Investigating the Organic Synthesis of Dityrosine

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.

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

New interest in peptides and proteins containing cross-linked dityrosine residues is increasing due to fascinating biological properties they have. Dityrosine is the dimerized form of the amino acid L-tyrosine. Some examples of said biological properties include increased stabilization of structures, conformational restriction of cyclic peptides, and variation in the electronic properties seen in enzyme active sites (Skaff et al., 2005). The possibilities of application in biological systems make tyrosine dimers a molecule of great intrigue. Dityrosine cross-linking also has a plethora of applications in the realm of biomaterials that mimic cellular environments with great potential for developing ground-breaking techniques in regenerative medicine and tissue engineering (Partlow et al., 2016). Dityrosine research also provides great insight into the progression of Alzheimer’s disease, which can be defined as the aggregation of insoluble amyloid plaques in the neuropil composed of highly stable, self-assembled Amyloid-beta42 fibrils, the neurotoxic dimerization of which is a direct result of dityrosine cross-linking (Al-Hilaly et al., 2013).

Furthermore, protein tyrosine and tryptophan have been identified as targets of photo-oxidation in milk and in other food products that contain proteins with high concentrations of these residues. The flexible structure of β-casein in milk favors radical–radical termination of tyrosyl radicals to produce dityrosine (Dalsgaard et al., 2011). Research on chitin also explores the possibilities of tyrosine. Chitin is one of the most abundant renewable nitrogenous materials accessible to humans, obtainable through the shells of crustaceans. However, the direct conversion of crustacean shell waste-derived chitin into nitrogen-containing chemicals is challenging using standard catalytic processes. To address this challenge, researchers are developing an integrated biorefinery process to upgrade shell waste-derived chitin into two aromatic NCCs, L-DOPA and tyrosine (Ma et al., 2020).

The research of the Sturgeon group is focused on phenolic compounds, most notably lignin. Lignin is the second most abundant natural polymer that exists on earth (Dimmel, 2010). There are three main lignin monomers: coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol. These are all characterized by a phenol group with methoxy substituents occurring at the C-3 and C-5 positions. This phenol contributes to stability of the assumed radical forms due to resonance stabilization (Dimmel, 2010). Tyrosine is also a phenolic compound with a relatively similar structure to that of the lignin monomers. Thus, there may be connections in the molecules' interactions. Both lignin monomers and tyrosine also have the ability to dimerize via oxidative coupling to form lignan dimers and dityrosine, respectively (Dimmel, 2010). Like dityrosine, lignan dimers have been shown to demonstrate a wide array of biological applications. Historical use of lignan has been traced back to a variety of folk and traditional medicines, and more current research has found lignan to have therapeutic effects on tumors in the digestive, reproductive, and endocrine systems (Dimmel, 2010). Unfortunately, lignin has proved complicated to isolate from natural products without alterations to convert to consumer-grade products (Dimmel, 2010).

The monomers couple together as radicals after they have undergone a one electron and one hydrogen oxidation process. Radicals occur at the O-4, C-1, C-3, C-5, and C-β positions (Dimmel, 2010). Upon the loss of an electron and a hydrogen, an unstable radical is formed. To gain stability, it undergoes oxidative coupling with another radical, which results in the formation of dimers. This process is then repeated to form trimers, tetramers, pentamers, and so on. In lignin monomers and in tyrosine, this radical is able to move freely about the molecule due to its conjugated structure. Consequently, there are many positions at which dimerization can occur, resulting in a variety of possible products (shown above). To make dityrosine, dimerization is needed at the 3 and 3' positions of the aromatic ring:

One of the ways dimerization of these phenolic monomers can be initiated is through enzymatic reactions. The Sturgeon research group has conducted many of these enzymatic reactions on lignin monomers and other phenols. These enzymatic reactions are not selective in nature, and this is a problem. The Sturgeon group would like to conduct various experiments on dityrosine, and this is difficult to do if it is only a small part of a larger mixture. Enzymatic reactions produce not only various dimers but also various trimers, tetramers, and even larger polymers as the reaction progresses. In order to isolate one specific product (or a specific dimer in the case of dityrosine) for testing, a great deal of time would have to be spent purifying the resulting mixture after the fact. The purpose of this project is to explore a way to avoid this rigorous separation process. An organic synthesis pathway has the potential to be much more selective than enzymatic reactions through using specific reagents and conditions. If pure dityrosine is synthesized via organic chemistry, it would theoretically save the Sturgeon research group a great deal of time in purification steps and allow for more precise experiments.

Project Goals

The goals of this project are as follows:

1. Optimize a synthesis pathway for dityrosine.

In order to study dityrosine, it is necessary to synthesize it rather than buy it. Dityrosine is only available to buy as a standard, and even the smallest quantities of it are quite expensive. For the Sturgeon group's intents and purposes, a larger amount of this compound is needed.

2. Apply dityrosine synthesis pathway to synthesis of dityrosine derivatives.

Once the organic synthesis of dityrosine has been optimized, it can be applied to the synthesis of dityrosine derivatives. Some of these analogs could be made by simply brominating or chlorinating an aromatic ring, the addition of a nitro group, etc.

3. Test dityrosine and derivatives for biological properties via Kirby-Bauer Disk Diffusion Assay.

Once the molecules of interest have been synthesized and purified, they will be tested for various biological properties, such as antifungal and antibacterial properties, in the hopes that some of the analogs will show some significant or interesting biological activity.

Methods

Organic Synthesis Pathway

Below is the proposed synthesis for dityrosine from Skaff et al. (2005):

The starting molecule is 3-iodo-L-tyrosine. This compound is reacted first with benzyl chloroformate and sodium carbonate followed by benzyl bromide and potassium carbonate. This step adds three protecting groups to the molecule. After the protecting groups have been added, this protected intermediate undergoes a Suzuki coupling reaction, which creates a bond between the two aromatic rings. This is accomplished using the Suzuki reagent, bis-pinacolato diboron. Once the coupling is complete, the molecule is deprotected using palladium on charcoal and hydrogen, yielding the final molecule of dityrosine. Chromatography is performed between each step to purify intermediates as well as the final product.

N-Cbz-4-O-Benzyl-3-iodo-L-Tyrosine Benzyl Ester 3

Potassium carbonate (2.60 mL, 48.8 mmol), 3-iodo-L-tyrosine (2) (2.03 g, 16.3 mmol), and deionized water (25 mL) were added to a round bottom flask submerged in an ice bath. A mixture of 50% benzyl chloroformate in toluene (1.71 g, 25.0 mmol) was gradually add to the reaction over a period of 30 minutes while the flask remained in the ice bath. A white precipitate fell out of solution as the 50% benzyl chloroformate solution was added. The reaction was removed from the ice bath and left to mix at room temperature for 24 hours. The reaction mixture was acidified to pH 4 using HCl, and aqueous extraction was performed using ethyl acetate (3 x 100 mL). The organic extracts were washed with saturated sodium chloride solution (2 x 100 mL). The organic extracts were dried with magnesium sulfate and concentrated under vacuo to yield Cbz-3-iodo-L-tyrosine as a white crystalline substance submerged in a yellow oil. Potassium carbonate (2.58 g, 49.2 mmol) and tetrabutylammonium iodide (18 mg) was added to Cbz-3-iodo-L-tyrosine in dry acetone (100 mL). Nitrogen was bubbled through the reaction for 15 minutes, and benzyl bromide (1.7 mL, 35.9 mmol) was added. The reaction was refluxed in the dark for 24 hours. Aqueous extraction was performed using dichloromethane (2 x 100 mL) and water (100 mL). The organic extract was concentrated under vacuo yielding Intermediate 3 as a pale yellow oil (9.01 g). The oil was chromatographed in reverse phase on a silica gel column eluting ethyl acetate/hexane (1:9 - 9:1 gradient). Fractions 31-44 were combined and concentrated under vacuo to yield a white crystalline solid (2.42 g, 25% yield).

Protected Dityrosine 4

Intermediate 3 (2.42 g, 0.00378 mol), potassium carbonate (0.200 g, 0.00151 mol), Pd(dppf)Cl₂ (5 mg), and bis(pinacolato)diboron (50 mg) were added to DMSO (50 mL) in a round bottom flask. The reaction was maintained at 80 °C and stirred under nitrogen for 48 hours. Aqueous extraction was performed on the reaction mixture with ethyl acetate (100 mL x 2) and a saturated NaCl solution (100 mL x 2). The organic extracts were combined, dried with magnesium sulfate, and concentrated under vacuo to give Protected dityrosine 4 as a dark brown oil (2.42 g).

Dityrosine 1

To Protected Dityrosine 4 (0.30g, 0.30 mmol) was added methanol (12 mL) and 10% palladium on charcoal (0.03 g, 0.03 mmol) in a round bottom flask. The reaction was stirred under hydrogen at room temperature for 48 hours. H₂ gas was generated using zinc and 3 M HCl. The resulting mixture was filtered through celite and washed with 1 M HCl (50 mL). Aqueous extraction was performed using ethyl acetate (150 mL x 2). The organic extracts were concentrated under vacuo to yield crude Dityrosine 1 (1.72 g) in the form of a pale yellow oil. The reaction was chromatographed on silica eluting H₂O/CH₃CN/TFA (95:5:0.05).

Reaction set up:

Results and Discussion

Figure 1: An HPLC analysis was conducted on Intermediate 3 following synthesis. Full HPLC method details can be found 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.

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

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

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

The Kirby Bauer Disk Diffusion Assay is a common, reliable, and simple technique for examining antimicrobial properties of potential drugs (Ho et al., 1998). It is a sensitive, convenient and inexpensive method of screening (Manganyi et al., 2018). Kirby Bauer Disk Diffusion assays will be carried out using a nonvirulent strain of E. coli on glucose minimal media plates. The plates are completely coated with the bacteria and disks containing compounds of interest will be placed onto the plate. After the bacteria’s incubation period, the zones of inhibition surrounding each disk containing the compound of interest will be recorded. The larger the zone of inhibition, the more effective the compound is as an antibiotic. This test can also be performed with fungi.

References

1. Synthesis of the side chain cross-linked tyrosine oligomers dityrosine, trityrosine, and pulcherosine

Ojia Skaff, Katrina A. Jolliffe, and Craig A. Hutton, The Journal of Organic Chemistry 2005 70 (18), 7353-7363, DOI: 10.1021/jo051076m

2. Dityrosine cross-linking in designing biomaterials

Benjamin P. Partlow, Matthew B. Applegate, Fiorenzo G. Omenetto, and David L. Kaplan, ACS Biomaterials Science & Engineering 2016 2 (12), 2 108-2121, DOI: 10.1021/acsbiomaterials.6b00454

3. A central role for dityrosine crosslinking of Amyloid-β in Alzheimer’s disease

Youssra K. Al-Hilaly et al., PubMed 2013, DOI: 10.1186/2051-5960-1-83

4. Reliability of the Kirby-Bauer Disc Diffusion Method for Detecting Methicillin-Resistant Strains of Staphylococcus aureus, W. Lawrence Drew,

A. L. Barry, Richard O'Toole, John C. Sherris, Applied Microbiology Aug 1972, 24 (2) 240-247

5. P L Ho, K H Chow, K Y Yuen, W S Ng, P Y Chau, Comparison of a novel, inhibitor-potentiated disc-diffusion test with other methods for the

detection of extended-spectrum beta-lactamases in Escherichia coli and Klebsiella pneumoniae., Journal of Antimicrobial Chemotherapy, Volume 42,

Issue 1, Jul 1998, Pages 49–54, https://doi.org/10.1093/jac/42.1.49

6. M.C. Manganyi, T. Regnier, A. Kumar, C.C. Bezuidenhout, C.N. Ateba,

Biodiversity and antibacterial screening of endophytic fungi isolated from Pelargonium sidoides, South African Journal of Botany, 2018, 192-199, ISSN

0254-6299, https://doi.org/10.1016/j.sajb.2018.03.016.

Document History

- Project started by K. Richter in Fall 2020