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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy
jou rn al hom epa ge: www.elsev ier .com/ locate /saa
tudy on the structure and vibrational spectra of efavirenz conformers using DFT:omparison to experimental data
oni Mishraa, Poonam Tandonb,∗, A.P. Ayalac
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore, IndiaDepartment of Physics, University of Lucknow, Lucknow 226007, IndiaDepartamento de Física, Universidade Federal do Ceará, C. P. 6030, 60.455-900 Fortaleza, CE, Brazil
r t i c l e i n f o
rticle history:eceived 25 August 2011ccepted 4 December 2011
eywords:favirenzRaman spectroscopy
a b s t r a c t
Efavirenz, (S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one, is an anti HIV agent belonging to the class of the non-nucleoside inhibitors of the HIV-1 virus reversetranscriptase. A systematic quantum chemical study of the possible conformations, their relative sta-bilities and vibrational spectra of efavirenz has been reported. Structural and spectral characteristics ofefavirenz have been studied by vibrational spectroscopy and quantum chemical methods. Density func-tional theory (DFT) calculations for potential energy curve, optimized geometries and vibrational spectrahave been carried out using 6-311++G(d,p) basis sets and B3LYP functionals. Based on these results, we
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have discussed the correlation between the vibrational modes and the crystalline structure of the moststable form of efavirenz. A complete analysis of the experimental infrared and Raman spectra has beenreported on the basis of wavenumber of the vibrational bands and potential energy distribution. Theinfrared and the Raman spectra of the molecule based on DFT calculations show reasonable agreementwith the experimental results. The calculated HOMO and LUMO energies shows that charge transfer occurwithin the molecule.
. Introduction
The Highly Active AntiRetroviral (anti-HIV) Therapy (HAART)as largely reduced the morbidity and the mortality of HIV-infectedatients, but a serious metabolic syndrome combining insulinesistance, dyslipidemia, central adiposity, and peripheral lipoat-ophy has arisen in treated individuals. HAART generally includesucleoside reverse transcriptase inhibitors (NRTI) and protease
nhibitors (PI). HIV reverse transcriptase (RT) is a multifunctionalnzyme that catalyzes RNA-dependent DNA polymerase, DNA-ependent DNA polymerase, and RNase H activities as well aspecifically binding its physiological primer, tRNA Lys3. These func-ions are all required in the replication of HIV, making RT centralo the virus life cycle, thus providing a primary target for anti-IV drugs widely used in the treatment of AIDS [1]. Efavirenz ishemically described as (S)-6-chloro-4-(cyclopropylethynyl)-1,4-ihydro-4-(trifluoromethyl)-2H-3,1-ben-zoxazin-2-one. Efavirenz
s a second-generation non-nucleoside inhibitor of HIV-1 reverse
ranscriptase and is responsible for the conversion of single-tranded viral RNA into double stranded DNA prior to integrationnto the genome of the human host [2]. The nonnucleoside
inhibitors of the HIV-1 virus reverse transcriptase (NNRTI) haverecently received a lot of attention [3–5]. The nonnucleoside ana-logue inhibitors (NNRTIs) are now established as part of multidrugcombinations for treating HIV infection [6,7]. This is partly due tothe fact that some of these compounds (nevirapine [8], delarvir-dine [9] and efavirenz [10]) have been approved for use in humans.Despite the great structural differences between these allostericinhibitors, all the available data indicate that they present similarmodes of action [4]. This is because they bind to the same site onthe enzyme [3,4,11] through similar interactions with amino acids,and present coincident patterns of resistance to punctual mutation[12].
NNRTIs bind in a region called the non-nucleoside reversetranscriptase binding pocket (NNIBP) which lies 10 A from theenzyme’s polymerase active site, causing a displacement of cat-alytic aspartate triad [13–18]. The NNRTIs nevirapine, delavirdine,and efavirenz have been approved for treatment of HIV-1 infectionin combinations with other RT and non-RT drugs. As with all classesof anti-HIV-1 drugs, resistant viral strains evolve which severelyimpair the long-term efficacy of the NNRTIs [19]. The NNRTIs ana-log such as nevirapine and efavirenz are noncompetitive inhibitors
that lock the polymerase active site in an inactive conformationand cause inhibition by allosteric modifications [20–23]. Secondgeneration NNRTIs can employ alternative strategies to minimizethe effects of many drug resistance mutations within RT [24].
The crystalline structure of one polymorphs of efavirenz (Fig. 1)nd its monohydrate have been reported by Ravikumar and Srid-ar using single crystal X-ray diffraction [25]. The report concludes;oth forms crystallize in an orthorhombic lattice (space group212121) with four molecules per unit cell (Z′ = 1). The crystalacking is determined by efavirenz molecules forming helicalydrogen-bonding catemers linked through the amide groups.owever, this structure does not reproduce the X-ray diffraction
PXRD) pattern of the form reported by DuPont (form I) [26]. Thebservation is similar also the case of the polymorph reported byuffini et al. [27], which exhibit two well defined conformers inhe unit cell (Z′ = 2). Mahapatra et al. [28] have recently reportedhe crystal structure of the form I, as well as two cocrystals. Form
is characterized by the orientational disorder of the cyclopropyloiety, which give rise to a Z′ value (Z′ = 3). Despite the relevance
f NRTIs and NNRTIs in the HAART therapy, vibrational spectro-copic investigations of these drugs are not fully explored [29].n continuation to our work on spectroscopic studies of efavirenz30] in the present communication, the conformational stability ofhe efavirenz molecule is investigated through quantum mechan-cal calculations by DFT with B3LYP functionals having extendedasis set 6-311++G(d,p) which are valuable information for theuality control of medicines including. A complete vibrational anal-sis of efavirenz is performed by combining Raman and infraredata with quantum mechanical calculations. IR and Raman spec-roscopic studies along with HOMO and LUMO analysis have beensed to elucidate information regarding charge transfer within theolecule. Vibrational spectroscopic methods are especially impor-
ant for characterization of supramolecular complexes becauseydrogen-bonding patterns and other “weak” interactions differmong forms and the functional groups affected will display shiftsn the energy of the vibrational modes. The calculated vibrationalpectra are analyzed on the basis of the potential energy distribu-ion (PED) of each vibrational mode, which allowed interpretationf the infrared and Raman spectra.
.1. Experimental details
The FT Raman spectrum is recorded on a Bruker IFS 55 EQUINOX
ith Raman attachment that uses a 1064 nm Nd–YAG laser line
s the excitation source for recording the Raman spectrum in theegion 80–3400 cm−1. The samples are measured in the hemi-pheric bore of an aluminum sample holder. The spectral resolution
ta Part A 88 (2012) 116– 123 117
of the Raman spectrometer is 4 cm−1. Raman spectra are acquiredwith 512 scans at a laser power of 500 mW.
Infrared spectrum is recorded on a Bruker IFS28 FT-IR spec-trometer with a spectral resolution of 4 cm−1. KBr pellets of solidsamples are prepared from mixtures of 100 mg KBr with 2 mg ofsample in laboratory press.
2. Computational analysis
Molecular geometries of the efavirenz are optimized by DFTusing 6-311++G(d,p) basis set and Becke’s three parameter (local,non-local and Hartree–Fock) hybrid exchange functionals withLee–Yang–Parr correlational functional (B3LYP). Wavenumber cal-culations are conducted at the same level for all the conformersto test the stability of their computed molecular structures. Pos-itive values in all cases confirmed the stability of the minimumenergy molecular structure. A complete vibrational assignmentis also conducted for efavirenz. For this purpose the vibrationalwavenumbers in the harmonic approximation were calculatedby B3LYP/6-311++G(d,p) method using computer software Gaus-sian 03 [31], which also provides weightage values of internalcoordinates for vibrational assignments. Since the DFT and HFvibrational wavenumbers are known to be higher than the experi-mental wavenumbers due to neglect of anharmonicity effects, theyare scaled down by the dual scaling procedure. Halls et al. [32]made a critical analysis of experimentally measured and calculatedwavenumbers at different levels of theory and divided the normalmodes in the two regions. The region below 1800 cm−1, is calledfinger print region and the region above 1800 cm−1 includes domi-nantly X–H stretching modes. The division is based on the fact thatin X–H stretching mode, the anharmonicity is large, whereas below1800 cm−1, it is quite small. Further as demonstrated in their reporta dual scaling factor gives better agreement between the calculatedand observed wavenumbers, in comparison to uniform scaling fac-tor. In view of these forgoing discussion dual scaling method inwhich the scaling factors are 0.9927 and 0.9659 for the fingerprint(below 1800 cm−1) and X–H stretching (above 1800 cm−1) regions,respectively are used in this study to offset the systematic errorcaused by neglecting anharmonicity and electron density [33,34].
Potential energy distributions along internal coordinates arecalculated by software Gar2ped [35]. The graphical presentationsof the calculated Raman and IR spectra were made using GaussView program [36]. By combining the results of the Gauss Viewprogram with symmetry considerations, vibrational mode assign-ments are made with a high degree of accuracy. There is alwayssome ambiguity in defining internal coordinates. However, theinternal coordinates defined in this study form a complete set andthe atomic motions of all the normal modes were observed usingthe Gauss View program. Visualization and correlation of calcu-lated data are performed by using the CHEMCRAFT program [37].The normal mode analysis is performed and the PED is calculatedalong the internal coordinates using localized symmetry [38,39].
3. Results and discussion
3.1. Conformational studies
Since the molecular geometry plays a very important role indetermining the structure–activity relationship, a conformationalanalysis provides meaningful information relevant to drug actionbecause, in the case of flexible molecules, the receptor is likely
to alter the solution conformation upon binding. The majority ofHIV-1 RT inhibitors that act on the same NNIBP (efavirenz, nevirap-ine, delarvirdine) show a pronounced dependence for their actionon seemingly major changes in molecular conformation [40]. On
118 S. Mishra et al. / Spectrochimica Acta Part A 88 (2012) 116– 123
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Fig. 3. Potential energy curve of efavirenz as a function of the dihedral angle�(C13C14C17C19). Symbols represent the dihedral angle observed in the poly-
Fig. 2. Equilibrium conformers of efavirenz.
he other hand, the NNBIP is very flexible and changes conforma-ion when different NNRTIs are bound [41]. Thus, determining theossible conformers of a NNTRI is very important in order to under-tand its inhibition mechanism. Several authors have investigatedhe conformational flexibility of NNTRI [42], but, at the best of ournowledge, there are no reports about the conformers of efavirenz.he different conformers of efavirenz are shown in Fig. 2.
The conformational landscape of efavirenz is investigated bycanning the relative orientation of the benzoxazine and cyclo-ropyl rings. The potential energy is determined by calculatinghe variation in the total energy of the molecule with change inihedral angle �(C13C14C17C19) in intervals of 10◦ by DFT/6-31Gethod (Fig. 3). Two minima are observed in the potential energy
urve, which correspond to the antiparallel (conformer I) and par-llel (conformer II) alignment of the bonds C13C14 and C17C19;he former is deeper than the latter and thus represents the moretable conformation to be called conformer I. The enthalpy differ-nce (�H) between the conformers I and II using more accurate
FT/6-311++G(d,p) calculation show after complete geometry opti-ization is 0.226 kcal/mol. At room temperature, the Boltzmann
ccupation ratio of the two conformers is 0.68 showing the con-ormer I should be the most populated one.
morphs of efavirenz. Arrows indicate the experimental values for the complexeswith the wild-type (1FK9 and 1IKW) and three mutant (1FK0, 1JKH and 1IKV) RTenzymes.
The most stable conformer is compared with those observedin the reported polymorphs by superimposing them using a leastsquares algorithm that minimizes the distances of the correspond-ing non-hydrogen atoms. The experimentally observed conformerscan be classified in two groups related to the orientation of the2-cyclopropylethynyl residue relative to the six-membered hete-rocycle. Either, this residue approaches an orthogonal dispositionor more flattened conformation. The first set was discussed in aprevious publication [30]. Therefore we will focus our discussion inthe second group as it will be shown that it is closely related to theconformation adopted by EFV in the enzyme–inhibitor complexes.
As stated previously, form I is characterized by an orientationaldisorder of the cyclopropyl group, which give rise to six non-equivalent conformers in a low temperature polymorph (form Ia)observed below 180 K [28]. Fig. 4a shows the conformer I comparedwith the equivalent conformer of form Ia. This comparison showsthat there are small differences between the experimental and cal-culated conformations. However, if the remaining conformers areconsidered, it may be noticed that they mainly differ in the orien-tation of the cyclopropyl group, as it can be observed in Fig. 3. Itcan be noted that only the two conformers of the form Ia and thepolymorph reported by Ravikumar and Sridhar [25] are in the moststable conformation. On the other hand, both orientation observedby Cuffini et al. [27] lie in an unstable region which is favored bythe crystal packing of this polymorph.
The optimized structures of the conformers I and II are alsocompared with the experimental conformation of efavirenz inenzyme–inhibitor complexes. Five complexes are compared withthe calculated conformations: efavirenz complexes with the wild-type (1FK9 [43] and 1IKW [44]) and three mutant (1FK0 [43], 1JKH[45] and 1IKV [44]) RT enzymes. The comparison of the optimizedstructure of the conformers I and II with the ones obtained fromX-ray crystallographic data of the enzyme–inhibitor complexes(Fig. 4b) shows that the geometry optimization mainly differs inthe orientation of the cyclopropyl ring. However, in most of theexperimental cases, the dihedral angle �(C13C14C17C19) exhibitsvalues very close to the one of the conformer II (Fig. 3). In the caseof the wild-type enzyme and in some of the mutation, the dihedralangle lies at values very close to the potential minima. The orien-tation of the cyclopropylethynyl in the complex is a consequenceof a variety of hydrophobic contacts. This group is located in the
top of the sub-pocket surrounded by the aromatic side chains ofTyr181, Tyr188, Trp229, and Phe227 [43]. In order to favor theseinteractions, in the wild-type complex (1FK9 and 1IKW), efavirenz
S. Mishra et al. / Spectrochimica Acta Part A 88 (2012) 116– 123 119
Fig. 4. Comparison of the optimized and experimental conformations of efavirenz:(a) conformer I and solid form (green), (b) conformers I and II (gray) overlapped totmtv
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Fig. 5. Experimental and calculated (scaled) infrared absorbance spectra in theregion, 2000–3700 cm−1 and 400–1900 cm−1.
hose from the complex with wild-type, 1FK9 (orange) and 1IKW (red), and threeutant RT enzymes, 1FK0 (blue), 1JKH (yellow) and 1IKV (brown). (For interpreta-
ion of the references to color in this figure legend, the reader is referred to the webersion of the article.)
dopts geometry similar to that of the conformer II, which is not theost stable one, but, being a metastable conformer, it contributes to
educe the inhibitor–enzyme binding energy. In the case of mutantomplexes, the conformational flexibility of the cyclopropylethynylroup allows for the fitting of efavirenz in the modified hydropho-ic pocket. The new geometries are moved away from the localinimum. The most striking difference is observed in the complexith the drug resistant mutation K103N (1FK0), where the cyclo-ropyl ethynyl group is rotated ∼100o relative to the position in theild-type structures. This orientation is energetically unfavorable
ecause it corresponds to a point near to the potential barrier maxi-um (Fig. 3) and could contribute to the observed slow association
ates with K103N [46]. Thus, the fact that efavirenz adopts confor-ations which are close to a conformational minimum provides aide range of energetically favorable conformers that allows the
inding of this inhibitor to mutant RTs. This property could be aelevant factor to explain the improved resilience of efavirenz toertain drug-resistant mutations.
.2. Vibrational analysis
No attempt seems to have been made in the past to providessignments to the vibrational modes of efavirenz. The total num-er of atoms in this molecule is 30; hence it gives 84 (3n − 6) normalodes. The calculated wavenumbers of the vibrational modes and
otential energy distribution (PED) along the internal coordinatesf the conformers I and II of the molecule, together with thexperimental wavenumbers are given in Table 1. Localized symme-ry was used in the analysis of the PED obtained using the program
Fig. 6. Experimental and calculated (scaled) Raman scattering spectra in the region,2000–3700 cm−1 and 200–1900 cm−1.
GAR2PED [35]. PED values <10% have not been included in the table.The assignments of the infrared and Raman bands of efavirenz arebased on a comparison of the experimental spectra and theoreti-cally obtained wavenumbers and PED values.
The Raman scattering cross-sections, which are proportional tothe Raman intensities may be calculated from the Raman scatter-ing amplitude and predicted wavenumbers for each normal modes[47,48].
3.3. Vibrational wavenumbers
Comparison of the wavenumbers calculated at B3LYP withexperimental values (Table 1) reveals the over estimation of thecalculated vibrational modes due to neglect of anharmonicity inreal system. Inclusion of electron correlation in density functionaltheory to a certain extent makes the wavenumber values smallerin comparison with the HF wavenumber data. Experimental (con-former 1) and calculated (scaled) Raman scattering spectra andinfrared absorbance spectra are shown in Figs. 5 and 6, respectively.
3.3.1. Benzoxazine group vibrations
In the aromatic ring (R1) C–H stretching vibrations are usually
strong in the Raman spectrum. The �(CH) ring modes are assignedat 3071, 3051 and 3029 cm−1 in the Raman spectrum. The corre-sponding calculated values are 3104, 3097 and 3067 cm−1 and thus
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Table 1Theoretical and experimental vibrational wavenumbers (cm−1) of conformers of efavirenz.
Conformer I Conformer II Observed Assignment (%PED)
Proposed assignment and potential energy distribution (PED) for vibrational normal modes.Types of vibration: �, stretching; ı, deformation; oop, out-of-plane bending; ω, wagging; � , twisting; �, rocking; �, torsion, puck, puckering.
1 ica Acta Part A 88 (2012) 116– 123
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oincide well with the experimental observations. R1[�(CC)] sym-etric stretch are calculated to be 1634 and 1620 cm−1 and are
ssigned to the band at 1603 cm−1 in the IR spectrum and at 1615nd 1603 cm−1 in the Raman spectrum.
Ring deformations are calculated to be 1491 and 1297 cm−1 andre well correlated with observed bands at 1490 and 1317 cm−1 inhe Raman spectrum and at 1498 and 1302 cm−1 in the IR spectrum.ut-of plane deformations of R1[ıoop(CH)] are calculated at 952 and97 cm−1 and matches well with IR and Raman spectra. Puckeringodes of ring are calculated to be 744, 705 and 690 cm−1. The tor-
ions of the ring are calculated to be 461 and 383 cm−1. These arelso in good agreement with the experimentally Raman values. Atonformer I to conformer II the 383 cm−1 band undergo a upwardhifts to 392 cm−1 accompanied by an abrupt intensity decrease inntensity.
In the oxazine ring (R2) N–H stretching vibration is usuallytrong in the IR spectrum. The �(NH) wavenumber in ring isssigned at 3317 cm−1 in the IR spectrum. It is calculated to be506 cm−1. R2[�(CO)] stretch is calculated to be 1767 cm−1 andre assigned to the bands at 1743 cm−1 in the IR spectrum and at729 cm−1 in the Raman spectrum. An additional significant fea-ure of efavirenz binding to HIV-1 RT is the presence of a hydrogenond to the main chain C O of residue 101, which is not present inhe nevirapine complex [43]. R2[�(CC)] symmetric stretch is calcu-ated to be 1279 cm−1 and assigned to the bands at 1277 cm−1 inhe IR spectrum and at 1275 cm−1 in the Raman spectrum.
Ring deformation ı(NH) is calculated to be 1482 cm−1 and it isbserved at 1472 cm−1 in the Raman spectrum and at 1454 cm−1
n the IR spectrum. Out-of plane deformation of R2[oop(NH)] isalculated to be 525 and 516 cm−1 and matches well with Ramanpectrum.
.3.2. Cyclopropyl ring vibrationsThe �(CH2) stretching vibration of the cyclopropyl ring (R3),
hose is assigned to 3093 cm−1 in the Raman and IR spectra. Thealculated value is 3121 cm−1. ı(CH2) deformations are observed at490 cm−1 in the Raman spectrum and 1498 cm−1 in the IR spec-rum. The in plane CH bending corresponds to well define bandt 1388 cm−1. Ayala et al. [48] assigned this band at 1358 cm−1 inevirapine. CH2 wagging mode occurs at 1076 and 1057 cm−1 inhe IR spectrum and at 1078 and 1065 cm−1 in the Raman spec-rum. The corresponding calculated values are found to be at 1075nd 1051 cm−1. The rocking modes of CH2 group give rise to theedium intensity bands at 824 and 818 cm−1 in the IR and Raman
pectra respectively. The corresponding calculated wavenumbersre 812 cm−1, as shown in Table 1.
The fluoromethyl group is present in the molecule which isirectly connected to the oxazine ring. The CF3 group has sev-ral modes such as symmetric and asymmetric stretches, bends,ock and torsional modes are associated with it. Assignments ofll these fundamentals modes are given in Table 1. The symmet-ic CF3 stretching mode �s(CF3) appears at a lower energy, inhe range 700–800 cm−1, compared to its asymmetric stretchinga(CH3) which appear in the range 1100–1200 cm−1. The rockingodes of CF3 appear to have variable magnitudes in CF3 contain-
ng benzene. The 341, 317 and 284 cm−1 bands are assigned to theocking modes of CF3. Comparing conformer I and conformer II it isbserved that 317 cm−1 band shifts to 307 cm−1 and 284 cm−1 bandhifts to 278 cm−1. The former band decreases abruptly in intensity,hile the latter increases in intensity. The CF3 rocking modes are
bserved at 336, 305 and 285 cm−1 in the Raman spectrum.
.3.3. Alkyl chainThe stretching mode �(C C) at the calculated wavenumber
265 cm−1 correspond to the observed bands at 2252 cm−1 in the IRpectrum and at 2250 cm−1 in the Raman spectrum. The vibrational
Fig. 7. Selected orbital transitions for efavirenz.
modes calculated to be 937 and 864 cm−1 have a major contribu-tion from the internal coordinate corresponding to the deformationof CC14C15. These modes are also observed at 942 and 853 cm−1 inthe Raman spectrum and approximately at the same positions inthe IR spectrum.
The calculated and experimental vibrational wavenumbers forthe conformer I and conformer II are given in Table 1. A satisfactory
agreement between the scaled and experimental wavenumbersmay be noted. A comparison shows that the vibrational wavenum-bers of the conformer I lie very close to those of the conformerII.
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By comparing the rest of the vibrational spectra of efavirenzith the help of PED distribution presented in Table 1, we find a
ery good overall agreement. The difference between the observednd scaled wavenumber values of most of the fundamentals is quitemall.
.4. Absorption spectra
The electronic absorption energies and oscillator strength (f) forhe efavirenz is computed at the time dependent density functionalheory (TD-DFT) methods. The atomic molecular orbital plots werebtained by Gauss View [34] and are presented in Fig. 7. The com-ound exhibits two absorption bands in gaseous state calculations.he first, second and third transitions originates from the �–�*
ransitions on the whole molecular plain. The first peak, dominantlyescribable with the HOMO to LUMO excitation, and has moderatetrong oscillator strength (f ) of 0.0439. The second excitation origi-ates from HOMO to LUMO + 1 with strong oscillator strength (f ) of.2167. The third moderately transition originates from HOMO − 1o LUMO with oscillator strength (f ) of 0.0354. The LUMO as anlectron acceptor represents the ability to obtain an electron, andOMO represents the ability to donate an electron. Moreover, a
ower HOMO–LUMO energy gap explains the fact that eventualharge transfer interaction is taking place within the molecule.
. Conclusion
Vibrational spectroscopy and density functional theoretical cal-ulation have been applied to the structural and spectroscopicnvestigation of efavirenz. The equilibrium geometries and har-
onic vibrational wavenumbers of all the 84 normal modes of theolecule were determined and analyzed with DFT level of theory
mploying 6-311++G(d,p) basis set, giving allowance for the loneairs through diffused functions. Geometrical optimizations showhat the efavirenz molecule in the crystal structure suffers smalleformations, whereas the conformation observed in the inhibitorIV-1 RT complexes agrees very well with a metastable structure.he existence of a metastable minimum in the energy landscapeould be contributed to the conformational flexibility of efavirenznd could explain partially its improved resilience in some drug-esistant mutations. Raman and infrared spectra were recorded andhe vibrational bands were assigned on the basis of the potentialnergy distribution obtained from the DFT calculations.
cknowledgements
The financial support from the CNPq and DST under Indo-Brazilroject is gratefully acknowledged.
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