Beta-lactam Antibiotics: The Past, Present, and Future

Authors: Allen Chen, Brian Fu, Andrew Liang, Karankumar Mageswaran, Ayeeshi Poosarla

Abstract

In 1928, the discovery of penicillin revolutionized the field of medicine. As the first reported beta-lactam antibiotic, penicillin laid the foundation for modern antibiotics. Beta-lactams, 4-membered cyclic amides, can be used for the prevention and treatment of bacterial infections. Since the discovery of penicillin, many beta-lactams have been discovered from natural sources, and modern methodologies in chemical synthesis have powered the design for synthetic beta-lactams. This is significant in supplying the continued need for novel antibiotics in the treatment of antibiotic-resistant bacteria. 

Beta-lactam History

Penicillin, a beta-lactam antibiotic, was discovered by Alexander Fleming. Fleming was growing a culture of Staphylococcus and observed that Penicillium notatum, a fungus, had contaminated the dish; a compound secreted by the fungus, later identified as penicillin, inhibited the growth of the Staphylococcus culture. Years later, the beta-lactam was isolated as the active component of penicillin, responsible for its antimicrobial activity. Today, as a result of the discovery of penicillin and the reactivity of beta-lactams, many antibiotics contain the beta-lactam moiety.¹

Since the initial discovery of penicillin, others have sought to synthesize, purify, and characterize it. Penicillin continued to advance throughout the twentieth century. Specifically, its antiseptic capabilities, initially discovered by N.G. Heatley using mice, were researched and developed.² In 1941, during World War II, penicillin’s antibiotic capabilities proved to be crucial as it saved millions of lives. Without effective antibiotic treatment, people were dying because of common bacterial infections. At this time, Florey and Heatley enlisted the help of the U.S. government to take on the mass production of penicillin, which was made possible with deep-fermentation tanks.³

Following the discovery of penicillin, other beta-lactam-containing natural products including penicillin derivatives, cephalosporins, monobactams, carbapenems, and carbacephems have been discovered (Figure 1). 

Cephalosporins are beta-lactams that have historically been used for treatment against gram-positive bacteria, but recent developments have shown that they can also be effective against gram-negative bacteria. Together with cephamycins, cephalosporins form a beta-lactam antibiotic subgroup called cephems.⁴

Monobactams are monocyclic, bacterially-produced beta-lactam antibiotics that can act against aerobic gram-negative bacteria. In contrast to other beta-lactam antibiotics, the beta-lactam ring on monobactams is not fused with another ring. Monobactams did not show impressive antimicrobial activity, but the side-chain variation resulted in potent compounds.⁵

Carbapenems are a class of beta-lactam antibiotics used for the treatment of severe, high-risk bacterial infections, as they are uniquely resistant to hydrolysis by many bacterial enzymes.

Carbacephems are a class of synthetic antibiotics based on the structure of cephalosporins. Carbacephems are similar to cephalosporins, but with a carbon substituted for the sulfur.⁶ All of these antibiotics were results of the discovery of penicillin and contain the beta-lactam ring first found in penicillin (Table 1).

Chemical Structure	Year of Discovery	Common Antibiotics	Significance	Species
	1928	Penicillin, Ampicillin, Azlocillin	Prevents peptidoglycan from cross-linking properly in the last stages of bacterial cell wall synthesis	Penicillium mold
	1948	Kefazol, Ancef, Ceftin, Zinacef	Effective against gram-negative bacteria	Fungus Acremonium
	1985	Aztreonam, Tigemonam, Nocardicin A, and Tabtoxin	Effective against aerobic gram-negative bacteria	Chromobacterium
	1976	Imipenem, Panipenam, Doripenam	Largely resistant to hydrolysis by bacterial enzymes	E. coli, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter freundii, Proteus mirabilis, and Serratia marcescens
	1967	Cefixime, Cefdinir, Cefotaxmine	Prevents bacterial cell division by inhibiting cell wall synthesis	E. coli, K. pneumoniae, and Enterobacter

Beta-lactam Mechanism of Action

A discovery in 1949 by Cooper and Rowley showed the irreversible binding of penicillin to the penicillin-binding proteins, or PBPs, of sensitive bacteria (Figure 2).⁷

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Soon after, in 1956, Lederberg discovered that penicillin converted rod-shaped E. coli bacteria into spherical protoplasts, thereby concluding that penicillin interfered with cell wall biosynthesis;⁸ further work by Wise, Park, Tipper, and Trominger et al. demonstrated that penicillin targeted the cross-linking of peptidoglycan strands.⁹ Ultimately, Timmer and Strominger proposed that the effectiveness of penicillin came from its structural similarity to the D-Ala-D-Ala residue of peptidoglycan, the native substrate of DD-transpeptidase (Figure 3).

DD-transpeptidase cross-links peptide side chains of peptidoglycan strands in the cell wall, providing structural rigidity for bacterial cells. This is followed by the breakdown of the acyl-enzyme intermediate and the formation of a new peptide bond between the carbonyl of the D-Ala moiety and the amino group of another peptidoglycan molecule, thereby crosslinking the two peptidoglycan molecules (Figure 4). 

Beta-lactam antibiotics disrupt cell wall biosynthesis via covalent inhibition of the transpeptidase enzyme. The four-membered beta-lactam ring is forced into a torsed diamond geometry, as opposed to the natural 109.5° bond angles of a tetrahedral carbon. A four-membered ring has approximately 25 kcal/mol of ring strain, prompting ring opening. As such, beta-lactams are often irreversible inhibitors of the enzyme because they permanently acylate the active site serine of DD-transpeptidase, effectively preventing the enzyme from interacting with its native substrate (Figure 5). This prevents the formation of the bacterial cell wall and promotes the osmotic lysis of the cell. 

Beta-lactam Resistance

Emergence of Beta-lactamases

From the initial discovery of penicillin as a beta-lactam antibiotic, bacterial resistance has trailed closely behind in a molecular arms race between antibiotics and bacteria. Bacterial resistance to beta-lactams is conferred by two major mechanisms: (1) inactivation of the beta-lactam by hydrolytic enzymes called beta-lactamases—shown in Figure 5—and (2) target site alterations to PBPs. Drug-resistant bacteria often express more than one of these mechanisms. The first instances of beta-lactam resistance came from beta-lactamases, which disrupt the amide bond of the beta-lactam. Molecular modeling of various serine beta-lactamases and PBP structures have demonstrated three-dimensional similarities with conserved folding patterns and preservation of topology at the active site, suggesting that beta-lactamases evolved from PBPs selected for their antibiotic resistance. First found in E. coli a year prior to the clinical release of penicillin, beta-lactamases have since been found in numerous gram-positive and gram-negative bacteria, often encoded as a mobile genetic element for plasmid-enabling horizontal gene transfer.¹⁰

Beta-lactamases have a similar structure to DD-transpeptidase but with one key difference: beta-lactams covalently bind to DD-transpeptidase while beta-lactamases hydrolyze the beta-lactam ring without binding to it. Thus, beta-lactamases prevent beta-lactams from inhibiting DD-transpeptidase.¹¹

Evolution of Beta-lactamases

Phylogenetic analyses and nucleotide substitution rates have determined that serine beta-lactamases are around 2 billion years old, while plasmid-encoded OXA beta-lactamases are millions of years old, both existing far before the discovery and usage of beta-lactam antibiotics.¹² The expression frequencies for these early beta-lactamases were low in bacterial populations and evolved in certain bacterial species as a mechanism for resistance against beta-lactam-containing compounds produced by fungi. However, the discovery and clinical development of beta-lactams as antibiotics resulted in the evolutionary selection for relevant beta-lactamases (Table 2).

Wave	Characteristics	Susceptible Beta-lactams	Unaffected Beta-lactams
1	Narrow-spectrum penicillinases TEM-1 and SHV-1 strands	Penicillin, Ampicillin	Cephalosporins, Carbapenems, Aztreonam
2	Extended spectrum cephalosporinases from point mutations	Cephalosporins	Ampicillin, Carbapenems
3	CTX-M family of beta-lactamases to mutations to TEM and SHV	Cephalosporins	Carbapenems
4	Carbapenemases: KPC class beta-lactamases, Metallo-beta-lactamases, OXA-type enzymes	Carbapenems, Cephalosporins, Penicillin, Ampicillin	Aztreonam (often ineffective)

Classification of Beta-lactamases

There are two classifications of beta-lactamases: the first of which is the comparatively older classification that distinguished beta-lactamases into classes A, B, C, and D based on amino acid sequencing.¹³ In a more novel classification, Bush and Jacoby distinguished beta-lactamases into Groups 1, 2, and 3, effectively combining classes A and D into a single group based on mechanism and evolutionary lineage (Table 3).¹⁴ The mechanism by which serine beta-lactamases hydrolyze beta-lactams is shown in Figure 6.

Group	Discovery	Description	Mechanism
Group 1 beta-lactamases	Beta-lactamases were first documented in 1940 as the first bacterial enzyme capable of facilitating the breakdown of penicillin.	Group 1 beta-lactamases include cephalosporinases, originally derived from Acremonium fungus.15, 16 The most clinically relevant cephalosporinases are AmpC beta-lactamases, mediating resistance to cephalothin, cefazolin, cefoxitin, and most penicillins. Overexpression of the AmpC gene confers resistance to broad-spectrum cephalosporins.17	The sequence of the AmpC gene was first recorded in E. coli in 1981,18 distinct from the genetic sequence of TEM-1 and TEM-2, but still with an active site serine nucleophile.19, 20
Group 2 beta-lactamases	TEM-1 was first documented in the early 1960s. The TEM-1 enzyme was originally found in a single strain of E. coli isolated from a blood culture from a patient named Temoniera in Greece, hence the designation TEM.	Group 2 beta-lactamases include broad-spectrum, inhibitor-resistant, and extended-spectrum beta-lactamases. The earliest serine carbapenemases began with the TEM-1 and SHV-1 strands which further evolved into extended-spectrum beta-lactamases against aztreonam and oxycilin-hydrolyzing beta-lactamases against carbapenem.	As with Group 1 beta-lactamases, Group 2 beta-lactamases have an active site serine.
Group 3 beta-lactamases	Metallo-beta-lactamases were initially discovered in the 1970s and attracted clinical attention in the 1990s with the spread of the IMP and VIM-type metallo-beta-lactamases.	Metallo-beta-lactamases have poor hydrolytic capabilities against monobactams—beta-lactams without fused rings—but have recorded high hydrolytic capabilities towards penicillins, cephalosporins, and carbapenems. Due to its reliance on the zinc cation, metallo-beta-lactamases are inhibited by metal ion chelators, such as EDTA.	In contrast to serine beta-lactamases, metallo-beta-lactamases utilize a zinc(II) cation to hydrolyze the beta-lactam ring.

An active site serine hydrolyzes the beta-lactam ring, creating a transition acyl-enzyme adduct which undergoes a general base-catalyzed attack by a hydrolytic water molecule to form a second tetrahedral intermediate, which then collapses to form a product complex.²¹ 

Drug Inactivation by Target-site Alterations: Mutations in PBPs

All beta-lactams have the same binding target for successful inhibition: bacterial PBPs. This makes the alteration of this binding pocket extremely significant in hindering beta-lactam activity. PBPs are membrane-bound DD-peptidases that evolved from serine proteases and are responsible for the crosslinking of peptidoglycan chains in bacterial cell wall formation. Due to the low expression of beta-lactamases in Staphylococcus bacteria, target site alterations of PBPs are responsible for almost all beta-lactam antibiotic resistance. The PBP targets in penicillin-resistant Streptococci are modified into low-affinity targets for beta-lactams, thereby reducing beta-lactam inhibitory activity.²² Meanwhile, certain bacteria such as methicillin-resistant Staphylococci express novel PBPs—termed PBP2a—encoded by the mecA gene with almost no binding affinity to beta-lactams. In the COL52 strain, E to K237 within the non-penicillin-binding domain, along with V to E470 and S to N643 near the SDN464 conserved DNA sequence of the penicillin-binding domain was important for resistance.^{23, 24} Thus, research towards inhibitors for PBP2a is of high priority. 

Beta-lactam Synthetic Routes

The battle against bacterial resistance has necessitated the continued development of new antibiotics and methodologies, thus synthetic access to beta-lactams has been of great significance.²⁵ The first synthetic beta-lactam was prepared by Hermann Staudinger in 1907—21 years before Fleming discovered penicillin—by reaction of the Schiff base of aniline and benzaldehyde with diphenylketene in a [2+2] cycloaddition.^{26, 27} This reaction, later coined the Staudinger [2+2] cycloaddition—as shown in Figure 7—still remains the most common method of beta-lactam synthesis to date.^{28, 29} 

There are two proposed mechanisms for the Staudinger 2+2 cycloaddition: a stepwise mechanism, wherein the nucleophilic nitrogen atom of the imine first attacks the sp-hybridized carbon atom of the ketene, followed by nucleophilic addition of the resulting enolate to the iminium; or a concerted mechanism which undergoes a pericyclic transition state to yield the beta-lactam ring. Many computational studies have suggested that the mechanism for the [2+2] cycloaddition is stepwise, as reported by Cossío et al., Bachrach and Halzner, and Sordo et al.^{30, 31, 32} Similar studies on the related reaction between ketene and alkenes by Burke, Houk and Wang, and Bottoni et al. have shown that the reaction has an asynchronous transition state with appreciable charge separations; however, no intermediates were isolated.^{33, 34, 35} Although more than 100 years have passed since the ketene-imine cycloaddition was first reported, the reaction mechanism is still unclear.

Variants of the asymmetric synthesis of beta-lactams have incorporated chiral auxiliaries to control enantioselectivity and diastereoselectivity.^{36, 37} Asymmetric syntheses of beta-lactams yields a mix of cis and trans isomers; however, stereochemical control is necessary if a particular isomer is desired. Imines, characterized by the C=N functional group, can be prepared via condensation of an amine and aldehyde or amine and ketone.^{38, 39, 40, 41} Ketenes are a highly reactive species that is characterized by the C=C=O functional group, which can be detected by infrared spectroscopy at around 2100-2200 cm^-1. The most common method of ketene preparation involves activation of the carboxylic acid as a leaving group, followed by deprotonation of the beta carbon and elimination of the leaving group. 

Modern Advancements

In an effort to produce better yields and improve reaction conditions and results, alternative synthesis methods have been produced in recent years as a substitute for the traditional Staudinger synthesis reaction. As reported by Dong et al., one such method involves a catalytic metal carbene insertion into C-H bonds. Another method consists of the activation of an unsaturated C-C bond in addition to a nucleophilic addition.^{42, 43} Transition metal-assisted Staudinger reactions have also been promising alternative synthetic routes devised in recent years.⁴⁴ 

Recent advancements have also been made to combat beta-lactam resistance. In 2015, a novel antibacterial treatment, Ceftazidime-Avibactam (Figure 8), was approved by the U.S. Food and Drug Administration, involving a combination of the beta-lactam antibacterial ceftazidime and the novel beta-lactamase inhibitor, avibactam. This combination has been shown to have significant activity against beta-lactamase-producing gram-negative pathogens.^{45, 46} 

Despite recent advancements in combating resistance to beta-lactam antibiotics, resistance to beta-lactam antibiotics remains a concern which requires continued research development. Specifically, more needs to be done regarding plasmid-mediated beta-lactamases, which transfer easily among groups of organisms and thus contribute to bacterial resistance. Induction of chromosomal beta-lactamases is also a continuing problem to be resolved in terms of beta-lactam antibiotic resistance.⁴⁷

Conclusion

Since the discovery and initial clinical uses of penicillin in the 1900s, beta-lactam antibiotics have undergone drastic improvements in order to combat the perpetual problem of bacterial resistance. The beta-lactam core moiety of several natural products has attracted the attention of chemists and biologists alike for its prevalence in natural products, structural simplicity and reactivity, and effectiveness in the mechanism of action against many strains of bacteria. The continued evolution of antibiotic resistant bacteria has made the development of new antibiotics all the more important. The development of new synthetic methodologies in accessing beta-lactam-containing compounds has the potential of giving rise to improved beta-lactam antibiotics that would be impactful not only in the medical field but also in the health and well-being of billions of people around the world. 

Acknowledgments

All authors contributed to the manuscript and declare no competing conflicts of interest in the work presented. The authors gratefully acknowledge Mr. Edward Njoo for his guidance and valuable aid throughout the writing process.