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Monobactams

The structure of the cell membrane of bacteria is unique and does not have any mammalian analogs. The cell membrane protects bacteria cells from lysis, which can occur as a result of different osmotic pressures between the cytoplasm and the surrounding medium.

The main component of bacterial cell membranes is a mixed polymer known as murein or peptidoglycan. Peptidoglycan is a long polysaccharide chain that is cross-linked with short peptides.

Polysaccharide chains are made up of two varying aminosugars—N-acetylglucosamine and N-acetylmuraminic acid. For example, Staphylococcus aureus (golden staphylococci), a tetrapeptide made of L-alanine, D-glutamic acid, L-lysine, and D-alanine, is joined to every one of the N-acetylmuraminic acid units, forming side chains of glycan chains. Many of these tetrapeptides are cross-linked with one another either directly or with short peptide chains. In S. aureus, L-lysine of one of the tetrapeptides is bound by a pentaglycine chain to D-alanine of the other. This kind of structure gives it a certain rigidity to bacterial membranes. The peptidoglycan layer of Gram-negative bacteria is thinner than that of Gram-positives, and it has fewer cross-(transversal) links.

The synthesis of peptidoglycan of bacterial cell membranes can be divided into three stages based on where the reaction takes place.

The first stage occurs in the cytoplasm, which results in the synthesis of precursor units—uridindiphospho-N-acetylmuramyl pentapeptide. Such an antibiotic, for example, cycloserine, the drug most frequently used to treat tuberculosis, blocks synthesis of cell membranes at this stage by competitive inhibition of the stage of introducing alanine into a pentapeptide.

Reactions in the second stage occur when precursor units move along the cytoplasmic membrane. In the first reaction, the N-acetylmuramylpentapeptide region binds (through a pyrophosphate bridge) to a carrier phospholipid that is bound to the cytoplasmic membrane. N-acetylglucosamine is then bound, forming a disaccharide-pentapeptide-P-P-phospholipid.

Further modification of the pentapeptide chain then occurs; for example, the binding of pen-taglycine in the case of S. aureus. The modified disaccharide is subsequently removed from the membrane-bound phospholipid and then bound to the existing region already containing the peptidoglycan. This reaction is mediated by the enzyme peptidoglycan synthetase. The primary repeating units of the peptidoglycan are thus collected, forming a glycopeptide polymer. This process can be disrupted by antibiotics such as vancomycine, which inhibits pepti-doglycan synthetase.

The third and final stage of synthesis of cell walls occurs outside the cytoplasmic membrane. Thus, the transpeptidation reaction results in transformation of the linear glycopeptide polymer into the cross-linked form. The enzyme transpeptidase, a membrane-bound enzyme, binds pentapeptide side chains by replacing terminal D-alanines.

As already noted, beta-lactam antibiotics interfere with biosynthesis of the primary component of cell membranes—peptidoglycan. Because of the fact that this process does not take place in human and other mammalian cells, beta-lactam antibiotics are relatively non-toxic to humans.

Beta-lactam antibiotics specifically bind with a number of proteins of cytoplasmic membranes known as penicillin-binding proteins (PBP). These proteins are enzymes involved in the reaction of transpeptidation during the break up of cell membranes during growth and division.

For example, Escherichia coli have six PBP. PBP-1a and -1b, which are transpeptidases, are involved in the synthesis of peptidoglycan. PBP-2 is necessary for supporting the "rod-shaped" form of bacteria. Selective inhibition of this enzyme causes production of other "non-rod-shaped" forms of bacteria, which eventually undergo lysis. PBP-3 is necessary to form the partition during division. Selective inhibition of this enzyme leads to the formation of a fibrous form of bacteria containing many units of rod-shaped bacteria unable to separate one from another, which results in their death. Various beta-lactam antibiotics have a selective affinity to one or a few PBP. Inactivation of certain PBP (PBP-1a, -1b, -2, or -3) causes cell death. Unlike these, inactivaition of low-molecular PBP (PBP-4, -5, and -6) is not lethal to bacteria.

Resistance of pathogenic microorganisms to beta-lactam antibiotics can result from one or a few of the mechanisms listed below: inability of the drug to directly find an active site; a change in PBP function, which is expressed in the reduction of affinity to the drug; or inactivation of the drug by bacterial enzymes.

Beta-lactam antibiotics must pass through the outer layer of the cell in order to get the desired PBP to the surface of the membrane. In Gram-positive bacteria, the cell membrane is the only layer covering the cytoplasmic membrane. In a few types of this bacteria, there is a polysaccharide capsule on the outer side of the cell membrane. However, not one of the described structures can serve as a barrier for the diffusion of small molecules such as beta-lactams. Therefore, the idea that the cause of possible resistance is the inability of beta-lactam antibiotics to get the desired PBP is not likely to be a possible mechanism of resistance for Gram-positive bacteria.

Gram-negative bacteria have a more complex cell surface. The peptidoglycan layer is also the outer layer with respect to the cytoplasmic membrane. However, besides this, they have another outer polysaccharide membrane. This outer membrane is built out of lipopolysac-charides and lipoproteins, and can be a serious barrier for permeating hydrophilic molecules.

Diffusion of beta-lactam antibiotics across this membrane is only possible through transmembrane channels made of proteins called porines. It has been shown that beta-lactam antibiotics diffuse through porine channels, and the ease of this process varies depending on their size, charge, and hydrophilic properties. Accordingly, the idea of the possible mechanism of resistance for Gram-negative bacteria being the inability of beta-lactam antibiotics to get desired PBP is also unlikely.

The second mechanism of resistance to beta-lactam antibiotics can appear as a change in target PBP, which is expressed in a reduction in the affinity to beta-lactam molecules.

Finally, the most important mechanism of resistance to beta-lactam antibiotics is the production of beta-lactamase by the bacteria. Beta-lactamases break the C-N bond in the beta-lactam ring of antibiotics. Since its existence is absolutely necessary for reacting with PBP, a break in the beta-lactam ring leads to a loss of antibacterial activity.

There are many beta-lactamases and they can be classified differently: by type of substrate, replacement of genes (chromosomes or plasmids), and place of production. A few of these enzymes directly hydrolyze penicillins (penicillinases), others hydrolyze cephalosporins (cephalosporinases), and others extend to a broad spectrum of substrates. A few bacteria have the ability to induce synthesis of beta-lactamase. Synthesis of beta-lactamase, which in a normal condition is suppressed, is induced in the presence of some beta-lactam antibiotics.

Thus, beta-lactam antibiotics can inihibit the process of synthesis of bacterial cell membranes in different ways, thus causing them to die quickly.

32.1.1 Penicillins

Penicillin was discovered in 1928 by Alexander Fleming, who noticed that one of his experimental cultures of staphylococcus was contaminated with mold, which caused the bacteria to lyse. Since mold belonged to the family Penicillium, he named the antibacterial substance penicillin.

About a decade later, a group of researchers at Oxford University isolated a crude substance made up of a few low-molecular substances, which were penicillins (F, G, K, O, V, X). Penicillin G (benzylpenicillin), the most active of these, was suggested for clinical trials in 1941.

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Structure of R radical

Name of Penicillin

Penicillin G Benzylpenicillin

Penicillin V

Phenoxymethylpenicillin

Penicillin X p-Oxybenzylpenicillin ch3ch2ch—ch—ch2—

Penicillin F 2-Pentenylpenicillin

CH3(CH2Î5CH2-

Penicillin K p-Heptylpenicillin ch^—ch_ch^_s_ch2—

PenicillinO

Allylmercaptomethylpenicillin

Drugs of the penicillin group are effective for infections caused by Gram-positive bacteria (streptococcus, pneumococcus, and others), spirochaetae, and other pathogenic microorganisms. Drugs of this group are ineffective with respect to viruses, mycobacteria tuberculosis, fungi, and the majority of Gram-negative microorganisms.

The production of penicillin was an extremely important milestone in the development of microbiology, chemistry, and medicine, signifying the creation of the powerful antibiotic industry and formation of modern biotechnology.

There have been attempts to chemically synthesize penicillins; however, no practical methods have been found.

An extremely important progress in the development of penicillins took place in 1959, when the penicillin nucleus, 6-aminopenicillanic acid (6-APA), was removed from the side chain and isolated from a culture of Pénicillium chrysogenum.

Subequent acylation of 6-APA by various acid derivatives led to the formation of a large number of semisynthetic penicillins.

Currently, penicillin (benzylpenicillin, penicillin G) is made in huge amounts (tens of thousands of tons) by the microbiological industry.

Penicillin can be made by many types of Penicillium fungi, and also by a few types of Asperigillus fungi. In industrial conditions, culture fluids are made that contain more than 30 mg/mL of penicillin. About two-thirds of the produced penicillin is used for making 6-APA. Despite the possibility of pure chemical deacylation, the most prospective way of making 6-APA is an enzymatic method of hydrolyzing benzylpenicillin molecules using immobilized penicyllinamidase, an enzyme isolated from practically all penicillin-producing fungi. It should be noted that 6-APA itself is practically devoid of antibiotic proterties. However, by acylating it with various acid derivatives, more than 50,000 semisynthetic penicillins have been made, of which less than 30 are currently used in medicine.

Variations of the acyl regions of the side chain in penicillin molecules produces significant changes in the properties of resulting compounds. It was discovered that the side

6-aminopenicillanic acid chain of the acyl region of the molecule determines the antimicrobial spectrum, sensitivity to beta-lactams, and the unique pharmacokinetic features of a specific penicillin. The unique feature of a few semisynthetic penicillins (meticillin, oxacillin, cloxacillin) is their efficacy with respect to a culture of microorganisms (staphylococcus) resistant to ben-zylpenicillins. Moreover, some semisynthetic penicillins (ampicillin) are active with respect to the majority of Gram-negative microorganisms.

From the chemical point of view, the first type of semisynthetic penicillins, undoubtedly, are considered relatively simple derivatives of 6-APA and aromatic or aryloxycar-boxylic acids (benzylpenicillin, phenoxymethylpenicillin, meticillin, naphicillin).

Another type of semisynthetic penicillins that are considered heteroylcarboxylic acid derivatives, in which the acyl group is represented as an aromatic heterocyclic acid derivative (oxacillin, cloxacillin, dicoxacillin).

The next type of semisynthetic penicillins are those in which the acyl group is represented by an amino acid, mainly a-aminophenylacetic acid (phenylglycine) or p-oxy-a-aminophenylacetic acid, and correspondingly, ampicillin and amoxicillin.

Finally, the fourth type of replacement in the side acyl region of penicillins is the replacement by dicarboxylic acid derivatives (carbenicillin, ticarcillin).

All penicillins are used as sodium or potassium salts.

Benzylpenicillin: Benzylpenicillin, [2S-(2a,5a,6^)]-3,3-dimethyl-7-oxo-6-(phenylac-etamido)-4-tio-1-azabicyclo[3.2.0]-heptan-2-carboxylic acid (32.1.1.1), is the gold standard penicillin, which is obtained biotechnogically using the fungus P. chrysogenum as the producer, and phenylacetic acid as the precursor. The sodium or potassium salts of this drug are used in medicine, and it is for this reason that the extract of the cultural liquid is treated with an aqueous solution of the respective base, and the commercial product is the corresponding lyophilized salt [1-5]. There are also purely synthetic ways of making benzylpenicillin [6,7].

Benzylpenicillin or penicillin G has a narrow antimicrobial spectrum. It is active with respect to Gram-positive bacteria (staphylococcus, streptococcus, and pneumococci), causative agent of diphtheria, and anthrax bacillus. Gram-negative bacteria are resistant to it. Benzylpenicillin is broken down by stomach acid and destroyed by staphylococcus penicillinase.

Benzylpenicillin is the drug of choice for infections caused by sensitive organisms. This includes streptococci infections (except enterococci), gonococci, and meningococci that do not produce beta-lactam anaerobes. Benzylpenicillin is used for croupous and focal pneumonia, skin infections, soft tissue and mucous membranes, periotonitis, cystisis, syphilis, diphtheria, and other infectious diseases. Synonyms of this drug are megacillin, tradocillin, bicillin, sugracillin, vicillin, and others.

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