DEVELOPMENT OF NOVEL COMPOUNDS FOR THE INHIBITION OF TOOTH AND GUM BACTERIA
BRIAN JAMES
COLLEGE OF NATURAL SCIENCE AND MATHEMATICS , INDIANA UNIVERSITY OF PENNSYLVANIA
Abstract:
There is a strain of bacteria known to be present within the oral cavity formally known as Streptococcus, whose mutants secrete glycosylated proteins that linger and congregate along the gum line over time due to their insolubility. These glycosylated proteins, if left untreated can have irreversible effects such as gum disease. Through previous studies, scientists have found out that there is a family of proteins within the Streptococcus mutants responsible for the production of these proteins. The proteins have been formally classified as glycosyltransferases and are known to be inhibited by molecules containing 5- and 6- membered cyclic structures. Microbiological measures are used to introduce the bacteria to a specific known cyclic compound and afterwards are diluted and spread on petri plates, incubated to produce colonies, then counted and divided by the number of colonies grown when uninhibited. Some compounds have exhibited notable inhibition of approximately 88
Introduction:
In 2010 the center for disease control released a statistic that said 47.2% of Americans have some form of periodontitis, which is a more advanced version of gum disease. If there is not adequate treatment, periodontitis could lead to tooth loss and subsequently expensive dental repair. Some procedures to replace lost or damaged teeth can range up to $4000 or more depending on the amount and location of the teeth necessary to be replaced. There are dental products that already aid with Periodontitis but the significantly effective ones are expensive and while serious investment in dental care can make a difference, some families do not have the adequate money to budget towards it. All the stress and expense associated with tooth upkeep, repair and replacement can be avoided if there was a cheap and simple product made to help.
Suzanne Walker has made numerous previous attempts at manufacturing a working molecule and has produced promising but not commercial grade yet. previous attempts at keying in on the most effective compound and the most effective exhibited 88% efficiency. Most of the compounds were complex, expensive and production was elaborate.
The aim for this study is to produce a more cost-efficient compound, so Dr. Keith Kyler began to study using simpler structures, and found this urazole ring structure bonded to a benzaldehyde with various substituents with a Para-nitro group exhibited the most favorable results thus far. This research proposal is to test three more substituent combinations. The first is the Bromine in the Para-position on the Phenyl ring. The second is a methyl group in the Para-position. The third is a nitro group at both the ortho and para- position of the molecule.
Figure 1: 3 different molecules being tested for effectiveness on S. mutants
Methods:
Preparation of product:
360 mg of 4-phenylurazole is dissolved in 20 ml of THF. 240 mg of triethylamine is added. Stir for 30 mins. Add 360 mg of para nitrobenzaldehyde over the course of 5 mins. Dissolve another 360 mg of para-nitrobenzoyl chloride in 8ml THF. Reflux for 1 hour. Pipet the pra-nitrobenzoyl chloride dissolved in 8ml THF into the reaction mix dropwise slowly over 1 hour. Reflux for two hours . Monitored via TLC every 2 hours. After approximately 4 hours the TLC analysis indicates that the reaction has come to completion. Evaporate until dry. purify by dissolving in 50 ml of ethyl acetate. wash with 20 ml of a 10% aqueous sodium bicarbonate solution. Dry over anhydrous magnesium sulfate. Decant the liquid portion into a 50 ml round bottom flask through a separatory funnel. Dry using the rotor-evaporator in a 60 degree bath. If necessary, in order to further purify the product recrystallized using methanol. This procedure should afford 200 mg of product. Characterized by H-NMR analysis.
Preparation of bacteria:
Add 20 ml of Tryptic Soy Broth to a sterile plastic test tube with a cap. Once the tube with the broth is prepared inoculate a flake of the commercial S. mutants 9102 bacteria into the broth with a sterile test tube. Cap and invert twice before placing into the incubator. Set incubator at 37 degrees Celsius and rock at 115 rpm , these conditions were previously determined by earlier research . grow in the incubator under these conditions for 18-20 hours.
Dilution:
Dilution:
Fill 20 small sterile capsules with 900 ul of water, these 20 capsules represent 4 sets of 5 capsules. One set of 5 dedicated to each of the drugs 1-3 and one as a control set, which is dedicated to methanol. Methanol is used as the control because it was necessary for dissolving the solid drugs 1-3 in order for them to be exposed to bacteria. The control is done to ensure that it was not the alcohol inhibiting the bacterial growth instead of the designed drug 1-3. Add 100ul of the stock bacterial solution to the capsule 1 of each of the 4 sets of 5. Invert twice and add 100ul to capsule2 with each set. This process was repeats until capsule 5 Once all dilutions are done ( all have 10^-6 cell concentration of the original solution) , Streak four separate tryptic soy agar plates were one with control capsule containing 100 ml of methanol and one for each of the three drugs with 100 ul of 10 mg of drug dissolved in 4 ml of methanol. Once streaked incubate for 24 hours . The first set of plates streaked represented the zero hour plates.
Figure 2: dilution method for plating of bacteria exposed to molecules.
Streaking:
Pipet 100 ml from the capsule into the center of the agar plate. streak with a sterile loop. Place a lid on top and wrap in plastic wrap to prohibit contamination. Place in incubator for 24 hours. Repeat every 4 hours until 24 hours of plates are incubated. Read plates and record results.