Investigating the Organic Synthesis of Dityrosine - Revision history

Bes at 13:17, 13 May 2021

2021-05-13T13:17:01Z

Krichter: /* Abstract */

2021-05-12T07:20:55Z

Abstract

Krichter: /* Conclusion */

2021-05-12T07:10:16Z

Conclusion

Krichter: /* Results and Discussion */

2021-05-12T06:57:49Z

Results and Discussion

Krichter: /* Results and Discussion */

2021-05-12T06:50:57Z

Results and Discussion

Krichter: /* Results and Discussion */

2021-05-12T06:39:02Z

Results and Discussion

Krichter: /* Results and Discussion */

2021-05-12T06:20:52Z

Results and Discussion

Krichter: /* Results and Discussion */

2021-05-12T06:19:59Z

Results and Discussion

Krichter at 06:00, 12 May 2021

2021-05-12T06:00:33Z

Krichter: /* Future Tasks for This Project */

2021-05-12T05:48:06Z

Future Tasks for This Project

← Older revision		Revision as of 13:17, 13 May 2021
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−	~~You have reached the page dedicated to research investigating an efficient organic synthesis~~ of ~~the dityrosine molecule~~. ~~This~~ research ~~is carried out by Monmouth College undergraduate Katelyn Richter~~ under the supervision of Dr. ~~Bradley~~ Sturgeon.	+	''<small>The following document was completed by Katelyn B. Richter in Spring of 2021 in partial fulfillment of her requirements for a Bachelor's Degree of Science in Chemistry, Monmouth College, Monmouth, Il 61462. The research was done under the supervision of Bradley E. Sturgeon.</small>''

	==Abstract==		==Abstract==

← Older revision		Revision as of 07:20, 12 May 2021
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	==Abstract==		==Abstract==

		+	Dityrosine, the dimerized form of the amino acid tyrosine, is a natural phenolic molecule that is biologically active and relevant in a number of ways. Previously, the Sturgeon Research group has synthesized phenolic dimers through enzymatic beaker reactions for various areas of study. While the enzymatic reaction is very fast-acting, the result is a mixture of many different products, from dimers to trimers to polymers. The enzymatic method requires rigorous separation in post to isolate a specific dimer in the pure form. Organic synthesis offers another avenue of approach, as reaction can be much more selective through the use of specific reagents and conditions. The main goal of this project was to optimize an organic synthesis of dityrosine to avoid tedious separation techniques. Once a successful synthesis pathway is known, it could then be applied to the synthesis of dityrosine analogs. These analogs would likely have their own biological properties, as they are structurally similar to dityrosine. Unfortunately, the organic synthesis explored in this project became more tedious and time-consuming than separation after an enzymatic reaction. Based on the findings in this research, the enzymatic method still prevails over the organic method in terms of both convenience and time.

	==Introduction==		==Introduction==

← Older revision		Revision as of 07:10, 12 May 2021
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	==Conclusion==		==Conclusion==
		+
		+	Overall, there is still a great deal of work and fine tuning to be done before this project can be a success. The organic synthesis pathway described by Skaff et al. did not yield the same results as those that were published. There were a number of struggles that Skaff et al. seemed not to have encountered based on their documentation. Perhaps the synthesis could be attempted again in the future. With more time dedicated and more rigorous purification, it is certainly possible to isolate dityrosine with this method. However, this researcher argues that an undergraduate who is not a full time researcher may actually save more time simply performing an enzymatic reaction using 3-iodo-L-tyrosine and braving the tedious separation procedures. A semester was spent on the organic synthesis alone, and a pure form of dityrosine was still not achieved. All in all, this was a educational project that produced more failures than it did successes. That said, it was still a valuable experience for an undergraduate to take part in, as it gave her the opportunity to learn about a variety of organic techniques and instrumentation. If this project were to be resumed by another student, it is suggested that the student take another route to synthesize dityrosine.

	==Future Tasks for This Project==		==Future Tasks for This Project==

← Older revision		Revision as of 06:57, 12 May 2021
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−	'''Figure 1:''' An HPLC analysis was conducted on Intermediate 3 following synthesis. Full HPLC method details can be found [http://esr.monmsci.net/wiki/index.php/File:Phenol_30m_02_methoddetails.pdf here]. Major peaks can be observed at 10 minutes, 14 minutes and 15 minutes. This experiment was run in reverse phase; therefore, more polar compounds eluted first.	+	'''Figure 1:''' An HPLC analysis was conducted on Intermediate 3 following synthesis. Full HPLC method details can be found [http://esr.monmsci.net/wiki/index.php/File:Phenol_30m_02_methoddetails.pdf here]. The blue spectrum represents the product, and it is overlaying the red spectrum, which represents the starting material. Major peaks can be observed at 10.5 minutes, 14 minutes and 14.5 minutes. This experiment was run in reverse phase; therefore, more polar compounds eluted first. The tallest peak at 14.5 minutes is the protected intermediate, as it is a nonpolar molecule. The peak at 14 minutes is likely a side product in which perhaps two out of the three of the protecting groups are attached in the desired locations. Finally, the peak at 10.5 minutes is thought to be a side product in which only one out of the three locations is protected. While this reaction was mildly successful, a better outcome than this is desired. Ideally, this reaction should be optimized to have less side products and a higher percent yield.

	[[File:suzukihplc.png\|frameless\|center\|750px]]		[[File:suzukihplc.png\|frameless\|center\|750px]]

← Older revision		Revision as of 06:50, 12 May 2021
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−	'''Figure 1:'''	+	'''Figure 1:''' An HPLC analysis was conducted on Intermediate 3 following synthesis. Full HPLC method details can be found [http://esr.monmsci.net/wiki/index.php/File:Phenol_30m_02_methoddetails.pdf here]. Major peaks can be observed at 10 minutes, 14 minutes and 15 minutes. This experiment was run in reverse phase; therefore, more polar compounds eluted first.

	[[File:suzukihplc.png\|frameless\|center\|750px]]		[[File:suzukihplc.png\|frameless\|center\|750px]]

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 [[File:P3ilt_hplc_inj1.PNG|frameless|center|750px]]
 [[File:suzukihplc.png|frameless|center|750px]]
-'''Figure 1:''' Thin Layer Chromatography (TLC) is typically the preferred method for mediating the progression of organic reactions. However, there is also opportunity to utilize High Performance Liquid Chromatography (HPLC) as well to observe the disappearance of reactants and the formation of products. HPLC was used to observe the Suzuki coupling reaction which forms the protected dityrosine intermediate. To prepare samples, an aliquot (three drops) of the reaction was added to a sample vial and brought to a volume of 1 mL with methanol with methanol. This was done five times as the reaction progressed at the initiation of the reaction, 1 hour, 12 hours, 36 hours, and 48 hours. As the reaction continued, the reactant peak (observed at 14 minutes) decreased. This can also be observed in this smaller red graph in the upper left corner. Various products also formed in small amounts (observed between 2 and 12 minutes). However, there does not appear to be one major product based on these spectra alone. This is because the protected dityrosine intermediate was insoluble in the reaction mixture. As a result, it was left at the bottom of the flask rather than being drawn up with sample aliquots taken for analysis. After aqueous extraction and concentration under vacuo of the reaction, the major product was a viscous brown oil. These results show that the Suzuki coupling reaction was indeed successful in producing a protected dityrosine intermediate. However, the presence of other smaller peaks other than the major peak at 14 minutes shows evidence of impurities, which needed to be separated through chromatography. Though side products did occur from this reaction, the amount of side products was not even comparable to those that result form an enzymatic reaction. The Suzuki coupling reaction used in combination with protecting groups was a success overall.
+'''Figure 2:''' Thin Layer Chromatography (TLC) is typically the preferred method for mediating the progression of organic reactions. However, there is also opportunity to utilize High Performance Liquid Chromatography (HPLC) as well to observe the disappearance of reactants and the formation of products. HPLC was used to observe the Suzuki coupling reaction which forms the protected dityrosine intermediate. To prepare samples, an aliquot (three drops) of the reaction was added to a sample vial and brought to a volume of 1 mL with methanol with methanol. This was done five times as the reaction progressed at the initiation of the reaction, 1 hour, 12 hours, 36 hours, and 48 hours. As the reaction continued, the reactant peak (observed at 14 minutes) decreased. This can also be observed in this smaller red graph in the upper left corner. Various products also formed in small amounts (observed between 2 and 12 minutes). However, there does not appear to be one major product based on these spectra alone. This is because the protected dityrosine intermediate was insoluble in the reaction mixture. As a result, it was left at the bottom of the flask rather than being drawn up with sample aliquots taken for analysis. After aqueous extraction and concentration under vacuo of the reaction, the major product was a viscous brown oil. These results show that the Suzuki coupling reaction was indeed successful in producing a protected dityrosine intermediate. However, the presence of other smaller peaks other than the major peak at 14 minutes shows evidence of impurities, which needed to be separated through chromatography. Though side products did occur from this reaction, the amount of side products was not even comparable to those that result form an enzymatic reaction. The Suzuki coupling reaction used in combination with protecting groups was a success overall.
 ==Conclusion==

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 ==Results and Discussion==
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 '''Figure 1:''' Thin Layer Chromatography (TLC) is typically the preferred method for mediating the progression of organic reactions. However, there is also opportunity to utilize High Performance Liquid Chromatography (HPLC) as well to observe the disappearance of reactants and the formation of products. HPLC was used to observe the Suzuki coupling reaction which forms the protected dityrosine intermediate. To prepare samples, an aliquot (three drops) of the reaction was added to a sample vial and brought to a volume of 1 mL with methanol with methanol. This was done five times as the reaction progressed at the initiation of the reaction, 1 hour, 12 hours, 36 hours, and 48 hours. As the reaction continued, the reactant peak (observed at 14 minutes) decreased. This can also be observed in this smaller red graph in the upper left corner. Various products also formed in small amounts (observed between 2 and 12 minutes). However, there does not appear to be one major product based on these spectra alone. This is because the protected dityrosine intermediate was insoluble in the reaction mixture. As a result, it was left at the bottom of the flask rather than being drawn up with sample aliquots taken for analysis. After aqueous extraction and concentration under vacuo of the reaction, the major product was a viscous brown oil. These results show that the Suzuki coupling reaction was indeed successful in producing a protected dityrosine intermediate. However, the presence of other smaller peaks other than the major peak at 14 minutes shows evidence of impurities, which needed to be separated through chromatography. Though side products did occur from this reaction, the amount of side products was not even comparable to those that result form an enzymatic reaction. The Suzuki coupling reaction used in combination with protecting groups was a success overall.
 ==Conclusion==

← Older revision		Revision as of 06:19, 12 May 2021
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	'''Figure 1:''' Thin Layer Chromatography (TLC) is typically the preferred method for mediating the progression of organic reactions. However, there is also opportunity to utilize High Performance Liquid Chromatography (HPLC) as well to observe the disappearance of reactants and the formation of products. HPLC was used to observe the Suzuki coupling reaction which forms the protected dityrosine intermediate. To prepare samples, an aliquot (three drops) of the reaction was added to a sample vial and brought to a volume of 1 mL with methanol with methanol. This was done five times as the reaction progressed at the initiation of the reaction, 1 hour, 12 hours, 36 hours, and 48 hours. As the reaction continued, the reactant peak (observed at 14 minutes) decreased. This can also be observed in this smaller red graph in the upper left corner. Various products also formed in small amounts (observed between 2 and 12 minutes). However, there does not appear to be one major product based on these spectra alone. This is because the protected dityrosine intermediate was insoluble in the reaction mixture. As a result, it was left at the bottom of the flask rather than being drawn up with sample aliquots taken for analysis. After aqueous extraction and concentration under vacuo of the reaction, the major product was a viscous brown oil. These results show that the Suzuki coupling reaction was indeed successful in producing a protected dityrosine intermediate. However, the presence of other smaller peaks other than the major peak at 14 minutes shows evidence of impurities, which needed to be separated through chromatography. Though side products did occur from this reaction, the amount of side products was not even comparable to those that result form an enzymatic reaction. The Suzuki coupling reaction used in combination with protecting groups was a success overall.		'''Figure 1:''' Thin Layer Chromatography (TLC) is typically the preferred method for mediating the progression of organic reactions. However, there is also opportunity to utilize High Performance Liquid Chromatography (HPLC) as well to observe the disappearance of reactants and the formation of products. HPLC was used to observe the Suzuki coupling reaction which forms the protected dityrosine intermediate. To prepare samples, an aliquot (three drops) of the reaction was added to a sample vial and brought to a volume of 1 mL with methanol with methanol. This was done five times as the reaction progressed at the initiation of the reaction, 1 hour, 12 hours, 36 hours, and 48 hours. As the reaction continued, the reactant peak (observed at 14 minutes) decreased. This can also be observed in this smaller red graph in the upper left corner. Various products also formed in small amounts (observed between 2 and 12 minutes). However, there does not appear to be one major product based on these spectra alone. This is because the protected dityrosine intermediate was insoluble in the reaction mixture. As a result, it was left at the bottom of the flask rather than being drawn up with sample aliquots taken for analysis. After aqueous extraction and concentration under vacuo of the reaction, the major product was a viscous brown oil. These results show that the Suzuki coupling reaction was indeed successful in producing a protected dityrosine intermediate. However, the presence of other smaller peaks other than the major peak at 14 minutes shows evidence of impurities, which needed to be separated through chromatography. Though side products did occur from this reaction, the amount of side products was not even comparable to those that result form an enzymatic reaction. The Suzuki coupling reaction used in combination with protecting groups was a success overall.
		+
		+
		+
		+	[[File:P3ilt_hplc_inj1.png\|frameless\|center\|750px]]

	==Conclusion==		==Conclusion==

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 You have reached the page dedicated to research investigating an efficient organic synthesis of the dityrosine molecule. This research is carried out by Monmouth College undergraduate Katelyn Richter under the supervision of Dr. Bradley Sturgeon.
 ==Introduction==
@@ Line 88: / Line 91: @@
 '''Figure 1:''' Thin Layer Chromatography (TLC) is typically the preferred method for mediating the progression of organic reactions. However, there is also opportunity to utilize High Performance Liquid Chromatography (HPLC) as well to observe the disappearance of reactants and the formation of products. HPLC was used to observe the Suzuki coupling reaction which forms the protected dityrosine intermediate. To prepare samples, an aliquot (three drops) of the reaction was added to a sample vial and brought to a volume of 1 mL with methanol with methanol. This was done five times as the reaction progressed at the initiation of the reaction, 1 hour, 12 hours, 36 hours, and 48 hours. As the reaction continued, the reactant peak (observed at 14 minutes) decreased. This can also be observed in this smaller red graph in the upper left corner. Various products also formed in small amounts (observed between 2 and 12 minutes). However, there does not appear to be one major product based on these spectra alone. This is because the protected dityrosine intermediate was insoluble in the reaction mixture. As a result, it was left at the bottom of the flask rather than being drawn up with sample aliquots taken for analysis. After aqueous extraction and concentration under vacuo of the reaction, the major product was a viscous brown oil. These results show that the Suzuki coupling reaction was indeed successful in producing a protected dityrosine intermediate. However, the presence of other smaller peaks other than the major peak at 14 minutes shows evidence of impurities, which needed to be separated through chromatography. Though side products did occur from this reaction, the amount of side products was not even comparable to those that result form an enzymatic reaction. The Suzuki coupling reaction used in combination with protecting groups was a success overall.
 ==Future Tasks for This Project==

@@ Line 91: / Line 91: @@
 ==Future Tasks for This Project==
-The organic method of synthesis for dityrosine still requires a great deal of fine tuning. Once synthesis of dityrosine has been optimized, it can then be edited to create analogs of dityrosine with potential biological activity. Following the synthesis of dityrosine and dityrosine derivatives, the molecule will be tested for antibacterial properties against a nonvirulent strain of E. coli via Kirby-Bauer Disk Diffusion assay. Analogs of dityrosine will also be synthesized and tested for antibacterial properties to investigate whether derivatized forms of dityrosine have possible medicinal applications.
+The organic method of synthesis for dityrosine still requires a great deal of fine tuning. Once synthesis of dityrosine has been optimized, it can then be edited to create analogs of dityrosine with potential biological activity. Following the synthesis of dityrosine and dityrosine derivatives, the molecules will be tested for antibacterial properties against a nonvirulent strain of E. coli via Kirby-Bauer Disk Diffusion assay.
 ==Kirby-Bauer Disk Diffusion Assay==