Our project aims to innovate levothyroxine production, a critical drug for hypothyroidism treatment. Traditional methods involve low yields and environmental challenges, notably the need for racemate resolution. By integrating transaminase enzymes known for high substrate specificity and stereoselectivity, we intend to simplify the synthesis process, improve yield, and reduce byproducts. Our team's multi-disciplinary approach focuses on optimizing the L-tyrosine-based synthesis pathway for levothyroxine, with the ultimate goal of setting new standards in sustainable pharmaceutical production.

The Problem


Hypothyroidism is an endocrine disorder characterized by an underactive thyroid gland, leading to insufficient production of thyroid hormones—namely thyroxine (T4) and triiodothyronine (T3). These hormones are crucial for regulating metabolic processes, energy production, and overall homeostasis in the human body. Symptoms of hypothyroidism can range from fatigue and weight gain to sensitivity to cold and cognitive impairments. If left untreated, the condition can escalate into severe complications like cardiovascular diseases, infertility, and in extreme cases, myxedema coma, a life-threatening condition.

Levothyroxine as a Treatment

Levothyroxine is a synthetic analog of the natural thyroid hormone thyroxine (T4). It serves as the frontline treatment for hypothyroidism and functions by replenishing or replacing the deficient thyroid hormones. This helps in alleviating symptoms and normalizing hormone levels. While generally well-tolerated, the medication requires meticulous dose adjustment to mitigate potential side effects such as osteoporosis or cardiovascular issues.

Figure 1: Levothyroxine Fragment

Racemate resolution

  • Current synthesis pathway for levothyroxine

    The conventional synthesis of levothyroxine is a complex chemical process that typically involves multiple steps such as condensation reactions between phenols and acetic acid derivatives, cyclization to form a precursor aromatic ring structure, and iodination to introduce the requisite iodine atoms. This sequence of reactions often culminates in a racemic mixture of levothyroxine and its enantiomer, dextrothyroxine, necessitating a separation step.

    Figure 2: The racemate resolution highlighted within the synthesis process.
  • What is Racemate Resolution?

    Racemate resolution is a subsequent process employed to separate this racemic mixture into its individual enantiomers. In the case of levothyroxine, this step is indispensable for isolating the pharmacologically active levothyroxine from the inactive dextrothyroxine. Typically, chiral resolving agents or crystallization methods are utilized for this purpose.

  • Why is Racemate Resolution a Problem?

    The necessity for a racemate resolution step poses several significant challenges in levothyroxine production:

    1. Complexity: This additional separation process escalates the overall complexity of the synthesis pathway.
    2. Cost: The requirement for chiral resolving agents or other specialized techniques substantially inflates production costs.
    3. Waste and Environmental Concerns: The separated, inactive dextrothyroxine often becomes waste, exacerbating environmental burdens. Moreover, the use of chemical resolving agents can also be environmentally unfriendly.
    4. Efficiency and Yield: The racemate resolution step results in a 50% loss of yield, as only one of the two enantiomers is pharmacologically active, rendering the process highly inefficient.

Our Solution


Transaminases are pyridoxal phosphate (PLP)-dependent enzymes that catalyze the transfer of amino groups between amino acids and alpha-keto acids. Due to their high substrate specificity and stereoselectivity, transaminases have been identified as ideal candidates for chiral amine synthesis, including the synthesis of pharmaceutical compounds like levothyroxine.

Rationale for Transaminase Integration

The conventional synthesis pathway for levothyroxine faces challenges such as low yield and the generation of excessive byproducts. To overcome these hurdles, our project aims to integrate transaminases into the levothyroxine synthesis pathway. Specifically, we are focusing on the L-tyrosine-based synthesis pathway, which has the potential to accommodate transaminase-catalyzed synthesis of key intermediates. This strategy aims to increase overall yield while minimizing byproduct formation, thereby streamlining the entire synthesis process.

Transaminase Selection

Given the importance of transaminases in our project, the selection of suitable enzymes is crucial. The identification process will involve both computational methods and biochemical assays to evaluate various transaminases based on their substrate specificity, enzyme kinetics, and other functional attributes. Our ultimate goal is to find transaminases that are most effective for catalyzing the specific reactions needed for levothyroxine synthesis.

Mechanistic Insights

Transaminases function by facilitating the transfer of amino groups, a feature that can be leveraged to bypass the traditional racemate resolution step in chiral amine synthesis. Their ability to act on specific substrates while maintaining high stereoselectivity makes them uniquely suited for synthesizing levothyroxine intermediates.

Advantages of Transaminase-Catalyzed Synthesis

  1. Stereoselectivity: Transaminases offer high enantioselectivity, allowing for the production of chiral amines without the need for racemate resolution.
  2. Reduced Byproduct Formation: The enzymatic reactions catalyzed by transaminases are more controlled, leading to significantly fewer byproducts.
  3. Operational Flexibility: Transaminases can function effectively under a variety of conditions, making them adaptable for industrial-scale operations.


The integration of transaminases into the levothyroxine synthesis pathway offers a transformative solution to the prevailing challenges in levothyroxine production. By utilizing these highly specific and stereoselective enzymes, our project circumvents the need for racemate resolution, thus alleviating issues of complexity, cost, waste, and inefficiency. The enzymatic approach not only simplifies the overall synthetic route but also enhances yield and minimizes byproduct formation. In doing so, this innovative methodology paves the way for a more sustainable, cost-effective, and efficient process for levothyroxine production, promising a significant impact on the pharmaceutical industry and, consequently, on the management of hypothyroidism. Moreover, successful synthesis of levothyroxine would serve as a proof-of-concept for the use of stereoselective biocatalytic enzymes in pharmaceutical applications, paving the way for further development of transaminase and other enzyme mediated synthesis pathways.

Figure 3: The end product of our synthesis process.

Genetic Design

Candidate Transaminases

Candidate transaminases were chosen for their endogenous activity towards halogenated aromatic rings, which resembles the keto-acid precursor to minithyroxine. We identified three transaminases that fit such criteria: human-halogenated tyrosine transaminase (HHTA), Saccharomyces cerevisiae aromatic transaminase (AATA), and Chromobacterium violaceum transaminase (CVTA).

Figure 4: Candidate transaminases we considered.

Expression System

Since our final activity assay requires purified protein, we designed our genetic constructs to maximze the amount of protein expression. Thus, we used the IPTG-inducible T7 promoter system in E. coli BL21 cells. This expression system allows for the high production of transaminase enzyme when we add IPTG (Isopropyl ß-D-1-thiogalactopyranoside) to our cell culture, because the addition of IPTG blocks the production of LacI, which inhibits the expression of our transaminase. Thus under this system, the addition of IPTG causes the high production of transamianse. We chose E. coli BL21 cells to use as our expression strain because it endogenously contains the phage-derived T7 polymerase, which is the crucial regulatory component of our system

Figure 5: The IPTG-inducible T7 expression system we used.

Additionally, because we need to work with purified enzyme, we added a 6x histidine tag to the N-terminus of our transaminase so that we can purifiy our expressed transaminases using a nickel column.

Thus, our final genetic constructs took the form of the following plasmid:

  • Backbone: pQE81XN

    1. Contains the LacI operon, an ampicillin resistance selection marker, T7 promoter, ribosome-binding site (RBS), and 6x His tag
  • Insert: transaminase coding sequence

Our E. coli BL21 chassis endogenously supplied the last part of our expression system - the T7 RNA polymerase under the lacUV5 promoter control.

UV-Vis Activity Assay

Confirming the catalytic activity of transaminases

UV-Vis spectroscopy was instrumental in confirming the catalytic activity of our selected transaminases. We monitored the unique UV-Vis absorbance peaks of key reaction intermediates, focusing on:

  • Internal Aldimine: Absorbance at 411 nm
  • External Aldimine: Absorbance at 330 nm
  • Pyridoxamine Phosphate (PMP): Absorbance at 314 nm

The ratio of the absorbance peaks at 314 nm to 411 nm was used to quantify the activity of the amine donor. Likewise, the ratio of 330 nm to 411 nm indicated the activity of the amine acceptor. These absorbance ratios were normalized to facilitate comparisons between different transaminases. A high 314:411 nm absorbance ratio identified the optimal amine donor, and an initial high 330:411 nm ratio that later shifted to a higher 411 nm absorbance confirmed successful catalytic activity with the amine acceptor.

Activity with Minithyroxine Substrate Confirmed

After confirming the transaminases' catalytic activity, we proceeded to validate their efficacy in synthesizing the 3,5-di-iodo-(L)-tyrosine subfragment of levothyroxine, commonly referred to as "minithyroxine." UV-Vis spectroscopy was pivotal in this validation phase. The successful catalytic activity was evidenced by the formation of an external aldimine, identifiable by its UV-Vis absorbance peak at 330 nm. The consistent presence of this 330 nm peak confirmed that the transaminase effectively catalyzed the reaction with the minithyroxine substrate.

By employing UV-Vis spectroscopy strategically, we were able to rigorously confirm both the catalytic activity of the selected transaminases and their effectiveness in synthesizing minithyroxine. This approach provided us with a robust and quantitative method to validate key aspects of our project.

Figure 6: A) Scheme of key intermediates of a transaminase reaction. B) UV-Vis spectra of intermediates, using S-MBA as the amine donor and pyruvate as the amine acceptor, collected by M. Saajid (Campopiano Group, U of Edinburgh)


To inform our decisions in the lab, we conducted simulations involving our candidate transaminases, helping us to understand the protein-ligand interactions that drive our project. Specifically, we began with docking simulations between our candidate transaminases and a levothyroxine fragment. This helped us confirm that all of our candidates would facilitate the reaction of interest as the affinity energies were conducive to an interaction in the enzymes’ active site. Upon selecting our transaminase, we began conducting molecular dynamics simulations to further understand the enzyme’s characteristics and behavior in a more realistic environment than that which is used in docking simulations. These simulations affirmed that we were on the right track, lining up with the results we saw day-to-day in the lab.

You can read more about our modeling here!


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