PENICILLIN: HISTORY AND

PROCESS TO MANUFACTURING WORLDWIDE

By Pamela Antkowiak, Rey Cipriano, and Jose L. Mendieta

PRESENTATION OUTLINE
Pre-World War II Alexander Fleming The Discovery Mould Product/ Identification Penicillin- The Name Fleming and Heatley During World War II First Official Document of Penicillin Structure Penicillin V: Retrosynthesis and Total Synthesis Conclusion and Impact on World

PRE- WORLD WAR II

Not much available:

Sulphonamides- antibiotic discovered by Dubos

Toxic and gave patients horrible reactions.

Majority of households used home remedies, bitter tonics, and any vitamins available.

Bacterial diseases were the cause of many fatalities. Diseases included:

Pneumonia Rheumatic fever Syphilis

Polio Staphylococcus aureus

Children were prime targets. Cleanliness and antiseptics was not well practiced as nurses and physicians would share equipment between patients

ALEXANDER FLEMING

Born in 1881 at Lochfield in Ayrshire, Scotland. 3rd child with 7 brothers and sisters Completed education at Regent Street Polytechnic, London in 1897 Studied medicine at St. Mary Hospital and was a member of British Royal Army Medical Corps Researched at the University of Oxford Died in 1955 due to a heart attack5

Fleming working in his laboratory in 1943.

DISCOVERY

University of Oxford- 1928

Fleming studied the effects of a contaminated culture plate filled with staphylococcal colonies. Was cleaning dirty culture plates when made the discovery and came across one with a peculiar mould growing on it.

MOULD PRODUCT

The mould became translucent and was in the process of lysis.

Fleming isolated the mould in pure culture to determine its properties. Figure 1 depicts Flemings findings in the culture plate.3

Figure 1: Process of Lysis

IDENTIFYING THE MOULD

Once isolating the mould, Fleming needed to identify the mould properly.
Fleming sent a sample of his mould to Harold Raistrick, the leading biochemist at the London School of Hygiene and Tropical Medicine. Raistrick was able to grow it reliably, but unable to classify the mould. Raistrick then sent the sample to his American friend, Charles Thom.

Thom was able to classify the mould as Penicillium notatum, and confirmed that this mould was actually first identified in 1911 by a Danish biologist named P. Westling.2

PENICILLIN

After identifying the mould, Fleming would submit the mould to the same technique he had created for the lysozymes to prove its capabilities to cure bacterial infections, and published his findings in the British Journal of Experimental Pathology in 1929.
Flemimg could not narrow down the specific chemical of the mould, so he called the mould penicillin as a whole.3

Penicillium notatum: penicillin

FLEMING AND HEATLEY

Fleming would then work with Howard Florey, Ernst Chain, and Norman Heatley to help synthesize the penicillin completely and to find a better form to create the mould in purer quantities.
Heatley created the apparatus solely for this purpose.
Separation of penicillin from juice exuded by mould Less well known than bedpan fermenters

Fleming and Heatley will leave Chain behind when continuing on the research.

Replica built by Heatley in 1986

DURING WORLD WAR II

Despite World War II happening, Fleming was able to transfer the experimentation with penicillin to the Northern Regional Research Laboratory in Peoria, Illinois with funding from the Rockefeller Foundation of New York. From here, penicillin would become mass produced in large facilities and soon be spread across the globe.
It was this initial act that started a rivalry between the British and the United States. No one was able to patent the vital antibiotic due to the enormous amount of people involved to create it. There was simply not one particular person or organization that created the penicillin.2

FIRST TESTED ON FRONT LINES

After first being tested on the front line in North Africa during World War II, penicillin would soon be distributed around the world to various places.
By June 1946, doctors were allowed to give penicillin to patients through painful injections.

TIME MAGAZINE

Fleming made the cover of Time Magazine in May of 1944.

Showed that Fleming was now an American celebrity. This greatly irritated the Oxford team.

FIRST OFFICIAL DOCUMENT OF PENICILLIN STRUCTURE

The structure of penicillin itself was not conceived until 1945. It was Professor Dorothy Crowfoot-Hodgkin of Oxford University that narrowed down the proper structure of penicillin by X-ray crystallography. The document to present the structure is in Figure 2.1 The total synthesis of penicillin did not come until 1957 by the work of Professor J. Sheehan and his group at MIT.4

Figure 2: Structure of Penicillin

HOW PENICILLIN WORKS

Structurally, penicillins are -lactam antibiotics. Bacterial cell walls are consisting of a protective peptidoglycan layer, which is continuously undergoing remodeling. The remodelling process involves the breaking of the -(1,4) linked N-acetylmuramic acid and N-acetylglucosamine; as well as the breaking of the cross-linking peptide chains. This cross-linking peptide chains is what provides the rigidity, to the otherwise fluid cell wall. The breaking of this peptide cross-linking is performed by an enzyme called transpeptidase.

The transpeptidase also helps in reforming the peptide bonds once the restructuring of the cell wall is done. The penicillins act by inhibiting this particular enzyme. By inhibiting this enzyme the penicillin prevents the reformation of the peptide bonds and thus makes the cell wall less strong.
This loss of cell wall integrity causes the bacteria to leak out its cellular contents and perish.

DIAGRAM OF PENICILLIN PROCESS

RETROSYNTHESIS PENICILLIN V

OF

Penicilloic acid (5) by direct cyclization or lactamization reaction.

At first, Sheehan came up with a few strategies that did not involve lactamization to create the -lactum ring.

A new mild method had to be formed in order to make the direct reaction between penicillin V and penicilloic acid.

Penicilloic acid is derived from the protective intermediate labeled 6.

Then, it is dissembled to create D-penicillamine hydrochloride and tertbutyl phthalimidomalonaldehydate.

Both are potential building blocks for total synthesis.4

TOTAL SYNTHESIS: 1) SYNTHESIS OF D-PENICILLAMINE

HYDROCHLORIDE

D-penicillamine hydrochloride and tert-butyl phthalimidomalonaldehydate are two intermediates that must be synthesized before continuing on with the rest of the synthesis as a whole.

The thiol and amino groupings in D-penicillamine hydrochloride can be induced to converge the electrophilic aldehyde in tert-butyl phthalimido-malonaldehydate.
This

can be done in order to create a thiazolidine 6 once the water molecule is removed. First, D-penicillamine hydrochloride (7- Scheme 2) needs to be synthesized.
Both

enantiomers that form can be created in their pure form. The reaction of an N-acylation using chloroacetyl chloride is applied to racemic valine to create 10.
The

acetic anhydride on 10 becomes a transient intermediate to produce a mixed anhydride that converges to oxazolone (11). This creates an isomerization process.

SYNTHESIS OF D-PENICILLAMINE HYDROCHLORIDE: CONTINUED

Next, 11 is a Michael acceptor that goes through conversion to create intermediate 12.

The

conversion is done by treating 11 with hydrogen sulfide and sodium methoxide. The thiol reacts chemselectively with the electrophilic carbon of 11.
The

heterocyclic ring is cleaved by the methoxide ion.

Then,

the hydrolytic cleavage of both N-acetyl and methoxycarbonyl groupings is done with the addition of aqueous hydrochloric acid.
The

contigious thiol and amino functions simultaneously protect by forming a thiazolidine ring with acetone.
From

here, add isopropylidene-DLpenicillamine (13) with formic acid and acetic anhydride to create Nformyl isopropylidene-DLpenicillamine (14).
The

racemic 14 is readily available to react with the brucine in water creates a diastereomeric mixture of salts.

SYNTHESIS OF D-PENICILLAMINE HYDROCHLORIDE: CONTINUED

The brucine salt of N-formyl isopropylidene-DLpenicillamine is crystallized from mixture and collected by filtration.

By adding an aqueous solution and concentrated hydrochloric acid to the burine salt, N-formyl isopropylideneD-penicillamine (15) is produced.

Add hot 2N hydrochloric acid to N-formyl isopropylidene-Dpenicillamine to create enantiomerically pure Dpenicillamine hydrochloride (7).

The physical properties are distinguished by comparing to derivation from natural penicillin.

2) SYNTHESIS OF TERT-BUTYL PHTHALIMIDO-MALONALDEHYDATE

This is done in one step from tert-butyl phthalimidoacetate (16).

Add t-butyl phthalimidoacetate to sodium tertbutoxide to create an enolate.

The enolate goes through conversion when added to tert-butyl formate.

The phthalimido and tertbutyl ester can be easily removed once in anhydrous conditions later in the synthesis.4

Combine

SYNTHESIS CONTINUED INTERMEDIATES:

WITH BOTH

both intermediates in an aqueous ethanol with sodium acetate.

The

D-PENICILLAMINE HYDROCHLORIDE AND TERTBUTYL PHTHALIMIDO-MALONALDEHYDATE

adjacent amino and thiol functions of D-penicillamine hydrochloride converge the electrophilic aldehyde carbonyl of tert-butyl phthalimidomalonaldehydate.
A

water molecule leaves and a mixture of diastereomeric thiazolidines (epimeric at C-6) is created.
Basically,

the racemic aldehyde of tert-butyl phthalimidomalonaldehydate joins the enantiomerically pure Dpenicillamine hydrochloride to produce a molecule with three stereocenters.
They

can theoretically form four distinct thiazolidine stereoisomers.

Actually,

only two diastereomers are formed (epimeric at C-6).

Desired

thiazolidine stereoisomer is D--6 and the undesired isomer is D--17.

The

undesired isomer creates equilibrium between the D- isomer and the D- isomer.
The

D--6 crystallizes when cooled, and the filtrate with D--17 can be recycled.

SYNTHESIS CONTINUED
INTERMEDIATES

WITH BOTH
D--6 converts to form amenable needed in lactamization reaction.

The addition of hydrazine removes the phthalimide protecting group to create D--18 once combined with diluted aqueous hydrochloric acid.

The acid-labile tert-butyl ester function withstands the latter step when solution acidified.

The addition of phenoxyacetal chloride and triethylamine to D--18 creates the phenoxyacetyl side chain of penicillin V in -tert-butyl Dphenoxymethylpenicilloate (19).

SYNTHESIS CONTINUED
INTERMEDIATES

WITH BOTH
By adding to anhydrous hydrochloric acid and recrystallizing the resultant carboxylic acid ammonia salt from aqueous acetone with one equivalent of pyridine, the key lactamization substrate 5 is created.

Add four equivalents of N,Ndicyclohexylcarbodiimide (DCC) to this substrate with dioxane water at 25oC to finally produce penicillin V potassium salt.

This yields about 10-12% of penicillin V potassium salt.4

CONCLUSION: THE WONDER DRUG

It was the reaction received from the patients and physicians about how effective penicillin was to bacterial infections that coined the drug the Wonder Drug. Physicians were free to use more and more quantities on ill patients as the production of penicillin would increase dramatically.

Cartoon shown in early 1950s to doctors at the Institute of Motivational Research.

CONSEQUENCES DUE TO POPULARITY

It was so popular that the U.S. government included penicillin on the restricted list of drugs that must only be prescribed by physicians.
The government was concerned that patients were self-diagnosing and not using the correct amount of penicillin needed. If the patient did not use enough penicillin, then the bacterial would become resistant and fail to work.

GROWTH OF PENICILLIN FAMILY

With some patients forming resistance or having allergic reactions to penicillin, companies with their own scientists would research for more forms of antibiotics among the penicillin family.
Some examples are ampicillin, amoxicillin, methicillin, carbenicillin, and ticarcillin. From here on out, all kinds of antibiotics would surface and start a new way to live longer.

RECOGNITION

As a tribute to the founders of penicillin, Fleming, Florey, and Chain shared the Nobel Prize on December 11, 1945 for their work in medicine and physiology.4

Nature makes penicillin; I just found it.

In addition, Flemings discovery was named the 6th most important story of the century and had the highest rated event not to have directly involved an American by the Newseum in Washington D.C. in 1999. Finally, a British poll done in 2002 announced that Fleming was the only twentieth century scientist to reach the top twenty Britons in the last thousand years.

CURRENT MANUFACTURERS

Penicillin V: West Ward (U.S.)

Penicillin VK:
Mylan Pharmaceuticals (U.S.) Teva Pharmaceuticals (U.S.)

Penicillin V Potassium:

Consolidated Midland (U.S.) Geneva Generics (U.S.) Mylan Pharmaceuticals (U.S.) Par Pharmaceuticals (U.S.) Solvay Pharmaceuticals (U.S.) Stada Pharmaceuticals (U.S.) Teva Pharmaceuticals (U.S.)

PRESS RELEASES- 2011

March 14 2011- Penicillin Susceptibility

Major heat shock protein ClpL is found to alter the cell wall biosynthetic enzymes and decrease penicillins susceptibility to resistance.

March 16, 2011- Scientist Robert Bud

Author of Penicillin: Triumph and Tragedy Speaks out at the Science Museum on how history and impact of penicillin

June 2011- Alternative to Antibiotics

Antimicrobial peptides (20 short chains) penetrate bacterial membranes. Anticipated to replace penicillin due to increasing resistance.

September 21, 2011- Penicillin is #7

Penicillin named in top ten most influential inventions of all time by Tim King.

November 18, 2011- Pre-Pen Test

Tests patients to see if they have outgrown their penicillin allergy.

WHAT WE DID
Pre-World War II Alexander Fleming The Discovery Mould Product/ Identification Penicillin- The Name Fleming and Heatley During World War II First Official Document of Penicillin Structure Penicillin V: Retrosynthesis and Total Synthesis Conclusion and Impact on World

REFERENCES
1. Bentley, Ronald. The Molecular Structure of Penicillin. Journal of Chemical Education: October 2004. Volume 81, 6 September 2011. <http://pubs.acs.org/doi/abs/10.1021/ed081p1462>. 2. Bud, Robert. Penicillin: Triumph and Tragedy. Oxford University Press Incorporated, New York: 2007. 26 September 2011.

3. Fleming, Alexander. Penicillin. 11 December 1945. 6 September 2011. <http:/www.nobleprize.org/nobel_prizes/medicine/laureates/1945/fleming-lecture.pdf>.

4. Fraunbofer-Gesellschaft. An alternative to Antibiotics. Research News: June 2011. 1 October 2011. <http://www.fraunhofer.de/en/press/research-news/2010-2011/18/alternative-toantibiotics.jsp>. 5. King, Tim. The 10 Most Influential Inventions of All Time. 21 September 2011. 1 October 2011. <http://www.listshere.com/the-10-most-influential-inventions-of-all-time>. 6. Li, Hoe-Shuen. Alexander Fleming. San Diego State University. 16 November 2011. <http://www.sjsu.edu/depts/Museum/flemin.htm>.

REFERENCES
7. Male, David. Professor Robert Bud. Open University:16 March 2011. 1 October 2011. <http://www.guardian.co.uk/science/video/2011/mar/16/miracle-tragedy-penicillinresistance>. 8. Mehta, Akul. Mechanism of Action of Penicillin. Pharmaxchange: 28 April 2011. 1 October 2011. <http://pharmaxchange.info/press/2011/04/mechanism-of-action-of-penicillin/>. 9. Penicillin. 27 November 2011. <http://www.antimicrobe.org/drugpopup/Penicillin%20-%20Brand%20names.htm>. 10. Sheehan, J.C. Penicillin V. VCH Publishers: 1957. 26 September 2011.

11. Simple Test Can Check for Outgrown Penicillin Allergies. KNTV/NBC. Las Vegas: 18 November 2011. 21 November 2011. <http://www.wsfa.com/story/16078473/simple-test-can-check-for-outgrown-pencillinallergies>. 12. Thao Dang-Hien Tran, et al. Decrease in Penicillin Susceptibility Due to Heat Shock Protein ClpL in Streptococcus Pneumoniae. American Society for Microbiology: 14 March 2011. 1 October 2011. http://aac.asm.org/content/55/6/2714.abstract>.