PCCP

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: Schneider@chemie.uni-erlangen.de 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 acidied 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 < n /cm1 < 1600 to either the A- or the BCD-chromophore could be established. In addition, DFT/BP86 calculations yielded normal mode frequencies and coordinates, as well as IR intensities. By comparison of these with the information contained in IR and Raman spectra (especially the deuterium isotope shift), a fairly unique correlation between experimental and theoretical spectra could be achieved. Inspection of the normal coordinate pictures also provides a rationale for which of the bands are shifted upon partial or complete deprotonation or upon derivatization of tetracycline. Altogether, the presented material provides strong evidence that the fully protonated tetracyclines investigated exhibit in aqueous environment a conformation which is very close to that found in the crystalline state (tetracycline hexahydrate).

1. Introduction
Since the discovery of the antibiotic action of tetracycline more than 50 years ago,1,2 a large number of derivatives have been synthesized.3 Many of them have also been successfully applied as antibiotics. More recently, non-antibiotic actions of tetracycline derivatives have attracted great interest.2 Examples are the treatment of periodontal diseases4 or the inhibition of metalloproteinase activity5 by doxycycline and others. Last but not least, tetracycline-dependent gene regulation has become a versatile tool in biology and medicine.6,7 For these reasons, much eort has been spent in the past to elucidate the protonation/deprotonation behaviour of tetracyclines, which represent three- or four-protic acids.810 The latter is of major importance in understanding their pharmaco-kinetical properties, because an orally administered drug will most likely change its state of protonation during its passage through the acidic stomach and the alkaline intestinal tract. This change of protonation inuences the drugs solubility in an aqueous environment and its permeability through membranes. Furthermore, it is known that tetracyclines bind to many proteins preferentially, if not exclusively, as complexes with divalent metal ions.11,12 In body uids, the predominant divalent metal ions are Mg2+ and Ca2+. Therefore, complexation sites of these two ions are heavily discussed topics in the literature with mostly divergent conclusions.13 There is also evidence in the literature that tetracyclines undergo a conformational change upon deprotonation.10,14,15 But here again, the conclusions are not unique because the involved techniques (UV/vis absorption and CD spectroscopy) provide only indirect information about the molecular structure. Therefore, we decided to employ vibrational spectroscopy in combination with DFT calculations to gain further information concerning the addressed topics. A major problem with investigating DOI: 10.1039/b210522e

tetracyclines is their low solubility in water at pH around 7 (dominance of a zwitterionic form). Consequently, experimental techniques are required which are applicable to solutions in low concentration (e.g. resonance-enhanced Raman spectroscopy). Alternatively, experiments have to be performed at high or low pH, where the fully protonated or fully deprotonated species predominate. In another publication, we have reported the structures of tetracycline and doxycycline in crystals that have been crystallised from dierent types of solutions.16 From the same crystals, NIR Raman spectra could be recorded. Thereby, we have created a unique correlation between molecular structure (including hydrogen bonded water molecules) and vibrational frequencies and relative band intensities. These can now be compared with the vibrational spectra of solutions. In this contribution, we report the vibrational spectra of tetracycline and several derivatives in acidied H2O and D2O. DFT/BP86 calculations are performed using the known crystal structure as starting geometry for geometry optimisation. It will be shown that the fully protonated species exhibit in aqueous environment a conformation which is close to that found for tetracycline hexahydrate in the crystalline state.

2. Materials and methods

2.1. Materials Tetracycline hydrochloride and derivatives (see Scheme 1 for structure and numbering of atoms) were generally used as supplied by the manufacturers (tetracycline, oxytetracycline: Fluka; doxycycline, methacycline, minocycline: Hovione). Only tetracycline hydrochloride was re-crystallised from H2O and D2O, respectively, to yield single crystals for X-ray Phys. Chem. Chem. Phys., 2003, 5, 11491157 1149

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3. Results
3.1. IR and Raman spectra In Figs. 1 and 2, the NIR Raman spectra (lex 1064 nm) of tetracycline hydrochloride and doxycycline hydrochloride in acidic H2O and D2O, respectively, are compared with the corresponding NIR Raman spectra of the solid material. Inspection of Figs. 1 and 2 immediately reveals that the spectra of both compounds exhibit many similar features as one expects because of the similarity in chemical structure. When looking into details, one recognizes band shifts on the order of some 20 to 30 cm1 and/or changes in relative intensities. The corresponding vibrations must therefore involve a movement of the carbon atoms C7, C6 and C5 and of the atoms of the functional groups attached to them (see Scheme 1). The second interesting aspect derived from a comparison of Fig. 1 and Fig. 2 is the pronounced change in spectral appearance upon switching to deuterated solvent. Knowing the pKa values of tetracycline, one concludes that at pD 1 three hydrogen atoms are exchanged by deuterium: OH3, NH4 and OH12. Secondary eects on the vibrational spectra can be induced by intermolecular hydrogen (deuterium) bonds from the solvent to the various carbonyl and/or hydroxyl groups. In view of the expected strong coupling between the various local modes, one must not be surprised that deuterium substitution does not lead to simple shifts towards lower wavenumber of only a few bands. On the other hand, it is quite interesting that in the NIR Raman spectra of the deuterated samples three bands of high relative intensity appear around 1443, 1512 and 1609 cm1. For the sake of completeness, it should be mentioned especially that a similar band pattern as for tetracycline is observed for other

Scheme 1 Chemical structure of fully protonated compounds investigated and numbering of the atoms.

analysis. For preparation of sample solutions, the compounds were dissolved in H2O (D2O). In order to assure complete protonation (deuteration), 1 n HCl (DCl) was added until pH 1 (pD 1) was reached. The pH (pD) of the solution was increased by adding the proper amount of NaOH (NaOD). 2.2. Experimental techniques For recording IR spectra (Bruker, Equinox 55, resolution 1 cm1), the crystalline material was diluted in KBr pellets. Solubility in H2O (D2O) was too low for recording IR spectra of solutions. Raman spectra with 1064 nm excitation were monitored on a Bruker RFS 100 FT-Raman spectrometer (resolution 24 cm1). Resonance-enhanced Raman spectra of the aqueous solutions with 400 nm excitation were recorded by means of a newly developed technique employing a fast Kerr-gate for uorescence rejection.17,18 For both 400 nm and 280 nm excitation, the Raman scattered light was dispersed by a spectrograph and monitored by a CCD camera (typical accumulation time 10 s, resolution ca. 8 cm1). Calibration of the Raman shift was done by using the known shifts of the solvents. 2.3. Quantum-chemical methodology All calculations were performed with the density functional programs provided by the Turbomole 5.1 suite.19 The BeckePerdew functional (BP86)20,21 was selected because it is known to yield good structural parameters and normal mode frequencies.2224 This good performance is due to a systematic error cancellation found for the BP86 functional by comparison with anharmonicity calculations.25 The resolution-of-the-identity technique has been used throughout.26,27 The TZVP basis set developed by Ahlrichs and coworkers was employed.28 For the vibrational analyses, the second derivatives of the total electronic energy were calculated as numerical rst derivatives of the analytic energy gradients.22,29 Based on our experience of previous assignments of experimental vibrational spectra by comparison with the results of calculations employing the same computational technique we estimate that the error in the calculated vibrational wavenumbers is on the order of 10 cm1 provided that the molecular geometry is well reproduced.30 1150 Phys. Chem. Chem. Phys., 2003, 5, 11491157

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

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

derivatives investigated, e.g. oxytetracycline and minocycline. This observation implies that the vibrations giving rise to the three strong Raman bands must be dominated by OD local vibrations, but must also contain a signicant contribution of CC skeleton vibrations. Furthermore, it can be noted that all bands found in the solution spectra of tetracycline have a counterpart, although sometimes with dierent relative intensities, in the spectra of the crystalline material. This suggests that the conformation in both environments is very similar, but not identical. Hereby we assume that interaction with crystal water is not the primary source for the dierences. In Figs. 3 and 4, the dependence of the Raman spectra on excitation wavelength is illustrated for tetracycline in acidic H2O and D2O, respectively. The number of bands with medium and high intensity is only slightly reduced when going from lex 1064 nm to lex 400 nm. With lex 280 nm, only a few bands can be identied beyond doubt. The most prominent one with ~ n 1420 cm1 in H2O (Fig. 3) has no counterpart in the 400 nm spectrum. In the 1064 nm spectrum, only a very weak feature can be seen. This nding can be understood as consequence of resonance enhancement. The UV/vis absorption spectra of tetracyclines are interpreted as superposition of the contributions of the so-called A-chromophore (comprising the diketone moiety around CO1, amide group and OH3) and the BCD-chromophore involving the p-electronic system of the phenyl ring D and the b-hydroxyketo system extending across CO11 and COH12. The latter chromophore is responsible for the long wavelength absorption band with maximum around 360 nm. The A-chromophore possesses its longest wavelength absorption band around 270 nm. One can therefore expect that the Raman spectra recorded with excitation wavelength 400 nm and 280 nm, respectively, are dominated by the vibrations located on the BCD- and A-chromophore, respectively. This information is quite valuable when assigning the experimentally observed Raman bands to calculated normal modes (see below). Included in Figs. 3 and 4 are also the IR spectra recorded from crystalline material diluted in KBr. To facilitate the comparison with Raman spectra and the theoretical spectra, the IR spectra of the samples under investigation are presented here

Fig. 3 Raman spectra of tetracycline in acidic H2O recorded with different 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 of the bars is proportional to the IR band intensities).

as absorption spectra rather thanas is usualas transmission spectra. In contrast to the Raman spectra, the changes induced in the IR spectra upon a transition from protonated to deuterated species are only minor. Especially most of the more intense bands appear fairly conserved both with respect to location and intensity. This implies that they must originate from atoms and functional groups, which are not engaged in intra- or intermolecular 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 geometry for DFT/BP86 structure optimisations aimed at the equilibrium geometry of tetracycline in the zwitterionic form. In the case of the hexahydrate complex, the energy optimized geometry of the tetracycline zwitterion diers only very little from the starting geometry. As can be seen from the overlay presented in Scheme 2, the dierences are mainly restricted to the conformation of ring A and the orientation of its substituents. 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 the structural dierences concentrate on rings B and A and on the substituents connected to these. A comparison of the vibrational frequencies calculated for the two geometries allows an estimate how sensitive the dierent normal modes react to structural changes around ring A and which of them can eventually be taken as marker bands for determining the conformation. In this contribution, the emphasis is on vibrational bands connected with the BCD-chromophore. This also justies why the theoretical results obtained for the zwitterionic form are used for the comparison with the experimental spectra Phys. Chem. Chem. Phys., 2003, 5, 11491157 1151

Scheme 3 Overlay of tetracycline geometry calculated as hexahydrate (a) and nonahydrate (b).

give a complete listing of all calculated normal mode frequencies and a graphic presentation of the corresponding normal coordinates. Instead, we present only a description of those modes (Table 1 and Fig. 9) which can unambiguously be assigned to experimentally observed ones. Noteworthy in this context is thatin accordance with the experimental ndingmost of the intense IR bands are calculated to be essentially independent of deuteration. This fact can be used as an additional criterion when attempting an assignment. 3.3.
Fig. 4 Raman spectra of tetracycline in acidic D2O recorded with dierent excitation wavelength. (A) lex 400 nm, (B) lex 1064 nm. Also included are the IR spectrum (KBr pellet of the compound crystallized from D2O) (C) and the results of the quantum chemical calculations (the height of the bars is proportional to the IR band intensities).

IR and Raman spectra of derivatives of tetracycline

recorded from the fully protonated form of tetracycline. In the following, we will use the results calculated for tetracycline hexahydrate geometry for the band assignment. This procedure can yield, however, larger deviations between experimental and calculated wavenumbers of modes which involve movement of O3 or atoms of the amide group. In Figs. 3 and 4, some results of the quantum-chemical calculations are also incorporated. The location and height of the bars represent wavenumber and IR absorption intensity of the calculated normal modes, numbered consecutively according to increasing frequency. (Only for the modes with high IR intensity is the mode number indicated to avoid congestion.) The density of modes in the wavenumber range 1100 < ~ n /cm1 < 1500 is so high that a simple correlation between calculated vibrations and IR bands on the basis of the two criteria (position and intensity) is impossible. Consequently, we do not

Scheme 2 Overlay of the calculated minimum energy geometry of the protonated/deuterated zwitterionic form (a) of tetracycline hexahydrate and the X-ray structure (b) which was used as the starting geometry.

Generally we needed a concentration of ca. 0.1 M in order to produce NIR Raman spectra of good quality. Since the solubility of methacycline was not suciently high, solution spectra could not be recorded for this derivative. For the other three derivatives investigated, the spectra recorded from acidic H2O and D2O solutions are displayed in Figs. 1 and 2 for comparison with the spectra of tetracycline itself. The spectra of crystalline samples of all four derivatives are also included in Fig. 1 for the sake of completeness. Above 1300 cm1, the solution spectra of tetracycline and the three derivatives are very similar in appearance. Pronounced dierences are found in the wavenumber range 10001300 cm1, where one nds the in-plane deformation modes. The NIR-Raman spectra of the crystalline samples exhibit groups of medium to strong bands above 1600 cm1 and around 1300 cm1. Although one nds a great number of similar features when comparing the spectra of two arbitrarily chosen pairs of compounds, there are less common features for all derivatives than in the case of the solution spectra. This could be an indication that in solution the derivatives adopt the same state of protonation and a similar conformation. In contrast, it is known that the crystal structures and hydrogen bonding patterns are dierent for the various derivatives. Due to the resonance enhancement, Raman spectra could be recorded with lex 400 nm from solutions of all four derivatives (Figs. 5 and 6). As mentioned in a previous section, the general appearance of the NIR Raman spectra is the same for all derivatives investigated. When looking more closely, one notices that the various derivatives possess characteristic features. For example, in the fully protonated species, it is ~ the range 1200 < n /cm1 < 1400, where pronounced dierences occur. In the fully deuterated systems, one notices band shifts for the two strongest bands around 1512 and 1450 cm1. In the ngerprint region between 1050 and 1250 cm1, more or less pronounced changes can be found in the spectra of both the protonated and deuterated species. This can be rationalized since the change in substitution pattern should aect the CH-in plane bending modes localized around C5 to C7 more than the modes, which involve OH bending modes localized around C10, C12 and C12a.

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Table 1 Assignment of experimentally determined vibrations of tetracycline (fully protonated form) to calculated normal modes. All wavenumbers are given in cm1. Band intensities are characterised as strong (s), medium (m) and weak (w). For description of local modes and more details see text and Fig. 9 Deuterated ~ n calc(H) 1657 s 1597 w 1557 m 1569 m 1543 w 1516 1502 1505 1440 s w w w 1507 m/1503 m/-/-/1481 w 1478 w 1451 s -/1503 w -/-/1452 m/1451 w 1519 1230 1198 1453 s w w m -/1547 w 1512 s 1505 w/1512 m -/1563 w 1564 m 1543 w 1549 w/1568 w -/1585 w 1584 m 1573 w 1581 w/1586 m 1640 m/1639 m -/1624 m 1621 s 1652 w 1609 s 1639 m/1639 m 1609 s/1652 s 1607 m ~ n n IR/~R crystal ~ n R (NIR) solution ~ n R (NIR) solution ~ n n R/~IR crystal ~ n calc(D) Description

Protonated

Description

P180, n(CO1),z n(amid-CO), d(amid-NH) P173, n(CO1), n(amid-CO), d(amid-NH), n(CO3), n(C2C3), d(OH10,12) P169, d(amid-NH), d(amin-NH), n(Amid-CO), n(C2C3), n(CO3) P170, d(OH10,12), n(D), n(C11aC12)

P168 d(CH7), d(OH10,12), n(CO11), n(D), n(C11C11a) P167, d(amin-NH), n(C2C3), d(amid-NH) P165, d(amin-NH), d(amin-CH3) P166, d(amin-NH), d(amin-CH3) P158, d(CH7,8,9), d(OH12), n(D), n(CO10,CO11,CO12), d(amin-CH3), d(CH36), d(C11a), d(OH10), d(amin-NH) P161, d(OH10,12), d(CH7,8,9), n(D), n(CO10,CO11), n(C11C11a, C11aC12), d(OH12a), d(CH36) 1458 m 1450 m/1455 w 1443 s 1445 m/1446 w 1456 m 1387 w 1418 w/1424 w 1420 w 1418 w 1418 w/1418 w 1429 w

1380 m

1394 m/1393 w

1394 w

1395 m

1392 w/1392 w

1381 m

1313 w

1315 w/1316 s

1318 s

1333 w

1319 s/-

1332 w

D180, n(CO1), n(amid-CO), d(amid-NH), n(CO3) D178, n(CO1), n(amid-CO), d(amid-NH), n(amid-NC), n(CO3), n(C2C3), d(OH10) D177, d(OH10), n(D), n(C11aC12), d(CH8,9), n(CO11), d(amid-NH) D176, d(OH10), n(D), n(C11aC12), n(CO11), d(CH8,9) D173, d(CH7), d(OH10), d(OD12), n(CO11), n(C11C11a,C11aC12) D174, n(C2C3), d(amid-NH) D143, d(amin-ND), d(amin-CH3) D140, d(amin-ND), d(amin-CH3) D168, d(OH10), n(CO11), d(CH8,9), d(CH25), d(OH12a), n(D), d(CH36), n(C11C11a), n(C12C12a), d(amin-CH3) D169, d(OH10), n(CO11), d(CH8,9), d(CH25), d(OH12a), n(D), d(CH36), n(C11C11a), n(C12C12a), d(amin-CH3) D163, n(C6aC10a,C6aC7,C9C10), n(C10aC11, C11C11a,C12C12a), d(OH12a), n(CO10,CO11,CO12), d(OH10) D158, n(amid-C,C2), d(amid-NH), d(CH4a), n(C3C4), d(OH10,12a), d(CH36), n(amid-CN), n(C6aC10a,C6aC7,C8C9) D153, d(OH10), d(CH4,4a,5,5a), n(CO12), n(C10C10a), n(C6aC7,C9C10,C12C12a), d(CH7,8,9), d(CH3)6, d(OH12a)

1295 w

-/1292 s

1298 s

1303 w

-/-

1309 w

D151, d(CH4,4a,5,5a), d(amid-NH), n(C5a11a,C1C2), n(CO3), d(OH10), n(CO12)

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

1290 w 1278 w/1280 m 1279 s 1251 w -/1249 m 1251 m 1271 m

P151, d(OH10), n(C6aC10a, C6aC7,C8C9), n(C10aC11, C11C11a), d(CH36), n(amid-CC2), n(C2C3), d(OH12a), d(CH5,5a), d(C5aC6C6a) P150, n(amid-C,C2), n(C3,C4), d(CH4a,5), d(CH36), d(OH12a), d(amid-NH), n(amid-CN), d(OH10), n(C6aC10a,C6aC7) P143, d(OH10,12), d(CH4,4a,5,5a), n(C5aC11a), n(C1C2), n(C9C10, C10C10a, C10aC11), n(CO11,12), n(CO3), d(CH7,8,9) P142, d(CH4,4a,5,5a), d(OH12), n(C1C2), n(CO3), d(amid-NH), n(C11aC12), d(OH10), n(CO10, CO11), n(C10C10a,C6aC10a) P141, d(CH4,4a,5,5a), d(OH12), d(amid-NH), n(CO10), n(CO3), d(CH7,8,9), n(amid-NC), n(C4aC5), n(D) P137, d(CH4,4a,5,5a), d(OH10), d(CH7,8,9), d(OH12), n(D) 1265 s/-

1282 w

D148, d(CH4,4a,5,5a), n(CO10,11,12), d(CH8,9), n(C6aC10a,C12C12a,C2C3)

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Table 2 Position (in cm1) of bands (a. . .h) marked in the Raman spectra recorded from H2O solutions of tetracycline and derivatives (A. . .E) (Fig. 5) a A B C D E 1617 1616 1615 1615 1610 b 1559 1562 1557 1560 1557 c 1479 1478 1478 1478 1476 d 1462 1462 1463 1466 1463 e 1315 1313 1312 1301 1309 f 1296 1288 1300 g 1280 1278 1265 1278 h 1255 1254 1247 1253

(Figs. 5 and 6 and Tables 2 and 3), because they contain more or less exclusively normal modes that are localized on the BCD-chromophore and involve a signicant amount of skeleton vibrations. 3.4. pH-dependence of the Raman spectra of tetracycline

Fig. 5 Comparison of the resonance-enhanced Raman spectra (lex 400 nm) of various derivatives of tetracycline in acidic H2O. (A) tetracycline, (B) oxytetracycline, (C) doxycycline, (D) minocycline and (E) methacycline.

Although introduction of the vinyl group at C6 in methacycline extends the p-electronic system of the BCD-chromophore, the longest wavelength absorption band shifts by ca. 18 nm towards shorter wavelengths and may thus explain the lower cross-section for Raman scattering of this derivative. Furthermore, one can expect that the substituents modify the mixing pattern of the various types of local modes thus giving rise to changes in band position and relative intensities. This eect is most clearly seen in the resonance-enhanced spectra

In the crystalline state (tetracycline hexahydrate), tetracycline is present in the zwitterionic form, whereas in solution at pH 2 (pD 2), the fully protonated (deuterated) species, tcH3+, is prevalent (Scheme 1). The great similarity of the spectra recorded from crystalline samples and from acidic solutions can be rationalized if the presence or absence of a proton (deuteron) on O3 has only a small inuence on the vibrational spectra. Since pKa1 , which characterises protonation/deprotonation of O3, is ca. 3.3, at pH 3.9 approximately 70% of the molecules should exist in the zwitterionic form, tcH . The NIR 2 Raman spectra recorded at this pH show essentially the same features as at pH 2, thus indicating that deprotonation at O3 has essentially no eect on band positions and only small eects on relative band intensities. The same conclusion is true for tetracycline in D2O (Fig. 7). In alkaline solution where the dianion tc2 prevails, the Raman spectra should, in principle, be the same in H2O and D2O because all three exchangeable protons (OH3, OH12, NH4) are released. Small dierences in band position should be detectable only for those normal modes that are subject to intermolecular hydrogen (deuterium) bonding. The spectra displayed in Fig. 7 are in accord with this hypothesis. The expected eect is even more clearly demonstrated in the resonance-enhanced Raman spectra (lex 400 nm) displayed in Fig. 8. They also provide clear evidence that the normal coordinates, which are localized on the BCD-chromophore, undergo a major change above pH 8, when the proton at O12 is released.

4. Discussion
4.1. Assignment of the vibrations of tetracycline in acidied solution The calculated normal coordinates were analysed with respect to the ve criteria mentioned already: (i) Involvement of hydrogen atoms, which are subject to deuterium exchange.
Table 3 Position (in cm1) of bands (a. . .f) marked in the Raman spectra recorded from D2O solutions of tetracycline and derivatives (A. . .E) (Fig. 6) a A B C D E 1604 1604 1605 1608 1604 b 1508 1513 1510 1514 1512 c 1442 1444 1448 1454 1448 d 1389 1394 1396 e 1334 1331 1324 1331 f 1269 1262 1260 1266

Fig. 6 Comparison of the resonance-enhanced Raman spectra (lex 400 nm) of various derivatives of tetracycline in acidic D2O. (A) tetracycline, (B) oxytetracycline, (C) doxycycline, (D) minocycline and (E) methacycline.

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Fig. 8 Resonance-enhanced Raman spectra (lex 400 nm) of tetracycline with dependence on pH and pD, respectively. (A) pH 2, (B) pH 5.3, (C) pH 8.0, (D) pH 11, (E) pD 11 and (F) pD 2.

Fig. 7 NIR Raman spectra of tetracycline with dependence on pH and pD, respectively. (A) pH 2, (B) pH 3.6, (C) pH 3.9, (D) pH 11, (E) pD 11, (F) pD 3.5 and (G) pD 2.

(ii) Involvement of substituents, which are changed in dierent derivatives. (iii) Extent of resonance enhancement (localisation on the A- or BCD-chromophore). (iv) Calculated IR intensity. (v) Similarity of the band intensities in the NIR Raman spectra of tetracycline in acidic solution and in the crystalline state. Accordingly, an assignment of the experimentally observed vibrations to the calculated normal modes was established. It is summarized in Table 1. To characterise the normal coordinates, the usual abbreviations are used: n, d denote stretching and deformation vibration, respectively. The vibrating groups are given in parentheses whereby use is made of the numbering of the atoms displayed in Scheme 1. CC stretching vibrations of the phenyl ring D are referred as n (D) and the CO stretching vibration of the amide group as n (amide CO). The ordering of the local modes is according to their contribution to the normal coordinate. For the sake of simplicity, only the wavenumbers of the Raman bands found in the NIR spectra are quoted. The intensities are characterised as strong (s), medium (m) and weak (w). The adopted assignment can explain why many of the conserved bands are fairly insensitive to H/D exchange. For example, modes P180/D180 (1657/1652 cm1) involve a stretching of the carbonyl groups of the A-chromophore and a NH-deformation of the amide group. Consequently, P180 is resonance-enhanced in the Raman spectra with l 280 nm excitation. The IR bands around 1505 cm1 are assigned to modes P167 (1516 cm1) and D174 (1519 cm1), respectively, which again comprise movements of atoms of the A-chromophore. The conserved band at about 1395 cm1 (modes P150 and D158, respectively) is also fairly localized

on the A-chromophore. In contrast, the band around 1455 cm1 (P161/D169) is calculated to contain contributions from d (OH10) and d (OH12a), but also from deformation modes of the substituents on C5 and C6. Therefore, the band position can be expected to dier in tetracycline and its derivatives, as it is most clearly seen in Fig. 6. The most pronounced feature in the IR spectra of tetracycline is the rather intense and broad band around 1600 cm1. This is rather surprising since the calculations produce no normal mode in this wavelength regime which exhibits a high IR intensity. The intense band should therefore represent the superposition of a large number of weaker bands with small H/D isotopic shifts. Alternatively, one could speculate that the calculated wavenumber positions of modes P180/ D180 are badly reproduced because of the assumption of the wrong isomer (rotation of amide group). The latter argument would also be valid for the pair of modes P173/D178 and P169/D177 and could explain the incorrectly predicted H/D shift. (The problem of distinguishing isomers is addressed in a forthcoming publication in more detail.) The Raman spectra of tetracyclines in acidic D2O are dominated by bands around 1609, 1512 and 1445 cm1 with only the latter two being signicantly enhanced upon excitation with lex 400 nm. According to the chosen assignment, the normal coordinate of the band at 1609 cm1 (D178) resembles closely that of the 1639 cm1 IR band except that it should be considered the out-of-phase linear combination of the two CO stretching vibrations versus the in-phase one (D180). Since it is also located on the A-chromophore, the low resonance enhancement for lex 400 nm is not surprising. In contrast, the normal mode D173 (1512 cm1) is essentially conned to motions of atoms of the BCD-chromophore. Because of a low contribution of d (OH12/OD12) the equivalent normal mode in the protonated system is found around 1545 cm1 (P168), but with very low intensity. The doublet at 1443/ 1451 cm1 is assigned to the modes D169 and D168 (1456/ 1453 cm1). It is, as already mentioned above, delocalised across the whole BCD-fragment and, therefore, subject to Phys. Chem. Chem. Phys., 2003, 5, 11491157 1155

strong resonance enhancement for lex 400 nm. In the theoretical spectrum of the protonated species, one also nds a pair of vibrations representing the out-of-phase and in-phase linear combination of certain local modes: P161 and P158 (1458/ 1440 cm1). Of the normal modes assigned to the three remaining bands in the D2O spectrum at 1395, 1333 and 1271 cm1, the latter two are also completely delocalised across the BCDchromophore. The band positions dier somewhat from those found in the crystalline material. The shift could be caused by dierent intermolecular hydrogen/deuterium bonding. Only for the 1395 cm1 mode (experimental), one nds a counterpart in the theoretical spectrum of the protonated species with essentially the same wavenumber (D158/P150). For the two lower energetic vibrations, it is possible to establish a correlation between the modes of the protonated and deuterated species (P143/D153 and P137/D148). They exhibit, however, a completely dierent mixing pattern of the local modes. This can be rationalized, because upon switching to the protonated solvent the concomitant D/H exchange at O12 results in a pseudo-resonance between d (OH12) with the corresponding local modes of OH10 and OH12a. The normal modes, which are obviously strongly enhanced in the Raman spectra of the protonated system, contain a signicant amount of d (OH12) local mode (P143/1318 cm1 and P142/1298 cm1). In addition, there are signicant contributions of the d (CH4, 4a, 5, 5a). Consequently, the calculated wavenumbers dier somewhat for the geometries calculated for the hexahydrate and the nonahydrate (e.g., 1313 vs. 1332 and 1295 vs. 1309 cm1). The given assignment provides a rationalization of the relative band shifts observed for the various derivatives investigated (Figs. 5 and 6). Those normal modes, which involve no signicant movement of C7, C6 or C5 are fairly conserved in their position (e.g. P173, P170, P161, D178, D173). In the protonated system, the most pronounced changes are seen in the range 1200 to 1350 cm1 (Table 2). The modes assigned to this region are P137, P141, P142 and P143. The description of these modes presented in Table 1 shows for all modes large contributions of d (CH4,4a,5,5a) and thus explains the sensitivity to substitution in this part of the molecule. In the deuterated system, the corresponding modes D153 and D148 are shifted because of the isotope eect on the contributing local modes d (OH12,10) (Table 3). Because of the contribution of d (CH4,4a,5,5a) their appearance changes with substitution. The very well separated mode D168 contains only smaller contributions of the relevant local modes d (CH25) and d (CH36). Therefore, its shift with substitution is less pronounced but clearly seen in the spectra of doxycycline, minocycline and methacycline. 4.2. Eect of deprotonation

Fig. 9 Corresponding pairs of normal coordinates of the protonated (left side) and deuterated (right side) zwitterionic form of tetracycline. The numbering of the modes is as in Table 1. Included are also the wavenumbers (in cm1) of the calculated and NIR-FT Raman spectra.

Inspection of Figs. 7 and 8 leads to the conclusion that two of the stronger bands observed in the Raman spectrum of tetracycline at pD 2 are conserved in position despite complete deprotonation at pD 11 (1438 cm1 and 1271 cm1), whereas another two are slightly shifted (1609 cm1 to 1600 cm1 and 1333 cm1 to 1316 cm1). The strong band around 1512 cm1 (pD 2) seems to disappear, as does the weaker band originally found around 1395 cm1. In case of the fully protonated species, tcH3+, the establishment of a similar correlation is much more dicult for some of the bands found in the spectrum of the alkaline solution. The situation seems to be identical to that in the deuterated molecule for the bands at about 1600 and 1258 cm1 (pH 11). The band at 1431 cm1 most likely corresponds to the band around 1455 cm1, the precursor of the band around 1306 cm1 could be that around 1318 cm1 (pH 2). Since the valleys between the stronger bands of the fully protonated systems are not very pronounced, one can speculate that there are more bands present than those mentioned. Hints

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in this direction can be found in the NIR Raman spectra. The assignment given for the normal modes of the fully deuterated system allows rationalization of the described behaviour. For example, mode D169, which represents the intense band around 1443 cm1 (pD 2), contains preferentially d (CH) and d (OH) contribution, but nearly no d (OD12). Therefore, release of the deuterium at O12 should leave the mode unchanged. The band around 1333 cm1 is assigned to mode D153, which contains some n (CO12) and d (CH). The observed small shift upon transition to the dianion at pD11 could be due to the frequency shift of the CO12 stretching vibration as consequence of deprotonation or to change of the d (CH) vibration as the consequence of a change of the overall conformation. The strong band around 1512 cm1 (D173) involves a signicant amount of d (OD12) next to n (CC) of the diketone moiety. This fact may explain the dramatic decrease of intensity when going to the dianion. It is also noteworthy that all three bands mentioned involve a large contribution of d (OH10). The degree of intramolecular hydrogen bonding to O11 could be modied with deprotonation at O12. In the protonated system, the modes that exhibit the corresponding vibration patterns as D169, D153 and D173 are P161 (1455 cm1), P143 (1318 cm1) and P168 (1547 cm1). In P161 the contribution of d (OH12) is increased, that of d (OH12a) decreased versus D169, thus explaining the 12 cm1 shift of this band upon deuteration. Modes P143 and P168 also contain a large contribution of d (OH12), as manifested by band shifts of 15 cm1 (upwards) and 35 cm1 (downwards), respectively.

acknowledged. We also appreciate a grant by the European Union TMR Large Scale Facility Access Programme. Furthermore, we thank Hovione, Spain, for the donation of samples.

References
1 B. M. Duggar, Ann. N. Y. Acad. Sci., 1948, 51, 177. 2 Tetracyclines in Biology, Chemistry and Medicine, ed. M. Nelson, W. Hillen and R. A. Greenwald, Birkhauser Verlag, Basel, 2001. 3 M. L. Nelson, in Tetracyclines in Biology, Chemistry and Medicine, ed. M. Nelson, W. Hillen and R. A. Greenwald, Birkhauser Verlag, Basel, 2001. 4 M. A. Ryan and D. M. Baker, in Tetracyclines in Biology, Chemistry and Medicine, ed. M. Nelson, W. Hillen and R. A. Greenwald, Birkhauser Verlag, Basel, 2001. 5 R. Hanemaaijer, N. van Lent, T. Sorsa, T. Salo, Y. T. Konttinen and J. Lindeman, in Tetracyclines in Biology, Chemistry and Medicine, ed. M. Nelson, W. Hillen and R. A. Greenwald, Birkhauser Verlag, Basel, 2001. 6 C. Gatz and P. H. Quail, Proc. Natl. Acad. Sci., 1988, 85, 1394. 7 M. Gossen and H. Bujard, in Tetracyclines in Biology, Chemistry and Medicine, ed. M. Nelson, W. Hillen and R. A. Greenwald, Birkhauser Verlag, Basel, 2001. 8 B. R. Martin, in Antibiotics and their complexes, Marcel Dekker, New York, vol. 19, 1985. 9 M. O. Schmitt and S. Schneider, Phys. Chem. Commun., 2000, 9, 359. 10 S. Schneider, in Tetracyclines in Biology, Chemistry and Medicine, ed. M. Nelson, W. Hillen and R. A. Greenwald, Birkhauser Verlag, Basel, 2001, p. 65. 11 L. A. Mitscher, in The Chemistry of Antibiotics, Marcel Dekker, New York, 1978. 12 W. Hinrichs, C. Kisker, M. Duvel, A. Muller, K. Tovar, W. Hillen and W. Saenger, Science, 1994, 264, 418. 13 J. M. Wessels, W. E. Ford, W. Szymczak and S. Schneider, J. Phys. Chem. B, 1998, 102, 9323. 14 L. A. Mitscher, A. C. Bonacci and T. D. Sokoloski, Antimicrobial Agents and Chemotherapy, 1968, 8, 78. 15 L. J. Hughes, J. J. Stezowski and R. E. Hughes, J. Am. Chem. Soc., 1979, 101, 7655. 16 C. Leypold, PhD Thesis, Universitat Erlangen-Nurnberg, Germany, 2003. 17 P. Matousek, M. Towrie, A. Stanley and A. W. Parker, Appl. Spectrosc., 1999, 53, 1485. 18 P. Matousek, M. Towrie, C. Ma, W. M. Kwok, D. Phillips and A. W. Parker, J. Raman Spectrosc., 2001, 32, 983. 19 R. Ahlrichs, M. Bar, M. Haser, C. Horn and C. Kolmel, Chem. Phys. Lett., 1989, 162, 165. 20 A. D. Becke, Phys. Rev. A, 1988, 38, 3098. 21 J. P. Perdew, Phys. Rev. B, 1986, 33, 8822. 22 J. Neugebauer, M. Reiher, C. Kind and B. A. Hess, J. Comput. Chem., 2002, 23, 895. 23 M. Reiher, J. Neugebauer and B. A. Hess, Z. Phys. Chem., 2003, 217, 91. 24 D. Sellmann, T. Gottschalk-Gaudig, D. Hauinger, F. W. Heinemann and B. A. Hess, Chem. Eur. J., 2001, 7, 2099. 25 J. Neugebauer and B. A. Hess, J. Chem. Phys., in press. 26 K. Eichkorn, O. Treutler, H. Ohm, M. Haser and R. Ahlrichs, Chem. Phys. Lett., 1995, 240, 283. 27 K. Eichkorn, F. Weigend, O. Treutler and R. Ahlrichs, Theor. Chem. Acc., 1997, 97, 119. 28 A. Schafer, C. Huber and R. Ahlrichs, J. Chem. Phys., 1994, 100, 5829. 29 C. Kind, M. Reiher, J. Neugebauer and B. A. Hess, SNF a program for vibrational analyses, Universitat Erlangen-Nurnberg, 19992001, http://www.chemie.uni-erlangen.de/hess/html/ downloads.html. 30 G. Brehm, M. Reiher and S. Schneider, J. Phys. Chem. A, 2002, 106, 12 024. 31 S. Schneider, G. Brehm, M. Schmitt, C. Leypold, M. Reiher, P. Matousek and M. Towrie, in Annual Report, Rutherford Appleton Laboratory, 20012002, p. 100.

5. Summary and conclusion

The NIR Raman spectra recorded from solutions of tetracycline at dierent pH provide evidence that deprotonation of the A-chromophore has only a minor eect on positions and relative intensities of bands located on the BCD-chromophore. This can be understood if this deprotonation does not induce a signicant change of the geometry of the latter. This assumption is conrmed by the results of the DFT/BP86 calculations. The energy optimised geometry of the zwitterion matches that of the hexahydrate as determined by X-ray crystallography. Consequently, the theoretical spectra determined for the zwitterionic forms (protonated and deuterated) were used for an assignment of bands observed in IR spectra of crystalline samples and Raman spectra of solutions employing dierent excitation wavelengths. The normal coordinate pictures produced allow one to rationalize the eects of H/D exchange, as well as the eects of dierent substitution patterns. The successful assignment of the resonance-enhanced Raman spectra of tetracyclines in alkaline solution also allows conclusions to be drawn concerning the site of complexation by divalent metal ions. Whereas upon complexation by Mg2+, the strong band around 1450 cm1 is essentially unaffected, it disappears nearly completely upon complexation with Ca2+ or Eu3+.31 Since, according to our assignment, this band corresponds to P161/D169 with contribution of d (OH12a), its disappearance provides evidence for Ca2+ complexation to involve OH12a. Because metal ion complexation is of utmost importance for biological action, experimental data and the results of further quantum chemical calculations will be presented in a forthcoming publication.

Acknowledgements
Financial support by Deutsche Forschungsgemeinschaft (SFB 473) and Fonds der Chemischen Industrie is gratefully

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