tetracycline spectra

8/3/2019 Tetracycline Spectra

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Tetracycline and derivativesassignment of IR and Raman spectra

via DFT calculations

C. F. Leypold,a M. Reiher,*a G. Brehm,a M. O. Schmitt,a S. Schneider,*a

P. Matousekb and M. Towrieb

a Institut fur Physikalische und Theoretische Chemie, Universitat Erlangen-Nurnberg,

Egerlandstr. 3, D-91058, Erlangen, Germany. E-mail: [email protected] Central Laser Facility, CLRC, Rutherford Appleton Laboratory, Didcot, Oxfordshire,

UK OX11 0QX

Received 28th October 2002, Accepted 23rd January 2003

First published as an Advance Article on the web 5th February 2003

IR and Raman spectra (lex 1064, 400 and 280 nm) were recorded for solutions of tetracycline and several

derivatives in acidified and alkaline H2O and D2O, respectively. Based on the observed resonance enhancement,an empirical assignment of most of the Raman bands in the wavenumber range 1000< ~nn/cm1


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analysis. For preparation of sample solutions, the compoundswere dissolved in H2O (D2O). In order to assure complete pro-tonation (deuteration), 1 n HCl (DCl) was added until pH 1(pD 1) was reached. The pH (pD) of the solution was increasedby adding the proper amount of NaOH (NaOD).

2.2. Experimental techniques

For recording IR spectra (Bruker, Equinox 55, resolution1 cm1), the crystalline material was diluted in KBr pellets.Solubility in H2O (D2O) was too low for recording IR spectraof solutions.

Raman spectra with 1064 nm excitation were monitored ona Bruker RFS 100 FT-Raman spectrometer (resolution24 cm1).

Resonance-enhanced Raman spectra of the aqueous solu-tions with 400 nm excitation were recorded by means of anewly developed technique employing a fast Kerr-gate forfluorescence rejection.17,18 For both 400 nm and 280 nmexcitation, the Raman scattered light was dispersed by a spec-trograph and monitored by a CCD camera (typical accumula-tion time 10 s, resolution ca. 8 c m1). Calibration of theRaman shift was done by using the known shifts of thesolvents.

2.3. Quantum-chemical methodology

All calculations were performed with the density functionalprograms provided by the Turbomole 5.1 suite.19 TheBeckePerdew functional (BP86)20,21 was selected because itis known to yield good structural parameters and normalmode frequencies.2224 This good performance is due to a sys-tematic error cancellation found for the BP86 functional bycomparison with anharmonicity calculations.25 The resolu-tion-of-the-identity technique has been used throughout.26,27

The TZVP basis set developed by Ahlrichs and coworkerswas employed.28 For the vibrational analyses, the second deri-vatives of the total electronic energy were calculated as numer-ical first derivatives of the analytic energy gradients.22,29

Based on our experience of previous assignments of experi-mental vibrational spectra by comparison with the results of

calculations employing the same computational technique weestimate that the error in the calculated vibrational wavenum-bers is on the order of10 cm1 provided that the moleculargeometry is well reproduced.30

3. Results

3.1. IR and Raman spectra

In Figs. 1 and 2, the NIR Raman spectra (lex 1064 nm) oftetracycline hydrochloride and doxycycline hydrochloride inacidic H2O and D2O, respectively, are compared with thecorresponding NIR Raman spectra of the solid material.

Inspection of Figs. 1 and 2 immediately reveals that thespectra of both compounds exhibit many similar features asone expects because of the similarity in chemical structure.When looking into details, one recognizes band shifts on theorder of some 20 to 30 cm1 and/or changes in relative inten-sities. The corresponding vibrations must therefore involve amovement of the carbon atoms C7, C6 and C5 and of theatoms of the functional groups attached to them (seeScheme 1). The second interesting aspect derived from a com-parison of Fig. 1 and Fig. 2 is the pronounced change in spec-tral appearance upon switching to deuterated solvent.Knowing the pKa values of tetracycline, one concludes thatat pD 1 three hydrogen atoms are exchanged by deuterium:OH3, NH4 and OH12. Secondary effects on the vibrationalspectra can be induced by intermolecular hydrogen (deuter-ium) bonds from the solvent to the various carbonyl and/or

hydroxyl groups. In view of the expected strong couplingbetween the various local modes, one must not be surprisedthat deuterium substitution does not lead to simple shiftstowards lower wavenumber of only a few bands. On theother hand, it is quite interesting that in the NIR Raman spec-tra of the deuterated samples three bands of high relativeintensity appear around 1443, 1512 and 1609 cm1. For thesake of completeness, it should be mentioned especially thata similar band pattern as for tetracycline is observed for other

Fig. 1 Comparison of the NIR Raman spectra (lex 1064 nm) oftetracycline (trace D) and various derivatives in acidified H2O(pH 1), oxytetracycline (A), doxycycline (B), minocycline (C) andof crystalline material: tetracycline hydrochloride (E), methacycline(F), minocycline (G), doxycycline (H) and oxytetracycline (I).

Scheme 1 Chemical structure of fully protonated compounds investi-gated and numbering of the atoms.

1150 Phys. Chem. Chem. Phys., 2003, 5, 11491157


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derivatives investigated, e.g. oxytetracycline and minocycline.This observation implies that the vibrations giving rise to thethree strong Raman bands must be dominated by OD localvibrations, but must also contain a significant contributionof CC skeleton vibrations. Furthermore, it can be noted thatall bands found in the solution spectra of tetracycline have acounterpart, although sometimes with different relative intensi-ties, in the spectra of the crystalline material. This suggests thatthe conformation in both environments is very similar, but notidentical. Hereby we assume that interaction with crystal wateris not the primary source for the differences. In Figs. 3 and 4,the dependence of the Raman spectra on excitation wavelengthis illustrated for tetracycline in acidic H2O and D2O, respec-tively. The number of bands with medium and high intensityis only slightly reduced when going from lex 1064 nm tolex 400 nm. With lex 280 nm, only a few bands can beidentified beyond doubt. The most prominent one with~nn 1420 cm1 in H2O (Fig. 3) has no counterpart in the400 nm spectrum. In the 1064 nm spectrum, only a very weakfeature can be seen. This finding can be understood as conse-quence of resonance enhancement. The UV/vis absorptionspectra of tetracyclines are interpreted as superposition of the

contributions of the so-called A-chromophore (comprising thediketone moiety around CO1, amide group and OH3) andthe BCD-chromophore involving the p-electronic system ofthe phenyl ring D and the b-hydroxyketo system extendingacross CO11 and COH12. The latter chromophore is responsi-ble for the long wavelength absorption band with maximumaround 360 nm. The A-chromophore possesses its longestwavelength absorption band around 270 nm. One can there-fore expect that the Raman spectra recorded with excitationwavelength 400 nm and 280 nm, respectively, are dominatedby the vibrations located on the BCD- and A-chromophore,respectively. This information is quite valuable when assigningthe experimentally observed Raman bands to calculated nor-mal modes (see below).

Included in Figs. 3 and 4 are also the IR spectra recordedfrom crystalline material diluted in KBr. To facilitate the com-parison with Raman spectra and the theoretical spectra, the IRspectra of the samples under investigation are presented here

as absorption spectra rather thanas is usualas trans-mission spectra.

In contrast to the Raman spectra, the changes induced in theIR spectra upon a transition from protonated to deuteratedspecies are only minor. Especially most of the more intensebands appear fairly conserved both with respect to locationand intensity. This implies that they must originate from atomsand functional groups, which are not engaged in intra- orintermolecular hydrogen bonding.

3.2. Results of quantum-chemical calculations for tetracycline

The previously determined X-ray structure of tetracycline16

crystallized from acidic H2O was used as starting geometryfor DFT/BP86 structure optimisations aimed at the equili-brium geometry of tetracycline in the zwitterionic form. Inthe case of the hexahydrate complex, the energy optimized

geometry of the tetracycline zwitterion differs only very littlefrom the starting geometry. As can be seen from the overlaypresented in Scheme 2, the differences are mainly restrictedto the conformation of ring A and the orientation of itssubstituents.

In Scheme 3, an overlay is shown of the obtained structures,if either six (as in crystal structure) or nine water molecules(purely computational) are considered. It is obvious that thestructural differences concentrate on rings B and A and onthe substituents connected to these. A comparison of the vibra-tional frequencies calculated for the two geometries allows anestimate how sensitive the different normal modes react tostructural changes around ring A and which of them can even-tually be taken as marker bands for determining the conforma-

tion. In this contribution, the emphasis is on vibrational bandsconnected with the BCD-chromophore. This also justifies whythe theoretical results obtained for the zwitterionic form areused for the comparison with the experimental spectra

Fig. 3 Raman spectra of tetracycline in acidic H2O recorded with dif-ferent excitation wavelength. (A) lex 280 nm, (B) lex 400 nm, (C)lex 1064 nm. Also included are the IR spectrum (KBr pellet) (D)and the results of the quantum chemical calculations (the height ofthe bars is proportional to the IR band intensities).

Fig. 2 Comparison of the NIR Raman spectra (lex 1064 nm) oftetracycline (trace D) and various derivatives in acidified D2O(pD 1), oxytetracycline (A), doxycycline (B), minocycline (C), andof tetracycline zwitterion (E) crystallized from acidic D2O solution.

Phys. Chem. Chem. Phys., 2003, 5, 11491157 1151


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recorded from the fully protonated form of tetracycline. In thefollowing, we will use the results calculated for tetracyclinehexahydrate geometry for the band assignment. This proce-dure can yield, however, larger deviations between experimen-tal and calculated wavenumbers of modes which involvemovement of O3 or atoms of the amide group.

In Figs. 3 and 4, some results of the quantum-chemical cal-culations are also incorporated. The location and height of thebars represent wavenumber and IR absorption intensity of thecalculated normal modes, numbered consecutively accordingto increasing frequency. (Only for the modes with high IRintensity is the mode number indicated to avoid congestion.)The density of modes in the wavenumber range 1100

tetracycline spectra

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