Graphical Abstract To create your abstract, type over the instructions in the template box below. Fonts or abstract dimensions should not be changed or altered. Halogenase engineering and its utility in Leave this area blank for abstract info. medicinal chemistry Amy E. Fraley, David H. Sherman Bioorganic & Medicinal Chemistry Letters !"#$%&'()"*+,&-+.(///0+'1+23+$04"* Halogenase engineering and its utility in medicinal chemistry Amy E. Fraleya,b, David H. Shermana,b,c,d,! aLife Sciences Institute, bDepartment of Medicinal Chemistry, cDepartment of Chemistry, dDepartment of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109, United States. ARTICLE INFO ABSTRACT Article history: Halogenation is commonly used in medicinal chemistry to improve the potency of pharmaceutical Received leads. While synthetic methods for halogenation present selectivity and reactivity challenges, Revised halogenases have evolved over time to perform selective reactions under benign conditions. The Accepted optimization of halogenation biocatalysts has utilized enzyme evolution and structure-based Available online engineering alongside biotransformation in a variety of systems to generate variants with optimal stability and selectivity. The stability of the halogenase-catalyzed reaction has demonstrated the Keywords: utility of these biocatalysts for industrial purposes, and their ability to achieve a broad substrate halogenase scope implies a synthetic tractability with increasing relevance in medicinal chemistry. biocatalysis Keyword_3 Keyword_4 2009 Elsevier Ltd. All rights reserved. Keyword_5 1.!Introduction involving the formation of chloramine on a catalytic lysine in the enzyme active site. Enzymatic fluorination requires unique Enzymes have evolved to catalyze reactions with high conditions that enable desolvation of the fluoride ion, which efficiency and selectivity, and can overcome inherent chemical subsequently performs a nucleophilic attack on the substrate. biases with complex substrates. Halogenation is particularly Numerous studies relating to the mechanism and function of FDHs important in medicinal chemistry and drug design, and with the aid have been reported, from which a foundation for halogenase of biocatalysts, chemists can implement efficient methods for engineering has been built. Each of these four classes will be halogenation that are environmentally benign. Since the discovery further discussed below. of halogenating enzymes (halogenases), an effort has been made to engage their selective reactivity in syntheses of complex 2.!Bioactive halogenated natural products and halogenating molecules such as natural products. Thus, the knowledge gained enzymes from the characterization of these enzymes has led to a major effort in halogenase discovery and engineering for biocatalyst Many of the over 4,000 known halogenated natural products 4 development. This has included methodologies in structure-based have therapeutic potential. The antitumor agent rebeccamycin, 5,6 7,8 engineering1 and directed evolution2 where optimized biocatalysts antibiotic vancomycin, and vasorelaxant malbrancheamide are with scalable capabilities3 have been generated. Additionally, structurally complex compounds, and strategies toward their halogenases have been implemented in vivo using various synthesis would benefit from new halogenation methods (Fig 1). microbial expression systems to optimize biotransformation of The challenges involved in synthetically preparing these materials complex molecules. have motivated researchers to investigate the biosynthetic machinery involved in the assembly and late-stage tailoring of Most halogenases are currently grouped into four classes these secondary metabolites. Current chemical halogenation including 1) haloperoxidases (heme-containing and vanadium- methods are rarely selective and tend to involve the use of toxic containing), 2) iron (II)/2-(oxo)-glutarate (FeII/2OG)-dependent reagents. The rebeccamycin halogenase RebH has been halogenases, 3) flavin-dependent halogenases (FDHs), and 4) extensively characterized9,10,11 and the wealth of biochemical fluorinases. Haloperoxidases tend to be nonselective due to the information has been utilized toward enzyme engineering efforts. freely diffusing hypohalous acid that acts as the halogenating RebH binds tryptophan, a biosynthetic precursor, and catalyzes agent, whereas FeII/2OG-dependent halogenases proceed through chlorination at the Trp C7 position prior to incorporation into the a radical mechanism to halogenate aliphatic carbons on a variety metabolite by additional biosynthetic enzymes (Fig 1a). The of substrates. FDHs utilize hypohalous acid similarly to the halogenase involved in vancomycin biosynthesis (VhaA) performs haloperoxidases, but they implement a selectivity mechanism a late-stage dihalogenation of the carrier protein bound ——— ! Corresponding author. Tel.: +0-000-000-0000; fax: +0-000-000-0000; e-mail: [email protected] H N O !" COOH O H2N :+#; # N N H " N Cl Cl OH ! H O OH <)6-/.-&!0 HO OH MeO OH NH2 OH OH O O #" O OH HO :+#+$$!56$*0 OH Cl O Cl O O HO OH HO OH O O O H H H O O O O N N N H H H N N N O N N N H H H N N N S O O %&!' H H H HN O O PCP O O NH2 NH2 OH OH (!))*+),-)./+*01#.203,&+4!-+-/*3+ HO OH %!0$.56$*0 $" H H N N Cl H H Cl O H 7!8' O H N N N N 9)+5!8#)!0$&+!5*3+ 7!8#)!0$&+!5*3+ Figure 1. Flavin-dependent halogenases involved in the production of therapeutically relevant natural products. PCP denotes peptidyl carrier protein. hexapeptide precursor prior to cyclization and offloading of the (edge-on) or interacts directly with the center of the aromatic final product (Fig 1b).12 Late-stage halogenation of complex ring(face-on).18,19 Structural analysis of proteins bound to molecules is particularly challenging, but tethering a substrate to a halogenated ligands has indicated that the edge-on interaction is carrier protein presents its own biosynthetic demands. MalA is a more common and tends to involve phenylalanine and histidine late-stage halogenase involved in malbrancheamide biosynthesis side chains. In contrast, the face-on interaction frequently involves with the ability to react with complex molecules that are not bound the side chains of tryptophan and tyrosine residues, potentially due to an accessory carrier protein, making it more suitable as a to their higher " densities.19 When the halogen atom is attached to potential biocatalyst (Fig 1c).13 Chloramphenicol is a well-known an aromatic region of the ligand, these bond strengths are antibiotic that is chlorinated by the FDH CmlS, which bears a rare potentiated. covalently bound flavin cofactor, and its biosynthetic halogenation The pharmacokinetic properties of drugs can be modulated by remains under investigation.14,15,16,17 Molecular dissection has the insertion of a halogen substituent such as fluorine, which is enabled engineering strategies directed toward improving commonly substituted for aromatic hydrogens that are prone to stability, expanding substrate scope, and altering site-selectivity of metabolism. In a study of N-benzylamide-based thrombin FDHs. inhibitors, para substitution of fluorine showed the highest 3.!Halogenation and drug design increase in potency presumably due to the decrease in metabolism.23 The halogen bond has also been utilized to facilitate One third of the drugs currently in clinical trials are the selective binding of certain cytochrome P450 enzymes halogenated, which underscores the significance of methods for (CYPs).18 Cl-" interactions between the cardiovascular drug introducing F, Cl, and Br atoms into pharmaceuticals.18 Halogens molecules ticlopidine and amlodipine and CYP2B4 stabilize a can aid in controlling metabolism and improving pharmacological properties such as lipophilicity and permeability. Additionally, the &'(')*(+,-.//+,+%0,*-!"#$1%& halogen functionality can facilitate target binding which was 2+34(-,$)53+0'(+506-78-9+0:79;:7 originally thought to be a consequence of electron-withdrawing <%,50=-,$)53+0'(+506-8>-9+0:79;:7 and steric effects. More recently, investigators have begun studying the “halogen bond” and its utility in generating more selective therapeutics.19 Due to the anisotropic nature of the ! halogen atom, a region of positive potential (sigma hole) can interact with a negatively charged region on the protein to form a #$% " halogen bond,20,21 the strength of which directly correlates with the size of the halogen itself. In fact, the strongest halogen bond (between iodine and a backbone carbonyl) is comparable in energy to the weakest hydrogen bond.22 Typically, halogen bonds have been observed either between the ligand halogen and aromatic 21 amino acids or a backbone carbonyl. The interactions with Figure 2. Cl-" interaction in MalA halogenase. The C9-chlorinated aromatic side chains are considered either edge-on or face-on, substrate is shown in blue (PDB ID 5WGW) and the C8 chlorinated where the halogen approaches the aromatic ring from the periphery substrate is shown in yellow (PDB ID: 5WGZ) conformation conducive to metabolism that is not observed when regenerated through a recycling system consisting of either glucose the molecules bind CYP2B6. The Cl-" interaction is not only dehydrogenase28 or alcohol dehydrogenase (Fig 3).3 One significant for therapeutic targets, but it can also potentiate hypothesis is that by bringing the enzymes in this system into close reactivity of some biosynthetic halogenases involved in producing proximity, they could exchange cofactors