Biocatalysis • General Principles - Stereoselectivity - Biocatalyst production - Biocatalyst immobilization - Biocatalyst modificatiln • Hydrolytic reactions • Redox reactions • Addition-/elimination reactions • Glycosyl Transfer • Industrial applications • Cascade processes Stereochemistry & Drug Synthesis • Enantiomers & Diastereomer Discrimination ft-Enantiomer S-Enantiomer HOnC SH toxic Penicillamine HS C02H NH2 antiarthritic Carvone caraway scent anise scent H02C i NH 2 sweet O Asparagine HoN COOH O NH2 bitter Stereochemistry & Drug Synthesis • Enantiomers & Diastereomer Discrimination (fi)-(-)-epinephrine enzyme activesite enzy me-s ubs tra te com p] e k natural epinephrine Stereochemistry & Drug Synthesis • The Thalidomid Incident an bei Thalidomid-Einwirkung Thalidomid: (R)-enantiomer: weak analgetic (S)-enantiomer: strong teratogenic side effects Augenmuskel- und Himnervenlähmung j - völliges Fehlen der Ohrmuschel (Anotie) f ) • fehlende Organanlage (Agenesie) des Daumens r---- grauer Star ) - Fehlbildung (Dysplasie) des Herfens ■r* unct der großen Gefäße y - Fehlbildung der Niei j - völliges Fehlen der Arme (Amelie) > n - Innen- und Mittelohrfehlbildungen ) -Fehlbddung der Ober- und —^ Unterarmkrochen (ElektrornelP' Hände sitzen an der Schulter (Phokomelie der Arme) ^ - Fehlbildung der Hüftgelenke 1- Zwölffingerdarmverschluß —■* ■ unvollkommene Bildung des Oberarmbeines ) - Füße sitzen am Becken -r^ - unvollkommene Bildung des Daumens - Fehlbildung der Verdauungsorgane Zwolffingerdarmverengung Verschluß des Afters Fehlbildung des Atmungsapparates - völliges Fehlen der Beine (Amelie) j - unvollkommene Bildung (Aplasie) ■s des Zeigefingers nvollkommene Bildung der Speiche ^) - Verdoppelung der Großzehe * fehlende Organanlage der Gallenblase ■ Fehlbildung der Genitalien unvollkommene Bildung des Schienbeins 35. 40. Leistenbruch J - unvollkommene Bildung y des Oberschenkelknochens - Fehlbildjng der Ober- und Unterschenkelknochen ) - Dreigliedrigkeit (Trlphalangie) S des Daumens 45. 50, Tag nach der letzten Regel Pros >^ high enantioselectivity >^ high regioselectivity (incl. diastereoselectivity) >^ high chemoselectivity >^ broad substrate tolerance >^ high efficiency >^ environmentally benign >^ mild reaction conditions >^ enzyme compatibility (reaction cascades) Cons enantiocomplementarity cofactors low flexibility in operational parameters aqueous reaction conditions (loss of activity in organic solvents) inhibition availability Enzymes & Transformations Lyases (viii) Transferases (ix) I so m erases (x) (i) Ester formation, -aminolysis, -hydrolysis; (ii) ester hydrolysis; (iii) ester and amide hydrolysis, peptide synthesis; (iv) nitrile hydrolysis; (v) hydrolysis of epoxides, halogens, phosphates, and glycosides; (vi) reduction of C=0 and C=C bonds; (vii) hydroxylation or C-H bonds, sulfoxidation of thioethers, epoxidation of alkenes, Baeyer-Villiger oxidation of ketones, dihydroxylation of aromatics, peroxidation; (viii) cyanohydrin formation, acyloin and aldol reaction; (ix) glycosyl and amino-group transfer; (x) Claisen-type rearrangement, isomerization of carbohydrates, racemization. Fig. 4.1 Frequency of use of particular biocatalysts in biotransformations Induced-Fit-Theory • Koshland 1961 - conformational influence by substrate & enzyme modification of the biological activity of proteins Induced Fit —>■ Active Enzyme Enantioselectivity • Three-Point Attachment Theory (Ogston 1948) B' B' Enantioselectivity • Three-Point Attachment Theory (Ogston 1948) - optical antipodes result in diastereomeric pairs upon interact, with enzyme - different energy levels of enzyme-substrate-complexes 3.14 199 99 4^50 1999 99.9 ee (%) = (P-Q)/(P+Q) ^/Re-íace Desymmetrization • Enantiofacial Differentiation Case VII S/-attack Chemical operator Case VIII fle-attack chemical operator C C Desymmetrization • Enantiofacial Differentiation Desymmetrization • Enantiotopos Differentiation C02Me -^pro-R (or Re) 'If!1______/ C02Me pro-S (or Si} Case V chemical operator Case VI chemical operator Substrate C a sequence rule order of A>B>C is assumed Desymmetrization • Enantiotopos Differentiation - Single step process selectivity a = enantiomeric excess e.e. = (a-1 )/(a+1) = (R - S) / (R + S) e.e. depends on conversion Kinetic Resolution • irreversible reaction • reversible reaction • sequential resolution • dynamische resolution racemic substrate separable enantiomers • recognition of existing chirality • yield limitation (50%; except dynamic process) Kinetic Resolution • Enantiomeric Ratio E + [EA]-- E+ P + [EB]-► E + Q Enantiomeric Ratio [Km] A ^cat [KfA_ B AAG^ = -RJ In E Kinetic Resolution _ rcA1__c D + A ^ |taj * b + H Enantiomeric Ratio E + [EB]-- E + Q - ideal case: kA/kB = oc »^ reaction stops at 50% conversion - real case: kA/kB = finite value reaction progresses beyond 50% transformation of both enantiomers depends on conversion e.e.(substrate) & e.e. (product) function of conversion (since ratio A/B & P/Q not constant during whole biotransformation) - Mathematical model by Sharpless & Fajans (irreversible kin. ses.) For the product For the substrate ln[1 -c(1 +e.e.P)] ln[(1 -c)(1 -e.e.s)] ln[1 - c(1 - e.e.P)] ln[(1 - c)(1 + e.e.s)j c = conversion, e.e. = enantiomeric excess of substrate (S) or product (P), E = enantiomeric ratio Kinetic Resolution • Irreversible Kinetic Resolution conversion [%] conversion [%] E=5 £=20 - e.g. hydrolysis: irreversible due to high water concentration - product with high e.e. obtained before reaching 50% conversion - beyond 50% decline in e.e. (high cone, of "undesired" substrate) - inverted trend for substrate e.e. - quality of resultion depends on E-value Kinetic Resolution • Irreversible Kinetic Resolution - Substrate recovery - Product isolation recovery of product substrate e.e. [%] 100 J 50- °\ 0 100 Kinetic Resolution • Problems in kinetic resolutions: - maximum yield of 50% for required enantiomer - remaining antipode often of no use - separation required (extraktion, distillation, etc.) - limitation of optical purity by finite E-value • Ideal industrial process: - 100% yield - single enantiomer Repeated Resolution • Racemization of unwanted antipode (mostly chemically) • Repetition of biocatalytic resolution (iterative) • Several additional steps • Decrease in yields due to (mostly) forced reaction conditions Kinetic Resolution • ln-situ Inversion: - reaction mixture after resolution consists of enantiopure product enantiopure substrate - single chiral center: inversion by chemical activation and reaction OL OAcyl OH Inversion Hydrolase L-X + -"-* + -** +, OAcyl kinetic resolution OAcvl activation T I - OAcyl " B [OH-] chemical hydrolysis Ri " R2 Retention OH R1/^R£ single enantiomer L = leaving group (e.g. tosylate, Inflate, nitrate, M itsu no bu-intermediate) Scheme 23 Kinetic resolution with in-situ inversion Kinetic Resolution Dynamic Kinetic Resolution classical resolution in-situ racemization of substrates dynamic process equilibrium constantly regenerated of the desired enantiomer always beneficial ratio in cSub^kR R, S = substrate enantiomers « P, Q = product enantiomers kRl kg = enzymatic hydrolysis of enantiomers R, S Sub kSpont< ON T7-RNA-polymerase production recognition of T7-promoter T7-promoter CHMO gene production of CHMO active CHMO protein approx. 25% active protein Protein Expression Whole-cell Biotransformations NADPH NADPH Enzyme #1 B Enzyme #2 Enzyme #3 Primary Metabolism cofactor recycling enzyme production enzyme in natural environment cheap C-source (glucose, saccharose) for stereoselective reactions toxicity of non-natural substrates transport effects side reactions Protein Expression Tabic 1.2 Pros and cons of using isolated enzymes vs. whole cell systems Biocatalyst Form Pros Cons Isolated Any Simple apparatus, simple workup. Cofactor recycling necessary, enzymes better productivity due to higher limited enzyme stabilities concentration tolerance Dissolved High enzyme activities Side reactions possible, lipophilic in water substrates insoluble, workup requires extraction Suspended Easy to perform, easy workup, Reduced activities in organic lipophilic substrates soluble, solvents enzyme recovery easy Immobilized Enzyme recovery easy Loss of activity during immobilization Expensive equipment, tedious workup due to large vol nines, low productivity due to lower concentration tolerance, low tolerance of organic solvents, side reactions likely due to uncontrolled metabolism Growing Higher activities Large biomass. enhanced culture metabolism, more byproducts, process control difficult Resting cells Workup easier, reduced Lower activities metabolism, fewer byproducts Immobilized Cell reuse possible Lower activities cells Whole Any No cofactor recycling cells necessary, no enzyme purification required Biocatalyst Immobilization ► Coupling Coupling to Carrier h® -® Adsorptive/ Ionic Covalent Cross-Linking Cross-Linking Co-Cross-Linking (B) = biocatalyst (enzyme or whole eel = carrier (Enz) = enzyme Biocatalyst Immobilization » Entrapment in Matrix Entrapment by Membranes Gel or Polymer O00 Reversed Micelle (B) = biocatalyst (enzyme or whole cell) ^>wwO Surfactant (polar head, lipophilic tail) d3> Hollow Fibre = carrier Membrane Reactor (MEEC-Technique) enzyme Biocatalyst Immobilization ► Covalent Linkage 0 c I—O—Si 1 o 0 1 O—Si' I o OEt OH + EtO- Si' I OEt \ EtOH 0 1 c I—O—Si' I o 0=CH-^\/^CH=0 N=CH CH = N cuc=s X N-C5)* Enz J— Nht. 0 1 O—Si O carrier enzyme NH, N=C=S Scheme 3.34 Covalent immobilization of enzymes onto inorganic carriers Biocatalyst Immobilization Entrapment - Whole cells Protein Modification 1-► 2-N—* 5'-1- 3" Site-directed —3 «—4 mutagenesis anCdThprimers1| | PCR with primers 2 and 4 5'—-*- 5' 3'—I-*- 3- 5' 3'- Denaturation and annealing result in heteroduplex formation Overlap extension: DNA polymerase fills in 3' recessed ends 5'-3"' 2nd PCR reaction amplifies mutagenic DNA Figure 2. Overlap extension PCR method: —» represents a primer, and x represents a mutagenic codon. - known structure & mechanism - usually: knock-out tests Protein Modification • Site-directed mutagenesis - rational design c PAMO X-ray structure 1W4X CHMO homology model Arg 337 w — Leu-443 Ala-442 BV- monooxygenase O A O Ph Ser-441 Ph BV- monooxygenase Protein Modification • Enzyme evolution promotion Protein Modification • Error prone PCR (epPCR) PCR mumm Taq dCTP, dTTP dGTP, dATP Mg2* Error prone PCR iUllUJ Taq dCTP, dTTP T dGTP, dATP i Mg2*T Mn2+ Figure 3.3 Differences in classical and error prone PCR. operating PCR under non-ideal conditions (also saturation possible) degeneration of Code different mutation frequencies distribution of mutations randomly (remote from active site) Protein Modification • Error prone PCR (epPCR) ch3 ch3 ch3 rec-1 (R = u-CgH17) (5)-2 3 enzyme £=11.3 variant A mutant generations Fig. 14. Increasing the E values of the lipase-catalyzed hydrolysis of the chiral ester 1 by cumulative mutations caused by four rounds of epPCR (16.22.24). Protein Modification • Combinatorial Active-Site Saturation Test - CASTing loop (J sheet 3io helix a helix ("♦1) {n+2) {/>+3) (n+4) Figure i. Structural guides in designing libraries of mutant enzymes for CASTing according to the secondary structure of proteins. - synergistic amino acids in spatial proximity Protein Modification Combinatorial Active-Site Saturation Test - CASTing Library Figure 2. CASTing of the lipase from Pseudomonas aeruginosa leading to the construction of five libraries of mutants (A-E) produced by simultaneous randomization at two amino acid sites. (For illustrative purposes, the binding of substrate 1 is shown.) 10 11 Protein Modification • Combinatorial Active-Site Saturation Test - CASTing - combination of best sub-library candidates •WT Figure 1. Schematic illustration of iterative CASTing involving (as an example) four randomization sites A, B, C, and D: Confined protein-sequence space for evolutionary enzyme optimization (redundancy in some cases is expected). CH3 rac-1 (R = n-CsHn) H;Q iipase-variants Y^OH + RV^0H + S0-^-NO1 CH3 (5)-2 CH 120 110 100 90 BO Uj 70 f. GO > 1 50 £=115 LW202 40 30 20- 10- £=49 LW126 AorF no substantial improvements £=21 LW086 £=14 - LW081 / q v= »1 IAJ- 24 j LW123' B WT-ANEH A or C no substantial improvements Figure3. Iterative CASTing in the evolution of enantioselective epoxide hydrolases as catalysts in the hydrolytic kinetic resolution of rac-1. Protein Modification • Summary of technologies Table 3: Main library creation technologies.1 Error-prone PCR Saturation mutagenesis Massive mutagenesis Gene shuffling Synthetic wf shuffling Need for physical starting gene 1 gene 1 gene 1 gene several genes no gene Large diversity/low cost mutant ? yes no yes yes yes Control over the diversity generated very little (mutation rate) complete complete little (starting sequences) complete Need for double strand cloning yes yes/no (different technologies) no yes yes Protein Modification • Library Screening - workflow mutagenesis expression colony picking oooooooooooo oooooooooooo OOOOOOOOQQQQ oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo screening for enantioselectivity' oooooooooooo oooooooooooo ooooiooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo library of mutant genes in a test tube bacterial colonies on agar plate bacteria producing mutant enzymes grown in nutrient broth visualization of positive mutants Scheme 2. Individual steps in the directed evolution of an enantioselectivc enzyme.'1**1 Protein Modification • Screening Techniques - Colorimetric Screens • double experiments • high throughput a) 1 Figure 3. Course of the lipascotalyzed hydrolysis of the (R)- and (S)-3I as a function of time."71 a) Wild-type lipase from P. aeruginosa, b) improved mutant in the first generation. Protein Modification • Screening Techniques - MS-based Screens - „Pseudo" Racemates OAc Ph (S)-52 OAc[D3] Ph^CH3 (R)-79 OH Ph^CH3 (S)-51 OH Ph"^CH3 (R)-51 + CH3C02H + CD3C02H 80 81 Scheme 27. Kinetic resolution of pseudo-cnantiomers (S)-52 and (R)-79. Screening was carried out by ESI-MS.'8'' 100% 60% t 60% 40% 20 %" [(S)-52+-Nal Ml [(R)-79+Na] 80 100 120 140 160 1fi0 200 22 0 240 2S0 230 300 mil—*- Figure 13. ESr-niass spectrum of a sample containing (.S)-52 and (fi)-79.^ synthesis ol pseudo-enantiomers enantioselective transformation chiral catalysts or reagents sample manager for microtiter plates ESI-MS PC and data processing Figure 14. Experimental setup of an ESI-MS tv-screening system.I8'1 Enzyme Groups • Esterases (cleavage of ester functionality) • pig liver esterase (PLE) • horse liver esterase (HLE) • acetyl choline esterase (ACE - Zitteraal) • Bacillus subtilis esterase • yeast (whole-cell system) • Proteases (cleavage of amid bond) • a-chymotrypsin • pepsin • subtilisin • thermolysin • Lipases (cleavage of triglycerids) • div. Candida lipases • Nitrilases & Nitrile Hydratases • Epoxid Hydrolyses Reaction Types • Hydrolysis O ^ R Jj^ -^ ^COOH R OR' Deprotection O J[ -^ HNR'2 R NR'2 Esterification O OR' COOH -^ V, K R ( Ser195 Enzyme Mechanisms • Serin-proteases - e.g. chymotrypsine catalytic triad Asp Step I His H—N JA jW Ser iAAW I His57 Asp102 Step II Asp His Nu Ser Acyl-enzyme intermediate O R ^Nu Enzyme Mechanisms • Thio-proteases V7T - Mechanism comparable to Ser-proteases - examples: Papain, Cathepsin - Minor modification of amino acids in catalytic triad upon retention of function Enzyme Mechanisms • Metallo-proteases - Zn2+ as Lewis-acid - no covalent intermediate - examples: thermolysine, acylases Enzyme Mechanisms • Aspartyl-proteases - 1 st carboxylate = base - 2nd carboxyl groupe - general acid catalysis - no covalent intermediate - example: pepsin Synthetic Applications • Various nucleophiles o RV OH hydrolysis O R'OPT acyl transfer H20 R4-C O Enz" "R Acyl-enzyme intermediate Nu = H20, R4-OH, R3-NH2, H202 R3 = alkyl, aryl, -NR52 R4 = alkyl, aryl, -N=CR52 O .iX R"" ^HN-R" ester aminolysis O r1-^0-OH peracid formation Amino Acid Synthesis • Esterase Method COOR1 ^wNHR2 COOH R esterase or protease buffer R2HN R DL COOR1 -NHR2 R D R = alkyl or aryl; R1 = short-chain alkyl; R2 = H or acyl - Ester hydrolysis via: • Protease (cleavage of ester & amid bond possible sequential biotransformation) • Esterase • Lipase - Most important enzyme: a-Chymotrypsin - Usual preferred cleavage of enantiomer most similar to natural a.a. - Since 1905 applied in chemistry Amino Acid Synthesis • Amidase Method coim /wviNH2 DL amidase buffer COOH Ft L CONH2 —NH2 R D R = alkyl, alkenyl, alkinyl, (hetero)aryl H+ cat. CONK CONK OH" cat. Ph N= + Ph-CH=0 \ Ph Schiff-base ex-situ racemization Enzymes: mikroorganisms (Pseudomonas, Aspergillus, Rhodococcus sp.) Negligible chemical hydrolysis of amide products separate chemical racemization possible Now also with N-acylaminocarboxylic acid racemase dynamic process Amino Acid Synthesis • Hydantoinase Method D-hydantoinase buffer L-hydantoinase C02H |—NH^ NH2 R o M H DL-hydantoiri LJ R = aryí: spont. racemization [OH"] cat. pH>8 R = alkyl: hydantoin racemase buffer D-A/-carbamoyl amino acid H20 NH3 + >/ C02 carbamoylase R H -N N H C02H h NH, R D C02H H2N H R L-A/-carbamoyl amino acid H20 co2 carbamoylase H2N^ C02H R L Ester Hydrolysis Substrate types for esterases 0 L R1 É H O — R3 Type I OR3 R2 O O R3 H Type II prochiraj substrates mesoforms R1, R2 = alkyl, ary!; Rs = Me, Et; * = center of (pro)chirality Ester Hydrolysis • Substrate types for esterases Cbz H N XOOMe .COOMe Cbz = Ph-CH2-0-CO- H Cbz—N. a-chymotrypsin buffer papain H Cbz—N .COOH ^COOMe COOH Ester Hydrolysis • Lipases vs. Esterases Esterase Lipase activity activity critical micellar concentration formation of second phase (=substrate) start of biotransformation Ester Hydrolysis • Lipases vs. Esterases intGrface +S E " [Ef [ESf [Ef+ P E = inactive lipase (closed lid conformation) [Ef = active lipase (open lid conformation) S = Substrate; P = Product - Lipases work best in solvent/water mixtures Ester Hydrolysis • Lipse flap / lid Active Site C-Terminal Domain N-Terminal Domain Flap Col i pase Esterification • Principle o o Hydrolase + R3-OH + OR2 OR3 strong nucleophile Problem: formation of water during reaction formation of aqueous interphase separation of enzyme & substrate incomplete conversion Excess acyl donor - removal of „bulk" water - pseudo-irreversible reaction Decrease of nucleophilicity of newly formed alcohol - electron withdrawing effects - subsequent reaction / tautomerization Esterification • Leaving group alcohol acyi donor Nu1 Porcine pancreatic lipase ^Et90 O A O R + leaving group Nu2 Acyl donor R Leaving group Nu2 Initial rate [%] Ethyl acetate Me EtOH 0.3 2-Chloroethyl acetate Me CICH2-CH2OH 1 Methyl butanoate n-Pr MeOH 5 Ethyl cyanoacetate N==CCH2- EtOH 6 Trichloroethyl trichloroacetate CI3C- CI3C-CH2OH 7 Methyl bromoacetate BrCH^- MeOH 14 Tributyrin n-Pr dibutyrin 34 Trichloroethyl butanoate n-Pr CI3C-CH2OH 58 Trichloroethyl heptanoate n"C6H1 gp CI3C-CH2OH 100 Esterification • Enol ester acyl transfer Enol Esters FT + R-OH Hydrolase O o R1^ O-R R1 = n-alkyl, aryl, aryl-alkyl, haloalkyl R2 = H, CH3 HO- > enol or CH3-CH=0 Nitrile Hydrolysis ► Enyzmes for Nitrile Hydrolysis - Nitriles are improtant Crbuilding blocks in chemical industry - Chemical methods for hydrolysis: • Strong acidic or bacis incompatible with functional groups • High energy demands • Side reactions Nitrilase j? R—C=N-w-R—« + NH3 7^ » o Nitrile hydratase D // Amidase 4 ~/f NH2 ~& H,(/ H20X Nitrile Hydrolysis • Acrylamid Production - 450,000 t/a global production - chem. process: hydration of acrylonitrile with Cu-catalyst - whole-cell process yields (also lyophylized cells) >99% • amidase inhibition • 400g/L Titer • >100,000 t/a by biocatalysis (1997) y CN microorganism hUO Nhi >400 g/L Rhodococcus rhodochrous Pseudomonas chlororapis Brevibacterium sp. C Reaction types R1 R* O A R1 R£ X prochiral substrate Dehydrogenase or Ene-Reductase Cof actor ' Recycling System i OH R1 R 2 or Ri OH x R1 X H R2i + - ■R1 ■H or R H * z * - •H |R2 chiral products Cofactors K .H NH2 ~tH J Nicotinamide Cofactors: Nature's Complex Hydrides O AW |_| NH * H 0 Flavin Scheme 2.110 Reduction reactions catalyzed by dehydrogenases Cofactor Recycling • General Principle Substrate Auxiliary Substrate ■--------i Substrate-H2 Substrate Enzyme A Substrate-H2 NAD(P)H NAD(P)- Auxiliary Substrate i r ' Enzyme B ' Auxiliary Substrate-H2 Cofactor Recycling • Recycling systems for reduced nicotinamids H-C02H Formate Dehydrogenase (mutants) >- CO, 0 II Q H-P-Ou Phosphite Dehydrogenase (mutants) G HO- NAD(P) NAD(P)H NAD(P)+ NAD(P)H NAD(P)+ NAD(P)H ®= PhosPhate Aldehyde Alcohol Dehydrogenase Dehydrogenase EtOH--CH3-CH=0 - J. -CH3-C02H NAD(P)+ NAD(P)H NAD+ NADH Cofactor Recycling • Photochemistry Photosensitizer CB =\1 * Donor Glutamate»' Dehydrogenase Tg^j L-glutamate a-ketogIutarate [Photochemical Cofactor Regeneration] Cofactor Recycling • Electrochemistry [Electrochemical Cofactor Regeneration] Cofactor Recycling • Whole-cell biotransformations Product Metabolism ADP+ Cofactor Recycling ► Whole-cell biotransformations - recombinant organisms - Genen overexpression - Gene knockout protospacer NADPH NADP+ Enzyme #1 zyme #3 B Cas9 5-II 111 I Mill IIP sgRNA G™3 3' Primary Metabolism Carbonyl Reductions • Stereochemistry L-Reductase D-Reductase Carbonyl Reductions • Prelog's rule Carbonyl Reductions oh 0 ► Dynamic Processes r^V^o*^ R2 OH O O O B R2 R" R1" ^OR3 R + S ♦_t in-siiu racemization OR3 r OH OH O R2 R2 C02R3 R1" T OR3 Pathway R1 R2 R3 Biocatalyst Yieid [%] d.8. [%] e.e. [%] Ref. A mg ally! Et baker's yeast 94 92 >99 [907] A Me Me /7-Octyl baker's yeast 82 90 >98 [908] B Me Me Et Geotrichum candidum 80 >98 >98 [909] B Et Me Et Geotrichum candidum 80 96 91 [910] c 4-MeOC6H4- CI Et Sporotrichum exile 52 96 98 [911] D 4-MeOCeH4- CI Me Mucor ambiguus 58 >98 >99 [912] Carbonyl Reductions • Reductive Aminations - Amino acid dehydrogenase o A. Amino acid dehydrogenase R C02H NH, H20 NAD{P)H NAD(P)+ I R C02H L © Lys—NH3 Lys^JH ffi O J R 0,0 Q HO / y-^r Lys —NH2 H20 \Hf Lys — NH, 0>V +NAD(P)H A CO, Lys —NHg t i © Lys —NH3 [HI +H+ X ->- R .\ L C< Lys—NHf Lys R"^C02H O A. FT "C02H Carbonyl Reductions • Imine Reductases - Problem: imine stablity in aqueous systems Alkene Reductions • Enoate Reductases (Old Yellow Enzymes) Isubunrl Bi R [H_] 6 EWG [Hi EWG = electron-withdrawing group: aldehyde, ketone, carboxylic acid ester, lactone, cyclic imide. nitro Flavin H2 Ox-HR Ene- Reductase ! Flavin NAD(P)+ NAD(P}H i i Recycling System I______________ EWG R 2 , 'R ■ or EWG R H R- H Ox-HR: oxidative half-reaction Red-HR: reductive half-re action [H~] = hydride delivered from N5 of the flavin cofactor [H4] = proton delivered via Tyr-resid je Alkene Reductions • Enoate Reductases (Old Yellow Enzymes) a.p-unsaturated ketone Alkene Reductions • Enoate Reductases (Old Yellow Enzymes) - Alkene substitution pattern (Si C°2Me Ene reductase ^C02Me NADH-Recycling ^e02C ^ C02Me or CO,Me _ 2 Ene-reductase C02Me NADH-Recycling (fl) C02Me C02Me Configuration YojM OPR1 c [%] e.e. [%] c [%] e.e. [%] Z E 93 >99{R) 91 >99(fl) 70 >99(S/) 99 >99(R) Alkene Reductions • Enoate Reductases (Old Yellow Enzymes) + H* NAD(P)* + H* NAD(P)* Scheme i. Reductions catalyzed by Old Yellow Enzymes (OYEs). 1: 4-phenyl-3-butyne-2-one; 2: (E)-4-phenyl-3-butene-2-one; 3: 4-phenyl-2-butanone; NAD(P)+: nicotinamide adenine (phosphate) dinucleotide. Oxidation Reactions Dehydrogenases SubH2 + Donor->- Sub + DonorH2 ^ cofactor-recyciing_| Oxygenases Mono-Oxygenases Sub + DonorH2 + 02 -SubO + Donor +H20 ^_cofactor-recyciing_| Di-Oxygenases Sub + 02 -* Sub02 Oxidases SubH2 + 02 -^ Sub + H202 02 + 2e"-022" + 2H+ » H202 02 + 4e"-202- +4H+ > 2H20 Peroxidases 2 SubH + H202 -2 Sub • + 2 H20 ->- Sub —Sub Sub + H202 -*~ SubO + H20 Alcohol Oxidations • HLADH Biooxidations - FMN as sacrificial substrate R R OH OH HLADH S cis-meso NAD+ recycli lg p 1st enzymatic oxidation OH ;h=o iponl R O OH ^CHg—S—CH£— R=Me Spontaneous chemical pr 3cess HLADH IMAD+ recycling o e.e. 100% 2nd enzymatic oxidation Monooxygenations Reaction types mono-oxygenase Sub + 02 + H+ + NAD(P)H -Sub0 + NAD(P)+ + h20 —C— H -*~ —C—OH R O A Rn-x Rn— X=0 o Ri R2 O X = N, S, Se, P. Substrate Product Type of reaction Type of cofactor AI kane alcohol hydroxy lat i on metal-dependent Aromatic phenol hydroxylation metal-dependent Alkene epoxide epoxidation metal-dependent Heteroatoma Ketone heteroatom oxide ester/lactone heteroatom oxidation Baeyer-Villiger flavin-dependent flavin-dependent aN, S, Se, or P similar to chemical oxidation using peracids (nucleophilic process) similar to chemical oxidation using hypervalent metals (electrophilic process) Monooxygenations • Flavin dependent enzymes Monooxygenations • Cytochrome P450 enzymes Monooxygenations ► Cytochrome P450 enzymes roh + h2o rh + o2 ADX: Adrenodoxin ADR: NADPH-Adrenodoxin Reductase nadph Bacterial/mitochondrial System Microsomal System NAD(P)H Sub + 0£ + 2H+ Heme NAD(P)+ f ^y^y - Sub O + H20 Self-sufficient BM-3 System NAD(P)H NAD(P)+ +H+ Sub + 02 + 2H+ SubO + H20 NAD(P)H NAD(P)+ +H+ Sub + Or + 2H+ Sub O + H20 FdFt = Ferredoxin Reductase FAD = Flavin adenine dinucleotide Fdx = Ferredoxin FeS = iron-sulfur cluster CpR - Cytochrome P Reductase FMN = Flavin mononucleotide P450= Cytochrome P-450 ^^./348B Monooxygenations • Baeyer-Villiger Oxidation X 58 X R Recomb. cells 59a S[S2] H 48%l95l 59b NMe H 50%^ 59c NCOMe H 39% (59%)(95] 59d NCOOMe H 40% (67%)^ 59e 0 H 59f 0 Me 79%/> 99% ee?[96] Baeyer-Vi Nig erase in E. coli host -> NADPH-recycling 0„ (S): R = Me, Et, n-Pr, APr3; e.e. up to >99% (fl): n-Bu; e.e. up to 60% Baeyer-Villigerase R in E. coli host -> NADPH-recycling 0„ rac 0 >R (A) R = Me, Et, n-Pr, allyla,n-Bu, E 200 aSwitch in sequence priority Monooxygenations ► Baeyer-Villiger Oxidation Baeyer-Vüligerase in E. coli host -* NADPH-recycling rac + antiperiplanar migrating group anti major minor R E Me 60 (S) Et > 200(fl) n-Pr >200 (fl) /-Pr >200 (S)a n-Bu 20 (ff) or (S) Substrate ketone "Normal" lactone 67 "Abnormal" lactone 68 .0 X 1a-g X=C, O ...../ 2a-g "Normal" lactone 3a-g "Abnormal" lactone o CD 44%/> 95% etf 36%/> 95% e 95% ee 41%/86%«r 52%/60% ee 42%/> 95% (1Ä,55)M<1I4J 31%/> 95%£>e 37%y> 95% 36%7> 95% ee (VS,6Rfmi 2S%/> 95% ee ncuuA riociouvji id Monooxygenations • Heteroatom Oxidation o2 di-oxygenase (P) @ mono- (S) 0 mono- Q ^\ / oxygenase e'' oxygenase ^ .s—x—x—> sb - —x—x—.s Ri R2 / \ R1 * R2 / \ R chiral mono- 02 H20 02 H20 oxygenase NAD(P)H NAD(P)+ NAD(P}H NAD(P)+ oxidase H202 H2Q H2Q2 H20 Monooxygenations • Biohydroxylations - Steroid hydroxylations • reactivity: sec. > tert. > prim, (compare to radical reactions) • primarily whole-cell biotransformations (enzymes difficult to isolate and/or unknown) • sources: esp. fungi (Beauveria sp., Cunninghamella sp., Aspergillus sp.) progesterone lithiocholic acid Rhizopus arrhizus Fusarium equiseti Aspergillus niger Monooxygenations • Biohydroxylations - Substrate engineering o oo o rac ifi -is (trace) 95%e.e. 85%e.e. 46%e.e. Dioxygenases Sub-H + O di"°XygenaSe > Sub-O-O-H redUCti°n > Sub-OH hydro-peroxide e.g. NaBH4 di-oxygenase Sub lf\ reduction ^ ellh^°H Sub + Oo ->~ Nis -^ bub ^ ° e.g. NaBH4 OH endo-peroxide Dioxygenases • Fe-S Cluster Enzymes Dioxygenases • Aryl dioxygenations R di-oxygenase R reductase T [H21* R oxidative further ring-cleavage metabolism ^ dihydrodiol H dehydrogenase NAD(P) NAD(P * Commonly NADH deficient mutant Dioxygenases • Aryl dioxygenations Toluene Dioxygenase (TDO) \ 8 Naphthalene Dioxygenase (ND0) Biphenyl Dioxygenase (BPDO) Dioxygenases • Aryl dioxygenations prodiastereotopic plane proenantiotopic plane 2 R = H H Pseudomonas sp. mutant 1 R 2 R =l <|0H Ho, Pd/C ^k* OH 'OH ,1 F, Me oxidative cleavage V [4 + 2] cycloadditions [2+1]" [2 + 2] [2 + 3] [2 + 4]^ oxidation, electrophilic addition cycloadditions R 'OH H, Me, Et, n-Pr, f-Pr, n-Bu, f-Bu, Et-O, n-Pr-O, Halogen, CF3, Ph, Ph-CH2, Ph-CO, CH2=CH, CH2=CH-CH2> HC=C. H sigmatropic rearrangements Dioxygenases • Aryl dioxygenations Dioxygenases • Ipso-Aryl Dioxygenases CQ2CH3 OBOM A. eutrophus C02H scale: >250g many diverse synthetic starting materials TBSOtk" C02CH3 TBSO Dioxygenases • Alkene cleavage C) lignostilbene-a-p-oxygenase (LSD) from S. paucimobilis OMe LSD-I, 02 buffer, pH 8.5 10%v/vMeOH 30 °C OMe OMe D) stilbene-u-fl-oxygenase (NOV-| and NOV2) from N. aromaticivorans .OMe OH (1 mM) E) isoeugenol oxygenase NOV1/NOV2 300 mM NaCI 10 mM Na-ascorbate 0.5 mM FeSQ4, Q2 buffer, pH 7.2, 30 °C * 5 min to 12 h HO OMe isoeugenol isoeugenol oxygenase, _Q2 buffer, pH 7.0 10%(v/v) EtOH 30 °C, 10 min HO HÖH Laccases • Enzymatic radical chemistry ncuuA ncauiiui io Laccases • Enzymatic radical chemistry Laccase Laccases ► Enzymatic radical chemistry Ca-Cp Cleavage 0--CH HX- ■_-o OH 1- (3/5-dimethoxy-44iyob"oxyphenyl)- 2- (3,5-dimetiioxy-4^thoxyphenyl) propane-l,3-diol Syringaldehyde Q CH. OH l-(33-dimeťhoxy-4^ ethoxyphenyl)-2-hydroxyeťhanone Alkyl aryl O CH l-(3,5-dimethoxy-4^thoxyphenyl)- 2/6-dimettioxy-p-benzoqtiinone 3-hydroxypropanal Ca oxydation H l-ÍB/S-dimeťhoxy^hydroxyphenyl)^^^-din\eťhoxy^eťhoxyphenyl)-3-hydroxypropanone Addition Reactions • Lyases: addition of small molecules (H20, NH3 etc.) across C=C or C=0 • Oxynitrilases cyano hydrin formation • Fumarases »^ water addition across activated double bonds • Haloperoxidases halogenations / dehalogenations (no true lyases) Addition Reactions • Oxynitrilases HCN HO^^CN in situ commercial + HCN HQ. C N R-oxynitrilase 2 R =H, Me 2 R =H S-oxynitrilase NC OH S Table 14.7-1. Oxynitrilases available for organic synthesis. Plant Enzyme availability Natural substrate Substrate acceptance Stereo-for syntheses selectivity Prunus amygdalus Almonds Linum usitatissi- Flax seedlings overexpression mum Sorghum bicolor Millet seedlings Hcvea brasiliensis Rubber tree leaves overexpression Manihot esculenta Manioc leaves overexpression (R)-Mandelonitrile Acetone cyanohydrin (R)-2-Butanone cyanohydrin (S)-4-Hydroxymandel-onitrile Acetone cyanohydrin All R' and R2 Aliphatic aldehydes and ketones Aromatic aldehydes All R1 and R2 Acetone cyanohydrin All R1 and R2 Addition Reactions ' Oxynitrilases Addition Reactions • Water/ammonia addition - Activated double bond - anti-mechanism nucleophile si-face, proton re-face Addition Reactions • Haloperoxidases substrate + H202 + X~ + H+ haloperoxidase hal-product + 2 H20 No lyases Halide generates electrophilic halo species upon consumption of H202 terrestrc organisms (e.g. fungi): X = CI marine organisms (e.g. algae): X = Br Usually broad substrate profiles haloperoxidase lactonisation halolactone nucleophilic attack + .-x halonium intermediate -OH -X halohydrin hydroxy-halogenation X - CI, Br, I Y = F,CI, Br,! -Y 1,2-dihalide alkane-halogenation Nitrogen Transfer • Transaminases (^Transaminase NH, buffer R" R1" \ r amine acceptor PMP PLP 0 V J NH2 JL * - y— JL R^R' R R' amine donor NH? NH2 NH2 Amine donor (examples): oo2H ^ R = Me, Ph Nitrogen Transfer ► Transaminases - kinetic resolutions NHS + NH, R' rac-amme co-Transaminase Jj^ O NH2 R'J ^C02H -^CO,H NH. R R' + H products - deracemizations o prochiral ketone co-Transaminase NH, ( R"'^^RI" R-'^^R'" ketone co-product Nitrogen Transfer ► Transaminases co-Transaminase R' "R1 NH2 - ■ o - Degradation of co-products I II NH, OH LDH NAD(P)H Decarboxylase CO,H ylase 1 Acetolactate- OH synthase - Recycling of amine-donor (o-Transaminase CO.: + 2 CO, X ■ R^^R" NH2 R^R' C02H NAD4)-Gal-OH p-Gal-(l- +4)-p-GlcNAc-(l- +4)-Gal-OH p-GlcNAc-(l~ +6)-Gal-OH p-Gal-(l- ->4)-p-GlcNAc-(l- -►6)-Gal-OH p-GlcNAc-(l- ->3)-Gal-OH P-Gal-(1- ->4)-p-GlcNAc-(l- ->3)-Gal-OH Glycosyl Transfer • N-Acetyllactosamine Production Plazierung der Abgangsgruppe 1 galactosyl transferase 4 phosphoglucomutase 2 pyruvate kinase 5 inorganic pyrophosphatase 3 UDP-glucose pyrophosphorylase 6 UDP-galactose epimerase (p)= phosphate UDP = uridine diphosphate UTP = uridine triphosphate Glycosyl Transfer ► Mixed-culture approach - several (recomb.) strains C> OH AcHN -O. COOH OH OH H NHAc -GlcNAc OH H HO Neu Ac; Sia R = Sialic acid OH in bioreactor _ - detergents for material transfer Recombinant e so// - example: sialic acid formation Gal- batch process: 123g/L Incorporation of leav ng group UP! -Gal productioi i Orotic acid UDP-Gal fgalT,K,U ppa UTP I UDP UTP- UDP-Gal GicNAc Recombinant E. coli ß1^4-GalT A UDP I -►UMP LacNAc C. ammoniagenes UTP production !: HIST XH o=c ,c-c N Orotat 0=C n PRPP PP, 0 V f II -a^t. ' -0-POCHj^ 0 Phosphoribosyl- 1 L-^ transferase "° l\H H / h\j i/h ho oh Orotidylat HN ^CH - IV hn^ ^ch 0- H* CO, 0 Orotidylat- " Decarboxylase I "0 f\H H> h\| l/h ho oh Uridylat (UMP) Applications in Medicinal Chemistry Anitvirals HO-m OH /—0 Aldolase ^Epimerase^ H0| ./ Yr-OH Pyruvate ^ NH 58 (NAG) N-Acetyl-D-Glucosamine HO' 0=( CH, 57 (NAM) N-Acetyl-D-Mannosamine HO 56 (NANA) N-Acetyl-D-Neuraminic Acid Abacavir (32) Applications • Cytostatics fjH H. polymorpha SC 13865 COzEt H. fabianii 65 CK NH C02Et Pseudomonas t . Acp y«as/ sp. SC 13856 Ac9, .*l XL lipase n 67 Racemic Acetate "nh J-tiH R-67 S-68 (3/?>-Acetate (3S)-Alcohol HO,, / ^Pnh (2fl,3S)-66 Paciitaxel Side-chain or 71 Open side-chain RQ» P OH OH OBzOA<= 63 Baccatin III, R=Acetate 64 10-DAB, R=H OH OBz Paciitaxel 62 Epothilone F t Sorangium cellulosum Fermentation Process Figure 19 Third Generation: Synthetic HMG-CoA Reductase Inhibitors HO Applications • Lipitor oh o o bioreduction 9H 9H i? OH OH OH QH PhNH CO„H 11 X n n 0=S=0 I Me OtBu OtBu OH OH O Atorvastatin Rosuvastatin bioesterification C>v^JLv^LNJls. 0 OtBu OtBu OH OH O Y o OtBu Xo 9 OtBu O^O 0 Y°XU X0 0 OtBu X o o R R = -CN (Atorvastatin) R = -OH (Rosuvastatin) ^ I o o CI, ,J, OH 0 -ci- +cr +CN' nc, OH 0 Third Generation: Synthetic HMG-CoA Reductase Inhibitors OH OH Applications • Lipitor OH OH PhNH CO,H OH O OH OH O Atorvastatin DERA DERA spontaneous OH OH O OR OH OH OH 0 Ck OH ^ 0 ^OH OR CO,H Rosuvastatin HjN O ^OR,