C−X···H Contacts in Biomolecular Systems: How They Contribute to

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C-X · · · H Contacts in Biomolecular Systems: How They Contribute to Protein-Ligand Binding Affinity Yunxiang Lu,†,§ Yong Wang,†,§ Zhijian Xu,† Xiuhua Yan,† Xiaoming Luo,† Hualiang Jiang,† and Weiliang Zhu*,†,‡ Drug DiscoVery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China, and School of Science, East China UniVersity of Science and Technology, Shanghai, 200237, China ReceiVed: July 6, 2009; ReVised Manuscript ReceiVed: August 6, 2009

The hydrogen bond acceptor capability of halogens has long been underappreciated in the field of biology. In this work, we have surveyed structures of protein complexes with halogenated ligands to characterize geometrical preferences of C-X · · · H contacts and contributions of such interactions to protein-ligand binding affinity. Notably, F · · · H interactions in biomolecules exhibit a remarkably different behavior as compared to three other kinds of X · · · H (X ) Cl, Br, I) interactions, which has been rationalized by means of ab initio calculations using simple model systems. The C-X · · · H contacts in biological systems are characterized as weak hydrogen bonding interactions. Furthermore, the electrophile “head on” and nucleophile “side on” interactions of halogens have been extensively investigated through the examination of interactions in protein structures and a two-layer ONIOM-based QM/MM method. In biomolecular systems, C-X · · · H contacts are recognized as secondary interaction contributions to C-X · · · O halogen bonds that play important roles in conferring specificity and affinity for halogenated ligands. The results presented here are within the context of their potential applications in drug design, including relevance to the development of accurate force fields for halogens. 1. Introduction Intermolecular interactions such as hydrogen bonds, salt bridges, and van der Waals (vdW) forces are of fundamental importance in the structures and functions of biological macromolecules. Hence, it is not surprising that great efforts have been made to understand how these interactions participate in protein folding, ligand binding, and enzymatic catalysis. In recent years, there are many other specific nonbonded contacts, e.g., halogen bonds,1-8 CH-π,9-11 and cation-π12-14 interactions, that have attracted increasing interest, due to their significant contributions to the molecular recognition in protein-ligand interactions. Interactions involving halogens as hydrogen bond acceptors, which play crucial roles in the stabilization of a large number of supermolecular assemblies, have been thoroughly investigated through the Cambridge Structural Database (CSD) searches and theoretical calculations.15-23 Now it is commonly accepted that these interactions have the characteristics of weak hydrogen bonds, especially when the acceptors X (X ) halogens) bonded to transition metals (M-X).15 This can be readily interpreted according to the strongly polarized character of M-X bonds relative to classical organic halides (C-X), which generally gives rise to an enhanced partial negative charge on halogens. In addition, halogens appear to become reasonably good hydrogen bond acceptors when bonded to boron (B-X) and, in this respect, are comparable in strength to metal-bound halogens.16 The hydrogen bond acceptor capability of halogens * To whom correspondence should be addressed. E-mail: wlzhu@ mail.shcnc.ac.cn. Phone: +86-21-50805020. Fax: +86-21-50807088. † Chinese Academy of Science. ‡ East China University of Science and Technology. § These authors contributed equally to this work.

has, however, received far less attention in the world of biology. Indeed, halogen substitution is an important approach in drug design; approximately half of the molecules used in highthroughout screening are halogenated. It is noteworthy that there has been specific interest in the hydrogen bond acceptor capability of C-F groups, ascribed to the common method of superseding a hydroxyl group by fluorine in enzyme substrate analogues.24-29 More importantly, the hydrogen bond patterns of organic fluorines in biomolecular systems were determined based upon an inspection of the Protein Data Bank (PDB).30 Nonetheless, the accurate chemical and structural basis for their contributions to ligand-protein affinity and recognition has been largely overlooked and, accordingly, has not been fully explored in the context of drug design. Knowledge of nonbonding features of covalently bonded halogens is of paramount importance to the correct treatment of any kinds of molecular modeling problems. Of noticeable interest is that heavier halogens (X ) Cl, Br, I) display both electrophilic character along the C-X bonds and nucleophilic character along vectors perpendicular to these bonds. This unusual property of halogens arises from the anisotropic distribution of electrostatic potentials (ESPs), as shown in Figure 1. It can be seen that a small positive ESP cap is presented at the end region of halogen atoms along the C-X axis, which is surrounded by an electroneutral area and, farther out, a large electronegative domain. Halogens can, consequently, form either halogen bonding interactions with electronegative atoms/or groups showing roughly linear arrangement or hydrogen bonds with electrophiles taking place in the side-on fashion. Recently, the positive cap of halogens has been referred to as “sigma-hole” that intersects the C-X axis, and moreover, this concept has been extended to atoms of Groups IV-VI to explore their potential to form directional noncovalent interactions.31-36

10.1021/jp906352e CCC: $40.75  2009 American Chemical Society Published on Web 08/26/2009

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Figure 1. Electrostatic potential surfaces for PhX.

It is worth noting here that the ESP surface of fluorine remains entirely electronegative so that organic fluorines are more likely to serve as hydrogen bond acceptors. In addition, the ESPs of halogens (even for fluorine) can be completely positive when the X atoms are bonded to very strongly withdrawing groups.37,38 For example, the surfaces of fluorine and bromine in fluoroand bromo-trinitromethane have totally positive ESPs with the fluorine’s being the least positive.38 Conspicuously, halogen bonding, a novel interaction including halogens as acceptors of electron density, has, nowadays, been incorporated into the design toolbox and utilized in parallel with traditional hydrogen bonding for both supramolecular selfassembly and biomolecular design. In a very recent article, we have surveyed short X · · · O contacts in protein complexes with halogenated ligands and found that the amount of such interactions increased by more than one time in comparison to those assembled in 2004.39 Intriguingly, Ho et al. have demonstrated the orthogonal relationship between the two analogous intermolecular forces in biological molecules; i.e., halogen bonding is shown to be geometrically perpendicular and energetically independent of hydrogen bonding that share a common carbonyl oxygen acceptor.40 Besides, as a result of the amphoteric character of covalently bonded halogens, we believe that halogen atoms in protein-ligand complexes might be involved in a double interaction: halogen bonding in the “head on” orientation and hydrogen bonding in the “side on” fashion. Therefore, understanding the detailed relationship between the two distinct types of interactions in biomacromolecules and clarifying the C-X · · · H interaction contributions to C-X · · · O halogen bonds that play important roles in inhibitor recognition and binding would be very helpful in aiding new protein design and drug discovery. Herein, we report a comparative study of the geometries of C-X · · · H contacts in biological systems on the basis of an exhaustive survey of the PDB. The observed geometrical features of such interactions are subsequently validated by using ab initio electronic structure calculations performed on simple model systems. In particular, the electrophile “head on” and nucleophile “side on” interactions of more polarizable halogens are analyzed via the examination of interactions in structures of protein-ligand complexes and a two-layer QM/MM ONIOM methodology. This study would help to elucidate how C-X · · · H interactions participate in ligand-binding affinity and how they can directly be utilized as a molecular recognition element in designed systems. 2. Materials and Methods PDB Surveys. The current PDB (up to May 2009) contains 7867 NMR structures and more than 2000 X-ray crystal structures in which the ligands are halogenated. From these structures, we collected those with halogenated ligands for NMR protein structures and those with hydrogen atom positions that are determined for X-ray crystal structures of proteins. In total, 130 structures of proteins in complex with halogenated ligands

Figure 2. Distributions of electrostatic potentials for PhX.

were extracted. Then, the search was restricted to C-X · · · H-Y (Y ) C, N, O) contacts with X · · · H distances less than ΣvdW + 0.2 Å but without constrained C-X · · · H and Y-H · · · X angles (0-180°).16 We found 1327 X · · · H contacts that occurred in 66 NMR and high-quality X-ray crystal structures. Through the small number of protein structures retrieved from the PDB, it is possible to accurately characterize the properties of hydrogen bonding interactions involving halogens as acceptors in biomolecular systems. Normalized distances, RHX ) d(X · · · H)/(rH + rX), were assessed on the basis of vdW radii: rH ) 1.20 Å, rF ) 1.47 Å, rCl ) 1.75 Å, rBr ) 1.85 Å, and rI ) 1.98 Å.41 Such distances have been broadly used to compare the relative hydrogen bond capability of different halogens and to estimate the relative strengths of different types of hydrogen bonding.15,16,42 Theoretical Calculations. The intermolecular potentials for small model complexes of monohalogenated benzenes PhX with three donors CH4, NH3, and H2O were obtained at the MP2 level43 with Dunning’s correlation-consistent basis set, aug-ccpVDZ. The interaction energy (∆E) was evaluated as the difference between the total energy of the complex and the sum of total energies of the two subunits. The basis set superposition error (BSSE) was eliminated by the standard counterpoise method of Boys and Bernardi.44 For large biological molecules, the ONIOM (our own N-layered integrated molecular orbital and molecular mechanics) methodology,45-47 which has been successfully applied in the study of several enzymatic systems,48-54 was adopted. To render the calculations tractable, two representative protein complexes in which the Cl atom of the ligands forms a bifurcated interaction with the same residue were selected. The structures of studied protein systems were built in terms of the corresponding structures deposited in the PDB (PDB codes: 1C1455 and 1QYE56). Since there are two almost identical protein complexes in the pdb file of 1C14, we have removed one of them by deleting the B chain. Then, the systems were partitioned into two layers: the ligands and the corresponding protein residues that form halogen bonding and hydrogen bonding with halogens were in the QM layer (see Figure 6), while the rest atoms were in the MM layer. For the QM layer, the hydrid density functional theory (DFT) of B3LYP57,58 in conjunction with the standard 6-31G(d) basis set was employed, while the MM layer of the systems was modeled with the Amber force field.59 The ONIOM(B3LYP:Amber) scheme used in this work has recently been demonstrated as suitable for studying proteins system in complex with halogenated ligands.39 The binding

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Figure 3. Intermolecular potentials for small model systems of PhX with (a) CH4, (b) NH3, and (c) H2O.

Figure 4. Frequency distributions of C-X · · · H angles for C-X · · · H-Y contacts ((a) X ) F, (b) X ) Cl, Br, I) in retrieved protein structures.

energies between the ligands and the corresponding protein residues in the QM layer of the model systems were computed via the protocol as described previously39 at a higher level of theory, MP2/aug-cc-pVDZ, to obtain more precise results. All of these calculations were carried out with the help of the Gaussian 03 suite of programs.60 3. Results and Discussion Distributions of ESPs of Halogens. The ESP surfaces for PhX, as graphically depicted in Figure 1, were generated by mapping the MP2/aug-cc-pVDZ ESPs onto the surfaces of molecular electron density (0.002 e au-3). Evidently, the size of the electropositive cap enlarges progressively with the radius or polarizability of halogens, manifesting that the iodine atom would form the strongest halogen bonding interaction, whereas the fluorine atom is a relatively good hydrogen bond acceptor. To provide a quantitative insight into the distributions of ESPs of halogens, the ESPs at some given points were calculated at the same theoretical level for PhX. Here, the points Z were considered with the Z-X distances that are equal to respective vdW radius of halogens,41 namely, d(Z-F) ) 1.47 Å, d(Z-Cl) ) 1.75 Å, d(Z-Br) ) 1.85 Å, and d(Z-I) ) 1.98 Å, as well as with the Z-X-C angles that range from 60° to 180°. The graphical illustration of the derived ESPs at these given points for PhX is displayed in Figure 2. As can be seen, the curve of PhF is shown to be quite different as compared to PhCl, PhBr, and PhI. When the Z-X-C angles approach 180°, the negative values of ESPs for PhF remain almost unchanged, while the other three counterparts possess positive ESPs that increase

along with the Z-X-C angles. These observations unveil that in the latter three cases halogens can form directional halogen bonds with electron donors, and the interaction strength decreases in the order I > Br > Cl, whereas fluorocarbons seem to behave only as hydrogen bond acceptors. Noteworthily, the maximum negative ESPs for PhX (X ) Cl, Br, I) occur at the same angle (100°), and therefore, heavier halogens should form hydrogen bonding interactions in the “side on” orientation. Small Model Systems of PhX with Hydrogen Bond Donors. Figure 3 shows the intermolecular potentials for the model systems of PhX with CH4, NH3, and H2O as a function of the intermolecular X · · · H distances. Particularly, the C-X · · · H angles are predicted to be within a range from 110° to 160° for the systems including PhF and from 90° to 105° for the complexes of PhX (X ) Cl, Br, I) with the donors. The wider scope of the C-F · · · H angles is not unexpected, considering the entirely negative electrostatic domain around the fluorine atom (see Figure 1). From Figure 3, it is seen that the C-X · · · H-Y interactions appear to be attractive even when the intermolecular separations are larger than 3.4 Å. Such interactions are by implication electrostatic attractive in nature and, as a consequence, should be best viewed as weak hydrogen bonds. In general, the fluorine atom serves as good hydrogen bond acceptors relative to the other three halogens; the interactions between PhX and the donor NH3 or H2O are shown to be stronger than those in PhX · · · CH4. The F · · · H interactions would, in this regard, contribute to protein-ligand binding affinity to a greater degree, especially when associated with N-H or O-H groups in proteins. The relatively stronger

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Figure 5. Plots of normalized distances RHX versus Y-H · · · X angles for C-X · · · H-Y interactions ((a) X ) F, (b) X ) Cl, Br, I).

Figure 6. Optimized structures of the QM layer of the models for the systems of 1C14 and 1QYE.

C-F · · · H-N(O) hydrogen bonds shown in simple systems probably result in the smaller values of mean RHX distances for these interactions found in biological molecules (vide infra). C-X · · · H-Y Contacts in Protein Complexes with Halogenated Ligands. The mean RHX values for C-X · · · H-Y contacts in structures of protein-ligand complexes are summarized in Table 1. Also listed are, for comparison purpose, previous results attained by Brammer and co-workers for C(M)-X · · · H-Y interactions in the CSD.15 As might be anticipated, the mean RHX values of the interactions in biomolecules follow the same tendencies as those of statistical C(M)-X · · · H-Y contacts from the CSD. For example, the mean normalized distances decrease with the increase of polarity of H-Y bonds, i.e., C-H · · · X > N-H · · · X > O-H · · · X; the values of mean RHX raise in the order H · · · F < H · · · Cl < H · · · Br

< H · · · I, when the halogen acceptors for a given donor are compared. However, these trends are not observed for C-X · · · H-Y contacts assembled from the PDB, except that the C-F acceptor exhibits the mean RHX values reducing in the order C-H · · · F > N-H · · · F > O-H · · · F. The irregular variations for C-X · · · H-Y interactions in biological systems reflect the markedly enhanced complexity of biomacromolecules. Interestingly, the mean RHX values for C-X · · · H-C interactions in the PDB are shown to be less than those in the CSD, and moreover, in the cases of C-Cl (Br and I) · · · H-C, the corresponding values are even smaller relative to M-X · · · H-C interactions in the CSD. Hence, the C-X groups seem to act as relatively strong hydrogen bond acceptors when interacting with C-H donors of protein residues. In this respect, C-X · · · H-C interactions, the most frequent C-X · · · H-Y

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TABLE 1: Mean Normalized Distances RHX for C-X · · · H-Y Contacts in Biological Systemsa X b

M-F C-Fb C-F M-Clb C-Clb C-Cl M-Brb C-Brb C-Br M-Ib C-Ib C-I C-Xd

C-H · · · X

N-H · · · X

O-H · · · X

0.943 (374) 0.976 (7579) 0.968 (361) 0.975 (7943) 0.995 (7729) 0.947 (537) 0.982 (3269) 0.998 (4018) 0.928 (296) 0.997 (2429) 1.006 (603) 0.979 (31) 0.942 (864)

0.776 (73) 0.923 (52) 0.939 (55) 0.853 (1341) 0.963 (55) 1.010 (17) 0.879 (205) 0.973 (12)

0.703 (37) 0.930 (19) 0.901 (10) 0.799 (416) 0.916 (21) 0.908 (7) 0.820 (30) 0.902 (17)

c

0.923 (83) c

0.998 (7) 0.998 (24)

c

0.868 (8) c

0.985 (6) 0.944 (13)

a Numbers of observations are in parentheses. b Taken from ref 15. c Fewer than five observations. d Data for the entire C-X · · · H (X ) Cl, Br, I) contacts.

contacts found in biomolecular systems, may play important roles in ligand recognition and binding. Overall, the mean normalized distances, RHX, for the entire C-X · · · H (X ) Cl, Br, I) interactions detected in biomolecules are calculated to be 0.942, 0.998, and 0.944, respectively, as also provided in Table 1. FrequencydistributionsoftheC-X · · · HanglesforC-X · · · H-Y contacts in the retrieved structures of protein complexes with halogenated ligands are described in Figure 4. It can be readily appreciated that the C-F · · · H angle distribution for F · · · H interactions exhibits disparate motifs as shown by three other kinds of X · · · H (X ) Cl, Br, I) interactions. First, very few C-F · · · H-Y contacts have been observed when the C-F · · · H angles are less than 70°, whereas in the same angle range a number of C-Cl (Br and I) · · · H-Y interactions are indeed presented. This can be, to some extent, attributed to the positive ESP value calculated at the Z-F-C angle of 70° for PhF, as shown in Figure 2. The C-X (X ) Cl, Br, I) groups can, nonetheless, form weak hydrogen bonding interactions even with the angles below 70° because of the negative ESPs predicted at the same angle for PhCl (Br and I). Besides, more C-F · · · H-Y contacts in relation to C-Cl (Br and I) · · · H-Y are found when the C-X · · · H angles are close to 180°, reflecting the distributions of ESPs for halogens with the Z-X-C angles approaching 180° (see Figure 2). Particularly notable is that the angular distribution for C-X · · · H-Y (X ) Cl, Br, I) contacts appears to be centered around the mean value of 100°, which is consistent with the fact that the maximum negative ESPs for PhCl (Br and I) take place at the same angle of 100° (vide supra). However, the angular distribution for C-F · · · H-Y interactions is somewhat complicated, with a first maximum of 80°, a second, wide maximum around 120°, and a third of 170°. This vast angular distribution also mirrors the ESP surface of PhF in which the ESPs hold negative when the Z-X-C angle varies from 80° to 180°. According to our calculations, the C-X · · · H-Y (X ) Cl, Br, I) interactions with the C-X · · · H angles greater than 150°, which occupy approximately 4% of all statistical C-X · · · H-Y contacts from the PDB, should be recognized as repulsive electrostatic forces. The occurrence of these interactions may be owing to steric hindrance and constraint in biomacromolecules. The plots of normalized distances RHX versus the Y-H · · · X angles for C-X · · · H-Y contacts in biological molecules are displayed in Figure 5. Here the scatter plots for interactions including O-H groups as donors are not shown, taking into

account the markedly smaller set of contact data (see Table 1). Although there were no angle criteria employed in the present survey, few C-X · · · H-Y contacts with the Y-H · · · X angles less than 80° were observed. As can be deduced from Figure 5, the shortest F · · · H contacts show a preference of approximate linearity, which agrees with typical distributions of hydrogen bonding.61 Furthermore, when elongating the distances, the directionality appears to be more flexible, thereby suggesting the weakness of these interactions. It is well documented that weak hydrogen bonds prefer to bend, and the directionality of the interactions is often disrupted by steric hindrance as well as competition with other interacting groups.15,16 The scatter plot for the C-X · · · H-C (X ) Cl, Br, I) interactions seems to be appreciably complex. Nevertheless, when the three shortest contacts that fall within 60° ∼ 120° were excluded, the interactions, in general, follow the trend observed in C-F · · · H-C, other than strikingly more cases occurring at longer distances. These results, together with those derived from ab initio calculations on small model systems, indicate the hydrogen bonding nature of C-X · · · H-Y interactions in biological molecules. Previous studies of C (B or M)-X · · · H-Y contacts in the CSD also pointed out that such interactions should be considered as weak hydrogen bonds rather than vdW forces.15,16 C-X · · · H-Y (X ) Cl, Br, I) Interactions in ProteinLigand Complexes Associated with Halogen Bonding. Recently, halogen bonding, a specific molecular interaction where halogens act as electrophiles, has been an active area of research. Detailed analysis of crystal structures of protein complexes with halogenated ligands has established such interaction as a potential tool for biomolecular engineering, including structurebased drug design.3,5,39 As mentioned above, heavier halogens are found to behave as either halogen bond donors or hydrogen bond acceptors. Thus, in complex biomolecular systems, the covalently bonded halogens are likely to form both halogen bonds in the “head on” orientation and hydrogen bonds in the “side on” fashion. Herein, it is interesting to ask what the relationship between the two parallel types of interactions in biomolecules is and how these forces contribute to protein-ligand binding affinity. To address these open questions, 37 out of 66 retrieved structures of proteins noted above were further surveyed by the halogen bond search criteria, as described in our recent literature.39 Three protein structures were found (PDB codes: 1GAC, 1GJD, and 2JST) that satisfy the criteria. In the case of 1GJD, for example, the iodine atom in the ligand forms both a halogen bond with carbonyl oxygen of residue Val41 (d(X · · · O) ) 3.42 Å, ∠(C-X · · · O) ) 164°) and two hydrogen bond interactions with the CR-H group of Gly193 (d(X · · · H) ) 3.23 Å, ∠(C-X · · · H) ) 103°, ∠(C-H · · · X) ) 128°) and Cβ-H of Ser195 (d(X · · · H) ) 3.06 Å, ∠(C-X · · · H) ) 102°, ∠(C-H · · · X) ) 143°), respectively. Besides, the 146 X-ray crystal structures of proteins associated with halogen bonding39 were also selected to survey. Hydrogen atoms were added to the crystal structures by using SYBYL,62 although protonation with this program may lead to inaccurate results. It is wellknown that locating the hydrogen atom in macromolecules is a long-standing challenge. According to our survey, halogen atoms in the ligands for most of the protein structures are involved in C-X · · · H-Y interactions. To gain a quantitative insight into the relationship between the two kinds of interactions, two systems of protein complexes with halogenated ligands were chosen to investigate via the QM/MM ONIOM methodology described above. Presently, the hybrid QM/MM approach has been proved useful as a tool to study binding phenomenon within drug discovery.63 Figure 6 shows the optimized structures

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TABLE 2: Geometrical Parameters of Two Selected Systems at Various Levelsa 1C14

1QYE

data

ONIOM

Tripos

Amber

X-ray

ONIOM

Tripos

Amber

X-ray

d(X · · · O) Å ∠(C-X · · · O)° d(X · · · N) Å d(X · · · H) Å ∠(C-X · · · H)° ∠(N-H · · · X)° rmsd (QM)

3.46 173 3.36 2.83 136 113 0.61

5.09 154 3.74 3.46 147 99 3.74

3.70 146 3.45 2.59 123 143 3.62

3.25 173 3.15

3.06 147 3.62 3.04 130 118 0.21

3.15 124 3.92 3.48 107 108 0.42

3.58 143 3.52 2.89 128 120 0.17

2.92 142 3.40

a In Tripos and Amber geometry optimizations, Amber FF99 charges were used for proteins, while for small molecule ligands, Gasteiger-Hu¨ckel charges were employed.

of the QM layer of the models for the two systems. The calculated rmsd values for the models (see Table 2) are considerably below the X-ray diffraction resolutions of studied protein crystals (2.0 Å for 1C14 and 2.2 Å for 1QYE), indicating that the optimized model structures should be reliable. At the MP2/aug-cc-pVDZ level, the binding energies between the ligands and the corresponding protein residues are computed to be -3.44 and -6.95 kJ/mol, respectively, for 1C14 and 1QYE. From Figure 6, it is evident that halogen atoms in the ligands form both a halogen bond and a hydrogen bond with respective protein residues. For example, for the system of 1QYE, in addition to a Cl · · · O interaction with carbonyl oxygen of Gly153 presented in the QM model, there might exist a hydrogen bond between the Cl atom and the N-H group of this residue. In fact, the Cl · · · H distance, computed with the ONIOM scheme, amounts to about 3.04 Å, which is slightly larger than the sum of the vdW radii of H and Cl (2.95 Å).41 A topological analysis of the electron density reveals the existence of a bond critical point (BCP) for this interaction. Moreover, the value of charge density at the BCP for the Cl · · · H interaction is predicted to be much smaller than that for the Cl · · · O interaction, implying the very weak hydrogen bond in comparison to the halogen bond in this model. The C-X · · · H contacts can be, therefore, considered as secondary interaction contributions to C-X · · · O halogen bonds that play important roles in conferring specificity and affinity for halogenated ligands. It must be pointed out that the statements presented here are based only upon quantum chemical calculations on two systems and, of course, have great limitations. Nevertheless, considering the very weak C-X · · · H-Y interactions and the significance of halogen bonds in ligand-protein affinity and recognition, the conclusions derived may be suitable for most protein-ligand systems associated with halogen bonding. Relevance to the Development of Force Fields for Halogens. By virtue of the unique properties of halogen atoms, current molecular mechanics simulations, which rely primarily upon empirical force fields that in general do not explicitly account for electronic polarization, appear to be not appropriate for describing the interactions of halogens. To achieve this, the above two systems were also optimized with the aid of classical Tripos and Amber force fields implemented in the SYBYL package. The optimizations were performed allowing residues with atoms within 10 Å of the ligands to move while the rest were held fixed. The geometrical data obtained with these methods are summarized in Table 2. Clearly, empirical force fields reproduce the interactions of halogens poorly, though only two systems are studied here. For the system of 1QYE, when using the Tripos force field, the halogen bond is shown to be repulsive, on account of the considerable departure from the linearity for this interaction (∠(C-Cl · · · O) ) 124°), while the hydrogen bond seems to be absent, as indicated by the quite

long Cl · · · H distance (3.48 Å). The Amber method describes the interactions in this model fairly well, but the computed Cl · · · O distance deviates significantly from that in crystal structure (3.58 vs 2.92 Å). As such, both Tripos and Amber force fields cannot yield reliable descriptions of the interactions in the model of 1C14, especially for the Cl · · · O interaction (see Table 2). Furthermore, the rmsd values calculated with the two methods are substantially larger than the X-ray diffraction resolution of this protein crystal. These unequivocally demonstrate that current molecular mechanics simulations and force fields must be used with great caution for halogenated compounds. Given the importance of halogen bonding in ligand recognition and binding as well as the huge number of halogenated biomolecules available by now, developing accurate force fields for halogen atoms is necessary. Recently, a new specific angular function of fluorine was defined and inserted into the program GRID to estimate the effect of fluorine hydrogen bonds on the protein-ligand binding.30 However, for more polarizable halogens, the situation should be more intricate owing to the anisotropic distributions of the ESPs (see Figure 2), which was also mentioned in a recent article regarding σ-hole bonding between like atoms.64 To develop reliable force fields for halogens, we can first carry out high level ab initio calculations on intermolecular potential energy surfaces (PES) of a series of small complexes of biological relevance. The calculated PES data can then be fitted into an analytical expression, and finally, the analytical PES can be transformed into accurate force fields and incorporated into molecular mechanics packages. More detailed works concerning force fields for halogens are currently underway in our laboratory. 4. Conclusions Despite the small set of contact data for C-X · · · H-Y in retrieved structures of protein complexes with halogenated ligands, these interactions are characteristic of weak hydrogen bonds. Similar to C(M)-X · · · H-Y contacts in the CSD, there are remarkable differences between the behavior of F · · · H interactions and that of three other kinds of X · · · H (X ) Cl, Br, I) interactions in the PDB. The observed geometrical preferences of C-X · · · H-Y contacts in biological molecules have been substantiated by using ab initio calculations performed on small model systems. Moreover, the electrophile “head on” and nucleophile “side on” interactions of more polarizable halogens have been thoroughly studied via the examination of interactions in protein-ligand structures and a two-layer QM/ MM ONIOM methodology. In biomolecular systems, C-X · · · H contacts are shown as secondary interaction contributions to C-X · · · O halogen bonds that play important roles in ligand recognition and binding by proteins. The systematic investigation of interactions including halogens, which has for a long time

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