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 Polio

Rheumatic fever Staphylococcus aureus

Syphilis

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 Fleming’s 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 β-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 β-(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 OF

PENICILLIN V

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 β-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 tert-butyl phthalimido-malonaldehydate.

Both are potential building blocks for total synthesis.4

TOTAL SYNTHESIS:

1) SYNTHESIS OF D-PENICILLAMINE

HYDROCHLORIDE

D-penicillamine hydrochloride and tert-butyl phthalimido-malonaldehydate 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 β 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-DL-

penicillamine (13) with formic acid

and acetic anhydride to create N-

formyl isopropylidene-DL-

penicillamine (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-DL-penicillamine is crystallized from mixture and collected by filtration.

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

Add hot 2N hydrochloric acid to N-formyl isopropylidene-D-penicillamine to create enantiomerically pure D-penicillamine 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 phthalimido-

acetate to sodium tert-

butoxide to create an

enolate.

The enolate goes through

conversion when added to

tert-butyl formate.

The phthalimido and tert-

butyl ester can be easily

removed once in

anhydrous conditions later

in the synthesis.4

SYNTHESIS CONTINUED WITH BOTH

INTERMEDIATES:

D-PENICILLAMINE HYDROCHLORIDE AND TERT-

BUTYL PHTHALIMIDO-MALONALDEHYDATE

Combine both intermediates in an aqueous ethanol with sodium acetate.

The adjacent amino and thiol functions of D-penicillamine hydrochloride converge the electrophilic aldehyde carbonyl of tert-butyl phthalimido-malonaldehydate.

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

Basically, the racemic aldehyde of tert-butyl phthalimido-malonaldehydate joins the enantiomerically pure D-penicillamine 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-α-6 and the undesired isomer is D-γ-17.

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

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

SYNTHESIS CONTINUED WITH BOTH

INTERMEDIATES

D-α-6 converts to form amenable

needed in lactamization reaction.

The addition of hydrazine removes

the phthalimide protecting group to

create D-α-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-α-18

creates the phenoxyacetyl side chain

of penicillin V in α-tert-butyl D-

phenoxymethylpenicilloate (19).

SYNTHESIS CONTINUED WITH BOTH

INTERMEDIATES

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,N’-

dicyclohexylcarbodiimide (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 1950’s 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, Fleming’s 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 penicillin’s 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-to-antibiotics.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-penicillin-resistance>.

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-pencillin-allergies>.

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>.