superimposed
(T. A. Lewis, Montana State University, unpublished). Genes that lie outside the
pdt
cluster play
roles in PDTC production as well (T. A. Lewis, Montana State University, unpublished) and a
thorough analysis of those functions may help to answer some of the remaining questions
regarding additional levels of control of PDTC production, and the PDTC biosynthetic pathway.
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