The Study of Triazine Antimicrobial Compounds

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15 Νοε 2013 (πριν από 3 χρόνια και 7 μήνες)

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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.

R1

Structure

R2

Structure

A

1

B

2

C

3

D

4

E

5

F

6

G

7

H

8


I

J

N
H
2
O
H
N
H
2
O
H
N
H
2
N
H
2
N
H
2
O
H
O
H
O
H
N
H
2
O
H
N
H
2
O
H
O
H
N
H
2
O
O
N
H
2
N
H
2
O
O
N
H
Bz
O
M
e
N
H
2
N
H
2
N
H
2
O
H
N
H
2
N
H
2
N
H
2
N
H
2
N
H
2
N
N
N
N
H
R
1
R
2
Positive Charge


Group 2

N
N
N
N
H
R1
R2
O
O
N
H
2
R1

Aryl/Alkyl

R2

Amine

A

1

B

2

C

3

D

4

E

5

F

6

G

7

H

8

9

Cl
N
N
H
2
N
N
H
2
N
H
N
N
O
N
H
2
N
N
H
2
N
N
H
2
N
N
H
2
N
N
H
2
N
N
H
O
H

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

A

B

C

D

E

F

G

H

I

J

1

*


73.1


4.1


77.3


54.2


4.3


38.3


28.0


40.8


44.3


2

*


99.4


88.5


*


29.3


84.1


33.7


96.6


44.9


39.2


3

3.9


19.1


42.0


11.5


*


97.2


82.5


97.8


12.6


10.5


4

0.7


39.2


*


44.0


5.6


72.4


67.7


*


27.8


27.4


5

9.3


*


*


33.9


26.3


7.5


9.2


6.7


44.7


30.2


6

*


1.5


10.5


23.9


94.1


3.9


*


*


15.6


18.3


7

*


5.5


97.2


23.7


17.1


29.9


7.2


100


15.2


10.4


8

50.1


8.1


98.1


69.1


22.3


99.6


99.6


*


7.6


4.4


*

= 0% Inhibition

% Inhibition at 100
m
M

Positive Results from Group 1

A

B

C

D

E

F

G

H

I

J

1

*


73.1


4.1


77.3


54.2


4.3


38.3


28.0


40.8


44.3


2

*


99.4


88.5


*


29.3


84.1


33.7


96.6


44.9


39.2


3

3.9


19.1


42.0


11.5


*


97.2


82.5


97.8


12.6


10.5


4

0.7


39.2


*


44.0


5.6


72.4


67.7


*


27.8


27.4


5

9.3


*


*


33.9


26.3


7.5


9.2


6.7


44.7


30.2


6

*


1.5


10.5


23.9


94.1


3.9


*


*


15.6


18.3


7

*


5.5


97.2


23.7


17.1


29.9


7.2


100


15.2


10.4


8

50.1


8.1


98.1


69.1


22.3


99.6


99.6


*


7.6


4.4



: >90% inhibition : >80% inhibition

Results from Group 2

1

2

3

4

5

6

7

8

9

A

5.6


8.1


0.4


*


5.5


9.7


99.7


9.6


4.6


B

99.6


6.2


14.2


4.4


8.5


*


99.5


*


*


C

84.3


15.2


83.4


21.3


100


32.6


99.9


10.6


72.1


D

23.6


16.4


15.0


12.5


7.3


*


*


8.3


6.2


E

99.0


46.7


97.9


78.5


97.9


98.4


98.1


*


7.6


F

85.2


10.6


99.0


*


97.3


*


98.9


1.4


*


G

*


*


*


*


1.0


1.8


*

*


*


H

15.8


2.0


6.3

*


7.1


*


99.4


*


28.6


% Inhibition at 100
m
M

Results from Group 2

1

2

3

4

5

6

7

8

9

A

5.6


8.1


0.4


*


5.5


9.7


99.7


9.6


4.6


B

99.6


6.2


14.2


4.4


8.5


*


99.5


*


*


C

84.3


15.2


83.4


21.3


100


32.6


99.9


10.6


72.1


D

23.6


16.4


15.0


12.5


7.3


*


*


8.3


6.2


E

99.0


46.7


97.9


78.5


97.9


98.4


98.1


*


7.6


F

85.2


10.6


99.0


*


97.3


*


98.9


1.4


*


G

*


*


*


*


1.0


1.8


*

*


*


H

15.8


2.0


6.3

*


7.1


*


99.4


*


28.6


Positive Results Highlighted


: >90% inhibition : >80% inhibition

Frequency of Effectiveness (G1)

R1

Structure

Freq.

R2

Structure

Freq.

A

0

1

0

B

1

2

4

C

3

3

3

D

0

4

0

E

1

5

0

F

3

6

1

G

2

7

2

H

3

8

3

I

0

J

0

N
H
2
O
H
N
H
2
O
H
N
H
2
N
H
2
N
H
2
O
H
O
H
O
H
N
H
2
O
H
N
H
2
O
H
O
H
N
H
2
O
O
N
H
2
N
H
2
O
O
N
H
Bz
O
M
e
N
H
2
N
H
2
N
H
2
O
H
N
H
2
N
H
2
N
H
2
N
H
2
N
H
2
Frequency of Effectiveness (G2)

R1

Structure

Freq.

R2

Structure

Freq.

A

1

1

4

B

2

2

0

C

4

3

3

D

0

4

0

E

5

5

3

F

4

6

1

G

0

7

6

H

1

8

0

9

0

Cl
N
N
H
2
N
N
H
2
N
H
N
N
O
N
H
2
N
N
H
2
N
N
H
2
N
N
H
2
N
N
H
2
N
N
H
O
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:


N
N
H
2
N
N
H
2
N
N
H
2
N
N
H
2
O
H
N
H
2
Cl
O
H
O
H
N
H
2
O
H
N
H
2
O
N
H
2
N
H
2
O
M
e
N
H
2
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.

R1

Structure

R2

Structure


A

A

B


B

C

D


C

1

2


D

3

4

5

6

7

8

9

N
N
H
2
N
N
H
2
N
N
H
2
N
N
H
2
O
H
N
H
2
Cl
O
H
O
H
N
H
2
O
H
N
H
2
O
N
H
2
N
H
2
O
M
e
N
H
2
N
N
H
2
N
N
H
2
N
N
H
2
N
N
H
2
N
N
N
N
H
R
1
R
2