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Biotechnol. Appl. Biochem. (2001) 33, 65–69 (Printed in Great Britain)
Synthesis and analysis of ethylated tetracycline, an antibiotic
derivative that inhibits the growth of tetracycline-resistant
XL1-Blue bacteria
Ronald Bartzatt1, Kelly Koziol, Theresa Benish and Jason Stoddard
Chemistry Department, College of Arts and Sciences, University of Nebraska, Omaha, NE 68182, U.S.A.
Bacterial resistance to antibiotics is a significant prob-
multi-faceted problem for medical research [4]. The threat lem in medical care facilities, causing increased fatalities
to quality medical care may be underestimated [3].
due to infection. The present study demonstrates that
In the present study the antibiotic tetracycline has had antibiotic structures can be selectively altered in a
its molecular structure altered and the new form was placed manner that revives their ability to inhibit bacterial
in tissue culture with a bacterial strain that is resistant to growth. The antibiotic tetracycline was ethylated at the
tetracycline (the bacterial strain is designated XL1-Blue). By position of the phenolic hydroxy group with the use of
utilizing this approach and observing the effects, some diazoethane, forming an ethyl ether functional group.
important information is obtained, such as : (1) greater This derivative was dissolved in Luria–Bertani (LB) agar
understanding of the mechanism of bacterial resistance, and medium, then placed in tissue culture for screening
(2) the means to produce new antibiotics, which can be used against a tetracycline-resistant bacterial strain. The
to control the spread of resistant bacterial strains.
growth of this bacterial strain, designated XL1-Blue,
The antibiotic tetracycline is in wide use, and its was inhibited by the ethylated form of tetracycline. The
medicinal application and action on bacteria is known.
procedure for
synthesizing
ethylated tetracycline
Tetracycline inhibits bacterial propagation by blocking the utilizes diazoethane and is presented with the mole-
binding of aminoacyl-tRNA to the aminoacyl site of ribo- cular structures and IR spectra. The ethylated form of
somes. The study of a tetracycline altered at the molecular tetracycline was stable at k20 mC for many weeks, and
level will aid in the understanding of the mechanism by which was soluble in LB agar plate medium. Ethylated tetra-
this antibiotic functions, and the action of the resistant cycline induced growth inhibition of XL1-Blue bacteria
within the first 24 h of incubation. The level of bacterial
growth inhibition was greater than 30 %. Calculation of
Materials and methods
the partition coefficient, log P, was accomplished and
indicates that ethylated tetracycline has an increased
Materials
lipophilic tendency relative to unmodified tetracycline,
Chemicals and reagents were obtained from Sigma Chemical and therefore has greater solubility in lipid bilayers.
Introduction
A Wheaton double-chamber device was used to generatediazoethane for derivatization (Wheaton, Millville, NJ, The wide-scale use of antibiotics has improved the quality of U.S.A.). Tetracycline was dissolved in an organic solvent many lives and provided potent tools for the treatment of (ethyl acetate\diethyl ether, 6 : 4, v\v), and was then allowed diseases. In response to the presence of antibiotics, bacteria to react with diazoethane. The tetracycline (0.1 g) was have gone through natural selection and developed mech- allowed to react with generated diazoethane by placing anisms to resist the action of antibiotics. Consequently, 0.15 g of 1-ethyl-3-nitro-1-nitrosoguanidine into 0.15 ml of antibiotic-resistant bacteria can pose dangerous infection 5 M NaOH and allowing gas evolution into the solvent. After threats in hospitals and daycare centres [1].
15 min of reaction the solvent was removed under nitrogen An increase in patient fatalities has resulted from the gas flow. This process was repeated up to three times in proliferation of antibiotic-resistant bacteria [1]. Formal order to optimize the yield of product. The product was studies have shown that the wide-scale and indiscriminate use of antibiotics will generate resistant strains of bacteria[2]. The cost financially, to the United States alone, is greater Key words : antibiotics, diazoalkane, microbes, pathogens.
than 100 million US$ per year [3]. The various mechanisms Abbreviation used : LB, Luria–Bertani.
1 To whom correspondence should be addressed (e-mail by which bacteria can form resistance to antibiotics poses a Tetracycline-resistant bacterial strain XL1-Blue
The tetracycline-resistant bacterium was provided by the
Biology Department, University of Nebraska, Omaha, NE,
U.S.A. This resistant bacterium is a strain of Escherichia coli
and is designated XL1-Blue. A spiral loop of the bacteria (that
had been stored as a liquid nitrogen glycerol stock) was
streaked on to prepared LB medium plates and incubated at
37 mC.
Molecular structure of tetracycline with carbon atoms numbered Medium plates for inhibition screening
Each plate contained 25 ml of medium. For 25 ml of medium The carbon atoms of tetracycline have been numbered and their chemical-shift 0.25 g of tryptone, 0.125 g of NaCl, 0.25 g of yeast extract (p.p.m.) values are as follows : 1, 120.8 ; 2, 134.1 ; 3, 112.8 ; 4, 157.2 ; 5, 124.4 ; 6, and 0.375 g of bacto-agar were added to 25 ml of water.
140.9 ; 7, 27.9 ; 8, 78.5 ; 9, 187.0 ; 10, 110.7 ; 11, 40.3 ; 12, 17.1 ; 13, 164.1 ; 14,96.4 ; 15, 27.4 ; 16, 40.0 ; 17, 40.0 ; 18, 60.7 ; 19, 200.7 ; 20, 100.9 ; 21, 178.8 ; 22, Tetracycline and its derivative were dissolved into warm medium and poured on to plates for solidification, giving aworking concentration of 12 µg\ml medium.
Calculation of partition coefficient log P
The algorithm developed by Moriguchi et al. [5] was utilized :
log P l1.244(CX)0.6k1.017(NO)0.9+0.406PRX k0.145(UB)0.8+0.511HB+0.268POLk2.215AMP+0.912ALK k0.392RNGk3.684QN+0.474NO2+1.582NCS+0.773BLMk1.041 where CX l total number of carbon and halogen atoms,NO l total number of N and O atoms, PRX l proximityeffect of N\O, UB l total number of unsaturated bonds, HB Molecular structure of ethylated tetracycline with carbon atoms l intramolecular hydrogen bond, POL l number of aro- matic polar substituents, AMP l amphoteric property, ALK The carbon atoms of ethylated tetracycline have been numbered and their l variable for alkane, alkene, cycloalkane or cycloalkene, chemical-shift (p.p.m.) values are as follows : 1, 119.8 ; 2, 133.3 ; 3, 111.3 ; 4, RNG l ring structures, QN l quaternary nitrogen, NO2 158.7 ; 5, 122.9 ; 6, 140.1 ; 7, 27.9 ; 8, 78.5 ; 9, 187.0 ; 10, 110.7 ; 11, 39.9 ; 12, 15.9 ; 13, 164.1 ; 14, 96.4 ; 15, 32.8 ; 16, 40.0 ; 17, 40.0 ; 18, 60.7 ; 19, 200.7 ; 20, 100.9 ; number of nitro groups, NCS l isothiocyanato or 21, 178.8 ; 22, 167.6 ; 23, 65.1 ; 24, 14.3.
thiocyanato, and BLM l B-lactam.
Results and discussion
resistant to the unaltered tetracycline. Growth inhibition ofXL1-Blue, induced by the ethylated tetracycline, was greater Antibiotics have become widely used since their discovery.
As a consequence bacteria have adapted, by natural selec- The ethylation of tetracycline was accomplished by the tion, a response to the antibiotics’ mechanism of action, reaction of diazoethane with tetracycline in an organic which induces growth inhibition. The various responses of solvent composed of ethyl acetate and diethyl ether (see the bacteria towards antibiotic action include chemical in- Materials and methods section). This mixture was suitable activation of antibiotics, alteration of biological targets and for the solubility of the initial reactant tetracycline and the alteration to cellular permeability [4].
resultant derivative product. Diazoethane is generated as a In the present study, the antibiotic tetracycline has had gas (see the Materials and methods section), which when its molecular structure modified in order to evaluate the dissolved in a suitable organic solvent is highly reactive effect against a tetracycline-resistant strain of bacteria.
towards, and specifically reacts with, acidic hydrogen atoms Results of tissue-culture screening showed that modifying [6]. The subsequent product will have sites of ethylation. The the molecular structure of this antibiotic resulted in a form reaction is fast and selective for sites of highest hydrogen that inhibited the growth of the bacteria, which were acidity. In the case of tetracycline, the site of highest acidity Ethylated tetracycline inhibits resistant bacteria
Tissue-culture plate showing the growth of tetracycline-resistant Tissue-culture plate showing that ethylated tetracycline inhibits bacteria, designated XL1-Blue, in the presence of tetracycline the growth of tetracycline-resistant bacteria XL1-Blue Bacteria growth is uninhibited and extensive. Tetracycline was at a concen- A reduced number of bacteria colonies is evident, and bacterial proliferation is tration of 12 µg/ml of agarose medium.
inhibited by greater than 30 %, compared with Figure 3. Ethylated tetracyclinewas at a concentration of 12 µg/ml of agarose medium.
is the hydrogen of the phenolic hydroxy group ; see the tetracycline inhibits the growth and proliferation of the same structure shown in Figure 1, with 13C NMR assignments strain of bacteria, with growth inhibition greater than 30 %.
given as p.p.m. values. The derivative form that results from The IR spectra of normal unmodified tetracycline is the reaction with diazoethane is the ethylated tetracycline shown in Figure 5 and that of ethylated tetracycline is shown (Figure 2). The chemical reaction of diazoethane is fast and in Figure 6. All IR spectra were obtained in dried DMSO. The efficient [6]. 13C NMR assignments for carbon atoms of the phenolic hydrogen peaks appearing in normal tetracycline derivative form are shown in Figure 2. (p.p.m. values are (Figure 5) at wavelengths of 1100 cm−1 to 1300 cm−1 are removed in the ethylated form of tetracycline (Figure 6).
The result of the reaction of diazoethane with tetra- Converting the phenolic hydroxy group of tetracycline cycline is the ethylated tetracycline (formation of an ether into an ethyl ether group of the ethylated tetracycline re- group) and nitrogen gas [6]. The remaining diazoalkane is duces the level of molecular hydrogen bonding (by removing driven off by flushing with nitrogen gas or argon gas. This can the hydrogen of the phenolic hydroxy group) and reduces also be accomplished by evaporation of the solvent. Con- overall molecular polarity (by addition of the aliphatic ethyl sequently, by using diazoethane, no undesired products chain). Various methods for estimating the value of this remain. Repeating the reaction of diazoethane with tetra- alteration exist, and one approach is to measure the cycline for as many as four passes resulted in an essentially partitioning of the compound between octanol and water 100 % yield of the desired ethylated form of tetracycline. The [7]. Computational methods to estimate this parameter are structure and yield of the alkylated tetracycline were widely utilized and the method described by Moriguchi et al.
confirmed by IR spectroscopy. The general reaction of [5] (see the Materials and methods section) was applied to diazoethane with tetracycline is represented by : these two compounds. For unmodified tetracycline log P l k2.706, and for the ethylated tetracycline log P lk2.28.
The positive increase in the log P value for ethylated tetracycline, relative to the parent compound, indicates thatthe level of lipophilicity is increased. This also indicates that To test for possible growth inhibition of tetracycline- ethylated tetracycline will have greater solubility in lipid resistant bacteria strain XL1-Blue, ethylated tetracycline was solubilized in LB agar medium, plated under sterile con- The results of the present study demonstrate the ditions, and bacteria strain XL1-Blue was streaked on to the ability of the ethylated tetracycline form to significantly plate. As a control, the same amount of normal unmodified inhibit the growth and proliferation of a tetracycline- tetracycline was plated on to LB medium and streaked with resistant bacteria strain. The ethylation of tetracycline may XL1-Blue. Figure 3 shows bacteria growing extensively on occur at the phenolic hydroxy functional group. The the control plate, with normal unmodified tetracycline.
alkylation at the phenolic site reduces the overall polarity of However, Figure 4 clearly shows that the ethylated form of the tetracycline molecule and the level of hydrogen bonding.
Ethylated tetracycline inhibits resistant bacteria
It is known that, in general, a higher polarity will reduce the Acknowledgments
ability of a molecule to cross a lipid bilayer. Also, reducingthe overall polarity of tetracycline by alkylation of the This work was funded by the Chemistry Department, phenolic site increases its permeability across lipid bilayers.
University of Nebraska, Omaha, NE, U.S.A.
In addition, the alkylation of the phenolic site eliminates thecapacity of hydrogen bonding at that site. This result alsoincreases the permeability of the molecule across a lipidbilayer. This observation suggests that a site-specific alter- References
ation of the molecular structure of an antibiotic may reviveits ability to inhibit the growth of an otherwise resistant Watson, D. A. and Bogaert, D. (1997) Lab. Med. 28, 392–395 Watson, D. A. and Bogaert, D. (1997) Lab. Med. 28, 324–328 The alkylation of phenolic hydroxy groups by use of Haberberger, R. L., Kallen, A. J., Driscoll, T. J. and Wallace, M. R.
diazoalkanes is a well characterized reaction. In the present study, the products include the ethylated tetracycline and Brock, T. D. and Madigan, M. T. (1991) in Biology of Micro- nitrogen gas. Any remaining diazoethane is easily removed by organisms (Aloisi, T. and Bracewell, C., eds.), p. 343, Prentice solvent evaporation, nitrogen degassing, or is destroyed by use of ethanol (the product of this approach is diethyl Moriguchi, I., Hirono, S., Liu, Q., Nakagome, I. and Natsushita, Y.
ether, which is easily evaporated away). This manufacturing approach is attractive, owing to the minimal generation of Knapp, D. R. (1978) in Analytical Derivatization Reactions, pp.
by-products and the ease of their removal. The ethylated tetracycline product is stable for long periods of time at Lipinski, C. A., Lombardo, F., Dominy, B. W. and Feeney, P.
room temperature and 4 mC, when kept dry. Consequently, (1997) in Adv. Drug Delivery Rev. 23, 3–25 the product could be easily transported. The use oftetracycline has been well studied.
Received 13 October 2000\15 December 2000 ; accepted 18 December 2000

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