*1.1. Electronical Properties of Halogen Moieties*

The presence of a halogen (Cl, Br, I) usually increases the bulkiness of a compound, blocking for instance active site pockets or increases membrane permeability, relevant for oral absorption, and blood–brain barrier permeability. Besides their bulkiness, halogens exhibit extraordinary effects on the polarization of a compound. On the one hand, the halogens of the upper periods (F, Cl, Br) have a large electronegativity, which leads to a considerable latent polarization in the molecule (see Figure 1A). On the other hand, the polarizability increases with increasing period, so that interactions with soft nucleophiles or electrophiles in particular are promoted (see Figure 1B). Although the latent polarization is depicted in Figure 1A as a homogeneous gradient the model must be refined. Due to the p-orbital architecture there is a hole in the electron density opposing the binding partner of the halogen which is called the σ-hole (Figure 1C). Considering this σ-hole, it offers the option to interact with heteroatoms (O, S, N) by so-called halogen bonds as well as hydrogen bonds [15]. The ability to form halogen bonds has been the focus of several pharmacologically-oriented groups in the past years, as it can serve as an alternative non-covalent interaction between atoms (see Figure 1). For a detailed insight into the nature and characteristics of halogen bonds, as well as their possible impact on drug discovery in the future, see the corresponding articles [4,15–19]. The importance of halogens for biological activity of compounds can be profound. Vancomycin (**1**, Figure 2), an antibiotic, was shown to exhibit 30% to 50% less activity, based on the chlorine substituents missing, which is remarkable considering how small the portion of the halogens with respect to the entire vancomycin molecule is [20].

**Figure 1.** Schematic representation of electron distribution in halogens. (**A**): Latent polarization of a carbon-halogen bond. (**B**): Polarizability of large halogens (Br, I) bonded with a carbon. The external electrical field, for example, caused by an approaching electrophile/nucleophile leads to the distortion of the electron density. (**C**): Schematic view on the "σ-hole". The electron density is drawn to the carbon-halogen bond, with the strength gradually increasing with the size of the halogen (I > Br > Cl >> F). This anisotropic distribution of electrons in the outer orbitals of the halogen creates an area of higher electron density around the belt of the halogen, allowing interaction with electrophiles or H-bonds. Orthogonal to the direction of the bond is an area of electron deficiency, creating a partially positively charged area in the halogen, allowing for nucleophilic attacks, commonly called "σ-hole".

In terms of drug discovery, halogen substituents are regularly found in promising drug candidates with 35% in the discovery stage, while they appear in 36% of the candidates in clinical phase II and 26% in the drugs launched into the market (data from 2014) [16]. This trend shows that halogens play an important role in the field of drug design and discovery, and usually find their way to the final product assigned for treatment. In the following paragraphs, relevant halogens and some associated

drug candidates containing halogen atoms will be discussed regarding their characteristic effects on bioactivity.

**Figure 2.** Examples for halogenated active agents.

The most prominent halogen introduced into active agents is fluorine with 57% [3]. Due to its similar size compared to hydrogen and the extreme electronegativity, C–F bonds are polarized in a distinctive manner and render fluorine a weak halogen bond acceptor in contrast to be a good hydrogen-bond acceptor [21,22]. The covalent fluorine bond is very strong (456 kJ/mol for CF4), so that these bonds can only be cleaved under extreme and costly conditions in the body, if at all [23]. This increases the half time of active agents within the body (and environment) compared to their non-fluorinated pendants. Besides the electronic effects of fluorine within a molecule, fluorine also provides stereochemical properties which is summarized as fluorine *gauche* effect. Briefly, it can be described as a non-bonding weak interaction of the fluorine orbitals and other interacting partners. This reduces the degrees of freedom in rotation and this determines the conformation of a particular fluorinated molecule or guides reaction pathways. A review concerning this topic can be found in reference [24]. Apart from altering molecular characteristics, 18F is used as a common radioactive isotope label for in vivo study of protein function and enzyme catalysis [25]. Of all halogenated active agents, ledipasvir (**2**, see Figure 2) is one of the top-selling drugs, administered for the treatment of hepatitis C. Another important compound is dacomitinib (**3**), a single-fluorinated drug, which has been in clinical trials for the treatment of non-small-cell lung cancer [26].

Chlorine is the second prevalent halogen with 38% in halogenated drugs. Due to its increased size, it is a moderate halogen bond acceptor, while still being stable when being introduced into a carbon bond (327 kJ/mol for CCl4) [23]. Its presence in a compound alters volume and shape, allowing for positioning in deep cavities within proteins. These characteristics make it an interesting option for the functionalization of heterocycles. One of the most prominent chlorine-based natural compounds is rebeccamycin (**4**), a weak topoisomerase I inhibitor, which showed significant antitumor properties [27].

Brominated compounds are rarely found in drugs, making up only 4% of all halogenated compounds. This seems contradictory at first, as most halogenated compounds originate from marine organisms and are brominated despite chlorine being the more abundant halogen in water. Due to the lower polarization of the carbon-bromine bond and the extended bulkiness, bromine usually forms longer and thereby more labile bonds, not suitable for most drug candidates for a proper inhibition (272 kJ/mol for CBr4) [23]. These characteristics however allow an easier oxidation of bromine and consequently an easier incorporation into molecules, compared to chlorine. Although there is a prevalence of chlorinated and fluorinated active agents in pharmacology, some brominated compounds are known to display relevant bioactivity like eudistomin K (**5**), viable for the treatment of polio and herpes [28].

Iodine is the rarest halogen used (1%), commonly exploited for the synthesis of the active agents. Having a higher size and lower electronegativity, its bonds formed with carbon atoms are more labile than those of bromine, being easily cleaved off. Iodine is, therefore, preferably suitable for short-lived applications. An example of the use of iodine in medicine is radioactively labelled 124I in positron emission tomography (PET) as a tracer [29].

#### *1.2. Halogens as Synthetic Tools*

Both, bromine and iodine, are rare as functional moieties in active agents due to their labile covalent bonds. But it is precisely these properties that make halogens of higher periods valuable instruments for the synthesis of active substances.

A patent application for the production of hypohalous acids was applied for in 1944. *C. C. Crawford* and *T. W. Evans* described a process to obtain halide-free solutions of hypochlorous acid. This halogenating reagents were used in industrial applications to produce e.g., halohydrins from unsaturated organic compounds [30]. In 1993 another patent to produce concentrated slurries of sodium hypochlorite [35% (*w*/*v*)] was accepted [31]. They describe a process for highly pure hypochlorite slurry production. All the processes have the same starting materials in common. The first step is the solvation of molecular chlorine in water to get hypohalous acid (**6**) or the solution of sodium hydroxide and chlorine in water to end up with sodium hypochlorite. However, contaminations of sodium chloride and remaining sodium hydroxide occur in most processes that are carried out in industrial scale. The chlorine is hereby acquired by the chloralkali process where the electrolysis of sodium chloride produces molecular chlorine gas. Similar processes are state-of-the-art for the production of sodium bromate, which has the drawback of being a strong oxidizing agent [32,33], but can be used for the bromination of aromatic compounds [34]. The production of stable hypobromous acid is rather difficult because it easily oxidizes to bromate. Here, the production is carried out starting from hypochlorous acid or a modified chlorite [35].

More common halogenating agents are *N*-bromo-succinimide (NBS) and *N*-chloro-succinimide (NCS). Interestingly, even these reagents are synthesized from molecular halogens or hypohalous acids [36]. As a conclusion, it is now rather obvious, that all halogenating reagents have their origin in molecular halogen gases that are produced by cost-intensive procedures like halogen alkali electrolysis from halide salts (Figure 3).

**Figure 3.** Workflow for the provision of halogenating reagents from alkali salts. The electrolysis process thus produces molecular halogens (X2), as well as hypohalous acids (HOX, **6**) and *N*-halogenated succinimides (NXS, **8**) in further steps.

Having these halogenated building blocks at hand, further synthetic steps can follow to build up active agents. Not only in academia but also in industry, the synthetic tool in terms of cross-coupling reactions is one of the most common C–C- and C–Y bond formations (Y is in this case N, O, S). With the use of different transition metals and activated carbon components, it is possible to generate large bioactive natural products and their derivatives. One prominent example is the use of palladium for the selective preparation of arenes and heterocyclic scaffolds with different substitution patterns [37]. However, also non-noble transition metals like copper [38], nickel [39], and nowadays even iron [40–42] are firmly anchored as suitable catalysts. Besides the high chemoselectivity, a profound functional group tolerance is a main advantage of these kind of reactions. Therefore, it is not surprising that industry has established approaches to produce pharmaceuticals and fine chemicals at the kilogram scale [43,44]. The following Figure 4 gives an overview of the most popular metal catalyzed named reactions, that slightly differ in their reactive moieties for both products or starting materials [37,45–48]. However, the catalytic cycle and thereby the reaction mechanism is very similar for all (Figure 5). Finally, conversions such as the *Appel*reaction and the *Hell-Volhard-Zelinsky* reaction, in which functional groups such as alcohols are converted to haloalkanes or carboxylic acids that become acid chlorides, must also be mentioned here.

**Figure 4.** Most common reactions in organic synthesis exploiting halogen moieties. [37,45–48] Besides organolithium reactions as well as Grignard/Barbier reactions all of them are Pd-based, but can in many cases be substituted by other transition metals such as nickel.

**Figure 5.** Scheme of the steps in cross-coupling reactions. After oxidative addition of the organo-halogen species, the transmetalation occurs. The ligands start rearrange before reductive elimination to the final product is carried out and the catalyst is regenerated.

#### *1.3. Halogen Chemistry is Energy-Demanding*

It is estimated that about 55% of chemical and 85% of pharmaceutical end products were processed with key components derived from the chloralkali electrolysis process [49,50]. These include hydrochloric acid to adjust the pH, or chlorinated solvents as part of the synthesis and subsequent isolation. However, this results in the production of the active compounds under hazardous conditions and high costs, due to toxic waste management. Using enzymes to halogenate pharmaceutical active compounds in a mild way and with a high efficiency is certainly a desirable aim for a greener chemistry. In general, the production and further processing of chlorine is mostly performed in the very same geographical region or facility in order to avoid the transportation of toxic and dangerous intermediates. This was reported for German companies and, presumably, this is also the case for other countries. The key component for halogenation (chlorine) is produced by electrolysis and is one of the most energy-consuming processes in the chemical industry. The process is responsible for about 2% of the total energy consumption yielding 5 million tons per year of chlorine in Germany [50,51]. Obviously, the energy reduction is an objective of the chloralkali industry, since 50% to 60% of the production costs is spend for the electrical energy [52].

#### **2. Halogenating Enzymes**

Although halogenated natural compounds are rare and only found within the regime of secondary metabolism, at least six types of halogenating enzymes were evolved. Many were evolved from monooxygenases, since hypohalous acids are the core intermediate of catalysis in these halogenating enzymes. As diverse the origins of halogenating enzymes are as diverse is their classification. In Figure 6 we tried to give an overview on the categories of halogenating enzymes. Although often used synonymously, it can be differentiated between haloperoxidases and halogenases. The first group forms hypohalous acid from the respective halide and hydrogen peroxide via heme-iron-, vanadium-coenzymes, or even without any coenzyme. The hypohalous acid is set free for most of the enzymes and the very halogenation reaction takes place outside the active site. In contrast, the halogenases generate or simply use halonium species for the halogenation without the use of hydrogen peroxide.

**Figure 6.** Overview on the categorization of halogenating enzymes.
