Antibiotics Research at the University of Debrecen

 

The Research Group of Antibiotics was established in 1961 at the Department of Organic Chemistry, University of Debrecen, by the late professor Rezsö Bognár. The research profile of the Group is essentially associated with antibiotics, and it rests on the establishment of the BIOGAL Pharmaceutical Works (the former "Penicillin Factory") in the city of Debrecen in 1952. Most of the studies in the antibiotic field were carried out in a close cooperation with the associates of the BIOGAL Pharmaceutical Works and of the University Medical School of Debrecen. Here was isolated the first Hungarian antibiotic agent primycin. Collaboration in the field of antibiotics research is done with scientists in the former Soviet Union, Great Britain, France, Germany, Belgium, Japan and in the USA. The major research topics are as follows:

  1. Isolation and purification of antibiotics
  2. Detection and qualitative determination of antibiotics
  3. Structural elucidation of antibiotics
  4. Synthesis, and chemical modification of antibiotics and their structural elements


1. Isolation and purification of antibiotics

From an Actinomyceta species the antibiotic primycin was isolated, which is marketed by the CHINOIN Pharmaceutical Works under the trade name Ebrimycin. It was shown that the carbohydrate component of primycin is D-arabinose, and others have shown that the antibiotic complex contains several components:

Primycin

The antibiotic flavofungin was isolated from Streptomyces flavofungini, and a second antibiotic, desertomycin, with a significant value in mycological differential-diagnostics was also obtained from this species. Isolation and purification of the antibiotic oxytetracycline was carried out and patented.

2. Detection and qualitative determination of antibiotics

Analytical procedures were elaborated for the qualitative determination of desertomyin and oxytetracycline in the fermentation samples during manufactoring. A biological method was introduced for measuring the 6-amino-penicillanic acid–content of fermentation broths. HPLC was employed for the detection of glycopeptide antibiotics, and a book chapter1 was published on the modern UV/VIS spectroscopic methods for the structural elucidation of antibiotic substances.

3. Structural elucidation of antibiotics

The beauty and difficulty of the structural research of antibiotics and related microbial substances are that almost each of the studied compounds calls for the development of an independent method of structural elucidation.

3.1 Polyene macrolide antibiotics – oxopentaene

The polyhydroxy-petaene macrolactone structure of the flavofungin components A and B was established by chemical derivatization, and application of modern one and two-dimensional NMR techniques, which allowed the determination of the conformational properties of flavofungin, as well.

Flavofungin

3.2. Giant macrocyclic lactones – the desertomycin family

Chemical degradation and mass spectrometry (LC/MS, MALDI-TOF) were employed to identify the components of the desertomycin complex. It was shown that in the molecule of the major component (desertomycin A) a D-mannose unit and a 5-amino-2-hydroxy-1-methylpentyl side-chain are attached to carbon C-22 and C-41, respectively, of the 42-membered non-polyene macrolactone ring. Based on this structural feature the desertomycins belong to the highest-membered non-polyene-type macrolides. The structure of desertomycin B was determined with the aid of FAB-mass spectrometric investigations. It is interesting to note that desertomycin E carries a disaccharide unit, composed of two D-mannose molecules.

dezertomicin

3.3. Glycopetide antibiotics

During the past 38 years most of the efforts in the structural research of the Group was spent for studies2 of the sugar–containing, vancomycin-type glycopeptide antibiotics actinoidin A and B, and ristocetin A and B (ristomycin A and B), and later of the structure of eremomycin and avoparcin.

Based on chemical degradation and NMR studies the structure of the aglycone portion of actinoidin A and B was established. Besides the neutral sugars D-mannose and D-glucose, L-acosamine and L-actinosamine were isolated and identified as the aminodeoxyhexose components of the actinoidins. It was recognized that the former aminosugar is included in a disaccharide moiety [acobiose; 2-O-(3-amino-2,3,6-trideoxy-α-L-arabino-hexopyranosyl)-β-D-glucopyranoside]. The chemical sythesis of both acosamine and acobiose was carried out.

Actinoidin

It was found that the antibiotics ristocetin (isolated in the USA) and ristomycin of Russian origin are identical. Detailed chemical degradation, NMR- and synthetic studies revealed the structure of the building blocks (actinoidinic acid, ristomycinic acid) of the aglycone portion, as well as of the two reducing disaccharides (rutinose and ristobiose), the two trisaccharides (ristotriose and ristriose), and of the new aminodeoxy sugar (L-ristosamine). In cooperation with American scientists the structure of ristomycinic acid, the whole aglycon portion and that of L-ristosamine was established. Each of the sugar blocks was prepared by chemical synthesis, and the first definitive synthesis of L-ristosamine (3-amino-2,3,6-trideoxy-L-ribo-hexopyranose) was also elaborated, which opened new synthetic activities4,5 in the Group in the field of carbohydrate and antibiotic research. In the frame of these works various deoxy and aminodeoxy sugar components of antibiotics, such as D- and L-diginose, sarmentose, rhodinose, and D-and L-daunosamine, ristosamine, and acosamine, and L-kedarosamine were synthesized from carbohydrate precursors or tartraldehyde mercaptals.

Ristomycin

Modern NMR-spectroscopy allowed to identify the structure of ristomycin A as 4. Ristomycin A (ristocetin A) is the active component of the diagnostical kits "Aggristin Kit" and "Aggristin Plus" developed in cooperation with researchers in the University Medical School of Debrecen. The kits are marketed by the REANAL Chemical Works (Budapest). NMR-methods have been used to study the linkage of glycopeptide antibiotics to cell wall-analog oligopeptides (Ac-D-Ala-D-Ala).

In cooperation with Russian and German researchers the full NMR signal correlation was given to the glycopeptide-type eremomycin. It was also recognized that this antibiotic contains a branched-chain aminodeoxy sugar; 3-C-epi-L-vancosamine. As a result of the collaboration with French chemists, the disaccharide building block (avobiose) of the antibiotic avoparcin was prepared by chemical synthesis.

 

4. Synthesis, and chemical modification of antibiotics and their structural elements

4.1. Antracyclin glycoside antibiotics

Carminomycin

By employing the intermediates of the synthesis of L-acosamine, as well as L-and D-ristosamine, new analogs of the anticancer anthracycline glycoside-type antibiotics daunomycin and carminomycin were synthesized. These derivatives involve O- and S-glycosides with new (amino)deoxy sugar components, as well as those carrying a heterocyclic moiety at the sugar-portion.
4.2. Trehalosamine analogs

By applying the synthesis intermediates of L-acosamine and L-ristosamine, new symmetrical and non-symmetrical α,α-trehalose derivatives were synthesized, which might influence the immune system. Isotopically labelled derivatives of trehalose were produced, and NMR relaxation technique was employed for the investigation of the conformational and molcular-dynamical properties of these derivatives

4.3. Anti-AIDS azidothymidine (AZT) nucleoside analogs

The C-3 azido analog of L-acosamine (3-azido-2,3,6-trideoxy-α-L-ribo-hexo-pyranoside) was used to produce a novel, hexopyranosyl derivative of the anti-HIV agent azidothymidine (AZT).

4.4. Aminocyclitol antibiotics and enzyme-inhibitory cyclitol

Functionalized hexopyranoside derivatives, produced in the syntheses of aminodeoxy sugars, were converted into deoxyinososes by means of the Ferrier carbocyclic ring-transformation procedure. The mechanism, and stereochemical aspects of this reaction was studied in detail, and the method was successfully extended to 2’-deoxydisaccharides. The above deoxyinososes, and those derived from D-(–)-quinic acid were employed to synthesize new pseudodisaccharide-type aminocyclitol antibiotic models6, and enzyme-inhibitory inositol derivatives acting as "second messengers" in the inositol phospholipid Ca2+ ion-transport system.

4.5. Lincomycin and clindamycin derivative

Lincomycin
In cooperation with Japanese scientists, biologically potent semi-synthetic analogs of the antibiotics lincomycin and clindamycin, including C-7 azido and imidazol-2-yl-thio derivatives were synthesized. The structure of the two sulfoxide-glycosides produced upon fermentation of lincomycin was assigned and converted into a sulphone.


4.6. Synthesis of conagenin-stereoisomers

Conagenin

D-Starting from D-xylose the synthesis of the C-3 diastereoisomers of the immuno-modulant antibiotic conagenin was performed. One of the new isomers possessed higher biological activity than the natural compound.


4.7. Heterocyclic antibiotics - Hydroxylated indolizidine

Swaisonin
Two independent methods, based on hetero-Diels-Alder reaction and 1,3-dipolar cycloaddition, were elaborated for the synthesis of hydroxylated indolizidines, which are potent inhibitors of tumor-metastases. These works and another reported methods for the production of swainsonine, castanospermine and slaframine derivatives have been reviewed7 in a monograph.7

4.8. Beta-lactam antibiotics

PenicillinThe Group is engaged with the research of beta-lactam antibiotics since the early ninetysixties.
With the aid of the Morin-Jackson rearrangement penicillin G acetoxymethyl ester (penamecillin) was converted into 7-amino-3-deacetoxycephalosporanic acid (7-ADCA), which was used for the production of novel semisynthetic derivatives including those carrying a sugar side-chain or an isoxazolyl mioety. The cycloaddition reactions of various cephalosporins were studied in detail, and from cephalosporin sulfoxides and sulphones cyclopropane and pyrazoline derivatives were obtained. 2,3-Exomethylene-cephalosporins could be readily converted into novel tricyclic beta-lactam antibiotic analogs via the Diels-Alder reaction.
Novel beta-lactamase and human leukocyta (HLE) enzyme inhibitory halogeno- and pseudohalogeno-penicillanic acids and 7α-alkoxycephalosporin sulfones were synthesized. Several new rearrangements of the cephalosporin sulphones were described.
In the field of the research of beta-lactam antibiotics three book-chapters8-10 and a book11were published.


References

1. Z. Dinya, F. Sztaricskai: Ultraviolet and Light Absorption Spectrometry, in Modern Analysis of Antibiotics. (Ed.: A. Aszalós) Drugs and the Pharmaceutical Science, Vol. 27. pp. 20-96. Marcel Dekker, Inc., New York and Basel (1986)

2. F. Sztaricskai, R. Bognár: The Chemistry of Vancomycin group of Antibiotics, in Recent Developments in the Chemistry of Natural Carbon Compounds (Ed.: Cs. Szántay) Vol. 10, pp. 90-209. Akadémiai Kiadó, Budapest, (1984)

3. R. Bognár, F. Sztaricskai: Chemistry of the Most Important Antibiotics (in Hungarian). Tankönyvkiadó, Budapest (1970)

4. I. F. Pelyvás, C. Monneret, P. Herczegh: Synthetic Aspects of Aminodeoxy Sugars of Antibiotics. xvi+224 pages. Springer-Verlag, Heidelberg (1988) ISBN 3-540-18877-0

5. F. Sztaricskai, I. F. Pelyvás: Chemistry of Carbohydrate Components, in Glycopeptide Antibiotics (Ed.: R. Nagarajan) Drugs and the Pharmaceutical Sciences, Vol. 63. pp. 105-193, Marcel Dekker Inc., New York (1994)

6. F. Sztaricskai, R. Bognár: Research Trends in the Chemistry of Aminocyclitol Antibiotics (in Hungarian). A Kémia Újabb Eredményei (Ed.: B. Csákvári) Vol 62, p 167. Akadémiai Kiadó, Budapest (1985/86)

7. P. Herczegh, I. Kovács, F. Sztaricskai: Chemistry of Biologically Important Hydroxylated Indolizidines: Synthesis of Swainsonine, Castanospermine and Slaframine, in Recent Progress in the Chemical Synthesis of Antibiotics and Related Microbial Products (Ed.: G. Lukács) Vol. 2. pp. 751-828. Springer-Verlag, Berlin (1993)

8. J. Cs. Jászberényi, E. T. Gunda: Functional Modifications and Nuclear Analogs of ß-Lactam Antibiotics, Part I., in Progress in Medicinal Chemistry (Eds.: G.P. Ellis and G.B. West) Vol 12, pp 395-477. Elsevier - North Holland Publ. Co., Amsterdam (1975)

9. E.T. Gunda, J. Cs. Jászberényi: Functional Modifications and Nuclear Analogs of ß-Lactam Antibiotics, Part II., in Progress in Medicinal Chemistry (Eds.: G. P. Ellis G. B. West) Vol 14, pp 181-248. Elsevier - North Holland Publ. Co., Amsterdam (1977)

10.Sztaricskai, F.: Beta-lactam Antibiotics, in Pharmaceutical Chemistry II. (in Hungarian) (Eds.: L. Tőke, L. Szeghy). Tankönyvkiadó, Budapest (1992)

11.Gunda T.: Penicillins, Cephalosporins, and Other Beta-Lactam Antibiotics (in Hungarian). A Kémia Újabb Eredményei (Ed.: B. Csákvári), Vol 84. Akadémiai Kiadó, Budapest (1998)