In the Classroom
The Molecular Structure of Penicillin Ronald Bentley Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260;
[email protected] The structure of penicillin, the prototypical member of the β-lactam group of antibiotics, was determined under unusual circumstances. By 1940, the potential of penicillin in chemotherapy was apparent. However, at that time it was in short supply owing to problems with the fermentation process. To overcome this difficulty, an obvious possibility was to determine the chemical structure with a view to achieving chemical synthesis. A little experimental work to this end was published in the usual way in the period 1941–1943. However, under wartime conditions, the U.S. and U.K. governments decided that penicillin was of great military value and secrecy was invoked on chemical work with penicillin. A major collaborative effort between scientists in both countries was established with 39 laboratories, both academic and industrial, being involved. Some 800 secret reports of experimental results and speculations (some brief, some long) were circulated among the collaborating groups. By about the end of 1945, the chemical structure was known. In December of that year, two identical, anonymous notes briefly describing the experimental work were published (1, 2). However, a conclusion as to structure was ambiguous, with the final sentence reading as follows: “At present it can be stated that the formulae which are receiving the most active attention contain respectively a β-lactam structure and an incipient azlactone grouping.” At the beginning of 1946, participants in the project were freed from the secrecy requirement, but a decision was taken against the usual form of publication in the open scientific literature. Rather, a monograph, The Chemistry of Penicillin (3), was to incorporate the evidence and conclusions. Participants were expected to refrain from any individual publication until the monograph was published. Owing to the enormous volume of material, this monograph of more than one thousand pages was delayed until 1949. It was edited by three distinguished chemists— H. T. Clarke, J. R. Johnson, and R. Robinson—with 63 other individuals contributing. In a lengthy Appendix (25 pages), information as to the date of issue of the various reports and of their receipt in the United States by the Office of Scientific Research and Development (OSRD) and in the United Kingdom by the Medical Research Council (MRC) was tabulated. While monumental, The Chemistry of Penicillin is a seriously flawed volume and presents difficulties to a reader. To understand one major problem, further background material must be considered. One of the major investigators, and as indicated an editor of the monograph, was Robert Robinson. Robinson, at the University of Oxford, was the most highly regarded organic chemist in the United Kingdom; he received the Nobel Prize in Chemistry in 1947. He and his colleagues (E. P. Abraham, E. Chain, W. Baker) had collaborated with Florey’s group from the earliest days. In a famous report, PEN 103, dated October 22, 1943, they had proposed the “thiazolidine–oxazolone” structure (4). During the preparation of this report, and in Robinson’s absence, his 1462
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colleagues had detected a possible flaw with the structure and as an alternative proposed the β-lactam structure (not so named at that time). To Robinson’s dismay and without his knowledge, the β-lactam possibility was added to PEN 103. In an addendum dated October 23, 1943, it was stated that “one of us considers the four-ring structure somewhat improbable”, the dissenter, of course, being Robinson (Figure 1). The β-lactam structure was also suggested by investigators at Merck in a report dated November 1943 (5). However, they remarked that “a four-membered lactam fused upon a five-membered heterocyclic ring appears unlikely unless the strain involved could account for the reactivity to methanol and benzylamine.”
Figure 1. The last page (p 3) of the report PEN 103. This historic document presents the first representation of the β-lactam structure for penicillin and indicates Robinson’s objection to it. The copy was obtained from the National Archives by the author (Nov 1989). Although marked “SECRET” the first page of the report indicated that the classification had been changed to “OPEN”. It was declassified per Memo, Acting Secretary of Defense, dated Aug 2, 1960. The report was part of Record Group (RG) 227, Records of the Office of Scientific Research and Development. Location: Stack Area 17W2, Row 17, Compartment 7, Box 69, Entry 165, Shelf 2. Box 69 contained Records of the Committee on Medical Research, General Records, 1940–1946.1
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The seemingly trivial incident at Oxford had historic consequences; Robinson never completely accepted the accuracy of the β-lactam structure. Even after the successful synthesis of a penicillin many years later by Sheehan and Henery-Logan (6), Robinson informed Sheehan, “...that compound cannot be a penicillin. It is a β-lactam” (7). Robinson’s attitude also influenced The Chemistry of Penicillin. Chapter I of this monograph, “Brief History of the Chemical Study of Penicillin”, written by the three editors had already been published essentially unchanged in 1947 (8) in view of the delays. It is a brief overall summary and did state that the βlactam structure was “fully confirmed” through X-ray crystallography. However, Chapter XV, The Constitution of the Penicillins, presents a different picture. Rather than being a reasoned, integrated consensus, this chapter contains three separate sections by three different contributors. They are as follows: “Introduction” by J. R. Johnson; “Theoretical Aspects of the Reactions of Penicillins” by R. B. Woodward and “The Interpretation of the Reactions of Penicillin and Remarks on the Constitution of Penicillin” by R. Robinson. Of the first two authors, Johnson refers to the “widespread acceptance of the β-lactam formulation” and Woodward refers to it as the “now accepted formula”. On the other hand, Robinson suggests that “the almost universal acceptance of the plain β-lactam structure is perhaps too complaisant.” In his final summary Robinson states that “the β-lactam is not a conceivably correct expression…but it could be modified…so as to accommodate some of the requirements.” Hence, there is no general agreement between these three experts and this chapter alone must be a problem for the uninitiated reader. As well as being repetitious (with each author emphasizing the importance of his or her own contributions) and somewhat disorganized, The Chemistry of Penicillin uses a variety of structural formulas and pays little attention to stereochemistry. A present day student would not find it easy to uncover a simple structural proof for penicillin. It is also striking that whereas the Merck Index (12th edition) provides literature references documenting “structure” for antibiotics such as erythromycin, rifamycin, and streptomycin, no such entry is present for penicillin. Objective This article presents an overview of the observations that constitute a structure proof for penicillin, specifically aimed at the general student population. This is done primarily without reference to individual contributors and without dates, so that priorities are usually not implied. The basic data can be unearthed, with considerable effort, by interested readers from The Chemistry of Penicillin. Many reactions and much information are not included here.
nally termed penicillin F (F for Florey) or penicillin I and is now known as 2-pentenylpenicillin. In the United States, material obtained by submerged fermentation of Penicillium chrysogenum strains, with supplementation by promoters such as corn steep liquor, was termed penicillin G (next letter in alphabet after F) or penicillin II; it is now known as benzylpenicillin. Hence the same degradation could lead to products of different composition, depending on the source of the material. A second problem was that early experiments indicated that the penicillin molecule did not contain sulfur; this conclusion was revised in July 1943. In the United Kingdom, only small quantities of material could be spared for chemical investigations and some work was carried out with preparations that perhaps contained only 50% of actual penicillin. Chain has lamented that the Oxford work was done with about 2 g of material; of this, only about 500 mg was 90% pure, the remainder being about 50% pure (9). In the United States, as production by large-scale fermentation improved, larger quantities could be used. Chain estimates that the Merck group alone “used up many hundred grams of pure crystalline penicillin” (9). A crystalline preparation of the sodium salt of benzylpenicillin was first obtained in July 1943, and a little later the sodium salt of 2-pentenylpenicillin was crystallized at Oxford.
The Evidence 1. The empirical formulas: The sodium salt of 2pentenylpenicillin was found to be C14H19N2O4SNa and for benzylpenicillin, C16H17N2O4SNa. 2. The thiazolidine moiety: Acid hydrolysis of penicillin gave an amino acid, penicillamine, C5H11NO2S, that was identified as ββ-dimethylcysteine2 (3-thiolvaline, 1) (Scheme I). The structure was confirmed by synthesis. The material was shown to have the D configuration, belonging to the “unnatural” series of amino acids. The same penicillamine was obtained from either 2-pentenylpenicillin or benzylpenicillin. Penicillamine reacted with acetone to form “isopropylidene penicillamine”, 2,2,5,5-tetramethyl-4thiazolidinecarboxylic acid, 2 (Scheme I). The reaction was comparable to that known to occur between cysteine and formaldehyde (forming 4-thiazolidinecarboxylic acid). The possibility that penicillin contained a thiazolidine nucleus seemed likely. 3. Position of the double bond: Under various hydrolytic conditions, penicillin gave CO 2 and an aldehyde named penilloaldehyde, in addition to penicillamine. The penilloaldehyde from 2-pentenylpenicillin was easily shown
H2O
HS
The Proof
O +
Difficulties A few difficulties may be noted at the outset. It became apparent, largely on the basis of column chromatography, that there was more than one penicillin. In the United Kingdom, much of the material was obtained by surface culture of the original Fleming strain of Penicillium notatum. It was origiwww.JCE.DivCHED.org
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H2N
S HN
COOH
1
COOH
2
Scheme I. Reaction of 1, a hydrolysis product of penicillin, with acetone to form 2, which helped confirm the presence of the thiazolidine moiety.
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In the Classroom to be hexenoylaminoacetaldehyde, C5H9–CO–NH–CH2– CHO; the corresponding product from benzylpenicillin was phenylacetylaminoacetaldehyde, C6H5–CH2–CO–NH– CH2–CHO. In addition, 2-pentenylpenicillin could be hydrogenated to a biologically active dihydro form and from this material a further penilloaldehyde, C5H11–CO–NH– CH2–CHO, was obtained. The double-bond position in hexenoylaminoacetaldehyde, 3, was determined by conversion to the corresponding acid, C 5H9–CO–NH–CH2– COOH, 4; on further oxidation of 4 with dilute KMnO4, propionaldehyde, 5, was obtained (Scheme II). Hence, the double bond was at the 2 position of the pentenyl group. 4. Different R groups: The degradation products so far described accounted for all of the carbon atoms, the two nitrogen atoms, and the sulfur atom of penicillin. C10H11NO2 + C5H11NO2S + CO2 → 2 H2O + C16H18N2O4S C8H13NO2 + C5H11NO2S + CO2 → 2 H2O + C14H20N2O4S penilloaldehyde penicillamine
penicillin
Table 1. Names and Empirical Formulas of Acids Derived from Penicillins as Degradation or Rearrangement Products
2-Pentenyl Series
Benzyl Series
Penicillin
07
C14H20N2O4S
C16H18N2O4S
Penillic acid
11
C14H20N2O4S
C16H18N2O4S
Isopenillic acid
30
C14H20N2O4S
C16H18N2O4S
Penicillenic acid
32
C14H20N2O4S
C16H18N2O4S
Penillonic acid
34
C14H20N2O4S
C16H18N2O4S
Penicilloic acid
23
C14H22N2O5S
C16H20N2O5S
Penilloic acid
24
C13H22N2O3S
C16H20N2O3S
Penaldic acid
a
C9H13NO4
C11H11NO4
26
a
Compound 26 is actually the methyl ester of penaldic acid.
The penilloaldehydes, R–CO–NH–CH2–CHO, indicated that the different penicillins contained a variable R group. Hence penicillin F became known as 2-pentenylpenicillin,3 dihydropenicillin F became pentylpenicillin (originally, amylpenicillin) and penicillin G became benzylpenicillin. 5. Nomenclature: Penillic acid was the first of several rearrangement products of penicillin to be isolated. The nomenclature of these materials is confusing since their names and those of some other degradation products are similar ( Table 1). Penillic, isopenillic, penicillenic,4 and penillonic acids have the same empirical formula as does penicillin. Since the penicillins are acids, salts and esters were referred to as penicillinates; thus a sodium salt could be named as sodium 2-pentenylpenicillinate and a methyl ester as methyl benzylpenicillinate. Note also that the natural product, penicillic acid, is not related to the penicillin series. 6. Possible structures: Rather than trying to follow the very tangled trail of evidence in a sequential fashion, it will be simplest to indicate possibilities with evidence for or against them. As information was developed, possible structures
Formula
Structure in Text
Name
began to be formulated towards the end of 1943. The thiazolidine–oxazolone, 6, and β-lactam possibilities, 7, were mentioned earlier; a third was termed the tricyclic formula, 8 (Figure 2). Each structure had its champions and opponents and discussion was prolonged and vigorous. An embarrassing situation was that there were almost too many degradation products to be accounted for easily. Many proposals were rejected and it is usually considered that only the three mentioned here survived the initial screenings. 7. The thiazolidine–oxazolone arrangement, 6: While the formation of several degradation products could be accounted for with this structure, there was a major difficulty. Titration data had indicated that penicillin had a pK of about 2.7 corresponding to the ionization of the single carboxyl group; no basic function was detected in the usual solvents. (Titration required care to ensure that degradation products were not formed.) Since the thiazolidine ring in 6
O
CH3–CH2–CH=CH–CH2–CO–NH–CH2–CHO
3
RCONH
S
S
O
N N
R
N H
O
COOH
COOH
6 CH3–CH2–CH=CH–CH2–CO–NH–CH2–COOH
4
O
7 S
S
NH O
N
N O COOH
R
CH3–CH2–CHO
( + other products )
8
5 Scheme II. Reactions used to determine the position of the double bond in 2-pentenylpenicillin.
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COOH
9
Figure 2. Possible structures of penicillin. In this and subsequent schemes and figures the variable side chain, R, is either C5H9 or C6H5CH2
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In the Classroom clearly contains a basic NH group, this one piece of evidence is sufficient to exclude this structure. Some attempts to rationalize the lack of basicity for the thiazolidine NH group were unconvincing. Moreover, IR absorption spectra gave no evidence for the presence of an oxazolone ring. One degradation product, however, did contain an oxazolone ring, penicillenic acid (structure 32 in Scheme XI). It is formed by a complex rearrangement that is discussed in detail in connection with the β-lactam structure. Penicillenic acid cannot be considered to support the occurrence of an oxazolone ring in penicillin. 8. The tricyclic formula, 8: It was very difficult to predict the basicity of the NH group in this structure and the lack of basicity in penicillin could not be used as an argument either for or against 8. One difficulty was that the possible stable existence of a molecule containing a carbon atom linked to three electronegative atoms (two nitrogens, one oxygen) was questionable. On the plus side, this structure readily accounts for the formation of penillic acid, 11 (Scheme III). However, the very simplicity of the process precludes the introduction of 2H, bound in a stable position, into penillic acid as was observed when the rearrangement was performed in 2H2O. This difficulty weighed strongly against 8. Moreover, the IR spectrum of penicillin shows three bands in the double-bond region; 8 contains only two double bonds, not counting those in the benzene ring (using benzylpenicillin). The Chemistry of Penicillin contains relatively little discussion of the tricyclic possibility. However, a very detailed exposition was given by R. B. Woodward (11). 9. The β-lactam structure, 7: Persuasive chemical evidence for this structure is the desulfurization of benzylpenicillin by Raney nickel to form a desthiobenzylpenicillin, 12, and as well, phenylacetyl-L-alanyl-D-valine, 13 (Scheme IV). The structure of the latter dipeptide derivative strongly suggested that the desthio material was actually a β-lactam structure, especially since the dipeptide was apparently formed directly from benzylpenicillin and by reactions competitive with the formation of desthiobenzylpenicillin. That desthiobenzylpenicillin was actually a β-lactam was confirmed by
the IR spectrum (sodium salt) that showed three bands in the double-bond region (1720, 1670, 1585 cm1) corresponding to the carbonyls of the β-lactam, the side-chain amide, and the carboxylate ion. To form the dipeptide, the bonds, A and B of benzylpenicillin had to be cleaved (Scheme IV). It seemed likely that the CN bond, A, was cleaved first with a second process, B, removing the sulfur. For formation of desthiobenzylpenicillin, only hydrogenolysis of the two CS bonds at B was needed. While 7 essentially accounts for all of the properties of penicillin, including formation of rearrangement products (see later), there was one argument used against it. Amides are usually rather stable owing to the likely formation of resonance structures: NCO ↔ N+CO−. However, penicillin was a relatively unstable compound, readily inactivated with loss of antibiotic activity. In striking contrast, the undoubted β-lactam compound, desthiobenzylpenicillin, 12, exhibited the expected stability. For the usual amide resonance situation, the structures for penicillin as a β-lactam are 7 and 14 (Figure 3). However, of the two resonance structures, 14 is relatively improbable for two reasons. One is Bredt’s rule prohibiting a double bond as the bridgehead of a small bicyclic system. The other is that consideration of models, and more definitively the X-ray evidence, indicates that the marked atoms, a, b, c, d, of 14 cannot be coplanar. Consequently, the resonance contribution of 14 is suppressed leading to the observed instability of penicillin. 10. The X-ray evidence: Work with X-rays was impeded, initially, by the difficulty of obtaining crystalline salts of penicillin, and also by the primitive state of computational facilities in 1945. Nevertheless, important data were ob-
RCONH
A B
N
RCONH
O
S
COOH
12 N O COOH O
S
H
ⴙ
O
NH O
NH O
N H
O
N
H COOH
R
8 O
Scheme IV. Desulfurization of benzylpenicillin, 7, by Raney nickel, R = C6H5CH2.
10 HOOC
S
S
ⴙ
NH
HO
N
RCONH
RCONH
S
N COOH
Hⴙ
9
COOH
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N
ⴚ
c
O
O COOH
11
Scheme III. Formation of penillic acid, 11, from the tricyclic compound 8, a possible structure of penicillin.
S ⴙ
N R
b
a
N R
COOH
13
COOH
R
CH3
7
S ⴙ
N
RCONH
7
d
COOH
14
Figure 3. Resonance structures for β-lactam compounds.
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In the Classroom tained. Although initial results (Feb 1, 1945) did not favor the β-lactam structure, further work with the sodium salt of benzylpenicillin provided a set of atomic coordinates that corresponded with the “curled-up” molecular arrangement of 7. While great credit is due to the principal investigators (Dorothy Crowfoot Hodgkin, Charles W. Bunn), their initial contribution was an ability to eliminate incorrect proposals, guided by the possibilities deduced from the chemical investigations and by the knowledge of linkages between certain atoms. Crowfoot et al. state that the course of their work was affected “by the nature of the chemical evidence on the structure of penicillin available at the time” (12). Finally, complete sets of atomic coordinates were obtained. By May 1945, Hodgkin regarded the structure problem as settled in favor of 7. When she informed Robinson he was incredulous and implied that the crystal structure had undergone change by exposure to the X-ray beam (7). For work on penicillin and other natural products, Hodgkin received the Nobel Prize in Chemistry in 1964 as was recently noted in this Journal (13). In justice to the chemists who had toiled so long and so hard, it should be noted that R. B. Woodward (Nobel Prize in Chemistry, 1965) had independently concluded from a careful analysis of the available facts, that penicillin could only have the β-lactam structure. His report (11) was received by OSRD on January 18, 1945, and by the MRC on February 7, that is, before Hodgkin’s conclusion. He stated: “Among the many other experimental facts which have emerged during the course of the work on penicillin, we know of none which is definitely incompatible with (XL)”. Structure XL in his report was the β-lactam, 7.
Thus, by the end of 1945, the problem was solved; however, Robinson continued to argue for modifications as he does in The Chemistry of Penicillin. Towards the end of 1945, his Oxford colleagues who had originally proposed the βlactam structure stated unequivocally that Robinson’s thiazolidine–oxazolone structure was incorrect and that they considered his attempts to modify the β-lactam to be improbable (14).
H NHR SR
HO
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HO
D,S
D,R
O
O
R = C6H5NHCO
14
15
Scheme V. Penicillamine as a diphenylisocyanate derivative, 14, hydrogenolysis reaction with Raney nickel to produce 15.
O
H
C6H5
N H
H N
H D,R
L,S
H COOH
O
16 Figure 4. Dipeptide, 16, obtained from desulfurization of benzylpenicillin. H
H H RCON
S R
L,R
N O
D,S
HOOC H
17 Figure 5. Configuration of the second chiral center in the β-lactam ring, 17.
COOH
CHO
H
C
OH
HO
C
H
C
H
COOH
OH
H
CH2OH
COOH ()
C
NH2
CH2OH
()
( , H2O; , HCl )
19
20
18 COOH
COOH
H
C
NH2
H
C
NH2
H3C
C
CH3
H3C
C
CH3
Additional Considerations
Stereochemistry The antibiotic activity of penicillin is critically dependent on the correct configurations at the three chiral centers. Penicillamine was found to belong to the “unnatural” D amino acid series. One important observation was that the (di)phenylisocyanate derivative of “natural” penicillamine, 14, R = C6H5NHCO, on hydrogenolysis with Raney nickel gave the phenylureide derivative of D-valine, 15, R = C6H5NHCO (Scheme V). Further, the dipeptide obtained from desulfurization of benzylpenicillin was phenylacetyl-L-alanyl-D-valine, 16 (Figure 4). The D-valine component of the dipeptide was derived from the thiazolidine ring, and thus was in agreement with the D assignment of penicillamine, 14, R = H. The L configuration of the alanyl unit provided the configuration for the carbon atom of the β-lactam ring linked to sulfur. From the relative stereochemistry from X-ray diffraction, the configuration of the second chiral center in the βlactam ring, 17, was available (Figure 5).
H NHR H
H
SH
( , H2O; , HCl )
( , H2O; , NaOH )
21
22
Figure 6. Fisher projections of various compounds.
However, at the time of the penicillin work, the D configurations of amino acids were only arbitrary arrangements represented as Fischer projection formulas (Figure 6). Absolute configurations did not become known for any compound until the classic work of Bijvoet et al. (15) on L-(+)-tartaric acid, 18, in 1951 using the anomalous dispersion of X-rays. Since the usual configurational standard, D-(+)-glyceraldehyde, 19, had already been correlated with D-(+)-serine, 20, and L-(+)-tartaric acid, it followed that Fischer’s initial assumptions with respect to L and D amino acids were, in fact,
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correct. Hence, D-valine and D-penicillamine had the projection formulas, 21 and 22, respectively (Figure 6). For the protein based L amino acids, the configuration at the α carbon atom is usually S. Because of the CIP sequence rule, Lcysteine is an exception with R configuration. The same sequence rule situation applies to D-penicillamine, where the α carbon atom has S configuration. Using correct absolute configurations, the stereochemistry of the penicillin β-lactam molecule is that shown as 17 (Figure 5). Note that the chiral center of the thiazolidine ring, although correlating with D-(R )-valine becomes D-(S ) on account of the CIP sequence rule. The Chemistry of Penicillin presents a ball-and-stick drawing of the structure of benzylpenicillin that actually shows the biologically inactive enantiomer (12). It is clearly stated, however, that the biologically active form could be the enantiomer of the depicted structure. Since Hodgkin was aware that penicillamine belonged to the D configurational series, it is strange that the drawing is not based on the usual assumptions of the time, later proved correct. A correct version in the original ball-and-stick style is given as Figure 7. The incorrect configuration in The Chemistry of Penicillin has misled a number of authors. Penicillin was a featured WebWare Molecule in the July 2003, issue of this Journal (16).
Rearrangement and Degradation Products of Penicillin While the facts and arguments just given constitute an adequate structure proof, any formula must account for the observed rearrangement and degradation products. In the preceding discussion, little attention was given to them, although the penillic acid rearrangement was noted in connection with the tricyclic formula. The formation of the more important products is discussed here in terms of the β-lactam structure. Penicilloic, Penilloic, and Penaldic Acids Alkaline inactivation of penicillin led to the dibasic, penicilloic acids. These materials further decomposed with loss of CO2 to form monobasic penilloic acids. In turn, penilloic acids were decomposed by HgCl2 to form penicillamine and penilloaldehydes. These reactions are represented in Scheme VI for benzylpenicillin. That penicilloic and penilloic acids were substituted thiazolidines, 23 and 24, respectively, was
quickly established and confirmed by synthesis. The conversion of penicillin, 7, to a penicilloic acid, 23, is a simple cleavage of the β-lactam link (Scheme VII). Decarboxylation of 23 yields the penilloic acid structure, 24. When penicillins were warmed with acid, they underwent decarboxylation yielding (as noted) penicillamine and penilloaldehydes. This suggested a “bound carboxyl” as a labile group, joined to the penilloaldehyde moiety. The structures, R–CO–NH– CH(COOH)–CHO, were termed penaldic acids; formally, they are acylaminomalonic semialdehydes. The free acids are very unstable. Evidence for such structures was that the dimethyl ester of benzylpenicilloic acid, 25, gave the malonic CO2
H2O C14H20N2O4S
C14H22N2O5S
C13H22N2O3S
penicillin
penicilloic acid
penilloic acid H2O
+ C5H11NO2S
C8H13NO2 penilloaldehyde
penicillamine
Scheme VI. Alkaline inactivation of penicillin.
RCONH
RCONH
S N
O
ⴚ
S N
ⴚ
O
OH
COOH
Hⴙ COOH
OH
7 RCONH
S
HOOC
N H
S
RCONCH2 COOH
N H
CO2
23
COOH
24
Scheme VII. Structures of the compounds involved in the alkaline inactivation of penicillin, R = C6H5CH2. C6H5CH2CONH dimethyl ester of 23 with R = C6H5CH2
CH3OOC
S N H
COOCH3
25 HgCl2 CH2OH
CHO C6H5CH2CON H
Figure 7. Ball-and-stick model for penicillin. This drawing duplicates the style used in The Chemistry of Penicillin but indicates the correct stereochemistry at the three chiral positions.
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C
H
C6H11CH2CON H
H
COOH
COOCH3
26
C
27
Scheme VIII. Formation of a penaldic acid as a methyl ester, 26.
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R
H Nⴙ H
R
NH
S
O
H
Oⴚ
N
N
O
O
N N
2
2
H2O, 2Hⴙ
2*
S
O
Hⴙ COOH 28
COOH
7
R
S
Hⴙ
O
7
H
H 2 * Nⴙ H S
R O
2
O
O
⬅
N
S
ⴙ
NH
O
N H
COOH
H
2
R
29
H
O
2
S
2*
ⴚ
N
S N
H
O
H
OOC
Hⴙ
NH O
Hⴙ
COOH
R
2*
COOH
S N H
O
N
COOH
29
N COOH
R
R
10
COOH
11
Scheme IX. Rearrangement of penicillin, 7, to penillic acid, 11. In 29, the carbon-bound hydrogen of the oxazolone ring is labile. It is in the alpha position to the carbonyl group so exchange by enolization is possible. The exchanged hydrogen is marked with an asterisk.
HS
N R
N H
O
COOH
O
32 HOOC
Scheme XI. Formation of penicillenic acid, 32, from penicillin, 7.
N
N
HOOC S
R
Ba(OH)2
SH COOH
30
N N R
HgCl2 COOH
11
CO2
N
N
SH COOH
R
31 Scheme X. Reactions of penillic acid, 11.
semialdehyde, 26, on treatment with HgCl2 (Scheme VIII). By reduction and saponification, N-cyclohexylacetyl-serine, 27, was obtained, confirming 26, as the methyl ester of a penaldic acid. Penillic and Isopenillic Acids The rearrangement, penicillin → penillic acid is of interest since the carbonyl group of the side chain is involved (Scheme IX). Note that essentially the same structure, 10, that was postulated earlier for the conversion of 8 to penillic acid is involved. As was indicated previously, the reactions suggested for the tricyclic situation were unlikely to lead to 1468
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incorporation of stable 2H if the rearrangement was carried out in 2H2O. However, in the pathway from the β-lactam there was another intermediate, 29, in which exchange of 2H was possible (The exchanged hydrogen is noted as H*). Penillic acid, 11, was converted to isopenillic acid, 30, by various methods; for example, action of barium hydroxide solution for 24 h (Scheme X). In another reaction, treatment of penillic acids with HgCl2 led to opening of the thiazolidine ring and decarboxylation. The products, 31, were termed penillamines. Penicillenic Acid Penicillenic acid, 32, is most simply considered as a rearrangement product formed from an intermediate, 29, that is essentially that postulated for the formation of penillic acid (Scheme XI). An alternative mechanistic possibility is shown in The Chemistry of Penicillin. Methyl Ester of Penillonic Acid When the methyl ester of benzylpenicillin, 33, R = C6H5CH2, was heated in solvents such as xylene or p-cymene at temperatures from 140 to 170 C, the methyl ester of penillonic acid, 34, R = C6H5CH2, was formed (Scheme XII). This is another rearrangement involving the intervention of the side chain.
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tivity were sometimes obtained in synthetic attempts to make a thiazolidine–oxazolone structure. By careful standardization of conditions, and most importantly by extensive use of counter-current distribution for purification, they obtained a very small quantity of synthetic penicillin (17). However, it was not until 1957 that rational syntheses of penicillin became possible. In that year, penicillin V (phenylpenicillin) was synthesized by Sheehan and Henery-Logan at MIT (6). However, fermentation methods became so efficient that industrial production by complete synthesis has never been undertaken.
S
RCO N O COOCH3
33
RCO
H
ⴙN
Acknowledgment
S
I am indebted to Marjorie H. Ciarlante, Civil Archives Branch, National Archives, for assistance in locating reports.
N
Hⴙ
ⴚ
O
COOCH3
Notes RCO S
N N O
COOCH3
34 Scheme XII. Formation of penillonic acid as a methyl ester, 34, from the methyl ester of penicillin, 33, R = C6H5CH2.
1. It was of interest that some documents had a very distinct odor of benzyl mercaptan even after 43 years of storage. This reagent had been much used in syntheses of penicillamine. 2. Penicillamine should not be confused with penillamine (C13H20N2O2S, or C15H18N2O2S), which is a product derived from penillic acid. 3. Historical note: A rearrangement product of 2pentenylpenicillin named penillic acid was isolated before secrecy was invoked (10). It had the same composition, C14H20N2O4S, as did the parent penicillin. 4. Penicillenic acid should not be confused with penicillanic acid, 9 (Figure 2). The latter name is now used for the stripped βlactam nucleus devoid of the characteristic R–CO–NH side chain; a penicillin is an acyl derivative of 6-aminopenicillanic acid.
Literature Cited Summary The chemical methods used in the penicillin work were those of the “golden age” of organic chemistry and penicillin may be regarded as the last of the major natural products to have been so investigated. Melting points and boiling points were criteria of purity and a crucial tool was microanalysis leading to empirical formulas. For the rest, reliance was placed on chemical analogy and intuition, and on chemical synthesis. While limited use was made of column chromatography, paper and gas chromatography were not available. Some very helpful information was provided by physicochemical methods, particularly potentiometric titration and IR spectroscopy. However, even to obtain a UV spectrum in those days required a special laboratory. The indispensable tools of mass spectrometry and NMR spectroscopy were not available until after World War II. While the structure was determined, the overall goal of a reliable chemical synthesis was not. However, the use of specially selected fungal strains and of modern methods of bulk fermentation soon made penicillin available in quantities unimaginable to those who had worked with small quantities of the precious material. In 1946, du Vigneaud and his colleagues exploited the fact that low levels of antibiotic acwww.JCE.DivCHED.org
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1. Anonymous. Science 1945, 102, 627. 2. Anonymous. Nature 1945, 156, 766. 3. The Chemistry of Penicillin; Clarke, H. T., Johnson, J. R., Robinson, R., Eds.; Princeton University Press: Princeton, NJ, 1949. 4. Abraham, E. P; Chain, E.; Baker, W.; Robinson, R. Report PEN 103, Oxford, dated October 22, 1943 and addendum, Oct 23, 1943. 5. Anonymous. Summary of Structure Studies on Penicillin; Merck & Co., Inc.; Nov 1943. 6. Sheehan, J. C.; Henery-Logan, K. R. J. Am. Chem. Soc. 1957, 79, 1262. 7. Sheehan, J. C. The Enchanted Ring. The Untold Story of Penicillin; MIT Press: Cambridge, MA, 1982; pp 114–115. 8. Clarke, H. T.; Johnson, J. R.; Robinson, R. Science 1947, 105, 653. 9. Chain, E. In Nobel Lectures, Physiology or Medicine, 1942– 1962; Elsevier: Amsterdam, 1964; pp 110–143. The actual lecture was delivered on Mar 20, 1946. 10. Duffin, W. M.; Smith, S. Nature 1943, 151, 251. 11. Woodward, R. B. Report Wo2, Harvard University, dated 1945. 12. Crowfoot, D.; Bunn, C. W.; Rogers-Low, B. W.; Turner-Jones, A. In The Chemistry of Penicillin; Clarke, H. T., Johnson, J. R., Robinson, R., Eds.; Princeton University Press: Princeton, NJ, 1949; pp 310–367.
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In the Classroom 13. Jensen, W. P.; Palenik, G. J.; Suh, I.-H. J. Chem. Educ. 2003, 80, 753. 14. Abraham, E. P.; Baker, W.; Chain, E. Report CPS 554. Not dated. Received by OSRD on Sep 17, 1945. 15. Bijvoet, J. M.; Peerdeman, A. F.; van Bommel, A. J. Nature 1951, 168, 271. 16. Coleman, W. F. J. Chem. Educ. 2003, 80, 778. 17. du Vigneaud, V.; Carpenter, F. H.; Holley, R. W.; Livermore, A. H.; Rachele, J. R. Science 1946, 104, 431.
Appendix This paper, to some extent, represents closure for me on penicillin chemistry. From 1943–1946, I was one of the many working on penicillin chemistry with the hope of achieving a chemical synthesis. I was a very raw graduate student at Imperial College, London. I have always maintained an in-
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terest in many aspects of the penicillin story and now I am somewhat of a survivor. Almost all of the “big names” and many of the juniors such as myself are now deceased. As I approached retirement in 1989, I hoped to write a monograph on the structural work on penicillin, basing it on the original reports issued during the collaborative USA–UK program. I wanted to develop a chronology of the work, to attribute credit to individuals, and to use modern structural drawings and notations. There were about 800 reports, but not all dealt with relevant material. I located most of them in the National Archives and spent a few days working there. Unhappily, the magnitude of the problem defeated me—it would have needed many weeks of work along with much copying and annotation. I came to realize that it might be possible—but only if the material was indexed and available in computer form. I finally was able to put some of the material to use in this Journal of Chemical Education article.
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