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 by incorpora- cose substrates 3 and 4 indicated that varying the anomeric tion of random mutations and screening to provide total turn- substituent could be used to identify suitable enzyme/substrate Ͼ over numbers (TTN, ca. 1,000) and regioselectivities ( 80%) combinations without the need for protein engineering. Indeed, sufficient for practical gram-scale preparation of regioselectively variant 9–10A F87I catalyzed the demethylation of both 3 and deprotected monosaccharides. This approach also was used to 4 with suitably high yield (Ͼ 1,000 TTN) and regioselectivity (Ͼ identify variants capable of removing the methoxymethyl 80%) to warrant investigation of these reactions on a preparative (MOM) group from MOM-protected monosaccharides to en- scale, as discussed later. able additional synthetic applications (Fig. 1B). Although these results were very promising, we were con- Initially, 4 substrates, 1,2,3,4,6-pentamethyl-␣-glucopyrano- cerned about the lack of reactivity observed with galactose side (1), 1,2,3,4,6-pentamethyl-␣-galactopyranoside (2), 2 1,2,3,4,6-pentamethyl-␣-mannopyranoside (3), and 1-benzyl- substrate , especially given the frequency with which this 2,3,4,6-tetramethyl-␤-glucopyranoside (4) (Fig. 2A) were exam- monosaccharide is found in natural systems (1). To address this ined. The last of these compounds, 4, was included to determine issue, we examined the activities of the compilation library if a substrate-engineering approach (16) could lead to alter- enzymes on 4 additional tetramethyl galactose derivatives with a ␤ native regioselectivities, and authentic samples of each of the variety of anomeric substituents in the configuration, including possible mono-demethylation products for 1–4 were synthesized methyl (Me, 5), benzyl (Bn, 6), benzoyl (Bz, 7), and acetyl (Ac, to serve as GC standards for product identification. We first 8). Once again, 9–10A F87I displayed high demethylation ac- screened the compilation library for activity on these substrates tivity, this time on the 1-␤-benzyl galactose substrate 6, and (Fig. 2B). Pleasingly, we found that a number of enzymes in this nearly quantitative conversion and high selectivity for the 4-po- small library were active on substrates 1, 3, and 4 in the HT assay, sition was confirmed by NMR spectroscopy. This result further and GC analysis of these same reactions conducted with purified established the utility of substrate engineering to achieve highly enzymes under optimized conditions (17) revealed that, except selective demethylation reactions and confirmed that derivatives for C-1, nearly every position of the monosaccharide substrates of 3 of the most common hexoses were compatible with our investigated was accessible (Fig. 2 C–F). This broad range of enzymatic demethylation method.

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908954106 Lewis et al. Downloaded by guest on September 27, 2021 OMe MeOOMe OMe OMe A Me O O OO MeO MeO MeO O MeO MeO MeO MeO OBn MeO MeO OMe OMe OMe MeO α-pmGlc (1) α-pmGal (2) α-pmMan (3) β-tmBnGlc (4)

B OMe 0.5 µM P450, 0.4 mM NADP+ OR2 MeO O 40 mM G6P, 2u/mL G6PDH R2O O MeO R2O MeO 1 2 OR 100 u/mL SOD, 25 °C, 12h R O OR1 1 DMSO/KPi (100 mM, pH8) R = Me or Bn R2 = Me or H C D 6000 Total Turnover Number -pmGlc (1) product distribution 100% C3-OH pmGlc (1) C4-OH 80% 4000 pmMan (3) C6-OH tmBnGlc (4) 60%

2000 40% 20%

0 0% 9-10A 9-10A 9-10A 9-10A 9-10A 9-10A 12-10C 9-10A 9-10A 9-10A 9-10A 9-10A 9-10A 12-10C A82L A82F F87A F87V F87I A82L A82F F87A F87V F87I

E -pmMan (3) product distribution F -tmBnGlc (4) product distribution 100% 100% C3-OH 80% C2/4-OH 80% C2-OH C3-OH C6-OH C4-OH C6-OH CHEMISTRY 60% 60%

40% 40%

20% 20%

0% 0% 9-10A 9-10A 9-10A 9-10A 9-10A 9-10A 12-10C 9-10A 9-10A 9-10A 9-10A 9-10A 9-10A 12-10C A82L A82F F87A F87V F87I A82L A82F F87A F87V F87I

Fig. 2. (A) Structures of substrates used in initial screens. (B) Optimized reaction conditions for demethylation reactions. (C) TTN for enzymes (see SI Appendix EVOLUTION for sequences) identified in HT screen on substrates 1, 3, and 4 and (D–F) product distributions (% of total product) for demethylation of substrates 1, 3, and 4 by BM3 variants determined by GC analysis of crude product mixtures.

To broaden the generality of the method further, we explored 7, and 8. Colorimetric analysis of formaldehyde produced in the activities of the active-site library enzymes on ␣- and these reactions confirmed the activity observed on 2 and 5 but ␤-pentamethyl galactose substrates 2 and 5 because galactose also indicated that 7 is a compatible substrate (see SI Appendix). derivatives had proven most resistant to demethylation by the Six variants catalyzed the demethylation of these substrates with small set of enzymes surveyed thus far (Fig. 3A). A number of moderate efficiency (ca. 30% conversion based on formaldehyde hits were obtained in the HT assay, and 8 of the most active concentration), indicating that active-site engineering could variants were purified and re-screened against substrates 2, 5, 6, extend further the scope of the reaction. GC analysis of the crude reaction mixtures obtained with 2 revealed that variant 2C6dr provided the highest conversion of OMe P450, 0.5 mM NADPH 2 2 MeO R OOR R1 = αMe (2), βMe (5), substrate, producing a 1:1 mixture of O-2 (39%) and O-3 (41%) A 0-5% DMSO/ KP O i O βBn (6), βBz (7), demethylation (compounds 9 and 10). Although the activity of 2 MeO R O and βAc (8) OMe OR1 (100 mM, pH8) 2 1 2C6dr on 5 and 7 was slightly lower under these reaction OR OR R2 = H, Me 1 mM -CH2O conditions, we observed nearly exclusive O-3 demethylation with these compounds to give 11 (50%) and 12 (57%), as confirmed B MeOOMe MeOOMe MeOOMe MeOOMe O O O O by NMR spectroscopy and x-ray analysis, respectively. These MeO HO HO OMe HO OBz results indicated that the remaining 2 secondary hydroxyl posi- HO MeO OMe OMe 9 OMe 10 OMe 11 12 tions of galactose could be accessed, but both the conversion and 100 100 100 regioselectivity of these transformations required improvement 80 80 80 for practical synthetic applications. 60 9 60 60 11 12 A combination of random and site-directed mutagenesis of the 40 10 40 40 20 20 20 gene for 2C6dr led to the identification of variant E12r12, which Conversion (%) Conversion (%) 0 0 Conversion (%) 0 had improved efficiency on substrates 2 (50% increase in

B1 E12 9C7 5E5 E12 regioselectivity) and 5 (100% conversion) because of active-site 2C6dr 2C6dr 2C6dr E12r12 E12r12 B1SYN mutation A180V (see SI Appendix). Variant 5E5 was obtained from an additional round of random mutagenesis of the gene for Fig. 3. (A) Reaction conditions used for colorimetric analysis. (B) Yields for conversion of 2 to 9 ϩ 10, 5 to 11, and 7 to 12 determined by GC analysis of E12r12 and recombination of improved variants, and this en- crude reaction mixtures using indicated BM3 variants. The 1 mM (0.1 mol%) zyme provided high regioselectivity in the conversion of 2 to 9 BM3 variant was used except for the conversion of 7 to 12 where 0.5 mM (0.05 (96%, single isomer) because of active-site mutation V87G. A mol%) B1SYN were used. similar engineering effort led to the generation of B1SYN, which

Lewis et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 27, 2021 A OMe 0.1 mol% BM3 variant (5 µM) 2 1 2 OR R = Me, Bn, Bz MeO MeO + R O R2O O 0.1 equiv NADP , 10 equiv G6P O R2 = Me or H MeO R2O OR1 2 units/mL G6PDH, 100 units/mL SOD OR1 o 0.1 M KPi, pH 8, 25 C, 12 h Substrate Conv. Selectivity Yield Enzyme Product Stucture Sugar (%, GC)a (%, GC)b (%)c OMe OH Me MeO OO 3 9- 10A O 13 MeO Man MeO MeO F87Idr MeO 86 92 71 OMe OMe OMe OMe 4 9- 10A 14 MeO O Gl c HO O 73 77 44 MeO OBn F87I dr MeO OBn OMe OMe

MeOOMe HO OMe 6 9- 10A 15 O Gal O 90 80 64 MeO OBn F87Idr MeO OBn OMe OMe OMe OMe MeO 7 MeO 12 O Gal B1SYN O 100 100 98 MeO OBz HO OBz OMe OMe OMe MeOOMe MeO 5 11 O Gal E12r12 O 78 80 65 MeO OMe HO OMe OMe OMe MeOOMe MeOOMe 2 9 O Gal 5E5 O MeO MeO 50 93 48 MeO OMe HO OMe

B OH F MeO DAST F BBr3, CH2Cl2 RO O MeO O MeO O RO MeO MeO RO CH2Cl2, 0 °C MeO OMe OR 56% OMe AcCl, pyridine R = H 13 16 DMAP, CH2Cl2 R = Ac, 55% (2 steps) ca. 3:2 α/β (17) C MeOOMe 1. PhOC(S)Cl, DMAP MeOOMe ROOR BBr , CH Cl O pyridine, CH2Cl2, rt. O 3 2 2 O HO OBz H OBz H OMe 2. Bu3SnH, AIBN OMe OR OR toluene, relux 85% (2 steps) AcCl, pyridine R = H, 90% (19) 12 18 DMAP, CH2Cl2 R = Ac, 85%, >95:5 α/β (20)

Fig. 4. (A) Regioselective deprotection of substrates 11–15. a, Conversion of starting material determined by GC analysis of crude reaction mixture. b, Percent of desired product relative to additional products determined by GC analysis of crude reaction extracts. c, Isolated yield of pure product. d, 2 mM substrate used; 0.05 mol% B1SYN used. (B) Fluorination and global deprotection of 13.(C) Deoxygenation and global deprotection of 12.

had improved activity on 7 dramatically and enabled quantitative Next, the synthetic utility of the engineered demethylases was demethylation of this substrate even at 0.05 mol% enzyme demonstrated by their application in preparative-scale reactions loading. Fig. 3B shows the improvement in the conversion of 2 (Fig. 4A). The high conversions and regioselectivities observed to a mixture of 9 and 10,of5 to 11, and of 7 to 12 using the previously in small-scale reactions translated well into the 100-mg evolved BM3 variants. In each case, the activity or regioselec- scale investigated, and the desired products, 9–15, were isolated in tivity of 2C6dr was improved sufficiently by mutations identified high yield using 0.05–0.1 mol% enzyme loading. These mono- from 1 or 2 rounds of error-prone PCR and screening to enable demethylated compounds could be converted to a variety of efficient demethylation of the 2- and 3-positions of suitable biologically relevant products using established chemical transfor- ␤-galactose derivatives. Given the range of activities and regi- mations. Fluorination frequently is used to modify the pharmaco- oselectivities observed for BM3 variants identified from screen- kinetic properties of molecules for biological studies or for PET ing both the compilation and active-site libraries (Fig. 2 C–F), imaging (18). The Arnold group previously reported the tandem directed evolution should provide a rapid means for optimizing demethylation/fluorination of a variety of small molecules (15), and demethylases well beyond the examples shown here. this procedure proved applicable for the fluorination of 13 to

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908954106 Lewis et al. Downloaded by guest on September 27, 2021 A 0.1 mol% 9-10A F87V (5 µM) MOMO O 0.1 equiv NADP+, 20 equiv G6P MOMO O MOMO HO MOMO MOMO OMOM 2 units/mL G6PDH, 100 units/mL SOD OMOM 21, 5 mM o 22, 40% 0.1 M KPi, pH 8, 2% DMSO, 25 C, 12 h B AcO O MOMO O HO O 22, aO b, c HO O AcO AcO O HO O OH AcO AcO MOMOOMOM HO HO O CCl3 AcO 23 24 25 NH

Fig. 5. (A) Regioselective removal of 3-MOM group from substrate 21.(B) Synthesis of QS21-A disaccharide 25. a, TMSOTf, 4Å MS, 88%; b, NaOMe; c, 2 M HCl, 55% (2 steps)

provide 16. The latter compound could be demethylated in quan- Unlike synthetic supramolecular catalysts (24), the activity and titative yield and per-acetylated to provide 17 in 55% yield over 2 selectivity of BM3 variants can be tuned readily to suit a steps. Deoxy sugars also are important components of a variety of particular application. Such control enables efficient hydroxyla- small-molecule therapeutics (19), and deoxygenation of 12 using tion of C-H bonds lacking any significant stereoelectronic dif- Barton conditions proceeded smoothly to provide 18 in good yield ferentiation and augments the means by which synthetic chemists (20). This product was demethylated in excellent yield (19) and was can target otherwise indistinguishable C-H bonds (25). acetylated (20) to facilitate handling and characterization. All these factors result in a procedure that greatly simplifies To augment the utility of our method further, we explored the the synthesis of selectively deprotected monosaccharide deriv- regioselective deprotection of MOM-protected monosacchar- atives compared with conventional procedures that require ides in the context of a chemoenzymatic disaccharide synthesis extensive synthetic manipulation. The extension of this technol- (Fig. 1B). The ability to remove the MOM group is highly ogy to additional synthetically useful protecting groups, such as valuable for the synthesis of complex molecules, because the the MOM ether, renders the method compatible with sensitive CHEMISTRY conditions required for its removal are much milder than those glycosidic linkages and greatly expands the scope of the reaction. required for removal of methyl groups and therefore are com- We anticipate that this chemoenzymatic approach could greatly patible with sensitive functionality (21). A number of substrates simplify the synthesis of a wide range of polysaccharides for with differing sugar scaffolds, anomeric substituents, and ano- studies in chemistry, biology, and medicine. meric configurations were synthesized, and the compilation library was screened for activity on these substrates. High activity Materials and Methods was observed on a number of MOM-protected pentoses without Experimental Procedures and Characterization. Complete experimental proce- EVOLUTION the need for any protein engineering, and GC analysis of reactions dures and characterization can be found in the online SI Appendix. catalyzed by the most active variants revealed that 9–10A F87V catalyzed the removal of a single MOM group from ␣-xylose BM3 Expression and Purification. For preparative bioconversions, P450 BM3 substrate 21. Importantly, the product, 22, could be isolated in demethylases were used in purified form. Enzyme batches were prepared as moderate yield on a preparative scale, and removal of the 3-MOM follows. We inoculated2LTBamp with an overnight culture (100 mL, LBamp)of ␣ group was confirmed by NMR spectroscopy (Fig. 5A). Compound recombinant E. coli DH5 cells harboring a pCWori plasmid encoding for the 22 could be glycosylated with trichloroacetimidate donor 23 to give P450 variant under the control of Plac promoter. At an OD600 of 1.8 (ca. 3–4 h), the incubation temperature was reduced to 25 °C (30 min), and the cultures disaccharide 24 in high yield (22). Global deprotection of 24 under were induced by adding isopropyl-beta-D-thiogalactopyranoside to a final mild conditions provided disaccharide 25, a component of the concentration of 0.1 mM. The cultures were allowed to continue at this promising immune adjuvant, QS-21 (Fig. 5B) (23). temperature for another 24 h. After the cells were harvested by centrifugation (4 °C, 15 min, 3000 ϫ g), the cell pellet was resuspended in 25 mM Tris-HCl Discussion buffer (pH 8.0), and cells were disrupted by sonication (4 ϫ 1 min, 50% duty Regioselective deprotection of monosaccharide substrates using cycle). Cell debris was removed by centrifugation for 20 min at 4 °C and 20,000 ϫ g; the resulting cell lysate was loaded onto a Q resin, and the column engineered P450BM3 demethylases provides a highly efficient means to access valuable intermediates that can be converted to was washed with 3 column volumes of 25 mM Tris-HCl (pH 8.0), 150 mM NaCl. a wide range of substituted monosaccharides and polysaccha- Bound protein was eluted with 25 mM Tris-HCl (pH 8.0), 340 mM NaCl and was rides. Demethylases with activity on a number of monosaccha- concentrated using Millipore Centricon tubes. After buffer exchange with 100 mM KPi (pH 8.0), protein samples were frozen and stored at Ϫ80 °C. Protein rides and different positions on a given monosaccharide were concentration was determined in duplicate from CO-difference spectra as identified using a combination of protein and substrate engi- previously described. Yields typically ranged between 100 and 500 mg pro- neering, suggesting that this procedure ultimately could be used tein/L, depending on the variant. to functionalize regioselectively any monosaccharide at any desired position. The ability of these enzymes (i.e., 9–10A Preparative-Scale Bioconversions. Potassium phosphate buffer (100 mM, pH 8) F87Idr) to catalyze efficient demethylation of various substrates and a solution of the desired substrate in phosphate buffer or DMSO (1 equiv, with high regioselectivity should greatly simplify the expansion 5 mM final concentration) was added to a 100 ϫ 50 mm crystallizing dish (i.e., of the method to additional substrates. ChemGlass #CG-8276–100). A solution of NADPϩ (0.1 equiv, 0.5 mM final These results also highlight the efficiency with which BM3 concentration), glucose-6-phosphate (10 equiv, 50 mM final concentration), variants can insert oxygen into the C-H bonds of methyl ethers and glucose-6-phosphate dehydrogenase (2 units/mL final reaction volume) to catalyze heteroatom demethylation under conditions far more was added. A solution of the appropriate BM3 variant (0.001 equiv, 5 mM final concentration) was added, the dish was covered loosely with aluminum foil, mild than those associated with chemical methods. Furthermore, and the contents were stirred using a magnetic stir bar. The moderate stir rate the ability of the demethylases to situate substrates in a particular was used to allow efficient O2 transfer to the reaction mixture while avoiding orientation to favor hydroxylation of a particular methyl group foaming. The reaction progress could be monitored by analyzing aliquots of over 3 or 4 others located within only a few angstroms indicates the reaction mixture as described for the medium-scale bioconversions. After the exquisite control imparted by supramolecular interactions. 12 h, the pH of the reaction mixture was adjusted to 3–4, and the solution was

Lewis et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 27, 2021 saturated with NaCl to precipitate most of the protein from solution. The ACKNOWLEDGMENTS. This work was supported by the Jacobs Institute for resulting suspension then was filtered through Celite (Aldrich), which was Molecular Medicine (F.H.A.). J.C.L. is supported by U.S. National Institutes of rinsed with methylene chloride to provide a biphasic mixture. The mixture was Health Fellowship 1F32GM079932–01. S.B. was supported by the Deutsche transferred to a separatory funnel and extracted with 4 ϫ 75 mL CH2Cl2. The Forschungsgemeinschaft (DFG) Grant no. BA3486/1–1. C.H.W, C.S.B., Y.F., and combined organic extracts were dried with sodium sulfate, filtered, and W.A.G. were supported by the U.S. National Institutes of Health Grants concentrated. The resulting residue was purified by SiO2 chromatography GM044154 and AI072155. C.S.B. also was supported by the U.S. National eluting with ethyl acetate/hexanes. Institutes of Health Fellowship 1F32GM073500–02S01.

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