the three compounds lincomycin ( a natural antibiotic produced by streptomyces lincolnensis and discovered in 1961 ) , clindamycin , and pirlimycin ( two semi - synthetic derivatives of lincomycin ) comprise a group of clinically important antibiotics known as lincosamides . the structure of lincomycin ( fig . 
1a ) can be divided into two parts , a pyrrolidine derivative and a six - atom sugar ring ( methylthiolincosamide ) , which are linked via an amide bond in the central part of the molecule .
1b ) is obtained by 7(s)-chloro - substitution of the 7(r)-hydroxyl group of lincomycin , and pirlimycin ( fig . 
1c ) is obtained by trimming the propyl group of clindamycin to get an ethyl group .
these compounds are soluble in water and chemically stable both in the dry state and in solution.fig .
1chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) lincosamides block bacterial protein synthesis , which takes place on the ribosomes .
the bacterial 70s ribosome can be separated into two subunits : large ( 50s ) and small ( 30s ) , named after their sedimentation coefficients .
the small subunit of prokaryotic ribosomes consists of 21 proteins and one 16s rna chain , while the large subunit contains over 30 proteins and two rna chains ( 23s rna and 5s rna ) .
lincosamides bind to the 23s rna ( interacting with the a- and p - trna binding sites ) of the 50s ribosomal subunit and inhibit the peptidyltransferase reaction , i.e. , peptide bond formation .
the main spectrum of action of lincosamides includes bacteria associated with skin infections , and lincosamides are first - choice antibiotics used in veterinary dermatology .
however , although they do have adverse effects , such as nausea , rash , or abdominal pain , lincomycin and clindamycin are also effectively used in human medicine .
lincosamides are effective against gram - positive bacteria , such as staphylococcus , streptococcus , most anaerobic bacteria ( e.g. , bacteroides fragilis ) , and some protozoa . in the latter case
gram - negative bacteria are , in general , resistant to lincosamides , with one important exception : capnocytophaga canimorsus .
lincosamides exhibit excellent pharmacokinetic properties ; they are well absorbed orally and can penetrate well into infected skin .
also , the minimum inhibitory concentration ( mic90 , the minimum concentration needed to inhibit growth overnight in 90% of organisms ) of clindamycin towards streptococcus pyogenes is over 120 times lower than that of tetracycline .
unfortunately , extensive use of these antibiotics has led to increased resistance in many strains of bacteria , which have been developing various mechanisms to counter these drugs .
one known mechanism is structural modification of the drug s target ( ribosome ) through the methylation of 23s rna ( e.g. , base no .
2058 ) and/or mutations ( e.g. , of bases g2057 , a2058 , a2059 , c2452 , and c2611 ) . other mechanisms of resistance are active efflux of the drug across the cell surface , or its enzymatic deactivation
. bacterial resistance together with side effects are the most important reasons for improving known lincosamides and designing modified compounds .
the conformational properties of free clindamycin and lincomycin have been studied using x - ray techniques [ 9 , 10 ] as well as h and c nmr spectroscopy and molecular dynamics . however , verdier et al .  
ribosome interactions by two - dimensional transferred nuclear overhauser effect spectroscopy ( trnoesy ) , and showed that the conformation of the lincosamide plays a crucial role in its binding to the ribosome .
they found that the free conformers of clindamycin and linb ( lincosamide nucleotidyltransferase ) enzyme - bound clindamycin are similar .
rajeswaran et al .   solved the x - ray structure of lincomycin hydrochloride and found that intra- and intermolecular hydrogen bonds stabilize the structure of the drug . on the other hand ,
there are four crystallographically resolved structures of clindamycin bound to a target : three in complex with the ribosome [ 1315 ] , and one with the linb molecule , the bacterial enzyme responsible for the inactivation of lincosamides by nucleotidylation .
their crystal structures as well as the structure of the native ribosome   are available through the protein data bank ( pdb ) .
these crystallographic data form the basis for our theoretical studies of chemical and physical properties of lincosamides .
two out of the three conformers of clindamycin , when bound to the ribosome , show significant differences between antibiotic conformations : in two structures , the pyrrolidynyl propyl group is rotated by 180 relative to the other conformer .
theoretical calculations provide a complementary way to study molecular systems containing an intramolecular hydrogen bond ( ihb ) .
although the accuracy of ab initio calculations is still below the state - of - art accuracy of experimental spectroscopic data , these calculations provide information about the shape of the potential energy surface ( pes ) without the need for any initial assumptions . characterizing the conformational changes and their possible effects on the encounter with and binding to the ribosome are important aspects of understanding the mechanism of action of lincosamides . to the best of our knowledge ,
no systematic and accurate study of the conformational behavior of clindamycin in the gas phase and in solution has been reported .
the aim of the work described in the present paper was to clarify the role of intramolecular hydrogen bonds in clindamycin using quantum chemical calculations .
these ab initio calculations consist of the following steps : the choosing the model structures , geometry optimization , natural bond orbital analysis ( nbo ) , atoms in molecules analysis ( aim ) , and spectroscopic nmr parameter calculations , which are currently among the most popular methods used for conformational analysis .
the methods applied in the calculations are described in the first section of the paper .
we then characterize the conformations and molecular properties , focusing on the intramolecular hydrogen bonds .
four structures of clindamycin are available in the protein data bank   ( as of may 2011 ) .
three of these structures are in complex with the bacterial ribosome [ 1315 ] , while one is in complex with linb .
the two ribosome - complexed structures show similar clindamycin conformers , and in one the pyrrolidynyl propyl group is rotated by 180. in this work , we used two significantly different conformers of clindamycin , which were taken from ribosome  clindamycin complexes of deinococcus radiodurans   ( pdb code 1jzx ) and haloarcula marismortui   ( pdb code 1yjn ) .
therefore , based on the known x - ray structures of clindamycin ( fig . 
2ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .
black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .
black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) the second step involved optimizing the geometries of all of the antibiotic models constructed , using density functional theory ( dft ) at the b3lyp level [ 18 , 19 ] with the 6 - 31 g basis set and a redundant coordinate algorithm .
, we optimized the investigated molecules without any constraints . in the second case , the two dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) , which are significantly different in both experimentally known conformers ( a and b ; see fig . 
2 ) , were kept constant in the optimization procedure to differentiate between the two conformers .
the values of the dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) for conformer a were set to 47 and 180 , while they were set to 119 and 178 , respectively , for conformer b , in accordance with known experimental results .
the corresponding frequency calculations were carried out at the same level in order to confirm the nature of the stationary points .
no imaginary frequencies were observed , which means that the structures of the antibiotics are true minima . these structures , which we called  exp - opt ,  were used during aim , nbo , and nmr calculations ( see below ) . in a third step , in order to explore the conformational landscape of the molecules , we performed a potential energy surface scan along the torsional coordinates mentioned above in a relaxed manner ( i.e. , all other geometrical parameters were optimized at each point ) for both conformers .
to describe the environment of the antibiotic , either the continuum solvation model or an atomic representation of the solvent can be used .
therefore , as a fourth step , in order to study the solvent effect , optimization and frequency calculations were also performed at the b3lyp/6 - 31 g level of theory in combination with the polarizable continuum solvent model based on the integral equation formalism ( ief - pcm ) . in this model ,
the solvent is described by a dielectric constant , which was set to 80 in our work .
the second model that was used to describe the environment of the antibiotic , and mimics the surroundings of the antibiotic in the ribosomal rna , was the model with point ions placed around the antibiotic [ 22 , 23 ] .
the positions of these point charges were obtained from the x - ray structures of the ribosome 
the coordinates of residues within 10  of each clindamycin atom were considered , and partial charges were assigned based on the g43b1 gromos96 force field . in this way , we studied the effect of the charged ribosome environment on the conformations of the lincosamides .
next , in order to gain a deeper insight into the nature of the conformational changes , nbo and aim electron density analyses were applied for the two analyzed conformers of clindamycin .
the bond critical points ( bcps ) were characterized in terms of electron density and their laplacian values .
nmr spectroscopy is one of the techniques used to investigate molecular structures and interactions . in the last part of our work , we calculated the nmr chemical shifts and spin  spin coupling constants for the gas - phase optimized geometries at the b3lyp/6 - 31 g level .
shielding constants were calculated using the b3lyp / aug - pcs-1   method and the giao routine [ 2630 ] .
the chemical shifts of the i - th nuclei were calculated as \documentclass[12pt]{minimal } 
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				 \begin{document}$$ \delta ( i ) = \sigma_i(x ) - \sigma_i(c ) , $ $ \end{document } where i(c ) and i(x ) are the isotropic parts of the shielding tensors of the i - th nuclei in clindamycin and the i - th nuclei in the reference x molecule , respectively .
spin coupling constants were calculated at the b3lyp / aug - pcj-0 [ 31 , 32 ] level .
the nonstandard basis sets were taken from the emsl basis set library [ 33 , 34 ] .
the nbo calculations were performed with the nbo 5.0 program , while aim calculations were performed using aim2000 .
data were analyzed using gabedit , and the visualizations were carried out with vmd   and xdrawchem .
the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .
the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .
3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 
e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .
our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n 
h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .
because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .
first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .
the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,
no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .
the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 
h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .
the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .
n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .
the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .
full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .
the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . 
both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .
table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .
the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .
4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .
natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section
analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .
the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .
such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .
the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 . the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol )
are presented in fig .  5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .
5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor 
acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2
is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .
other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .
additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .
the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand ,
the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) .
to summarize , nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .
therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .
the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers .
in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .
it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .
topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .
it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .
however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .
the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .
a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .
a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .
in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .
according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig .  6 ) . the numerical values for the electron density ( bcp ) and
figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .
the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . 
7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .
exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis . the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .
the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9 
7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both 
exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .
the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .
the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure . bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .
0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions
. however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .
this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .
the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .
the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure
, we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb .
as usual , the central part of the molecule is the most interesting part to consider for this purpose .
the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .
(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 
intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 
these chemical shifts have also been shown to depend strongly on the local properties of the electron density .
the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .
finally , it is clear that (o ) can be classified into two groups . in the first group , the oxygen acts as the roton acceptor for the oh group , and
its chemical shift is lower for the b conformer than for the a conformer of clindamycin .
the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references
the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .
some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer
. however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer .
spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin 
spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum
the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .
the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .
3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 
e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .
our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n 
h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .
because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .
first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .
the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,
no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .
the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 
h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .
the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .
n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .
the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .
full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .
the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . 
both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .
table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .
the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .
4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method
in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .
natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section
analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .
the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .
such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .
the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 .
the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol ) are presented in fig . 
5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .
5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor 
acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2
is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .
other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .
additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .
the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand , the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) . to summarize
, nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .
therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .
the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers . in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .
it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .
topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .
it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .
however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .
the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .
a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .
a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .
in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .
according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig . 
the numerical values for the electron density ( bcp ) and laplacian ( bcp ) are presented in table  4 .
figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .
the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . 
7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .
exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .
the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .
the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9  h39c152.3700.01460.0131cp7c17cl28 
7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both 
exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .
the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively . the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure .
bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .
0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions .
however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .
this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .
the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .
the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as
chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure , we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb . as usual , the central part of the molecule is the most interesting part to consider for this purpose .
the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .
(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 
intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 
these chemical shifts have also been shown to depend strongly on the local properties of the electron density .
the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .
finally , it is clear that (o ) can be classified into two groups . in the first group ,
the oxygen acts as the roton acceptor for the oh group , and its chemical shift is lower for the b conformer than for the a conformer of clindamycin . the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references
the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .
some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer .
however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer . this value is large enough to be measured experimentally.fig .
spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin 
spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum
we have quantum chemically characterized the two conformers of each of the known lincosamides clindamycin , lincomycin , and pirlimycin at the b3lyp/6 - 31 g level .
internal rotations in clindamycin were investigated in vacuum and within the framework of the ief - pcm model . using nbo analysis , and with the aid of the aim theory ,
we focused on the sensitivities of electronic structure parameters such as nbo atomic charges , bond critical points , nmr chemical shifts , and spin  spin coupling constants to the conformation of clindamycin .
the two most stable conformers of clindamycin exhibit c = oh  o intramolecular hydrogen bonds . according to nbo and aim analyses ,
the presence of this internal hydrogen bond between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) is the main influence on conformer stability in vacuum and in water .
 lincosamides are a class of antibiotics used both in clinical and veterinary practice for a wide range of pathogens . 
 this group of drugs inhibits the activity of the bacterial ribosome by binding to the 23s rna of the large ribosomal subunit and blocking protein synthesis . 
 currently , three x - ray structures of the ribosome in complex with clindamycin are available in the protein data bank , which reveal that there are two distinct conformations of the pyrrolidinyl propyl group of the bound clindamycin . in this work , we used quantum mechanical methods to investigate the probable conformations of clindamycin in order to explain the two binding modes in the ribosomal 23s rna . 
 we studied three lincosamide antibiotics : clindamycin , lincomycin , and pirlimycin at the b3lyp level with the 6 - 31 g * * basis set . 
 the focus of our work was to connect the conformational landscape and electron densities of the two clindamycin conformers found experimentally with their physicochemical properties . 
 for both functional conformers , we applied natural bond orbital ( nbo ) analysis and the atoms in molecules ( aim ) theory , and calculated the nmr parameters . 
 based on the results obtained , we were able to show that the structure with the intramolecular hydrogen bond c = o  h  o is the most stable conformer of clindamycin . 
 the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , was found to be the dominant factor influencing the high stability of this conformer.figuremolecular graph of more stable conformer of clindamycin . 
Introduction
Computational details
Results and discussion
Conformational analysis
Natural bond analysis
Atoms in Molecules analysis
NMR chemical shifts
Conclusions