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Lincomycin

Page history last edited by PBworks 12 years, 8 months ago

Lincomycin is a natural product lincosamide produced by the growth of a member of the lincolnesis group of the actinomycete Streptomyces lincolnensis (Fam. Streptomycetscese). Lincosamides are a group of drugs that bind to the 23s portion of the 50s subunit of bacterial ribosomes and inhibit peptide elongation. Specifically, lincomycin serves as an antibacterial agent used to treat a wide variety of systemic infections. However, use of lincomycin as a therapeutic has been accompanied with a myriad of side effects, some severe. Among the most common, treated patients have complained of nauseau, headaches, glossitis, and stomatitis. Lincomycin has been replaced by it’s analog Clindamycin which exhibits greater antibiotic potency.

 


History

Lincomycin was first discovered in 1962 and subsequently isolated from Streptomyces lincolnensis, at the time a new streptomycete species. 4 Soon after, Upjohn Pharmaceuticals registered the first patent (US Patent No. 3,086,912) describing lincomycin production. Modern application of Lincomycin includes treatment against many gram-positive acting specifically on anaerobes like Bacteroids.

 

Structure

 

 

Chemical nameMethyl 6,8-dideoxy-6-(1-methyl-trans-4-propyl-L-2-pyrolidinecarboxamido)-1-thio-D-erythro-α-D-galacto-octopyranoside
Common nameLincomycin
StructureC18H34N2O6S
CAS #154-21-2
Mol. Weight406.54 g/mol
Boiling Point889.53 K
Melting Point765.65 K
Critical Temp1110.72 K
Critical Pressure19.74 Bar
Heat of Formation-1149.88 kJ/mol
ColorWhite
FormCrystalline powder
OdorNone

 

Structural studies show that Lincomycin is an N-acylated amino sugar consisting of an amino octose thioglycoside (termed methyl thiolincosaminide) and L-trans-n-propylhygric acid. Specifically, the antibiotic consists of a carbohydrate moiety, methyl 6-amino-6,8-dideoxy-1-thio-D-erythro-α-D-galacto-octopyranoside, bound to an amino acid, L-trans-4-n-propylhygric acid, by an amide linkage.

 

Biosynthesis

In their account, Spazek and Rezanka support their biphasic lincomycin biosynthetic pathway originally proposed by Retzlaff et al. and state that lincomycin is not formed during the logarithmic growth phase in S lincolnensis. 5

 

Biosynthesis of amino acid moiety

 

Biosynthetic experiments suggest that glucose is converted via glycolysis and the hexose monophosphate shunt to phosphoenolpyruvate and erythrose-4-phosphate, respectively. These proceed through the shikimic pathway to give tyrosine (1) and then dihydroxyphenylalanine (2). The pathway continues through the 2,3-extradiol cleavage of the aromatic ring of dihydroxyphenylalanine followed by pyrrole ring formation by condensation to give propylproline (3) through an unknown mechanism.

 

Biosynthesis of carbohydrate moiety

 

The exact mechanism of the reaction to form aminooctose moiety methyllincosamide is unclear due to the inability to isolate intermediates of the methylincosamide biosynthetic pathway. Several different schemes have been postulated as to how the carbohydrate moiety is biosynthesized.

 

Scheme 1

Careful studies of the metabolic origin of methylincosamide (4) suggest that the C8 carbon skeleton of the aminooctose moiety arises from the condensation of a pentose unit and a three carbon unit. The pentose unit could come from glucose through the hexose monophosphate shunt or be a result of a condensation of glyceraldehydes-3-phosphate with a two carbon donor such as sedoheptulose7-phosphate by a transketolase reaction. The three carbon unit would probably come from a donor molecule and be added via a transaldolase reaction or itself be synthesized from a two carbon and one carbon unit. Condensation of the three carbon and five carbon units would give octose which could be converted to methylthiolincosamide. A proposed mechanism involved isomerization of octulose to octose, dephosphorylation and reduction of the C8 carbon followed by transamination of the precursor 6-ketooctose. Finally, thiomethylation of the C1 carbon would give the carbohydrate product (4).

 

Scheme 2

Analysis of the lincomycin gene cluster sequence lead to an alternative design route for the carbohydrate moiety. According to this hypothesis, eight genes, lmb-LMNZPOSQ, form a “sugar subcluster” coding for enzymes involved in sugar metabolism. Comparative analysis to the better understood NDP hexose synthases led to the presumption that methylthiolincosamide biosynthesis involves a nucleotide activation step and a series of modifications on the dNTP-activated sugar intermediates. C13 labeling studies suggest that an octulose-phosphate intermediate is formed first, which would support glucose as a starter unit.

 

Scheme 3

Although not shown in the figure, detection of gene lmbR in the “sugar subcluster” could support an alternative route with a pyranosidic octose intermediate which is NDP-activated and further modified. The gene encodes for a transaldolase-like enzyme. Finally, A thiomethyl unit, possibly transferred from 5’-thiomethyladenosine, could be added to the C1 position of the (NDP-)6-amino-6,8-deoxyoctose intermediate.

 

Final modifications

 

The next step is the formation of the amide bond between the amino acid and carbohydrate moiety. Specifically, the amide bond would form between the carboxyl group of the propylproline and the amino group of the methylthiolincosamide, catalyzed by D-methyllincomycin-synthetase, producing N-demethyllincomycin (4). Lastly, N-demethyllincomycin is N-methylated by S-adenosylmethionine:N-methyllincomycin methyl transferase.

 

Synthesis

Several methods of lincomycin synthesis have been proposed. In 1970, Howarth et al. at Queen’s university proposed that the carbohydrate portion of lincomycin can be synthesized using two different schemes, arbitrarily named Schemes 1 and 2. 6 In both of Howarth’s methods, methyl lincosaminide (13) is acylated with the L-trans-4-n-propylhygric acid portion to give lincomysin product. That same year, Magerlein at the Upjohn Company developed a different synthesis method, arbitrarily named Scheme 3. 7

 

Scheme 1

 

Using 1,2:3,4-di-O-isopropylidene-α-D-galacto-hedodialdopyranose-(1,5) as their starting material (1), Howarth utilized the Wittig reaction and ethylidenetriphenylphosphorane to produce cis-6,7,8-trideoxy-1,2:3,4-di-O-isopropylidene-α-D-galacto-oct-6-enose (2). Treatment of (2) with potassium permanganate degraded the di-O-methyl derivative to 1-deoxy-2,3-di-O-methyl-L-erythritol to form 8-deoxy-1,2:3,4-di-O-isopropylidene-D-erythro-α-D-galacto-octopyranose (3). An alternative method for producing (3) is to treat (1) with vinylmagnesium bromide to give an allylic alcohol (1a) followed by the oxymercuration-demercuration procedure to give the Markovnikov hydration product. Careful addition of benzoyl chloride in pyridine to (3) at 273 Kelvin gave 7-0-benzoyl-8-deoxy-1,2:3,4-di-O-isopropylidene-D-erythro-α-D-galacto-octopyranose (4). Next, ruthenium tetroxide or the Pfitzner-Moffatt reagent was used to oxidize (4) to 7-O-benzoyl-8-deoxy-1,2:3,4-di-O-isopropylidene-D-glycero-α-D-galacto-octos-6-ulose (5). Treatment of (5) with hydroxylamine hydrochloride and pyridine in aqueous ethanol gave isomers (6) and (7). Reduction of (7) with lithium aluminum hydride in refluxing tetrahydrofuran followed by N-acetylation with acetic anhydride in methanol gave (9) and (10). In an alternative method, (7) was debenzoylated with a catalytic quantity of sodium methoxide to give (8), a hydroxyimino-alcohol, followed by hydrogenation over Raney Nickel to produce (9) and (10). (9) was then reduced with sodium borohydride and acetylated with acetic anhydride in pyridine to give peracetylated octitol (11). When 6-acetoamido-6,8-dideoxy-1,2:3,4-di-O-isopropylidene-D-erythro-α-D-galacto-octopyranose is treated reacted with methanethiol and concentrated hydrochloric acid to give dimethyl dithioacetal (9a). Methyl N-acetylthiolincosaminide (12) was isolated after (9a) was treated with dilute acid and isolated using t.l.c on silica gel. Finally, heating (12) with hydrazine at reflux temperature resulted in de-N-acetylation to give methyl thiolincosaminide (13).

 

Scheme 2

 

Scheme 2 proposes an alternative method for arriving at compound (9). Howarth used the same starting material (1) and nitroethane in the presence of sodium methoxide followed by acetylation to afford a mixture of β-nitro-acetates (14). Treatment of the predominant isomer with triethylamine in refluxing benzene gave cis-6,7,8-trideoxy-1,2:3,4-di-O-isopropylidene-7-C-nitro-α-D-galacto-oct-6-enose isomers (15) and (16) in a 6:1 ratio, respectively. (15), the cis olefin, was further reacted with methanolic ammonia gave (17) which was immediately N-acetylated to give a 1:1 mixture of two 6-acetomido-6,7-dideoxy-7-C-nitro derivatives (18). Upon oxidative denitration with potassium permanganate, ketones (19) and (20) were isolated. Reduction of (19) and (20) gave (9) and (10), respectively.

 

Scheme 3

 

In his method, Magerlein uses D-galactose as his starting material which he reacts with methanethiol to give methyl-1-thio-α-D-galactopyranoside (21). Tosylation of (3) in pyridine produced methyl 6-p-toluenesulfonyl-1-thio-α-D-galacto-pyranoside (22) which was treated with acetic anhydride-pyridine to give the acylated product (23). Treatment with sodium iodide in acetone with heat gave the 6-iodide (24). A nitro ion was used to substitute for the 6-iodide using sodium nitrite in DMF to give methyl-6-deoxy-6-nitro-1-thio-α-D-galactopyranoside (25) in about 20% yield. Repeated additions of acetaldehyde and sodium methoxide in methanolic solution to (25) gave a 50% yield of (26) which was reduced using lithium aluminum hydride in tetrahydrofuran to give (27). Finally, (27) could be condensed with 1-methyl-4-n-propyl-L-proline to give lincomycin.

 

Mechanism of action

Lincomycin acts by inhibiting the production of enzymes required by bacteria for survival. Specifically, the lincosamide antibiotic acts on translational machinery, forming cross-links within the peptidyl transferase loop region of the 50S ribosomal RNA (rRNA) and inhibits it’s binding to the ribosome-messenger complex. 1 Lincomycin interacts with both the A-site and the P-site on the ribosomal subunit, hampering positioning of both tRNA molecules and indirectly inhibiting peptide bond formation. The lincosamide was found to specifically inhibit the formation of the 50S subunit in addition to its inhibitory effect on translation. The antibiotic binds of regions of the 23S rRNA at the peptidyl transferase cavity, interacting exclusively with the enzyme’s center. 2 It has the unique ability to inhibit protein synthesis in gram-positive bacteria without interfering with DNA and RNA synthesis. 3 Resistance is achieved by methylation of the lincosamide binding sites on the 23S rRNA.

 

The drug is absorbed quickly after oral administration with peak levels 2 to 4 hours after administration. It maintains a potency level above the minimum requirement for 6 to 8 hours. Pharmacokinetic studies show that the drug is well distributed among the body but is concentrated in the liver, spleen, kidneys, and lungs.

 

Clinical Use

In the US, brand-name Lincomycin is also referred to as Lincocin or Lincorex and is frequently used as a hydrochloride salt. As of now, generic lincomycin capsules are not available. It is one of the two lincosamides currently in the clinic. Modern drug delivery formulations include capsules and injections which are only available with a doctor’s prescription. Lincomycin currently serves as an antibiotic for humans, particularly for certain penicillin-resistant infections. The drug is used to treat blood, lung, skin, tissue, and pelvic infections caused by bacteria. It is effective against most gram-positive bacteria including staphylococci, several streptococci and clostridium. It is only used for the most severe infections due to prevalence of severe side effects. It is recommended that a patient finish the entire treatment. Missing a dose or stopping the treatment prematurely can lead to development of drug-resistant bacteria and render the current medication useless. Lincomycin is also used as a therapeutic for veterinary dermatology. Studies have shown that lincomycin possesses good in-vitro and in-vivo potency against gram-positive microorganisms 4.

 

Delivery Methods

The current drug delivery formulation into man is through capsules taken by mouth and injection.

 

Oral delivery

It is recommended that lincomycin be taken on an empty stomach and with water. Additionally, it is advised that patients do not lie down after ingestion to avoid agitation in the throat. Capsules are to be stored dry between 15 to 30 degree Celsius.

 

Subcutaneous Injection

 

Lincomycin has been shown to cross react with other drugs such as azithromycin, chloramphenicol, clarithromycin, erythromycin, etc. Additionally, the molecule’s potency may be affected if taken with alcohol, caffeine or are a frequenter of smoking and/or illegal drug use.

 

Side Effects

Common side effects are…

- difficulty breathing

- diarrhea

- pain upon swallowing

- gastrointestinal pain

- skin rash

- unusual bleeding or bruising

Some side effects do not require medical attention (itching and nausea). However, it is advised that patients consult their health care professional if problems persist for extended periods of time.

 

References

 

1 Kirillov, S. V., et al., Peptidyl transferase antibiotics perturb the relative positioning of the 3′-terminal adenosine of P/P′-site-bound tRNA and 23S rRNA in the ribosome RNA (1999) 5, 1003-1013

 

2 Spizek and Rezanka., Lincomycin, Clindamycin and their applications, Appl Microbiol Biotechnol 64: 455-464 (2004)

 

3 Chang et al., Lincomycin, an Inhibitor of Aminoacyl sRNA Binding to Ribosomes Biochemistry 55, 431-438, (1966)

 

4 Spizek. J and Rezanka., Lincomycin, cultivation of producing strains and biosynthesis, Appl Microbiol Biotechnol (2004) 63:510-519

 

5 Michalik et al. Monophenol Monooxygenase and Lincomycin Biosynthesis in Streptomyces lincolnensis, Antimicrobial Agents and Chemotherapy (1975) 8: 526-531

 

6 Howarth et al. The Synthesis of Lincomycin, J. Chem. Soc Perkin 1, (1970): 16: 2218-24

 

7 Magerlein, BJ. Lincomycin. X. The chemical synthesis of Lincomycin. Tetrahedron Lett (1970) 1:33-36

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