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Synthesis of LTA4H Enzyme Activators

Synthesis of LTA4H Enzyme Activators

Synthesis of Novel Small Molecule LTA4H Enzyme Activators for COPD

Suzie Bae
Thomas Jefferson High School for Science and Technology

Abstract

            Chronic obstructive pulmonary disease (COPD) is a progressive disease that leads
to reduced airflow to the lungs due to persistent inflammation. The leukotriene A 4
hydrolase (LTA 4 H) enzyme is a bifunctional enzyme that catalyzes hydrolysis of
leukotriene A 4 to leukotriene B 4 (epoxy hydrolase activity) as well as hydrolysis of  the
N-terminus of tripeptides such as Pro-Gly- Pro (aminopeptidase (AP) activity). The
hydrolysis of LTA 4 to LTB 4 is a pro-inflammatory pathway, and hydrolysis of Pro-Gly-
Pro is an anti-inflammatory pathway. The small molecule 4-MDM selectively activates
the LTA 4 H enzyme for AP activity.  
            However, 4-MDM suffers from undesirable physicochemical properties such as
poor water solubility and at room temperature the compound exists as an oil. We
hypothesized that introducing more oxygen atoms in the molecule could provide new
compounds with improved properties, but preserve the activation properties of 4-MDM
on the LTA 4 H AP activity. Acetic anhydride was reacted with 4-benzylphenol in the
presence of pyridine in dichloromethane to afford the acetylated product. The compound
was purified by column chromatography and characterized by NMR and GC/MS
analysis. Enzyme assays show the compound, 4-AcDM, with increased oxygen atom
content activated the LTA 4 H AP activity.

Introduction

            Chronic obstructive pulmonary disease (COPD) is a progressive disease,
primarily caused by cigarette smoking, that slowly and gradually makes breathing
difficult [1] . Currently, COPD is the third leading cause of death in the United States [2] , and
resulted in 134,676 deaths in 2010. [2] Symptoms of COPD include coughing, wheezing,
and expectoration [1] . Emphysema is a sub-type of COPD that involves irreversible
damage to the alveoli sacs [1] . Alveoli sacs are air sacs within the lungs at which oxygen
and carbon dioxide are exchanged in blood. In emphysematous patients, the alveoli sacs
expand due to inflammation [1] . Even when individuals quit smoking, they usually do not
regain lung capacity [1] . For emphysematous patients, the process is irreversible and
progressive. Emphysema involves the destruction of the alveolar walls in the alveoli sacs
when alveolar macrophages release their proteases. The macrophages are important for
normal lung function because proteases from the macrophages digest foreign material.
However, under the persistent inflammation associated with emphysema, the proteases
are harmful because they damage alveolar tissue and create emphysematous lesions [3] .
Emphysema is also marked by a loss of elasticity that leads to narrowing of the thorax,
contributing to greater airflow limitation [3] . As a result, elastin, an elastic protein in
connective tissue, is destroyed [3] . Since tissue destruction in emphysema is permanent, the
best solution to combating the disease is prevention before onset, through smoking
cessation as early as possible [3] .
            The leukotriene A4 hydrolase (LTA 4 H) enzyme has two functions closely
associated with pulmonary emphysema. The LTA 4 H enzyme is a bifunctional enzyme
with both aminopeptidase (AP) and epoxy hydrolase (EH) activities. The EH activity
participates in a pro-inflammatory response by converting leukotriene A4 (LTA 4 ) to
leukotriene B4 (LTB 4 ). The LTB 4 metabolite signals neutrophil and macrophage
chemotaxis, which results in alveolar destruction when the macrophages release their
proteases. The LTA 4 H AP activity degrades the N-terminus of peptides of small
tripeptides such as proline-glycine- proline (PGP). PGP was found to participate in
inflammatory responses by inducing neutrophil chemotaxis. However, Paige et al.
determined that the role of the LTA 4 H aminopeptidase in the pathogenesis of emphysema
involves LTA 4 H-mediated hydrolysis of PGP and clearance of the neutrophil infiltration,
which is an anti-inflammatory response. The proof-of- concept was shown using 4-MDM,
a pharmaceutical agent designed to selectively augment the AP activity of the LTA 4 H
enzyme. Cigarette smoke (CS) exposure in the absence of 4-MDM leads to CS-induced
emphysema presumably due to suppression of LTA 4 H AP activity. However, treatment
with 4-MDM rescues the lungs from emphysema by  restoring the LTA 4 H AP activity,
which eventually results in a protective anti-inflammatory response [4] .
            The LTA4H enzyme pathway has been studied as a target for emphysema because
of its role in the synthesis of LTB 4 . Shin et al. reported that the metabolite LTB 4 is a
neutrophil chemo-attractant and therefore linked to several inflammatory diseases,
including cystic fibrosis, sepsis, and emphysematous COPD [10] . Further, it was found that
inhibition of LTB 4 showed significant beneficial effects in suppressing emphysema.
Although previous research efforts focused solely on the inhibition of LTB 4 as a
treatment option, Oliveira et al.’s work suggested that the anti-inflammatory AP activity
of LTA 4 H enzyme could be essential to future emphysema drug discovery efforts. In their
drug synthesis, Oliveira et al. used the knowledge that 4-methoxyphenoxybenzene
increases the AP activity of the LTA 4 H enzyme. On the basis of Lai group’s model of 4-
methoxyphenoxybenzene, Oliveira et al. concluded that the central oxygen atom of the
drug was not involved in binding, and likely was responsible for decomposition to toxic
drug metabolites in animal models. Therefore, 4-MDM was designed by exchanging the
ether linkage of 4-methoxyphenoxybenzene to a methylene bridge to the two aryl groups.
The compound 4-methoxydiphenylmethane (4-MDM) showed increased stability. In a
murine model of pulmonary emphysema, 4-MDM resulted in no observable toxicity in
the mice over the entire study. Oliveira et al’s research is significant because of its focus
on the lesser studied AP activity of the LTA 4 H enzyme as an improved strategy for
treating inflammation associated with emphysema [5] .
            Based on the information from previous literature, we conducted our study on a
pathway for which research was lacking, namely the LTA 4 H AP pathway. All previous
drug discovery efforts targeting the LTA 4 H enzyme have focused on the EH pathway.
Therefore, we synthesized 4-AcDM as a new lead compound with enhanced
physicochemical properties. In place of the methoxy group, an acetyl group at the end of
the compound was used in order to impart a reduced log P partition coefficient and
increased expected water solubility [5] . Since the EH pathway of the LTA 4 H enzyme has
been largely ineffective for emphysema treatment, likely due to its multiple mechanisms
that promote inflammation, we focused our efforts on the AP pathway instead [5] . Existing
drugs have focused solely on treating the symptoms after emphysema has already
manifested itself. The problem lies in that these drugs do not halt the infiltration of
inflammatory cells, which results in tissue damage. Our drug speeds up the clearance of
inflammation, which we believe will be effective because we are targeting emphysema
from its onset.
            The EH activity of the LTA 4 H enzyme is responsible for the pro-inflammatory
response to emphysema and the AP activity of the enzyme is responsible for the anti-
inflammatory response. Inflammation can be initiated by the hydrolysis of leukotriene A 4
to leukotriene B 4 , which is mediated by the LTA 4 H EH activity. However, clearance of
inflammation is promoted by hydrolysis of the tripeptide proline-glycine- proline (PGP)
by the LTA 4 H AP activity [4] . Our research question was how do we synthesize a
compound that will target the AP pathway of the LTA4H enzyme? The hypothesis was
that the synthesis of the compound, 4-AcDM, would be efficient in accelerating the rate
of PGP hydrolysis, which is expected to correlate with an anti-inflammatory response in
vivo. The hypothesis was developed upon the prediction that the replacement of methyl
group from the already known compound 4-MDM with an acetyl group would create
greater drug stability and increased solubility.The product to be developed was 4-AcDM,
which was synthesized by acetylation of 4-benzylphenol in dichloromethane solvent in
the presence of pyridine and acetic anhydride.
            Emphysema is a disease worthy of study, as effective treatments to halt or even
reverse the disease have yet to be identified. Regarding treatment for emphysema,
solutions have focused on the use of leukotriene A 4 inhibitors, but the development of
leukotriene A 4 aminopeptidase activators is limited [5] . Currently, treatments for
emphysema are concentrated almost exclusively on lessening symptoms rather than
extending the lifespan of those living with the disease and clearing inflammation as a
whole. In 2013, 17.8% of adults residing in the U.S. smoked cigarettes [6] , magnifying the
great need to conduct further research on emphysema, as many are at high risk for
developing the disease and its associated complications. With numbers that emphasize
the increasingly damaging impacts of emphysema, our work is a critical stepping-stone
for future development in therapy and management.

Materials & Methods

            Within our research, the methodology was divided into two aspects: synthesis and
testing. Before drug synthesis was begun, Tris buffer was prepared by mixing 2.42 grams
of Tris and 2.34 grams of sodium chloride in 300 mL of H 2 O. Hydrochloric acid was
added until the pH was stabilized to 7.21, and H 2 O was added until the buffer amounted
to 400 mL. L-Alanine- p-nitroanilide (Ala-pNA), a fluorescent reporter group that served
to imitate PGP and its function by cutting into PNA, was diluted into multiple
concentrations for testing purposes. PGP was not utilized as it was difficult to detect.
Instead, Ala-pNA was used because it changes color when hydrolyzed, allowing reliable
detection of enzyme activity. Concentrations of 1.0 mM, 2.0 mM, 4.0 mM, 8.0 mM, 12.0
mM, 16.0 mM, 20.0 mM, and 40 mM Ala-PNA concentrations were created by diluting
Ala-PNA with water.
            Drug synthesis began when 0.103 mL of acetic anhydride, 0.004 mL of pyridine,
and 0.1 g of 4-benzylphenol in 5 mL of dichloromethane were mixed together in a bulb
flask. 4-benzylphenol was diluted in dichloromethane because the reactant was a solid,
and 5 mL of dichloromethane were used since 10 mL of dichloromethane was needed for
every millimol of 4-benzylphenol. The compound was placed on a stirring plate
overnight for 18 hours to ensure that the reactants were thoroughly mixed. After 18 hours
had passed, thin layer chromatography (TLC) was run on the compound. TLC is a
method for analyzing mixtures by separating in the mixture, and consists of three steps as
follows: spotting, development, and visualization. All of the reactants were diluted with
1:3 ethyl acetate, since pure chemicals produce hazy and unclear dots when run through
TLC. Spotters, glass capillary tubes utilized to “spot” the silica gel coated plates, were
made by applying heat to glass pipettes and pulling them when warm. Silica gel coated
TLC plates were cut into proportionate squares and marked accordingly at the starting
and ending points with a pencil. Each of the diluted reactants and product were spotted
once on each “lane” of the TLC plate, and immediately placed in a bowl topped with a
watchglass with 1:3 ethyl acetate in it. The spotted TLC plate was placed so that the 1:3
ethyl acetate did not pass the marked starting point, as the solvent would instantaneously
cause the spots to move along with it. After the 1:3 ethyl acetate traveled up to the ending
point, the plate was removed from the bowl and placed under a 234 nm wavelength UV
light. By comparing the lanes with diluted reactants to the lane with the compound, we
were able to compare the spots with those of the compound, and determine which spot
was new and likely represented the product. After clearly identifying the product, the
compound was attached to the Buchi Rotovapor® machine to remove the methylene
chloride solvent and isolate the product [7] .
            A separation process was conducted to serve as a purification step that removes
the residual impurities was performed utilizing 50 mL of ethyl acetate, 25 mL of H 2 O, 25
mL of 10% copper sulfate, and 25 mL of brine. The product was mixed with 50 mL of
ethyl acetate in a separatory funnel, and vigorously shaken until the two solutions
appeared to be separated. Then, the separatory funnel was turned open so that all of the
ethyl acetate phase could be removed from the flask. This process was repeated with the
water, copper sulfate, and brine solutions. After the copper sulfate solution was removed
from the flask, TLC was conducted on the remaining product ethyl acetate phase to
ensure that no pyridine was remaining in the product, as copper sulfate was accountable
for removing any excess pyridine. The brine solution ensured that all excess water was
removed from the ethyl acetate solvent. Upon the completion of the separation process,
the product was transferred to a beaker, where solid sodium sulfate was added to rid the
product of any residual water, serving as a drying agent. The solid sodium sulfate was
filtered out using a funnel, and the solvent mixture was concentrated using a Buchi
Rotovapor® machine to isolate the drug.
            Column chromatography, a process used to purify liquids through the separation
of the sample by partitioning between the stationary phase silica gel and 1:9 and 1:19
ethyl acetate-hexanes mobile phase, was conducted as the final purification step [8] . Silica
gel was packed to the rim of the separating funnel, and 100 mL of hexanes were poured
into the funnel. A tube attached to the pressure and air pumps was inserted into the funnel
to accelerate the rate at which eluents would flow out of the funnel and into test tubes.
After 100 mL of hexanes were filtered out of the funnel until the liquid reached the top of
the silica gel, the process was repeated with 1:9 and 1:19 ethyl acetate-hexanes after the
product was washed with hexanes and poured into the funnel. Increasing ratios of ethyl
acetate to hexanes were used since they would increase the rate of product elution. After
all ethyl acetate-hexanes solutions were filtered out, TLC was run on each of the test
tubes to identify where the product was located. By matching the lanes that consisted of
spots with the corresponding test tubes, the tubes that displayed spots were mixed
together in a bulb flask and attached to the Buchi Rotovapor® machine to isolate the
drug.
            The bulb flask consisting of the drug was washed with 1:3 ethyl acetate-hexanes
to ensure that the remaining product on the sides of the flask were included in the drug
remaining at the bottom of the flask. The mixture of 1:3 ethyl acetate-hexanes and the
drug was pipetted into a clean bulb and attached to the Buchi Rotovapor® machine to
remove excess 1:3 ethyl acetate-hexanes solvent. Then, the bulb flask was attached to a
Buchi vacuum pump to get any remaining solvent excess out of the product. After the
drug was vacuumed for 5-10 minutes, the final product was obtained. In order to ensure
that the final product was the desired drug, nuclear magnetic resonance (NMR)
spectroscopy was run through the Bruker 400 megahertz NMR on the drug to determine
the structure of the organic compound. Gas chromatography-mass spectrometry (GC-MS)
analysis was run on the drug to identify substances within the drug sample, and also to
identify the chemical formula of the drug. Both of these analyses confirmed the purity of
the drug, and ensured that the acquired product was the desired drug- C 15 H 14 O 2 , or 4-
AcDM.
            During the testing stage, a BioTek microplate reader was utilized to run enzyme
assays. Using a 96-well plate, each well consisted of 50 microL of LTA4H enzyme
solution, 50 microL of Tris buffer, 50 microL of drug solution, and 50 microL of Ala-
pNA solution. Each row consisted of a different drug concentration, starting from 0 mM,
1 mM, 5 mM, 10 mM, 20 mM, 40 mM, 80 mM, and ending with 160 mM. Various drug
concentrations were created by diluting the drug with 20% DMSO buffer. Every three
columns consisted of a different Ala-pNA concentration: one plate held 12 mM, 16 mM,
20 mM, and 40 mM of Ala-pNA, while another held 1 mM, 2 mM, 4 mM, and 8 mM of
Ala-pNA. The reporter groups were inserted last to ensure that the reaction would occur
in the microplate reader when the plate was read. After a plate was read and optical
density values were produced, data was interpreted accordingly by utilizing the GraphPad
Prism 5 application.
            The lab director trained the student researchers in running instrumentation in the
lab and supervised the research. All procedures for this project were conducted and
carried out by the student researchers.

Results

            When the 96-well plate consisting of enzyme, drug, buffer, and reporter group
was placed into the BioTek Plate Reader, each well produced an optical density value
that represented the absorption by the sample. For each drug concentration, graphs
comparing varying Ala-pNA concentrations (as the x values) and optical densities (as the
y values) were initially created, and the linear portions of each graph were isolated to
determine the graphs’ equations. With the linear formula, the y value represented optical
density, x represented Ala-pNA concentration, and m and b both served as constants. To
convert the optical density values to Ala-pNA concentrations instead, the formula, y =
1575 x + 0.9177, was rearranged to x = (y - 0.9177) / (1575). This formula was found by
isolating the graph of Ala-PNA concentration vs. averages of optical densities to ensure
that the formula was a display of collective data, rather than solely one drug
concentration. Using this formula, the optical density values were converted to PNA
concentration values.
            After attaining PNA concentration values, the GraphPad Prism application was
utilized to construct graphs for each drug concentration that displayed the trend between
time and PNA concentrations. Each kinetic trend represented time in minutes, and PNA
concentration values were obtained from the values that were calculated using the initial
formula. For each of the graphs, the initial velocities, V0, were calculated by finding the
slope of the graph from 0 to 5 minutes. To construct the final Michaelis Menten plot,
which displays the effect of Ala-pNA concentrations on the initial velocities for each
concentration of drug, the lower Ala-pNA concentrations (0.00 mM, 0.01 mM, 0.02 mM,
0.03 mM, 0.04 mM, 0.05 mM) were placed on the x-axis, while the initial velocities
found by calculating slopes of the individual time vs. PNA concentration graphs were
placed on the y-axis. Each color represented a different drug concentration, while the
ranges for the x and y axis were preset based upon the input Ala-PNA concentration and
initial velocity values.
            The resulting data, in the form of a Michaelis-Menten plot, led to the conclusion
that with a greater drug concentration, a greater initial velocity of the reaction resulted.
Additionally, with an increasing Ala-pNA concentration, more pNA resulted, indicating
that the greater the presence of Ala-pNA, the greater the velocity of cutting. Hence, if a
drug produces more pNA, increasing reaction velocities result.
            Next, several enzyme kinetics tests were run. Vmax (mol / sec), the maximum rate
of reaction, increased with increasing drug concentrations. Since Vmax is under the
conditions of a sufficient amount of substrate molecules to saturate the enzyme’s active
sites, the data confirmed that the 160.0 mM drug accelerated the rate at which the enzyme
catalyzed the reaction. Km (mol / L), the concentration of substrate needed to achieve half
of the Vmax , was greatest at the drug concentration of 160.0 mM. The larger Km value,
0.08869 mol / L, meant that great amounts of substrate are necessary to saturate the
enzyme, also indicating that the enzyme has a low affinity for the Ala-pNA reporter
group and is less specific. Kcat (1 / sec), the turnover number for the AP activity, was
greatest at the drug concentration of 160.0 mM. As Kcat represents the rate of the enzyme,
the value stands for how fast the enzyme “makes” pNA from Ala-pNA, and is expressed
per unit time.  The large Kcat value means that with 160.0 mM of drug, the enzyme
produces pNA at the fastest rate from Ala-pNA. Kcat / Km , the catalytic efficiency, was the
lowest at 160.0 mM of drug. The Kcat / Km value represents the number of times the
substrate is converted to product per unit of time. To place the interpretation of Kcat / Km
into context, a “perfect” enzyme would have a very high Kcat (high speed) but a low Km
(specific). However, in reality, reducing the Km will slow the Kcat , because the substrate is
bound tightly to the enzyme and slow down the turnover number. Although we look at Kcat
/ Km to determine a maximum number, our drug showed a low Kcat / Km value since a
higher value exists with drugs that block enzyme activity (involving EH activity); our
was contrary and activated enzyme activity, leading to a lower value with an effective
drug.
            The initial research question asked was: how do we synthesize a compound that
will target the aminopeptidase pathway of the LTA4H enzyme? The results presented
address this question by proving the efficacy of our compound, 4-AcDM. The rate at
which 4-AcDM cuts Ala-pNA increases with increasing concentrations of the drug,
confirming that the compound targets the AP pathway of the enzyme in a quicker and
more thorough manner. Within the graphs, we recognized that the highest V 0 curve was
found representing the highest drug concentration of 160.0 mM, and diminished as drug
concentrations lessened. In addition, enzyme kinetics prove that our drug accelerated the
rate at which the substrate binds to LTA4H. Since the hydrolysis of Ala-pNA  to p-NA
mimics that of the hydrolysis of PGP, the drug has potential to lower the progression of
EM and resolve PMN activity.

Illustrations

 
Figure 1. The Michaelis-Menton plot shows that with increasing concentrations of Ala-pNA, the initial velocity of the reaction increased.

Figure 1. The Michaelis-Menton plot shows that with increasing concentrations of Ala-pNA, the initial velocity of the reaction increased.

 
Chart 1. Vmax is the maximum rate of reaction, Km is the concentration of substrate needed to achieve half Vmax, and Kcat is the turnover number for the aminopeptidase activity. The Kcat/KM value is the catalytic efficiency.

Chart 1. Vmax is the maximum rate of reaction, Km is the concentration of substrate needed to achieve half Vmax, and Kcat is the turnover number for the aminopeptidase activity. The Kcat/KM value is the catalytic efficiency.

Figure 2. The chemical reaction that produced our product, 4-AcDM.

Figure 2. The chemical reaction that produced our product, 4-AcDM.

 
Figure 3. The initial compound tested by Oliveira et al., 4-MDM.

Figure 3. The initial compound tested by Oliveira et al., 4-MDM.

 

Discussion

            The Michaelis-Menton plot obtained showed that with increasing concentration of
our drug, 4AcDM, the rate of pNA cutting (and therefore, PGP cutting) is accelerated.
Further, the maximum rate of reaction increased with increasing presence of 4AcDM.
These enzyme kinetics tests supported the conclusion that the drug was efficient in PGP
hydrolysis.
            The molecule synthesized in our experiments, 4AcDM, was efficient and
activated the AP activity of the LTA4H enzyme. Our work sets itself apart from other
emphysema-related treatments because it targets the anti-inflammatory pathway of the
disease itself and serves to be an activator of a pathway, rather than an inhibitor. Since
most work that are centralized around developing enzyme inhibitors for the EH pathways
have proved to be inefficient when tested, enzyme activators for the AP activity were
developed instead. In one study conducted by Konstan et al., the effects of LTB4 receptor
inhibition on the reduction of airway inflammation proved to be trivial as an increase in
the “risk of infection-related adverse events” was possible [11] . Although the inflammatory
response could have been suppressed, unexpected infections could have occurred in
conjunction, making the lessened inflammatory response insignificant. In another study
conducted by Roberts et al., the effects of inhibiting leukotriene (LT) biosynthesis on
inducing remission in patient with ulcerative colitis were observed [12] . Although the 5-
lipoxygenase inhibitor inhibited LT biosynthesis, the inhibition did not greatly differ
from the use of placebo in clinical efficacy. It was Paige et al. who first studied the role
of the the LTA4H aminopeptidase in the pathogenesis of emphysema, finding that the AP
activity of the enzyme breaks down and clears PGP [4] . Our observations support those of
Oliveira et al. who studied the anti-inflammatory pathway. In their experiments, Oliveira
et al. observed the effects of the compound known as 4-methoxydiphenylmethane (4-
MDM) [5] . Although this compound does activate the AP activity of the LTA4H enzyme,
the compound itself is chemically unsound. As Lai et al noted, several properties of 4-
MDM make it an undesirable drug treatment for emphysema, including its poor water
solubility and an unstable central oxygen atom [9] .
            However, our work extends and enhances these findings because we have
synthesized a novel compound. In our drug, 4AcDM, we replaced the central oxygen
atom with a stable carbon atom and attached an acetyl group to increase the overall
stability of the compound. The drug, with its enhanced physicochemical properties, is
effective in enacting the AP pathway of LTA4H. Enzyme assays confirm that we have
synthesized an improved compound that promotes anti-inflammation, extending and
improving on Oliveira’s work. With more oxygen atoms and an acetyl group, our drug
proved to more efficient at the enzyme level. Further, with increased concentrations of
4AcDM, the rate of AP activity increased, confirming the efficacy of our drug as
treatment for the inflammation associated with emphysema.
            The final product developed was C15H14O2, comprised of 4-benzylphenol in
dichloromethane solvent, pyridine as the catalyst, and acetic anhydride. Our drug is a
significant step towards novel emphysema treatment once tested within in-vitro and in-
vivo studies because it is efficient at clearing inflammation, ultimately preserving the
protective properties of 4-MDM while existing as a more stable compound.

Conclusion

            Our research concluded that 4AcDM was efficient when administered in 160.0
mM, as the greatest initial velocity (V 0 ) at which the reaction occurred resulted when the
drug was tested upon enzyme assays. Our work addressed the initial research question by
synthesizing a compound that was improved through the addition of a carbon atom and
acetyl group, which succeeded in producing a well functioning LTA4H enzyme activator.
Our results are fully supported by the results described in the report, as other research
focused upon the enzyme present the results found in in vivo and in vitro studies. Since
our research looked at the drug’s effects in the enzyme level, our results were strongly
supported by enzyme kinetics concepts versus those of others’ research.
            Experiments performed in the future should work towards improving the water
solubility of the drug, and determine the best formulation for in vivo and in vitro studies.
If we had more time, we would look into both water solubility and in vivo/in vitro
studies, while also looking towards utilizing methyl chloroformate and ethyl
chloroformate in place of acetic anhydride. We believe that the addition of a methyl
group or ethyl group could have potential benefits, and since both were tested in the
preliminary stages of our experiment, reasons behind why the chloroformate solutions
failed to react with other reactants would need to be investigated. If we were to start the
work today, we would work towards improving the percent yield by being more attentive
to amounts of reactants mixed to ensure that the greatest amount of product can be
collected at the end. We would run more enzyme assays to validify our results, and run
more TLC to assure that the final product is purified to its maximum potential. During
column chromatography, varying solutions of ethyl acetate hexanes could be used, rather
than simply 1:9 or 1:19. Using more solutions of ethyl acetate hexanes would allow more
of the product to come out at faster rates. Questions that still remain to be answered
include, what other reporter groups, other than Ala-pNA, could be used to portray the
activity of PGP in a more noticeable and researchable manner? What other roles could
the LTA4H enzyme have other than its function in lung diseases? Could it have a
significant role in common diseases such as cancer, and could our drug have multiple
uses? How could we develop our drug into one that is in crystallized form, for use
especially in clinical trials?


References

1. Doyle, Rodger. "Chronic Obstructive Pulmonary Disease." Scientific American Oct. 1997: 40. Web. 16 Aug. 2015.

2. Trends in COPD (Chronic Bronchitis and Emphysema): Morbidity and Mortality. (n.d.). Retrieved August 20, 2015, from http://www.lung.org/finding-cures/our-research/trend-reports/copd-trend-report.pdf

3. Stulbarg, Michael S. (2014). Emphysema. InAccessScience. McGraw-Hill Education. http://dx.doi.org/10.1036/1097-8542.230900

4. Paige, M., Wang, K., Burdick, M., Park, S., Cha, J., Jeffery, E., . . . Shim, Y. (2014). Role of Leukotriene A4 Hydrolase Aminopeptidase in the Pathogenesis of Emphysema. The Journal of Immunology, 5059-5068.

5. Oliveira, E., Wang, K., Kong, H., Kim, S., Miessau, M., Snelgrove, R., . . . Paige, M. (n.d.). Effect of the leukotriene A4 hydrolase aminopeptidase augmentor 4-methoxydiphenylmethane in a pre-clinical model of pulmonary emphysema. Bioorganic & Medicinal Chemistry Letters, 6746-6750.

6. Current Cigarette Smoking Among Adults in the United States. (2015, January 23). Retrieved August 20, 2015, from http://www.cdc.gov/tobacco/data_statistics/fact_sheets/adult_data/cig_smoking/

7. Thin Layer Chromatography. (n.d.). Retrieved August 24, 2015, from http://www.chem.umass.edu/~samal/269/tlc.pdf

8. Column Chromatography. (n.d.). Retrieved August 27, 2015, from http://www.wfu.edu/chemistry/courses/organic/CC/index.htm

9. Jiang, X., Zhou, L., Wei, D., Meng, H., Liu, Y., & Lai, L. (n.d.). Activation and inhibition of leukotriene A4 hydrolase aminopeptidase activity by diphenyl ether and derivatives. Bioorganic & Medicinal Chemistry Letters, 6549-6552. Retrieved September 11, 2015.

10. Shin, E., Lee, H., & Bae, Y. (n.d.). Leukotriene B4 stimulates human monocyte-derived dendritic cell chemotaxis. Biochemical and Biophysical Research Communications, 606-611. Retrieved September 11, 2015.

11. Konstan, M., Döring, G., Heltshe, S., Lands, L., Hilliard, K., Koker, P., . . . Hamilton, A. (n.d.). A randomized double blind, placebo controlled phase 2 trial of BIIL 284 BS (an LTB4 receptor antagonist) for the treatment of lung disease in children and adults with cystic fibrosis. Journal of Cystic Fibrosis, 148-155. Retrieved September 14, 2015.

12. Roberts, W., Simon, T., Berlin, R., Haggitt, R., Snyder, E., Stenson, W., . . . Berger, M. (n.d.). Leukotrienes in ulcerative colitis: Results of a multicenter trial of a leukotriene biosynthesis inhibitor, MK-591. Gastroenterology, 725-732. Retrieved September 14, 2015

Generative Networks for Deep Learning

Generative Networks for Deep Learning

3D Printed Scaffolds and Stem Cells: Study

3D Printed Scaffolds and Stem Cells: Study