Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450BM3 demethylases Jared C. Lewisa, Sabine Bastiana, Clay S. Bennettb,1,YuFub, Yuuichi Mitsudaa,2, Mike M. Chena, William A. Greenbergb, Chi-Huey Wongb,c, and Frances H. Arnolda,3 aDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125; bDepartment of Chemistry, The Scripps Research Institute, La Jolla, CA, 92037; and cGenomics Research Center, Academia Sinica, Nankang, Taipei 115, Taiwan Contributed by Frances H. Arnold, August 10, 2009 (sent for review July 2, 2009) Polysaccharides comprise an extremely important class of biopoly- migration to produce other products (11), non-selective migra- mers that play critical roles in a wide range of biological processes, tion remains problematic, and the generality of the lipase but the synthesis of these compounds is challenging because of method seems to be limited. their complex structures. We have developed a chemoenzymatic The generality and utility of a chemoenzymatic method for the method for regioselective deprotection of monosaccharide sub- synthesis of monosaccharide derivatives would be expanded strates using engineered Bacillus megaterium cytochrome P450 greatly if each site of a given substrate could be deprotected (P450BM3) demethylases that provides a highly efficient means using a unique catalyst. This approach would require not only a to access valuable intermediates, which can be converted to a protecting group resistant to migration under the deprotection wide range of substituted monosaccharides and polysaccharides. conditions but also a catalyst capable of removing this group with Demethylases displaying high levels of regioselectivity toward a high levels of chemo- and regioselectivity. We hypothesized that number of protected monosaccharides were identified using a cytochrome P450-catalyzed heteroatom demethylation might combination of protein and substrate engineering, suggesting that provide a solution to both these problems. Although chemical this approach ultimately could be used in the synthesis of a wide removal of methyl groups requires harsh and unselective con- range of substituted mono- and polysaccharides for studies in ditions, this enzyme-catalyzed reaction proceeds via insertion of chemistry, biology, and medicine. oxygen into a methyl C-H bond to generate a hemiacetal CHEMISTRY intermediate that spontaneously decomposes to release form- biocatalysis ͉ demethylation ͉ evolution ͉ P450 ͉ polysaccharide aldehyde and an alcohol under ambient conditions (Fig. 1A) (12). One of our groups (F.H.A.) has used variants of Bacillus omplex carbohydrates comprise an extremely important megaterium cytochrome P450BM3 (BM3) to hydroxylate a wide class of biopolymers that play critical roles in a number of range of non-natural substrates with high levels of regioselec- C tivity (13). If this enzyme could be engineered to accept perm- biological processes, including inflammation, cancer metastasis, ethylated monosaccharides, the challenging task of regioselec- and bacterial and viral infection (1). Unlike nucleic acids and tively demethylating these substrates could be possible. Because EVOLUTION proteins, oligosaccharides can be highly branched, and their BM3 is amenable to protein engineering, the activity, regios- biosynthesis proceeds in a non–template-directed fashion to electivity, and substrate scope of these reactions could be produce complex mixtures of closely related structures that are optimized using directed evolution (14) to generate BM3 dem- difficult, if not impossible, to separate (2). Because subtle ethylases for the production of any desired monosaccharide changes in the structures of these compounds can alter their derivative. properties dramatically (3), detailed studies of oligosaccharide- mediated events require the use of homogeneous synthetic Results material (4). Oligosaccharide synthesis traditionally has been We therefore screened libraries of BM3 variants on hand in our accomplished through the installation and removal of various laboratory to determine the scope of their reactivity toward a orthogonally reactive protecting groups to differentiate each number of common pentamethyl hexoses (Fig. 1C). A compi- position of a monosaccharide substrate, and a similar procedure lation library comprised of 96 BM3 variants previously engi- is used to synthesize glycosides and substituted monosaccharides neered for a variety of applications was used to survey functional in which 1 hydroxyl group is used as a handle for the introduction enzymes rapidly (15). Because many of these variants were of small molecules or other functional groups (5). Despite related along an evolutionary lineage by mutation of 1–3 resi- numerous advances in the synthesis of these materials during the dues, we also used a targeted library of 1,024 BM3 variants past decade, their preparation remains an arduous task (6). having up to 10 mutations in the active site of the parent variant, Researchers have sought to simplify the preparation of suit- 9–10A. Demethylase activity first was determined using a high- ably protected monosaccharides with 1-pot synthetic procedures, but these approaches require considerable synthetic expertise and are limited in terms of substrate scope (7). Elimination of Author contributions: J.C.L., W.A.G., C.-H.W., and F.H.A. designed research; J.C.L., S.B., protecting groups altogether also has been demonstrated using C.S.B., Y.M., and M.M.C. performed research; J.C.L., C.S.B., and Y.F. contributed new enzymes to affect the glycosylation of various substrates (8). The reagents/analytic tools; J.C.L., S.B., Y.M., and M.M.C. analyzed data; and J.C.L. and F.H.A. utility of this approach could be expanded greatly by identifying wrote the paper. or engineering additional enzymes (9), but the preparation of The authors declare no conflict of interest. substituted monosaccharides lies beyond its scope. Chemoenzy- Data deposition: The atomic coordinates have been deposited in the Cambridge Structural Database, Cambridge Crystallographic Data Centre, Cambridge CB2 1EZ, United Kingdom matic regioselective deprotection of a globally protected (CSD reference no. 673244). monosaccharide is a potentially practical alternative to these 1Present address: Department of Chemistry, Pearson Chemistry Laboratory, Tufts Univer- approaches. This approach has been demonstrated using lipases sity, Medford MA, 02155. to deprotect peracylated monosaccharides, but the range of 2Present address: Discovery Research Laboratories, Shionogi and Co. Ltd., 2–5-1 Mishima, products reported has been limited thus far to those accessible Setu, Osaka 566-0022, Japan. with known enzymes, and no attempt has been made to improve 3To whom correspondence should be addressed. E-mail: [email protected]. or alter the biocatalyst activity or regioselectivity (10). Although This article contains supporting information online at www.pnas.org/cgi/content/full/ careful control of reaction conditions permits regioselective acyl 0908954106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908954106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 27, 2021 A B P450 -CH O P450 -2 CH2O O O OH 2 O O O O OH R Me R ROH R Me R ROH O2 O2 C O OR 1. BM3 variants: PO 2. Substrates: E. coli - expressed in + PO OP - Glc, Man, Gal, etc. - 96 well plate format OP - different anomeric groups (R) - different protecting group (P) 4. Improve promising variants - random mutagenesis high activity 3. no and Screen variants for removal of P - rational design selectivity - HT colorimetric screen for activity - GC screen for regioselectivity yes 5. Preparative-scale reactions O ORn PO - mono-deprotected products isolateded - converted to value-added products PO OP OP P450 P450 P450 P450 O OR1 O OR2 O OR3 O OR4 HO PO PO PO PO OP HO OP PO OP PO OH OP OP OH OP O OH O OH O OH O OH R HO HO HO HO OH R OH HO OH HO R OH OH R OH R = F, H, monosaccharide, etc. Fig. 1. (A) P450-catalyzed removal of methyl (Me) and (B) methoxymethyl (MOM) groups. (C) Method used to screen and optimize activity of BM3 demethylases. Libraries of BM3 variants were screened for activity on Me- or MOM-protected substrates. The activity of BM3 variants in cell lysate was assayed by colorimetric detection of formaldehyde (CH2O) released during the deprotection, and regioselectivity was determined by GC analysis of the reaction mixtures. Promising enzymes were optimized by directed evolution to provide enzymes suitable for preparative scale. Mono-demethylated products were converted to a variety of value-added products. throughput (HT) colorimetric assay for formaldehyde released regioselectivities contrasts with the narrow scope of existing during the reactions (Fig. 1A; see supporting information (SI) chemoenzymatic methods and suggested that BM3-catalyzed Appendix). The most active enzymes then were re-expressed, demethylation could provide a general approach to monosac- purified, and used to catalyze the same reaction under controlled charide elaboration if the yields and regioselectivities observed conditions to ensure the accuracy and precision of catalyst for these enzymes could be improved using directed evolution. loadings. Finally, the reaction mixtures were analyzed by gas Furthermore, the significant differences in both activity and chromatography (GC) to establish the regioselectivity of de- regioselectivity between the methyl- and benzyl-protected glu- methylation. Promising variants were optimized