The Influence of Molecular Structure Modifications on Vibrational Properties of Some Beta Blockers : A Combined Raman and DFT Study

We report results of a systematic Raman, SERS, andDFT study on four beta blockingmolecules: Atenolol, Metoprolol, Propranolol, and, for the first time reported in the literature, Bisoprolol.The choice of thesemolecules wasmotivated by the structural similarities between Atenolol, Bisoprolol, and Metoprolol on one hand and by their differences relative to Propranolol. The density functional theory (DFT) approach, using the B3LYPmethod at the 6-311+G(d,p) level of theory, has been employed for geometry optimization and vibration bands assignments.The obtained results highlight themajor role played by the central aromatic ring whose vibrations dominate the Raman spectra in all compounds. While the phenyl group vibrations dominate the Raman spectrum in the case of Atenolol, Bisoprolol, and Metoprolol, the spectrum of Propranolol presents high intensity vibrations of the naphthyl group. SERS performed on gold and silver colloids, at various pH conditions, revealed a higher sensitivity for Propranolol detection. The pH dependence of the spectrum indicates that the studied beta blockers attach themselves to the metal nanoparticles in a protonated form.Themolecular adsorption geometry onmetal nanoparticles surface has been evaluated by using the experimental SER spectra and the quantum chemical calculations.


Introduction
Raman spectroscopy and its more recent development Surface Enhanced Raman Spectroscopy (SERS) represent very powerful vibrational spectroscopy techniques allowing highly sensitive detection of low concentration analytes, in different media [1].As a consequence of the fact that a single Raman scan can provide molecular specific spectral data in the 40-4000 cm −1 range, Raman spectroscopy gained a lot of popularity in different areas of pharmaceutical industry.Its ability to provide a high degree of specificity in analysis and its capacity to attain a high degree of lateral and depth resolution represent other powerful arguments for its future use in pharmaceutical industry.In the case of SERS, high amplifications are observed for the Raman modes which are perpendicular to the metallic surface, thus offering the possibility to investigate molecular orientation at the metallic surface and eventually the functional groups involved in the molecular attachment to the metallic surface.However, the complete interpretation and understanding of experimental Raman/SER spectra of molecules of pharmaceutical interest under different experimental conditions are not straightforward, the subject being under constant debate in the scientific literature.
The beta blockers are primarily used in the therapy of cardiovascular diseases [2].Propranolol (PRNL) (1-isopropylamino-3-(1-naphthyloxy)-2-Propranolol) was the first beta blocking molecule used in cardiovascular therapy.It is a nonselective beta blocking agent, having a membrane stabilizing activity owing to the fact that it is the most hydrophobic compound among all beta blockers.Bisoprolol (BIS) (1-{4-{[2-(1-Methylethoxy) ethoxy]methyl}phenoxy}-3-[(1-methylethyl)amino]-2-propanol), Atenolol (ATE) ((±)-4-[2-Hydroxy -3-[(1-methylethyl)amino]propoxy]benzeneacetamide), and Metoprolol (MET) ((±)1-(Isopropylamino)-3 -[p-(-methoxyethyl)phenoxy]-2-propanol (+)) represent other three drugs belonging to the class of beta1-adrenergic receptor blockers, able to specifically block adrenergic receptors from heart muscle and kidneys, with little activity against beta2-adrenergic receptors of the lungs and vascular smooth muscle, having a very similar chemical structure.As a common structural feature, all currently available beta blockers possess an amino-alkanol side chain and an aromatic group: phenyl in the case of ATE, BIS, and MET and naphthyl in the case of PRNL.The amino-alkanol side chain is characterized by the presence of an asymmetric carbon atom, resulting in the existence of a d-and an lenantiomer (in the case of propanolamine type compounds, like the ones studied in this paper, the d-enantiomers show the (R)-configuration and the l-enantiomers show the (S)-configuration), the latter being more potent in blocking the adrenergic receptors.These drugs are used in clinical practice as racemates, although in vitro studies have demonstrated in the early 60s that l-enantiomers are orders of magnitude more potent than their d-enantiomer counterparts [3].Over a long period of time it was considered that the d-enantiomers have no side effects, but during the years, more and more studies reported highly specific side effects of the d-enantiomers of beta blockers.Therefore, a "chiral switch," that is, replacing the currently clinically used racemic mixtures with optically pure l-enantiomers, was proposed [4].On the other hand, beta blockers are prohibited in some sports according to the List of Prohibited Methods and Substances, released every year by the World Anti-Doping Agency [5].As a consequence there is a need for a sensitive and reliable technique able to detect and measure the beta blocker concentration in body fluids.
The first use of Raman and SERS for beta blockers detection was reported by Rupérez and Laserna in 1996 [6].More recently Levene et al. [7] were able to determine the best experimental parameters leading to the detection of PRNL down to a concentration of 2.36 ng/mL (7.97 nM), well within the physiological concentrations.In previous papers published in our group [8] the SERS, Raman, and DFT were synergistically employed for elucidating the adsorption geometry of PRNL molecules on metallic nanoparticles surface.The nanoparticles mediated enantioselective recognition of PRNL enantiomers through native cyclodextrin complexation by using SERS was also reported [9,10].Cozar et al. [11] reported vibrational studies together with DFT simulations for ATE and MET and proposed possible adsorption geometries for these molecules on metallic surface.
In this paper we present a systematic Raman, SERS, and DFT investigation of four widely used beta blockers: ATE, BIS, MET, and PRNL.By extending the object of this study to four beta blocking molecules having very similar structures it was possible to better understand the relationship between the chemical structures and the vibrational properties of the molecules in a holistic manner.The conventional Raman (solid phase) and SERS spectra of BIS are also reported for the first time.We investigated the structural and vibrational similarities between ATE, MET, and BIS and compared them with PRNL.The choice of the four molecules is motivated by the fact that the main structural difference between ATE, MET, and BIS on one hand and PRNL on the other hand is represented by the central aromatic group which is a phenyl in the case of the first three and a naphthyl in the case of PRNL.Thus, it was possible to detect the main changes in the Raman spectra induced by this replacement.For a better understanding of the vibrational spectra and for a correct assignment of the vibrational frequencies the density functional theory (DFT) approach using the B3LYP method at the 6-311+G(d,p) level of theory has been employed.The calculations have been performed for the ground state geometry of the most stable species.The SERS experiments have been performed on both silver and gold colloids in various pH conditions for assessing the availability of the SERS method for detecting the beta blockers and to compare the SERS sensitivity among the studied molecules.The molecular adsorption geometry on noble metal nanoparticles surface has been evaluated by using the experimental SER spectra and the quantum chemical calculations.

Preparation of Colloids.
Silver colloids were prepared according to the Leopold and Lendl method [12].An amount of 0.017 g of silver nitrate was dissolved in 90 mL of double distilled water.In a separate recipient, 0.017 g of hydroxylammonium chloride was dissolved in 10 mL of water, followed by the addition of 250 mL of aqueous sodium hydroxide solution (0.1 M).This solution was then added rapidly to the silver nitrate solution under vigorous stirring.After few seconds, a grey-brown colloidal solution was obtained which was further stirred for 10 minutes.The pH value of the silver colloid, measured immediately after preparation, was 6.9.The UV-Vis spectrum of the silver colloidal solution exhibits a single narrow absorption peak at 418 nm, indicating the formation of spherical silver nanoparticles.
Gold colloid has been prepared by dissolving 0.0485 g of tetrachloroauric(III) acid trihydrate in 100 mL of water and boiled under vigorous stirring.A volume of 3 mL of 1% sodium citrate aqueous solution was then added dropwise while boiling and stirring.The solution was allowed to boil for further 20 minutes with continuous stirring.A significant color shift of the aqueous solution to a dark red color was observed [13].By the end of the synthesis the total volume of the colloidal solution was adjusted to 100 mL by water addition.The pH value of the gold colloid, measured after cooling to room temperature, was 6.The UV-Vis spectrum of the gold colloidal solution presents a single narrow absorption peak at 522 nm, indicating the formation of spherical gold nanoparticles.

Methods and Instrumentation.
Raman spectra (solid phase) and some SER spectra were recorded on a Renishaw micro-Raman spectrometer, using 3 wavelengths laser lines (532 nm, 633 nm, and 785 nm with powers up to 200 mW, 17 mW, and 300 mW, resp.).The system is integrated with a Leica optical microscope with 50x objective lens.The powder samples or a droplet of colloidal solution were placed on an aluminum plate in front of the lens.Each spectrum represents the average of minimum 5 recordings, the acquisition time for each spectrum being usually 5 s.A part of the SER spectra has been acquired in back-scattering geometry in the 200-2000 cm −1 range using a DeltaNu Advantage spectrometer (DeltaNu, Laramie, WY) equipped with a laser diode emitting at 785 nm.The laser power was 100 mW and the spectral resolution was 5 cm −1 .The spectra were recorded for 1 mL glass vials filled with 540 L of colloid and 60 L of the analyte.In some experiments, as indicated in the figure legends, 100 L of 1 M NaCl was added in order to produce the aggregation of the nanoparticles and the creation of "hot spots."The pH of the solutions was adjusted with solutions of HCl and NaOH before the addition of the aggregating agent.Each SER spectrum is the average of five recordings taken with an acquisition time of 30 seconds.The UV-Vis absorption spectra of colloidal solutions were recorded using a T92+ UV-VIS Spectrophotometer from PG INSTRUMENTS, using standard 1 cm quartz cells at room temperature, over a spectral range between 190 nm and 900 nm with a spectral resolution of 2 nm.

DFT Computations.
All the calculations related to molecular geometry optimization and generation of theoretical vibrational spectra were performed using Gaussian 09 software package [14] based on density functional theory (DFT) methods at the B3LYP/6-311+G(d,p) level of theory [15,16].No symmetry restrictions were applied during the optimization process.The calculated Raman activities (  ) were converted into relative Raman intensities (  ) using the relation given as follows [17,18]: Here  is a normalization factor for all peak intensities,  0 represents the exciting laser wavenumber,   is the wavenumber of the th vibrational mode,  stands for the speed of light, ℎ and   are Planck's and Boltzmann's constants, and  represents the temperature.Since the vibrational wavenumbers obtained by quantum chemical calculation are typically larger than their experimental counterparts, the computed wavenumbers for the frequencies larger than 1000 cm −1 have been scaled by 0.967 as proposed by Scott and Radom [19].

Results and Discussions
The structures of the most stable conformers of the 4 beta blockers studied in this paper are presented in Figure 1 together with the atoms numbering.The chemical structure and the optimized geometry of the molecules were calculated at B3LYP/6-311+G(d,p) level of theory.The vibrational frequencies were computed for the optimized geometries in order to avoid the occurrences of imaginary frequencies.Consequently, the optimized geometry corresponds to a local minimum on the potential-energy surface.At a closer look of their structure one can observe that the common structural features of these molecules are the amine alkanol side chain containing the asymmetric carbon atom, the amino groups, and the OH groups.The central part of the molecules consists of an aromatic group which is a phenyl one in the case of ATE, BIS, and MET and a naphthyl one in the case of PRNL.No other side chain exists in PRNL.ATE possesses an acetamide group bound to the central phenyl group, while BIS and MET possess propoxyethoxy and methoxyethyl groups, respectively.
The experimental Raman spectra of the 4 compounds are presented in Figure 2. One can easily see the likeness of the Raman vibration patterns in the case of ATE, BIS, and MET on one side and the complete different pattern recorded for PRNL.In the case of PRNL the Raman spectrum is dominated by two strong vibrations at 1385 cm −1 and 737 cm −1 while in the case of the other three beta blockers there are several high intensity common bands at ∼1612 cm −1 , ∼ 850 cm −1 , and ∼638 cm −1 .These differences can be attributed to the substitution of the phenyl group of ATE, MET, and BIS with the naphthyl group in the case of PRNL, as we will demonstrate bellow.
For getting more insights of the relation between the molecular chemical structure and the Raman spectra, the simulated and experimental Raman spectra of the studied beta blockers are shown in Figure 3.The vibrational bands assignments obtained from DFT for all beta blocking molecules are indicated in Tables 1 and 2. For BIS (Figure 3(a)) we obtained a good correlation between the experimental and the simulated spectra.The most intense experimental Raman bands are at 638 cm −1 (652 cm −1 in DFT), assigned to the (CCC ring), 823 cm −1 and 849 cm −1 (835 cm −1 and 859 cm −1 in DFT), both assigned to ring breathing, 1612 cm −1 (1597 cm −1 in DFT), assigned to ](CC ring), and 1173 cm −1 (1182 cm −1 in DFT), assigned to the (CC, ring).It is worthy to mention that all these vibrations involve the central phenyl ring of the molecule.
For ATE (Figure 3(b)) we experimentally measured the same intense bands as in the case of BIS, attributable to ring vibration modes at only slightly different wavenumbers (637 cm −1 , 828 cm −1 , 859 cm −1 , 1183 cm −1 , and 1611 cm −1 ) (Table 1), and vibrations occurring at 1457 cm −1 , 1205 cm −1 , and 1301 cm −1 with the same assignments as in the case of BIS: (CH 2 ) + (CH ring), ] as (CO), and different CH 2 vibration modes, respectively.The major differences in the Raman spectra of the two beta blockers were assigned to the acetamide group present in the ATE molecule.At 368 cm −1 an intense vibration band was assigned to the rocking mode of N 5 H 2 group, specific for this molecule, an increase in the intensity of the vibrational band from 722 cm −1 (assigned in the case of BIS only to a CCC ring wagging mode) due to a supplementary contribution for the rocking of N5H 2 group, and a weaker but clearly noticeable vibration mode at 1680 cm −1 assigned to a ](C 19 O 3 ) + (N 5 H 40 ).The peak from 1420 cm −1 can be assigned to a combined (C 18 H 2 ) and (N 5 H 2 ) vibration mode.
In the case of MET (Figure 3(c)) the major vibrations obtained in the Raman spectrum are again, like in the case of ATE and BIS, attributed to vibration modes of the ring atoms, that is, 848 cm −1 ring breathing, 640 cm −1 (CCC ring), and 1614 cm −1 and 1585 cm −1 (CC ring).Other intense bands were recorded at 1461 cm −1 assigned to (C 18 H 2 ) and (C 19 H 2 ) and similar to BIS and ATE the bands at 1210 cm −1 , 1180 cm −1 , and 1164 cm −1 assigned to (O 2 H 35 ), (C 13 H 34 ), and (CH ring), respectively.As it was the case for ATE and BIS, in the 900 cm −1 -1000 cm −1 region two supplementary medium intense bands are observed in the Raman spectrum of MET at 935 cm −1 and 966 cm −1 , which are assigned to (C 11 H 2 ) + (C 10 H 3 ) and (CH ring) + (C 18 H 2 ), respectively.
The experimental and simulated spectra for PRNL are very different as compared to ATE, BIS, and MET.The Raman spectrum (Figure 3(d)) is dominated by the 1383 cm −1 and 737 cm −1 bands assigned to the naphthyl ring vibration modes.Other medium intensity vibration modes were recorded at 1578 cm −1 (1560 cm −1 in the simulated spectrum) and at 1439 cm −1 (1423 cm −1 in the simulated spectrum) which are also associated with different modes of naphthyl group vibration.As a general observation we notice a very good correlation of the simulated spectrum with the experimental one both from the point of view of the wavenumbers   and relative peak intensities, as can be seen in Figure 3(d) and in Table 2.
Finally, the mean of the absolute wavenumber deviations between experiment and theory for the most important bands are ± 11 cm −1 for ATE, ±9 cm −1 for BIS, ±12 cm −1 for MET, and ±16 cm −1 for PRNL.Moreover, the root mean square standard deviation (RMSD) of residuals is 8 cm −1 in the case of ATE, 6 cm −1 for BIS, 9 cm −1 for MET, and 11 cm −1 in the case of PRNL.These discrepancies can be observed in Table 1 where one can also find the assignments of the normal modes.The assignments of experimental vibrational bands were made by visual correlation with calculated vibrational bands using Gauss View 5.0, taking into account the wavenumber values and relative intensities.
The SER spectra of PRNL on both silver and gold colloids are represented in Figure 4 and they are fully consistent with data previously reported by Stiufiuc et al. [8].We notice that the addition of PRNL to the colloidal solutions changes the color of both gold and silver colloids from red to blue and light grey to dark grey, respectively.This phenomenon can be attributed to the aggregation of the colloid due the presence of Cl − anions since PRNL was used as a hydrochloride salt.This aggregation leads to the formation of "hot spots" having increased Raman enhancements factors and higher sensitivity.The general pattern of the SER spectra of PRNL is the same for both silver and gold colloids.However, small differences can be observed.The spectra are dominated by the naphthyl ring vibration at 1383 cm −1 and by the ring breathing at 737 cm −1 with the wavenumbers being very close to the pure Raman vibrations.This small difference in the wavenumbers (∼2 cm −1 ) indicates a weak interaction of the molecule with the metallic surface.Other medium intensity bands were recorded at 1440 cm −1 and 1577 cm −1 , assigned to the ring CC stretching and ring CH bending vibrations, respectively.However there is a difference in the relative intensity of these Raman intensity (a.u.) Raman intensity (a.u.) two bands for the two colloids; the 1577 cm −1 band is more intense for Au colloid, whereas the 1440 cm −1 peak is more intense for Ag colloid.These differences can be explained by small differences in the orientation of the molecule at the metallic surface and by different interaction strengths with the metals, thus emphasizing the role of the metallic surface in SERS measurements.
Stiufiuc et al. have demonstrated that the adsorption of PRNL on the metallic surface occurs through the oxygen atoms with the naphthalene ring lying perpendicular to it [8].Their explanation was based on experimentally acquired SER spectra, Raman assignments, Molecular Electrostatic Potential (MEP) contour map, and SERS selection rules stating that the normal modes having a change in the polarizability component normal to the surface are enhanced [20,21].Our results, based on the used DFT method are in good agreement with these findings.The SER spectra of ATE, MET, and BIS, obtained on silver colloids, are presented in Figure 4 (upper frame).It is worth mentioning that for analytes concentrations of 10 −3 -10 −4 M no specific SER spectra could be recorded without the addition of an aggregating agent.ATE, BIS, and MET used in this study were free bases, whereas PRNL was a hydrochloride salt.The different behaviour of PRNL on one side and ATE, BIS, and MET, on the other side, can be attributed to the presence/absence in the colloidal solution of Cl − anions.
In order to increase the Raman signal for MET, BIS, and ATE, NaCl was added up to a concentration of 10 −4 M to the colloid-analyte mixture.Previously, several other salts (NaF, KBr) were tested, in order to assess the eventual fake vibration peaks belonging to the capping agents, used in the synthesis process, or to the impurities in the colloidal suspension strongly enhanced by colloid aggregation.In agreement with previous reports [22,23], where either NaCl or LiCl were added to the colloid suspension, any additional vibrational bands were not detected in the SER spectra, apart from the well-known metal-Cl − band, situated in the low wavenumber region (227 cm −1 for Ag-Cl and 250 cm −1 for Au-Cl).Therefore, the Cl − anions are the most suitable to increase the Raman signal.The SER spectra of all three beta1 blockers are dominated by the breathing vibrations of the phenyl ring at 848 cm −1 for BIS, 859 cm −1 for ATE, and 849 cm −1 for MET.The small difference between the Raman vibrational bands and the SERS ones indicate a small interaction between the analyte and the metallic surface.Other vibrations belonging to the ring are also enhanced: 635 cm −1 , 821 cm −1 , and 1609 cm −1 for BIS, 638 cm −1 and 824 cm −1 for ATE, and 637 cm −1 , 823 cm −1 , and 1609 cm −1 for MET.It is interesting to note that the 1612 cm −1 CC ring stretching vibration is not enhanced in the SER spectrum of ATE but the 1549 cm −1 band, assigned to a combined CC ring stretching and the bending of N 5 H 2 has a medium intensity, while in the case of MET and BIS the vibrational bands at 1612 cm −1 are enhanced.This different behaviour of ATE with respect to the other two similar beta blockers (MET and BIS) can be explained by a different orientation of the molecule to the surface, the enhancement of the 1549 cm −1 band being due to the N 5 H 2 bending (a group specific only for ATE).Also, as it can be observed from the SER spectrum of ATE, the amide I vibration band at 1681 cm −1 is lacking.This is an indication that the carbonyl group from acetamide lies in a configuration parallel to the metallic surface.ATE presents a clearly distinguishable vibration band at 725 cm −1 , corresponding to 722 cm −1 in the Raman spectrum with a higher intensity as compared to the other two beta blockers, attributed to a supplementary contribution due to a rocking vibration of the same N 5 H 2 group.
Other group of medium intensity vibration bands were recorded at 1172 cm −1 for BIS and 1183 cm −1 for both ATE and MET.These bands were assigned to different CC aliphatic chain vibration modes.The weak vibrations at 1448 cm −1 for BIS and ATE and 1446 cm −1 for MET were assigned to CH bending vibration modes.The mechanism of molecular attachment to the metallic nanoparticles surface was proposed to take place through the amino group of the amino alkanol side chain up to a pH value of ∼9.5 which is in fact the pKa for this amino group [8,11].Bellow this pH value the molecule is mostly protonated and can be electrostatically attracted by the nanoparticles.The pH dependence of the SER spectra for ATE, BIS, and MET (data not shown) demonstrates that as the pH is increased from acidic values the intensity of the SERS peaks increases up to pH = 9.For pH values greater than 11 the SER spectra could not be detected as the molecules became deprotonated.The same behaviour was proved experimentally for Propranolol [8].The main information concerning the molecules orientation at the surface can be obtained from the SER spectra.The possible groups involved are either the protonated amino groups, the lone electron pairs of N and O atoms, or the  electrons of the phenyl ring.A flat orientation of the ring to the surface would imply a dramatic decrease in the SERS intensity corresponding to the ring vibrations, which is not the case.The fact that the ring vibration dominates the SERS spectra for the studied beta blockers implies that the aromatic rings possess a tilted position with respect to the metallic surface.The relatively low enhancement of the vibration bands in SERS for BIS, ATE, and MET indicated that the adsorption onto the silver surface is done through a weak physisorption process.The MEP maps data (not shown) indicates that the most negative part of the molecules is around the oxygen atoms; therefore we cannot preclude their involvement in the attachment.

Conclusions
A systematic Raman, SERS, and DFT study was performed on four beta blocker molecules.The used DFT theoretical methods allowed us to obtain the optimized geometries, the assignment of vibrational bands, and the MEP maps.The Raman spectra revealed the major role played by the central aromatic group, the ring vibrations dominating the overall spectra in the 200-2000 cm −1 region.We also noticed a great degree of "vibrational" similarity for BIS, ATE, and MET.The replacement of the phenyl ring (existing in ATE, BIS, and MET) with a phenyl one (for PRNL) leads to a dramatic change in the Raman pattern.In this case an increased domination of the ring vibrations has been recorded on the Raman spectrum.The SERS results show a higher sensitivity for PRNL as compared to BIS, ATE, and MET.The SERS mechanism rely mainly on molecular physisorption to the metallic surface through weak electrostatic forces acting between the protonated form of the molecule and the metallic nanoparticles.The molecular orientation relative to the surface is most probably determined by the oxygen atoms with the central ring having a tilted orientation relative to the nanoparticles surface.

Figure 3 :
Figure 3: Experimental and simulated Raman spectra of the studied beta blocking molecules.In the case of Propranolol the experimental intensities at high wavenumbers were multiplied 10 times.

Figure 4 :
Figure 4: SER spectra for 10 mM Atenolol, Bisoprolol, and Metoprolol obtained on silver nanoparticles with 0.1 M NaCl used as aggregating agent (upper frame).SER spectra for 0.1 mM Propranolol on silver and gold nanoparticles (lower frame).
s (O 3 C

Table 2 :
Experimental and simulated vibrational bands in the Raman spectrum of propranolol.