Halogen Bonding in Nucleic Acid Complexes

Michal H. Kolar∗,†,¶ and Oriana Tabarrini‡

†Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences,Flemingovo nam. 2, 16610 Prague, Czech Republic

‡Department of Pharmaceutical Sciences, Universita of Perugia, Via del Liceo 1, I-06123Perugia, Italy

¶Current address: Department of Theoretical and Computational Biophysics, Max PlanckInstitute for Biophysical Chemistry, Am Fassberg 11, D-37077 Gottingen, Germany

E-mail: michal@mhko.science

Abstract

Halogen bonding (X-bonding) has attracted no-table attention among noncovalent interactions.This highly directional attraction between ahalogen atom and an electron donor has beenexploited in knowledge-based drug design. Agreat deal of information has been gatheredabout X-bonds in protein-ligand complexes, asopposed to nucleic acid complexes. Here weprovide a thorough analysis of nucleic acid com-plexes containing either halogenated buildingblocks or halogenated ligands. We analyzedclose contacts between halogens and electron-rich moieties. The phosphate backbone oxygenis clearly the most common halogen acceptor.We identified 21 X-bonds within known struc-tures of nucleic-acid complexes. A vast major-ity of the X-bonds is formed by halogenatednucleobases, such as bromouridine, and featureexcellent geometries. Noncovalent ligands havebeen found to form only interactions with sub-optimal interaction geometries. Hence, the firstX-bonded nucleic-acid binder remains to be dis-covered.

Introduction

Bringing a new drug to the market consumesenormous intellectual and financial resources.It typically takes more than a decade from thediscovery of an active compound (lead) to the fi-

nal approval of a drug, which is based on it. Thedrug discovery and development declined fromthe trial-and-error approach when the numberof recognized diseases steeply rose. Nowadays,the workflow stems from knowledge gained in avariety of studies believing that this can speed-up the whole drug development.

The number of known drug targets is slightlyhigher than 300.1 This amount is not extraor-dinarily high bearing in mind the number ofrecognized human genes (less than 20,0002).Among the targets, there are mostly proteinsand only a few nucleic acids (NAs). Given thatNAs are ubiquitous biopolymers with a myriadof cellular functions though, they represent aclinically prominent class of targets.1

Many strategies have appeared to optimizethe lead compound into a therapeutic sub-stance. The use of halogens is in this sensetraditional. Xu et al. estimated that about25 % of approved drugs are halogenated, andthe portion is similar in all stages of drug dis-covery and development.3 Halogen atoms mod-ulate physicochemical properties of the molec-ular scaffolds; they affect the polarity and hy-dro/lipophilicity, which in turn changes mem-brane and blood brain barrier permeation of themolecule. Also, carbon-halogen covalent bondis difficult to metabolize, so the halogenationprolongs the lifetime of the active compound,but at the same time might increase its livertoxicity.4

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Apart from nonspecific effects, it was abouta decade ago recognized that halogens mightpartake in a structurally specific and direc-tional noncovalent interaction called a halogenbond (X-bond).5 The X-bond is an interac-tion between a halogen and a Lewis base oran electron-rich moiety. The electron densitydonors may be represented by electronegativeatoms such as oxygen, nitrogen, sulfur, but alsoby aromatic rings or conjugated π-systems.

The X-bond has been found in many protein-ligand complexes including pharmaceuticallyrelevant ones (for review see Refs. 6, 7, and8). There have been only a few studies onX-bonds, where NAs played a role. The dis-tinguished exceptions are the efforts of ShingHo and co-workers. They focused on halogenbonding in so-called Holliday junction, a four-stranded (branched) complex of deoxyribonu-cleic acid (DNA).9,10 Using bromouridine as abuilding block, they directed a DNA into oneof the several nearly isoenergetic conformers.11

The DNA model system also demonstrated thatamong the halogens it is the bromine that hasthe most favorable entropy/enthalpy compen-sation. Consequently, bromine was claimed anoptimal element for X-bonding in DNA Holli-day junctions.12,13

To the best of our knowledge, no ligand hasbeen reported so far to form an X-bond in anyNA complex. The lack of information on X-bonding in NAs is somewhat surprising becauseNAs are naturally rich in electronegative atomswhich make them (in theory) prospective X-bond acceptors. It seems that there is a miss-ing relation between the worlds of NAs and X-bonds. This work aims at building such a rela-tion. Hence, we continue with the introductionsof the two worlds trying to find some overlap be-tween them and highlight the pharmaceuticalsignificance. Later, we analyze known struc-tures of NAs and reveal main features of theircomplexes. Finally, we discuss all of the fewexamples of low-molecular compounds whosehalogens are involved in interactions with elec-tron donors.

Halogen Bonding Features

The attraction of halogens with other elec-tronegative atoms was observed as early as the1950s in the crystallographic studies of Has-sel et al.14 although the synthesis of X-bondedcomplexes dates back to 19th century.15 Thepuzzling nature of the attraction between twoelectron-rich chemical groups was attributedmerely to charge transfer effects until Politzeret al. came up with a simple model explain-ing many of the X-bond features.16 Based onquantum chemical calculations of the molecularelectrostatic potentials (MEPs) they proposedthat the surface of halogens contains regions ofboth positive as well as negative electrostaticpotential (ESP).16 The positive region was la-beled a sigma-hole (σ-hole) (Fig. 1), and in-terestingly enough this label appeared only 15years after its first evidence.17 The X-bond hasbeen exploited in many areas of chemistry andmaterial science (reviewed e.g. in Refs. 5 and18) and a great amount of work has been doneon the theoretical aspects of X-bonds too.6,19

Figure 1: Molecular structure of bromouracil(a), the intersection of a molecular surface (b),and full ESP in atomic units (au) projectedonto the molecular surface (c). The blue discof positive ESP in the forefront is the σ-hole.

X-bonds are similar in strength to the morecommon hydrogen bonds (H-bonds). The X-bond stabilization energy typically amountsto 5–25 kJ/mol20,21 and it increases with theincreasing atomic number of the halogen in-volved.22 The reason for this is the increas-ing size and magnitude of the halogen σ-hole.23

Another factor is the halogen polarizability,24

which also increases with the halogen atomicnumber. Contrary to traditional view, mod-ern theoretical studies suggest that the roleof charge transfer in the X-bond stabilizationis modest.25 The strongest X-bonds are found

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in complexes of iodinated molecules; bromi-nated and chlorinated molecules form weakerX-bonds. Fluorine is the least polarizable andthe most electronegative halogen. It possessespositive σ-holes in rare cases and mostly in in-organic molecules,26 so it is of lower importancefor biological applications.

The structural trends of X-bonds inbiomolecules have been inferred from severalProtein Data Bank (PDB)27 surveys and the-oretical analyses.7,28–30 All of the studies fo-cused on protein-ligand X-bonds. The X-bondsare mostly established with the protein back-bone. Its carbonyl oxygen is the most frequentelectron donor.29 No preference for backbonesof α-helices, β-sheets or loop structures hasbeen found.31 Lu et al. reported that aboutone-third of protein-ligand X-bonds involve anaromatic ring of the amino acid side chains.29

It was also reported that the X-bonds mightnot disrupt existing networks of H-bonds.32 In-stead, an orthogonal pattern of X- and H-bondsis preferred involving the same electron donoratom.33 Another effect of ligand halogenationis a shortening of proximal H-bonds.34

Nucleic Acids as Drug Tar-

gets

The deoxyribonucleic and ribonucleic acids(DNA and RNA) appear in living organisms inseveral forms. Whereas the DNA adopts only afew conformational classes, the structural diver-sity of RNA is much broader. DNA mostly oc-curs in the B-form helical conformation. A keyRNA motif is the A-form double helix. How-ever, loop regions, bulges, and other forms ofmismatched nucleobases often disrupt the mo-tif. Such a conformational variability is mir-rored in the rapidly expanding variety of RNAfunctions recognized, especially in the last twodecades. Apart from the classic roles of riboso-mal, transfer and messenger RNAs, RNA wasalso shown to store genetic information, regu-late gene expression, or act as enzyme.35,36

Many NA-binders are halogenated. The DNAalkylating agents often contain halogen atomsbecause halogens facilitate the alkylating reac-

tions on NAs. This way, anticancer cisplatinand its analogues37,38 covalently modify DNA.The reaction products block replication or tran-scription processes. The effect of alkylation isnon-specific in terms of DNA sequence and alsocell type. The modifications occur in both nor-mal and cancer cells, but the higher prolifera-tion rate of the cancer cells makes these drugseffective.

Alternatively to covalent modifications, var-ious classes of compounds bind to double-stranded DNA (dsDNA) in a noncovalent man-ner. Examples include the anticancer anthra-cycline type antibiotics daunomycin and adri-amycin, and the polypeptide antibiotic dac-tynomicin, which works mainly by intercalat-ing into DNA. The intercalation, describedas an insertion of a planar molecule betweenconsecutive base pairs, interferes with DNA-processing enzymes.39 Other small moleculesbind to dsDNA interacting with base pair func-tional groups on the floor of the minor or ma-jor groove. Much of the research has concen-trated on the DNA minor groove recognitionleading to improved sequence selectivity of thecompounds.40,41

Important classes of drugs target ribosomalRNA (rRNA). In fact, most of the RNA-targeting drugs on the market act on ribo-somes.42 The ribosome is a biomachine whichsynthesizes proteins. As such, it representsa critical center of cellular life. The differ-ences between bacterial and eukaryotic ribo-somes have allowed developing specific antibac-terial agents that are often based on naturalproducts.43–45 There is a variety of binding lo-cations within the ribosome; the most frequentsites are the peptidyl-transferase center on the50S subunit and the decoding center on the 30Ssubunit. Aminoglycosides and tetracyclines arethe best-known classes of small molecules whoseprimary target is the 30S subunit. Oxazoli-donones instead exert their antibacterial actionby binding to the 23S rRNA present withinthe 50S subunit. Macrolides and related com-pounds (lincosamides and streptogramins), aswell as chloramphenicol and clindamycin alsotarget the 50S subunit. Several ribosome-binding molecules have been prepared contain-

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ing a halogen atom stemming mostly from thepioneering case study of chloramphenicol.46

In the past decade, other non-coding RNAs(ncRNAs) have emerged as prospective drugtargets.42,47 Highly conserved ncRNAs providenew opportunities to expand the repertoire ofdrug targets to treat infections. Viral regula-tory elements located in untranslated regions ofmRNA often form folded structures that harborpotential binding sites for small molecules. Theabsence of homologous host cell RNAs makesthem attractive for the development of innova-tive antiviral compounds.

There are a plethora of ncRNAs under in-tense pharmaceutical research due to their abil-ity to interact with low-molecular ligands. Forinstance, human immunodeficiency virus type-1(HIV-1) Trans-activation response (TAR) RNAplays an essential role in HIV-1 replicationthrough its interaction with the viral trans-activator of transcription. Such interactionmight be disrupted by ligands (reviewed inRefs. 48 and 49), where some of them containhalogen atoms.50

Another example is an internal ribosome en-try site (IRES) from Hepatitis C virus (HCV).51

The IRES RNA contains several independentlyfolding domains that are potential targets forthe development of selective viral translationinhibitors. A diverse set of ligands includ-ing oligonucleotides, peptides as well as smallmolecules have been reported to block IRESfunction by distinct mechanisms.52 Like in thecase of other ncRNAs, the drug candidates oc-casionally contain halogens. Nevertheless, nodrug targeting a ncRNA has been approved forthe market.

Many lines of evidence are linking mutationsand dysregulations of ncRNAs to neurodegen-erative disorders.53,54 The presence of expandedCNG repeats in 5’ and 3’ untranslated regionsis related to important diseases such as My-otonic dystrophy type 1, spinocerebellar ataxiatype 3, and fragile X-associated tremor-ataxiasyndrome. For example, the pathological ex-pansion of CAG repeats (>35 consecutive CAGcodons) in huntingtin exon 1 encodes a mutantprotein whose abnormal function determinesHuntington’s disease. Finding compounds able

to bind pathogenic CNG repeats with highspecificity may be a valuable strategy againstthese devastating diseases. Their design is stilllimited by the lack of structural information,although some small molecules have emergedthrough various strategies.55,56

Both DNA and RNA can also adopt anon-canonical higher-order structure calledG-quadruplexes (G4s) that are involved inregulating multiple biological pathways suchas transcription, replication, translation andtelomere structure.57 The building blocks ofG4s are guanosine-rich quartets that self-associate into a square-planar platform througha cyclic Hoogsteen H-bonded arrangement. G4sare found in oncogene promoters, in telomeres,as well as in introns of mRNAs. These re-gions have been recognized as potential targetsfor anticancer drugs.58 A large number of smallmolecules are able to bind the quadruplex struc-tures. They are characterized by polycyclicheteroaromatic scaffolds, or by cyclic/acyclicnon-fused aromatic rings. Thus far, only a fewmolecules have been found to selectively bindthe telomeric G4s, although their therapeuticpotential appears high.59

Nucleic Acids as X-bond Ac-

ceptors

In the X-bond, the σ-hole on a halogen repre-sents a Lewis acid that interacts with a Lewisbase. NAs seem to offer an abundance of ba-sic chemical groups. The backbone containsphosphate groups with two oxygens carrying acharge of −1. Likewise, ribose and deoxyribosecontain oxygens with lone electron pairs thatcould serve as σ-hole acceptors too. Further,the nucleobases form H-bonds that could be po-tentially replaced or augmented by X-bonds.

Nucleobases themselves show certain electro-static diversity, where the negative sites mayplay a role of halogen acceptors. Fig. 2 de-picts MEPs of five most common nucleobasesprojected onto the plane of their aromatic sys-tems. Cytosine and guanine contain areas ofmore negative ESP than the other nucleobases.Thymine and uracil resemble each other having

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two negative sites on the oxygens separated bya small positive site. Perhaps the most hetero-geneous ESP is around adenine which exhibitssix areas of zero ESP near the molecular sur-face, as compared to four (G, T, U) and two(C). Apart from this, all of the nucleobases arearomatic, which allows them to act as electrondonors via their π-electrons above and belowtheir rings (not shown).

In nucleic acids, the situation is more com-plicated than in nucleobases: the three-dimensional structure of DNA and RNA iselectrostatically diverse due to the presenceof the phosphate backbone. For instance, theDNA minor groove was shown to be more elec-tronegative than the major groove.60 Thanksto its plasticity, RNA may create folds witheven more unusual electrostatic characteristics.Indeed, regions of strong negative electrostaticpotentials were exploited in designing efficientTAR binders.61 Electrostatic interaction is alsothe main driving force of aminoglycoside bind-ing to the ribosome.62 Overall, NAs seem tooffer favorable electrostatics to attract positiveσ-holes on halogens. It remains elusive, how issuch ability employed in ligand recognition andNA self-assembly.

Structural Survey Yields

Two Data Sets

To understand interaction preferences of halo-gens we analyzed known NA structures. Westarted with a broad set of X-ray structuresfrom the PDB (September 2016).27 Apart fromX-ray structures, the PDB also contains NAstructures determined by nuclear magnetic res-onance (NMR) techniques. For two reasons,we deliberately omitted those from our analy-ses. First, we wanted to be consistent with thestrategies adopted by previous structural sur-veys of X-bonds in protein-ligand complexes.Second, there are indications that it may bedifficult to assess the quality of the depositedNMR structural ensembles,63 which could com-plicate the geometric characterization.

Within the X-ray structures, we searched forcomplexes containing a nucleic acid and a halo-

gen atom (Cl, Br, or I). Fluorine was excludedfrom the search due to its extremely low abil-ity to form X-bonds in biological systems. Theselected structures comprise nucleic acids andtheir complexes with other nucleic acids, low-molecular ligands, and/or proteins. To get re-liable geometric characteristics, only data withthe resolution better than 3.0 A were consid-ered. Note that the previous PDB surveys ofX-bonds in protein complexes used the sameresolution threshold.28,30

From the PDB, we obtained 672 files whichwere subsequently filtered. We excluded struc-tures containing halogens in the form of ions.Following the recommendation of the Interna-tional Union of Pure and Applied Chemistry,64

we selected X-bonds as the contacts betweenhalogens and electronegative atoms (N, O, P)shorter than or equal to the sum of van derWaals (vdW) radii65 (Tab. 1). The interac-tions involving at least one of the two inter-acting atoms with the crystal occupancy lowerthan 0.5 were omitted. Because of the X-bonddirectionality,23 we also required the angle of R–X· · ·Y to be higher than 120◦. Same or similargeometric criteria were used previously to definebiological X-bonds with proteins,28,29 althoughthe wider angular range was used in other stud-ies as well.7,30

Table 1: Sums of van der Waals radii65 ofatomic pairs in A.

N O PCl 3.30 3.27 3.55Br 3.40 3.37 3.65I 3.53 3.50 3.78

We also searched for the X-bonds that areformed with the aromatic systems of the nucle-obases. To this aim, we considered only halogencontacts closer than 5 A to the aromatic planethat make an angle between the plane normalvector and the X–C bond smaller than 60◦.

In the end, we obtained a set of 21 X-bondsthat satisfy the data quality, chemical and ge-ometric criteria. This amount is a rather low.Scholfield et al. reported 760 protein complexeswith an X-bond in 2012, which stands for about

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Figure 2: 2D projections of the electrostatic potential (ESP) of the most common nucleobases A, C,G, T, U onto the planes of their aromatic rings. Note the ESP in atomic units (au) was calculatedfor the N-methylated analogs.

1 % of the 80,000 protein structures depositedin the PDB at that time. The 21 X-bonds hererepresent about 0.2 % of ca 9,000 structurescontaining NA.

Hence to better capture possible geometricproperties of halogen interactions, we collecteda more extended set of complexes with a longerinteraction distance. An arbitrary threshold of4 A was chosen such that it is higher than theX-bond length but still short enough to hintfor an attractive interaction. Within the ar-ticle, the interactions are referred to as linearcontacts and they comprehend the X-bonds aswell. We found 72 linear contacts. Table 2 sum-marizes various subsets of the PDB query.

The X-Bonds Favor Nucleo-

base· · ·Phosphate Pattern

Within the X-bond set, the variety of interact-ing partners is low; 20 X-bonds involve a halo-genated nucleobase, one X-bond is formed bycisplatin chlorine. The set contains 19 X-bondswith a phosphate oxygen as the electron donor;further, there is one X-bonds with an aromaticring, and one with cytosine oxygen. Overall,only two X-bonds do not concur with the dom-inant nucleobase· · ·phosphate interaction pat-tern.

The X-bond geometries are close to ideal.The X-bond lengths are shorter than the sumof the vdW radii by about 8 %, which con-forms with the contractions reported for theprotein X-bonds.7 About 90 % of the X-bondsare straighter than 160◦.

The shortest X-bond in the set belongs to astructure of a Holliday junction; the X-bond

is found between a bromodeoxyuridine and aphosphate oxygen of two neighboring residues(Fig. 3a) (PDB: 2org, resolution 2.0 A).11 TheX-bond was shown to stabilize particular DNAassembly in competition with H-bond. Thestraightest X-bond also involves a brominateduridine and phosphate oxygen (Fig. 3b) (PDB:2bu1, resolution 2.2 A) in a complex of RNAand phage MS2 coat protein66 and the overallgeometry is remarkably similar to the one foundin Holliday junction. Whereas the study onHolliday junction fully appreciated the role ofX-bonding, in the latter case the specific inter-action of the bromine remained unrecognized.

Figure 3: The shortest (a) and the straightest(b) X-bonds found. Hydrogens were omittedfor clarity. Residues are labeled as BRU: 5-bromouridine, dBRU: 5-bromo-2-deoxyuridine,U: uridine, dA: deoxyadenosine.

What is important, we have identified no non-covalent ligand involved in X-bonding. Theonly non-nucleobase residue that participatesin an X-bonds is cisplatin covalently bound toan adenine (PDB 5j4c, resolution 2.8 A67). Itis also the only halogen donor that forms anX-bond with a π-system, although the interac-tion with one of the guanine nitrogens wouldalone classify the interaction as X-bond. TheX-bond features superb geometric characteris-tics (Fig. 4). Unlike proteins, in nucleic acids,

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Table 2: Analysis of the PDB. The contact is defined by the interatomic distance between ahalogen and oxygen, nitrogen, or phosphorus shorter than 4 A. The linear contact has the angleR–X· · ·O/P/N higher than 120◦. X-bonds is a linear contact shorter than the sum of vdW radii.

Cl Br I sumFilesPDB search count 402 204 66 672Contains contact(s) 43 162 34 239Contains contact(s) with NA building block 31 135 22 188Contains linear contacts(s) with NA building block 22 29 2 53Contains X-bond(s) with NA building block 2 17 2 21InteractionsNumber of contacts 611 1,319 205 2,135Contacts with NA building block 43 315 41 399Linear contacts with NA building block 22 48 2 72X-bonds with NA building block 2 17 2 21

the halogen-π interaction competes with π − πinteractions more often. Especially in dsDNA,it is hardly conceivable that there is a space fora halogen to attack the nucleobases from aboveor below of their aromatic planes. The situ-ation in RNAs might be more favorable, butthe single occurrence of such X-bond is hard togeneralize.

Figure 4: X-bond with an aromatic system be-tween cisplatin (CPT) and a guanine (G). Thedistance stands for the perpendicular distancefrom the aromatic plane.

Interactions Longer Than X-

Bonds

Table 3 summarizes counts of various types ofinteractions, and the statistics of the interac-tion geometries of the set of 72 halogen linearcontacts.

In the NA complexes, the geometric qualityof the halogen interactions increases in the or-der of Cl < Br < I. The interaction angles aremore linear for heavier halogens. Especially thecontacts of chlorine are rather bent with themedian angle of 141◦. The medians of the in-teraction lengths (Tab. 3) decrease with theincreasing atomic number of the halogen. Thesurveys on protein-ligand X-bonds28,29 revealedthe opposite trend, i.e. the increasing length ofX-bonds with the increasing atomic number ofthe halogen involved. In this work (but also inRef. 28), the statistical sample might be insuffi-cient to provide reliable statistics, which is trueespecially for the two iodine X-bonds.

Fig. 5 shows the histograms of the interactionlengths and angles. In the histogram of lengths,the minor peak near 3.0 A, which is compara-ble with the sum of the vdW radii, stands foralmost ideal X-bond length. There is a majorpeak near 3.5 A too. Two peaks also appearin the histogram of interaction angles; around140◦ and 170◦.

Each of the halogen subsets contributes to thehistograms with a different weight. Chlorine-and bromine-containing complexes span thewhole range of interaction lengths. The two io-dine complexes feature short interactions. Thesituation with angles is different. Chlorine com-plexes appear only in the region of lower inter-action angles. The highest angle in the chlorine

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Table 3: Medians (med) and interquartile ranges (iqr) of geometric characteristics of interactionsinvolving various types of partners.

Halogen Count Length [A] (med±iqr) Angle [deg] (med±iqr)chlorine 22 3.49±0.31 141±8bromine 48 3.45±0.30 165±22iodine 2 3.07±0.06 173±3Electron DonorN 21 3.47±0.28 140±11non-backbone O 9 3.69±0.42 135±25backbone O 42 3.41±0.45 166±8Halogenated ResidueHalogenated nucleotide 51 3.43±0.43 165±23Low-molecular ligand 21 3.50±0.29 140±7

Figure 5: Histograms of interaction lengths (a,c) and interaction angles represented by the R–X· · ·Y angle (b, d); color-coded according to thehalogen involved (a, b), or electron donatingatom (c, d).

subset is 154◦, so chlorine interactions are likelyweak. On the other hand, bromine complexesare scattered across the whole range of angles(Fig. 5b) with a cumulation around 170◦ (Fig.6). The two iodine X-bonds are very straight(X-bond angle higher than 170◦) suggesting astrong interaction.

Figure 6: Interaction geometries in polar co-ordinates color coded according to the halogen(a), or the electron donor atom (b).

We analyzed the electron donors of halogeninteractions found in the NA complexes. Thebackbone oxygen atom is the most commonone. We identified 51 linear contacts of halo-gens with oxygen (71 %), and 21 with nitrogen(29 %). Although we included phosphorus as anelectron donor in the search, no linear contactwas found. The actual occurrence of the oxygenand nitrogen in NAs is roughly 2:1 (O:N), whichlikely contributes to the dominance of the lin-ear contacts with oxygen. Most of the nitrogeninteractions were found with chlorine, whereas

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bromine preferentially interacts with oxygen.82 % of the all interacting oxygens belong tothe phosphate backbone. Unlike other oxygensand nitrogens in NAs, the phosphate oxygenscarry a negative charge, which explains theirhigher propensity to halogen σ-holes.

According to the electron-donating atoms,the geometric quality of the interactions in-creases in the order of non-backbone oxygen <nitrogen < backbone oxygen (Tab. 3). The in-teractions which employ a backbone oxygen aretypically shorter and straighter than the others.

We conclude that different interaction atomslikely occur in different interaction geometries.It is also apparent on a projection of the inter-action geometries to the polar coordinates (Fig.6). We observed no correlation between inter-action lengths and the corresponding angles.

Halogenated Ligands Show

Sub-Optimal Interaction

Geometries

We did not find any X-bonded noncovalent lig-ands in NA complexes. Nevertheless, in the setof linear contacts we have found 15 unique lig-ands – 14 chlorinated and one brominated. Theremaining interactions involve halogenated nu-cleobases as halogen donors. The halogen in-teractions of ligands are longer and less linearcompared to the interactions of halogenated nu-cleobases (Tab. 3). The interaction angles de-viate notably from linearity, which suggest thatsuch interactions do not play a critical role inthe NA-ligand recognition.

From the pharmaceutical point of view, thelow-molecular ligands are of higher interestthan the halogenated building blocks. Belowwe discuss all of the instances among the linear-contact data set which involve a low-molecularligand. Structural formulas of the ligands dis-cussed are shown in Fig. 7.

Several halogenated compounds bind to thebacterial ribosome. Chloramphenicol (1)46 isa classic antibiotic compound that targets theribosomal A-site crevice in the 50S subunit(PDB 1nji, resolution 3.0 A;68 4v7w, resolution

Figure 7: Structural formulas of several low-molecular ligands or their building blocks thatparticipate in halogen interactions.

3.0 A69). 1 contains two aliphatic chlorines thatare activated by the nearby carbonyl group.Two distinct binding orientations in two differ-ent ribosomal system were proposed (T. ther-mophilus 68 and H. marismortui 69) (Fig. 8).The lengths of both chlorine interactions arebeyond the respective sums of the vdW radii(about 3.3 A, Tab. 1), and quite bent. Oneof the chlorines approaches either the ribose in-ring oxygen (3.9 A, 154◦), or a guanine nitrogen(3.8 A, 126◦), respectively. The geometries sug-gest that the halogens interactions contributeweakly to the complex stability.

Clindamycin (2)70 is another halogenated ri-bosome binder71 (PDB 1yjn, resolution 3.0 A)that binds into the 50S subunit (Fig. 9a) andforms linear contacts. 2 contains one chlorinethat directs towards the sugar edge of guano-sine. There is an interaction with guanine ni-trogen (3.5 A, 148◦). At the same time, thereis a shorter but more bent contact with sugarO2’ oxygen (3.1 A, 112◦).

7-chlorotetracycline (3)72 belongs to a classof tetracycline antibiotics that bind into the ri-bosomal 30S subunit. This class acts by pre-venting correct processing of aminoacyl-tRNA.Although no complex of 3 with the ribosome

9

Figure 8: Chloramphenicol 1 (CLM) interac-tions in the 50S ribosomal subunit with guano-sine (G) and uridine (U). Hydrogens were omit-ted for clarity. The overlay of the two bindingposes found in T. thermophilus (PDB 1nji) andH. marismortui (PDB 4v7w) into a single ref-erence frame is shown in red and black, respec-tively. Only residues within 7 A from each ofthe halogens are shown.

satisfies the data-set criteria, a complex withan RNA aptamer does. The aptamer was de-signed to bind 3 with sub-nanomolar affinity(PDB 3egz, resolution 2.2 A73). It features aninteraction between a chlorine and a ribose oxy-gen of a cytidine (Fig. 9b) (3.7 A, 151◦). Dueto the sub-optimal geometry, the role of halo-gen interaction in complex stabilization is likelymarginal. Moreover, there are many other in-termolecular interactions between the 3 and theaptamer, such as a stacking of a planar part of 3with a guanine, and an oxygen-magnesium co-ordination (not shown). Those reduce the rel-ative importance of the halogen contact evenmore.

Recently, crystal structures of T. ther-mophilus ribosomes with cisplatin37 (4 inFig. 9c) were resolved (PDB 5j4b, resolution2.6 A; 5j4c, resolution 2.8 A67). Nine moleculesof cisplatin are covalently bound to the ribo-some in place of one of the chlorines. Two ofthe cisplatin residues were identified to interferewith the mRNA tunnel and the GTPase site –the places critical for the ribosome functions.67

One cisplatin out of the nine forms an X-bondwith a guanine π-system (Fig. 4). Another cis-platin directs with its chlorine in between twostacked adenines with the shortest distance to

a nitrogen of 4.0 A and angle 151◦. Anothernitrogen is about 4.2 A far away and forms theangle of 148◦ (Fig. 9c).

Our data set contains several ligands thatbind into the prototype foamy virus (PFV) in-tasome, i.e. a complex of a viral integrase andDNA. Such a system serves as a model forHIV-1 integrase inhibition. Several inhibitorswere developed with a halogenated phenyl ring(5 and 6) that form X-bonds with NAs.74–76

No difference in the interaction geometries wasfound between 5 and 6 analogs. 5-analog in-teractions show the best geometric characteris-tics among the PFV ligands (but generally stillmodest); the chlorine on 5 interacts with twoadenine nitrogens (Fig. 9d). While the contactlength of 3.3 A is comparable with the sum ofvdW radii, the angle of 142◦ is far from the ideal180◦. The other contact is 3.5 A-long and evenmore bent (angle about 114◦). We add thatan aromatic fluorine activates the neighboringchlorine by making its σ-hole more positive.23

The effect of fluorination on the ligand bindingaffinity is not straightforward, however.77

We found only one brominated ligand with acontacts shorter than 4 A. A brominated im-idazole analog (7) binds into a G-quadruplexformed within an RNA aptamer called Spinach(PDB 4q9q, resolution 2.45 A).78 The Spinachmodule allows a green-fluorescence-protein-likefunctionality by selectively activating fluores-cence of 7. 7 binds in a planar conformationwith a strong stacking interaction with adenine.Besides this, it forms a contact with a phos-phate oxygen of 3.5 A under the angle of 137◦

(Fig. 9e).

Summary and Outlook

The role of X-bonding in drug development hasbeen emphasized for a decade. Nonetheless, theknowledge-based design of halogenated drugsthat feature an X-bond with their biomoleculartargets is still a tedious task. Although therehave been published several pioneering studiesthat involved protein targets,77,79,80 the nucleicacids still lack a successful application.

In this work, we focused on X-ray structures

10

Figure 9: Selected halogen interactions of ligand complexes. Clindamycin 2 (CLY) bound to theribosome (a), chlorotetracycline 3 (CTC) bound to an RNA aptamer (b), cisplatin 4 (CPT) boundto ribosome (c), an intasome binder (ZZX) (d), a brominated imidazole analogue 7 (2ZZ) in anRNA aptamer (e). Hydrogens were omitted for clarity. The interactions lengths and R–X· · ·Yangles are provided.

of biomolecular complexes containing halogeninteractions with nucleic acids. The generalability of nucleic acids to provide electron do-nating groups to X-bonding has been confirmedhere. Using criteria recommended by IUPACfor X-bond definition, we found 21 NA com-plexes with the X-bond. We also analyzed in-teractions of the halogen atoms longer than X-bonds.

All but one of the X-bonds involved a halo-genated nucleobase (preferably uridine). Halo-genated nucleotides are often utilized in X-raycrystallography, which explains their high oc-currence in the NA complexes. The incorpora-tion of heavy atoms such as bromine into thestructure helps to overcome the phase prob-lem. Moreover, the halogenated nucleotides al-ter the physico-chemical properties of the NAsthus modulate the crystallization conditions; inthe classical theory this is due to their stackingproperties,81 but recently X-bonding has alsobeen shown to play a role.11 What is more, halo-genated nucleobases may be also used in radi-ation anti-cancer therapy as radiosensitizers.82

Although, the exact mechanism of the increasedradiosensitivity is not known, the changes in theionization potential brought by the halogens or

global structural changes associated with modi-fied nucleobase pairing have been proposed.83,84

An important feature of X-bonds in NA com-plexes is their preference towards backbone oxy-gens. This is true also for the longer halogeninteractions. The phosphate oxygens tend toform straighter and shorter X-bonds than otherelectron donors, possibly due to their negativecharge.

In NA· · ·ligand noncovalent recognition, a va-riety of interactions plays a role. H-bondingand London dispersion-driven stacking interac-tions belong to the classic interactions, comple-mented by salt bridges, water-network disrup-tions, or metal-ion interactions. Strictly speak-ing, no noncovalent ligand has been found toemploy X-bonding in the NA binding (accord-ing to the IUPAC recommendations). No X-bonded NA ligand found is an intriguing fact,which may be explained in two ways:

i) The X-bonding ligands may be inefficientin NA recognition. If so, the halogenated lig-ands that bind into NAs bind due to other kindsof interactions that are stronger than X-bonds.Our data support this hypothesis. All of thehalogen interactions of ligands found in the sur-vey here show sub-optimal geometries. Given

11

the high X-bond directionality,85 the stabiliza-tion energy of a complex reduces notably whendeviating from the ideal geometry. Hence, thecontribution to the stabilization of the NA com-plexes is likely not dominant. Of course, thereare exceptions where X-bonding is the drivinginteraction. For instance, it was proven thata single X-bond/H-bond exchange might trans-form the DNA conformation completely.11

ii) Medicinal chemists may under-appreciatethe role of X-bonding in the drug-design strate-gies. Our data support also this hypothesis, be-cause we found only a few halogenated ligands:20 chlorinated, 8 brominated and one iodinated.

The current study on its own is unable todissect which reason is more likely. The start-ing set of PDBs was biased towards the lighterhalogens, which are, however, less suitable forstrong X-bonds. For a better understandingof the role of X-bonding, structural and func-tional studies are required, especially of theless-frequent brominated and iodinated com-pounds.

The effects of halogens are not limited tothe X-bonding, though. The halogenation af-fects the global electron distribution and conse-quently many of the molecular properties. Forinstance, Fanfrlık et al. demonstrated that sol-vation/desolvation may compensate for favor-able σ-hole· · ·lone pair interaction in halogen-to-hydrogen substitution in a protein-ligandcomplex.86 Also, a PDB survey of two proteinfamilies revealed shortening on H-bonds proxi-mal to ligand halogens.34 This corresponds withthe notion that halogenation increases the acid-ity of proximal H-bond donors. Still, these ef-fects in NAs are difficult to inspect with thelimited statistical sample of X-bonds here, andof halogenated NA ligands in general.

Nevertheless, the current analyses may helpin designing novel X-bonded NA binders. Suchbinders should contain a bromine or iodine toform a strong X-bond. The ligand bindingshould be directed to the vicinity of sugar-phosphate backbone, for example to grooveor bulge regions. Consequently, such a lig-and should likely not contain extensive aro-matic systems that are prone to intercalationinto canonical helices. Systematic halogenation

of known pharmacologically active compoundsmay be an optimal strategy to identify newagents with enhanced activity, presumably sup-ported by the specific X-bonds.

Biographies

Michal H. Kolar received his Ph.D. in 2013from Charles University in Prague, and fromthe Institute of Organic Chemistry and Bio-chemistry, Academy of Sciences of the CzechRepublic. With Pavel Hobza he focused ontheoretical and computational description ofnoncovalent interactions. He is a recipient ofthe Humboldt Research Fellowship for Postdoc-toral Researchers. From 2014 he worked withPaolo Carloni in Forschungszentrum Julich,Germany, on RNA-ligand recognition. In 2016he moved to Helmut Grubmuller’s group at theMax Planck Institute for Biophysical Chem-istry, Gottingen, Germany, pursuing his inter-est in non-coding RNAs and computer simula-tions.

Oriana Tabarrini is a Professor of Medici-nal Chemistry at the Department of Pharma-ceutical Sciences (University of Perugia, Italy).After the degree in Pharmaceutical Chemistryand Technology she has worked for several yearswith research grants from pharmaceutical in-dustries. In 2002 was promoted to AssociateProfessor. Her research has mainly aimed atdeveloping small molecules as pharmacologi-cal tools and potential chemotherapeutics withparticular focus on nucleic acid binders as an-tivirals and more recently for the treatmentof neurodegenerative disorders. She has pub-lished over 85 research articles in leading peer-reviewed journals, including some invited re-views and patents. She has also received severalproject grants and is an ad-hoc reviewer for sev-eral top journals.

Abbreviations

BRU 5-bromouridine

CLM chloramphenicol

CLY clindamycine

12

CPT cisplatin

DNA deoxyribonucleic acid

dsDNA double-stranded DNA

G4 G-quadruplex

H-bond hydrogen bond

HCV Hepatitis C virus

HIV-1 human immunodeficiency virus type 1

IRES internal ribosome entry site

NA nucleic acid

ncRNA non-coding RNA

PDB Protein Data Bank

PFV prototype foamy virus

RNA ribonucleic acid

TAR trans-activation response RNA element

X-bond halogen bond

Acknowledgement MHK is thankful to N.Leioatts and A. Obarska-Kosinska for valuablediscussions on the manuscript, and acknowl-edges the support provided by the Alexandervon Humboldt Foundation. Most of the analy-ses prospered from Numpy87 and Matplotlib88

python libraries.

Corresponding Author In-

formation

Email: michal@mhko.science

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