Joseph Kim 902494525 Combinatorial Chemistry: Determination of a Possible Drug Structure Abstract: The combinatorial chemistry technique was used to find an antibiotic against E. against E. coli. coli. Six solutions using three hydrazines and aldehydes were prepared and tested on E. on E. coli. coli. to find the hit compound. A positive effect against E. against E. coli was shown in the hit compound through the formation of a clear circle around the cups. The hit compound, based on the agar plates, was the compound produced from aminoguanidine bicarbonate and 5-nitro-2-furaldehyde. The structure of the hit compound was determined to be (2E)-2-[(5-nitro-2-furyl)methylene] (2E)-2-[(5-nitro-2-furyl)methylene] hydrazinecarboximidamide using the structures of the reactants and mechanism of the reaction. Using a marker, two agar plates were divided into three sections each, labeled M1-M6. coli. The bacterial culture E. culture E. coli TG1 1/100 (5 mL) was used to cover two agar plates with E. with E. coli. With a transfer pipette, the extra liquid was carefully removed into a waste tube. Using the extra third plate, the cupping procedure was practiced. After a few practices, a small cup was made in the middle of each section on the two prepared agar plates. Six Eppendorf tubes were taken and labeled M1-M6. Different volumes of hydrazines and aldehydes, wh ich are listed in Table II, were added into six tubes to produce a series of structurally similar drugs. All six tubes were shaken rigorously for 10-15 seconds. The observations of each tube were recorded. With a new transfer pipette, 2 drops of each solution were placed into the cups of the appropriate section. The finished agar plates were incubated at 37 °C for 24 hours. hou rs. Images of the agar plates were taken after 24 hours in order to determine which solution had an antibiotic effect on E. on E. coli. coli. Data and Observation: Table I. Material Used Material E. coli TG1 1/100 2-nitrobenzaldehyde 3-nitrobenzaldehyde 5-nitro-2-furaldehyde 4-bromophenylhydrazine hydrochloride 4-cyanophenylhydrazine hydrochloride Aminoguanidine bicarbonate
Formula --------C7H5 NO NO3 C7H5 NO NO3 C5H3 NO NO4 BrC6H4 NHNH NHNH2 * HCl
MW (g/mol) --------151.12 151.12 141.08 223.50
Volume (mL) 5 .0 ---------------------------------
C7H7 N N3 * HCl
169.61
---------
CH6 N N4 * H2CO3
136.11
---------
Table II. Volume of Each Compound in Eppendorf Tubes Compound M1 2-nitrobenzaldehyde 15 3-nitrobenzaldehyde ----5-nitro-2-furaldehyde ----4-bromophenylhydrazine hydrochloride 5 4-cyanophenylhydrazine hy h ydrochloride 5 Aminoguanidine bicarbonate 5
Volume (drops) M2 M3 M4 M5 ------ -----5 5 15 -----5 5 -----15 5 5 5 5 15 -----5 5 -----15 5 5 ------ ------
M6 5 5 5 ------------15
Joseph Kim 902494525 Table III. Structure of Aldehydes and Hydrazines Compound 2-nitrobenzaldehyde
Structure O H
NO2
3-nitrobenzaldehyde O H
NO2
5-nitro-2-furaldehyde -
O
N+ O
O
O
H
4-bromophenylhydrazine hydrochloride
Br
NH2 N H
4-cyanophenylhydrazine hydrochloride N
HCl
NH2 N H
Joseph Kim 902494525 Table VI. Antibiotic Effect Result Section M4 M1 Neg M2 Neg M3 Pos * Orange = the hit compound *
M5 Neg Neg Pos
M6 Pos Pos Pos
Table VII. Structure of Hit Compound Compound (Aldehyde + Hydrazine) (2E)-2-[(5-nitro-2-furyl)methylene] hydrazinecarboximidamide
Structure H NH N
-
O N+
H N
O NH2
O
Figure I. Plate One (M1-M3)
Figure II. Plate Two (M4-M6)
Discussion: Six soltuions containing different aldehydes and hydrazines were prepared in order to test their effect on E. coli. The positive effect on E. coli would have a clear circle around a cup as can be seen in the figures above. The clear circle represents the inhibition of E. coli growth. The solutions that do not have an effect on E. coli would not have any change around the cups. Based on two agar plates that were treated with E. coli and six solutions, only two sections show a positive effect, indicating that there is exactly one solution that possesses an antibiotic effect against E. coli. The cups on sections labeled M3 and M6 have clear circles. In the M3 solution, three hydrazines and 5-nitro-2-furaldehyde were mixed. In M6 solution, all three aldehydes and aminoguanidine bicarbonate were mixed. Therefore, based on two sections, the compound that has an effect on E. coli as an antibiotic is the one produced from aminoguanidine bicarbonate
Joseph Kim 902494525 and 5-nitro-2-furaldehyde. The name of the hit compound is (2E)-2-[(5-nitro-2furyl)methylene]hydrazinecarboximidamide. This compound works as a bacteriostatic agent (Masui); it inhibits the growth of bacterial cells without killing them (Erfe). The bacteriostatic compounds usually hinders the protein synthesis, nucleic acid synthesis, and other cellular metabolism (Erfe). The mechanism invovled in the reaction between 5-nitro-2-furaldehyde and aminoguanidine bicarbonate is simple. First, the carbon-oxygen double bond shifts its electron pair to the oxygen atom, due to the lone pair on the nitrogen atom from the aminoguanidine attacking the carbon atom. This creates a carbon-nitrogen single bond and puts a positive charge on the nitrogen atom. Then, the oxygen atom utilizes one of its lone pairs to take a hydrogen atom from the nitrogen, resulting in protonation. The protonated oxygen will then take another proton from a hydronium ion with one of its other lone pairs. The nitrogen atom then moves an electron pair to form a carbon-nitrogen double bond; the nitrogen again obtains a positive charge. The formation of a carbon-nitrogen double bond causes the protonated oxygen to leave readily as a water molecule. The water molecule lastly takes a hydrogen atom from the nitrogen a tom using its electron pair, thereby recreating a hydronium ion for other reactions to take place. An electron pair from the bond between the nitrogen and hydrogen atom moves to the nitrogen atom, removing the positive charge. The shift of an electron pair concludes the reaction, and the final sturcture is produced. Figure III. Gerneral Reaction
H N
O
N
+
R1 H
R2
H2 N
H N
+ R2
R1
H
H2O
Joseph Kim 902494525 References:
Erfe, Jasmine, Ericka Senegar-Mitchell, and Sandra Slivka. Combinatorial Chemistry and Drug Discovery Lab [PDF document]. Retrieved from bridgestoliteracy.com/biotech/assignments/CombiChemLab.pdf Masui, Masamiki, et al. (1955). Mechanism of bacteriostatic action of nitrofuran compounds. Osaka City Medical Journal, 1, 47-55.
