Zachary M. Carrico

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Writer's comment: Sometimes researchers make their most interesting discoveries when they decide to take a shortcut from a standard procedure, or in the case of Alexander Fleming, take a vacation and come back to find the origin of modern antibiotics contaminating a Petri dish. My choice to write about tetracycline for this review paper was a result of routine planning ahead. I knew that I would be working with inhibitors over the summer, so I used this paper as an excuse to research something I should understand, but would not learn about without encouragement. This paper uses the tetracycline antibiotic to illustrate the rise of antibiotic resistance in organisms and the possible routes to overcoming this resistance. I chose tetracycline as a paradigm for antibiotics because of recent advances in the synthesis of tetracycline from common chemicals. Such advances in antibiotic synthesis are essential for the improvement of medicine in general, and are specifically needed to overcome the rapid development of bacterial resistance.
—Zachary M. Carrico

Instructor's comment: Zac Carrico’s scientific review paper successfully appeals to a wide span of readers, both inside and outside of the technical disciplines. One of the reasons this paper is accessible to many, rather than a few, is his prudent choice of topic and scope, given modern consumers’ expectations that pharmaceutical interventions will cure everything from an infant ear infection to a potentially fatal staph infection, and their corresponding feeling of mass panic and betrayal when a previously effective antibiotic begins to lose its effectiveness. This review explores medical science’s dynamic efforts to hold bacterial risks in check, given the robust, cleverly adaptive nature of these microorganisms. In framing this paper rhetorically, Mr. Carrico displays excellent structure as well. He begins with an open-ended thesis, enters quickly into an evaluative mode, catalogs his way through a tightly devised network of developmental modules, explicates the risks, the stakes, and the avenues for change using hard facts as evidence, and then cres-cendos into a high note ending, which acknowledges his reasonably grounded hopes for a future score card of significantly more wins than losses for research scientists battling bacteria using the new weapon of total synthesis.
—Brad Henderson, University Writing Program

Introduction
Infecting organisms’ resistance to antibiotics has increased to an alarming level due to the overuse of antibiotics such as pe-nicillin, vancomycin, and tetracycline. The absence of new broad-range antibiotics over the past three decades is, in large part, responsible for this increasing threat. For antibiotics to win this microscopic war we must radically improve our chem-ical understanding and, more importantly, our synthetic tech-niques. Most antibiotic syntheses have disappointingly low yields and their products have low antimicrobial activities due to our limited cache of synthetic methods. Nature has proved to be a most unsympathetic teacher when it comes to this, because our most advanced synthetic techniques cannot si-mulate the customizability and precision of enzymes in the synthesis of antibiotics. For this reason drug research has turned to biosynthesis, that is, the use of naturally occurring enzymes to alter antibiotics. Unfortunately, many enzyme mechanisms are poorly understood, so the range of present-day antibiotic biosynthesis is very limited.
      The most complicated and powerful type of antibiotic syn-thesis is a “total synthesis,” which is simply synthesis from common chemicals instead of biologically prepared chemicals. Tetracycline is an excellent example of a broad-range antibi-otic that has only recently been totally synthesized. The knowledge gained from tetracycline synthesis is invaluable to scientists involved in designing antibiotics. This review paper describes the method of tetracycline’s production in the past, its novel synthesis, and the advantages of a customizable te-tracycline production method oriented towards overcoming bacterial resistance.

Background
Tetracycline’s History
Tetracycline was first extracted from streptomycin in 1948, and in the 1950s tetracycline was proclaimed a “wonder drug” for its ability to inhibit a large range of bacteria. Tetracycline has been used as a broad-range antibiotic since its production by Pfizer Pharmaceutical® in the 1950s, meaning that for the last fifty years bacteria have been developing a resistance to it. It was also the first semisynthesized antibiotic, and was the most prescribed drug for three years after its first appearance in pharmacies (1). It is used for the treatment of bacterial infections such as pneumonia, acne, Rocky Mountain spotted fever, lyme and venereal disease, and eye, urinary, and respiratory infections (2). The side effects of tetracycline are mild, generally consisting of a slight allergic reaction, but an unusual characteristic of tetracycline is to cause teeth to turn yellow due to tetracycline’s absorption into the enamel. This is especially evident in infants and small children because of the rapid growth of their enamel.

Tetracycline Manufacturing
The four primary methods of tetracycline production are as follows: the abstraction of tetracycline from microorganisms, biosynthesis, semisynthesis, and total synthesis. Mass pro-duction of tetracycline is accomplished by fermenting micro-organisms in nutrient -rich broths; the broths are then strained of microorganisms and the antibiotics produced are filtered and extracted through chemical means. Biosynthesis edits the pathway that normally produces tetracycline to modify its structure. For example, the initiation enzyme might be changed to produce a structurally altered final product. Se-misynthesis takes the extracted natural tetracycline and alters it in a way to overcome certain resistances that invading or-ganisms have developed. This customization method is li-mited because many chemical alterations cannot be done di-rectly to the natural tetracycline product while still retaining its antimicrobial activity. The most versatile and expensive pro-duction technique is a total synthesis. Total synthesis essen-tially starts from scratch, producing a complex antibiotic from standard chemical reagents. It is the most difficult way to produce tetracycline, but the advantages are impressive. To-tal synthesis increases manufacturers’ ability to customize te-tracycline, enabling it to target specific bacterial pathways and making it a much more potent drug (3).

Bacterial Targeting
The most common targets of antibiotics are the cell wall and ribosome of infecting organisms. Antibiotics can interfere with proteins constructing cell walls, resulting in weaker walls that rupture due to osmotic pressure. In ribosome targeting, the antibiotic binds to the ribosome and blocks the transcriptional enzyme from binding. This inhibits the production of en-zymes, effectively killing the organism. Tetracycline does not target cell walls, but it does bind to the 30S ribosomal subunits present in most bacteria (4).

Infecting Organism’s Resistance
The three types of tetracycline resistance are efflux, ribosome protection, and tetracycline deactivation. Efflux enzymes facilitate the removal of tetracycline from the cells through the exchange of a proton for tetracycline against a concentration gradient. An example of such an enzyme is the TetA enzyme found in the majority of the resistant strains of bacteria (5). A ribosomal protection protein, such as TetM, will bind to the ri-bosome and alter its shape so that tetracycline cannot bind (6). Tetracycline can generally be made inactive if an enzyme reduces it, causing the loss of one of tetracycline’s key func-tional groups (7).

Overcoming Resistance
The most common way to overcome bacterial resistance is to make a small change to one of tetracycline’s functional groups. This results in making tetracycline invisible to enzymes that would target it, such as TetM/A. Total synthesis greatly increases the number of changes that can be made, increasing the chances of finding a new drug.

Synthesis
The tetracycline class of molecules is composed of four li-nearly fused six-membered carbon rings labeled A through D (see Figure 1). Attempts to achieve a total synthesis have been made, but most of these started with D or CD precursors, resulting in very low yields, the highest being 0.06% in the Muxfeldt synthesis of (+/-) -5-oxytetracycline (commonly known as terramycin) (2). A total synthesis of (+/-) -12a-deoxytetracycline was done by the Stork lab with an im-pressively high yield of 25%, but the tetracycline had greatly reduced antimicrobial activity due to the absence of a hydroxyl group on an AB carbon (9).

Figure 1. Natural tetracycline structure (reprinted from Charest et al., 2005) (8).

      Figure 2 shows tetracycline’s possible modifications di-vided into three categories: those functional groups that can be modified by biosynthesis (the amide, the amine, and the R2 group), those parts modifiable by total synthesis (carbons 7-10, and R7), and those functional groups that can be modified by both biosynthesis and total synthesis (R1, R3, R4, R5, and R6) (4). Comparing the functional groups modifiable by the individual syntheses, one can see the importance of both types of production methods. Total synthesis can alter the carbon backbone on the D ring and add functional groups, but biosynthesis can alter the amine and amide functional groups that are inaccessible by way of total synthesis. Biosynthesis and total synthesis differ in their ability to synthesize various functional groups because of biosynthesis’ dependence on enzymes. Enzymes can synthesize, at high yields, what to-day’s total synthesis could never approach, but biosynthesis is restricted by the enzymes available. Biosynthetic techniques to date have not yet developed a way to alter the carbons along the D ring. This weakness in biosynthesis is because of its reliance on enzymes, which primarily alter functional groups and not the carbon backbone.

Figure 2. Variable tetracycline structure (re-printed from Khosla & Tang, 2005) (4). Groups R1, R3, R4, R5, and R6 can be modified by both total syn-thesis and biosynthesis. The R2, amine, and amide can be modified by biosynthesis. Carbons 7-10, and R7 can be modified by total synthesis. The shaded area is the region bound to the 30S ribosomal subunit (4).


      The most practical total synthesis of tetracycline was ac-complished by Charest et al., with a yield of 5 to 7% for this 14 to 15 step synthesis (8). As Khosla and Tang put it, “The synthesis presented in [Charest et al.] is not just a synthetic tour de force; it enormously expands the armamentarium of the tetracycline medicinal chemist” (4). Starting with benzoic acid the B ring is created, and then the A, C, and D rings are attached. One advantage of this synthesis is the addition of the C ring at a later stage because this preserves the properties of the AB ring, which are known to play a role in binding to the 30S subunit.
      Since the D ring is added last, it is possible to make structurally variant D rings. Large structural variance of the D ring has shown an increased ability to overcome bacterial re-sistance, and Figure 2 shows that biosynthesis has not yet developed a way to modify the carbons on the D ring. Crystal structures of the tetracycline-bound 30S subunit show that certain faces of the tetracycline molecule do not interact with the subunit (see Figure 2). Alterations to these fac-es—specifically the D ring—have been shown to overcome resistance. One tetracycline derivative, tigecycline, has a modified D ring and is currently being evaluated by the U.S. Food and Drug Administration (8).
      Six drug candidates were synthesized by Charest et al., the most promising being a pentacycline derivative—a D ring derivative—with an activity greater or equal to that of tetracyc-line in Gram-positive strains that already have resistance to standard tetracycline, methicillin, and vancomycin. Charest has this to say about the new synthesis: “Although this finding is noteworthy, it is very likely that antibiotics with even greater potencies and/or improved pharmacological properties will emerge with further exploration,” the point being that the new synthesis protocol opens many doors for the improvement of tetracycline (8).
      Using data from clinical tests of semisynthesized tetracyc-lines, Charest et al. have incorporated some antimicrobial ac-tivating features into their synthesis. Semisynthesis of tetra-cycline has shown that derivatives missing the hyrdroxyl group on carbon 6 of the C ring have an increased likelihood of overcoming resistance because they are more resistant to de-gradation. Another beneficial feature of this synthesis is the preservation of the AB rings’ characteristics. The AB rings’ polar nature is necessary to ensure tight binding to the ribo-some, because a large portion of the AB rings are bound within the ribosome, as can be seen by the gray area in Figure 2 (8).
      One of the remarkable aspects of this synthesis is its ste-reo¬selectivity. In theory, two different diastereomers should have been produced by the stereochemical variation possible in the alteration to carbon 6 on the C ring. Two more diaste-reomers should have been produced by the alteration to car-bon 5a, a fusion point between rings B and C. The total theoretical number of diastereomers is four, in contrast to re-sults which show the predominant product being the desired 6-deoxytetracycline and a small dia¬stereomeric impurity (10).

Conclusion
Since tetracycline’s discovery over fifty years ago, the resis-tance to tetracycline has increased, but thanks to the continu-ing attempts by all flavors of scientists, tetracycline’s bacterial resistance has been rejuvenated. Because the structural sig-nificance is poorly understood, and because functional groups are difficult to retain, total synthesis remains a rarity. For this reason, the successful total synthesis efforts of tetracycline that do occur are all the more impressive. Today, the primary manufacturing method of antibiotics is large fermentations, but this may change. Humans are already heavily reliant on se-misynthetic and biosynthetic antibiotics; the next step required is a change in production methods to that of total synthesis. Without the discovery of new antibiotics, the race against re-sistance is a losing one, and the total synthesis of tetracycline is a much needed boon for the many hundreds of thousands of victims suffering and dying each year from bacterial infections.

References