The Study of Triazine Antimicrobial Compounds Jillian Greenaway New York University Dr. Neville Kallenbach

Objective The goal of this project is to synthesize Triazine compounds using biomimetics and test how well they inhibit the growth of Bacillus Subtilis.

Problem Peptides, when used in our bodies to kill bacteria, can be digested by protein digesting enzymes. In a result of this, we had to think of another strategy to inhibit bacterial growth. This is when we thought of biomimetics.

Biomimetics Biomimetics also known as Bionics, is the implementation of methods found in nature to the study of modern technology. We used biomimetics, specifically to make a synthetic material mimic a naturally occurring substance by giving it positively charged and hydrophobic properties.

Hypothesis Using biomimetics, an organic molecule can act the same way as a protein with similar properties. The properties for antimicrobial activity are an overall 2+ charge and 2 bulky hydrophobic groups. If enough of this compound with these characteristics is associated with the membrane, it can break the membrane and kill the bacteria.

Triazine Libraries The lab workers synthesized triazine libraries to test on Bacillus Subtilis by using Combinatorial Chemistry, which is the act of mixing products. Libraries are groups of the same compound synthesized in various ways. The following charts depict the two groups of triazine libraries used.

Group 1 Compounds This plate was chosen for the many bulky hydrophobic groups. The third functional group used in this library is adamantane. R1StructureR2Structure A1 B2 C3 D4 E5 F6 G7 H 8 I J

Positive Charge – Group 2 R1Aryl/AlkylR2Amine A1 B2 C3 D4 E5 F6 G7 H8 9 This group was chosen to have sufficient positive charge and also, hopefully, to have enough hydrophobic bulk to be active.

Methods and Materials In order to determine which compound kills bacteria efficiently, we ran assays or tests. 1. First we grew the bacteria in an incubator shaker for several hours. 2. Secondly, we set up plates containing twenty-four wells.

Each well contained 800 microliters of plain media, Bacillus Subtilis, sodium phosphate buffer, and a different concentration of the triazine compound. The amount of phosphate buffer and compound together equaled 150 microliters. All the wells contained 50 microliters of cell culture, which included Bacillus Subtilis and Tryptic Soy Broth. I allowed all the plates to grow for six hours.

3. After six hours I ran all the samples through the spectrophotometer. With a push a button we were presented with the samples’ light absorbance in a numerical value. I took this number and computed it into the following equation.

1- (Average Absorbance from the compound/ Average absorbance from the control) X 100 This equation results in the inhibition percentage. Therefore, if the percentage is high then the compound worked efficiently. On the other hand, if the percentage inhibition is low, the compound did not stop the bacteria from growing.

Results from Group 1 ABCDEFGHIJ 1 * * * * * * ** * ** * * * = 0% Inhibition % Inhibition at 100  M

Positive Results from Group 1 ABCDEFGHIJ 1 * * * * * * ** * ** * * : >90% inhibition : >80% inhibition

Results from Group A * B *99.5** C D ** E *7.6 F *97.3* * G **** * ** H *7.1*99.4*28.6 % Inhibition at 100  M

Results from Group A * B *99.5** C D ** E *7.6 F *97.3* * G **** * ** H *7.1*99.4*28.6 Positive Results Highlighted : >90% inhibition : >80% inhibition

Frequency of Effectiveness (G1) R1StructureFreq.R2StructureFreq. A B C D E F G H I 0 J 0

Frequency of Effectiveness (G2) R1StructureFreq.R2StructureFreq. A B C D E F G H

Analysis Group one did not have much positive results because of the lack of positive charge in the library. Group 2 had more positive results than Group one because it had both positively charged and hydrophobic groups. But it is possible that we’ve gone too far in the other direction and that there is now enough positive charge, but no longer sufficient hydrophobic bulk.

Conclusion We analyzed the data and designed a new library. The following chart depicts the functional groups for these libraries.

“Desirable” Functional Groups Hydrophobic Groups: Plate 1, # 2: Plate 1, # 3: Plate 1, # 8: Plate 1, # C: Plate 1, # F: Plate 1, # H: Plate 2, # C: Plate 2, # E: Plate 2, # F: Charged Groups: Plate 2, # 7: Plate 2, # 1: Plate 2, # 5: Plate 2, # 3:

Proposed New Library In the new library, we would like to use a triazine with adamantane, then use the selected positive groups for the R1 position, and cross them with both the hydrophobic and charged moieties in the R2 position. This should help us to determine the best balance of charge vs. hydrophobicity. R1StructureR2Structure A A B B C D C 1 2 D