Enzymatic and Chemoenzymatic Carbohydrate Synthesis Group Meeting Presentation April 20, 2016 Kelly Craft

Main Enzymatic Approaches Glycosidase-catalyzed glycoside bond formation – Transfers glycosyl moiety to sugar acceptor Glycosynthase-catalyzed glycoside bond formation – Mutated glycosidase lacking catalytic nucleophilic residue – Transfers glycosyl-fluoride to sugar acceptor

Catalytic Mechanisms Glycosidase-catalyzed glycoside bond formation – Retaining glycosidase – Inverting glycosidase

Catalytic Mechanisms Glycosynthase-catalyzed glycoside bond formation

Main Enzymatic Approaches Glycosyltransferase-catalyzed glycoside bond formation – Catalyzes bond formation between sugar nucleotide donor and appropriate sugar acceptor

Catalytic Mechanisms Glycosyltransferase-catalyzed glycoside bond formation – Inverting glycosyltransferase – Retaining glycosyltransferase

Glycosidases Advantages – Uses simpler, more easily accessible donors – More readily available and robust enyzmes Disadvantages – Less regioselective – Potentially lower yielding (product subject to hydrolysis)

Glycosidase-Catalyzed Syntheses Two-step syntheses of trisaccharides Crout, D.H. and Vic, G. Curr. Opin. Chem. Biol. 1998, 2, 98.

Glycosynthases Advantages – Uses simpler, more easily accessible donors – Lower rates of product hydrolysis Disadvantages – Less regioselective – Must mutate appropriate glycosidase

Glycosyltransferases Advantages – High regio- and stereoselectivity – High yields Disadvantages – Uses complex, costly sugar nucleotide donors – Enzymes not as readily accessible and subject to feedback inhibition

Commercially Available Glycosyltransferases Schmaltz, R.M., Hanson, S.R., and Wong, C.H. Chem. Rev. 2011, 111, 4259.

Sugar Nucleotide Donors UDP: Uridine diphosphate GDP: Guanosine diphosphate CMP: Cytidine monophosphate

General Sugar Nucleotide Synthesis NTP: nucleoside triphosphate PPi: pyrophosphate Enzyme: nucleoside diphosphate sugar pyrophosphorylase

Chemical Synthesis of Sugar Nucleotides Wagner, G.K., Pesnot, T., Field, R.A. Nat. Prod. Rep., 2009, 26, 1172.

Chemical Synthesis of Sugar Nucleotides Moffatt, J.G. and Khorana, H.G. J. Am. Chem. Soc. 1958, 80, Moffatt, J.G. and Khorana, H.G. J. Am. Chem. Soc. 1961, 83, 649. Original Khorana method Improved Khorana method (most common form)

Chemical Synthesis of Sugar Nucleotides 1H-tetrazole catalyzed phosphomorpholidate couplings – Shorter reaction times (1-2 days) – Increased yields (>75%) – Tetrazole both a Brønsted acid (pK a =4.9) and a nucleophile Drawbacks of original Khorana method – Long reaction times (days-weeks) – Material lost to side reactions or hydrolysis degradation Wagner, G.K., Pesnot, T., Field, R.A. Nat. Prod. Rep., 2009, 26, Wittmann, V. and Wong, C.H. J. Org. Chem. 1997, 62, 2144.

Wagner, G.K., Pesnot, T., Field, R.A. Nat. Prod. Rep., 2009, 26, 1172.

Chemical and Enzymatic Combined Syntheses Liu K.C. and Danishefsky, S.J. Chem. Eur. J. 1996, 2, Synthesis of GM 3

Liu K.C. and Danishefsky, S.J. Chem. Eur. J. 1996, 2, 1359.

Chemical and Enzymatic Combined Syntheses Wenlond, Y., Cao, H, et. al. Carbohyr. Res. 2015, 401, 5. Synthesis lacto-N-tetraose and sialyl lacto-N- tetrasaccharides

Chemical and Enzymatic Combined Syntheses Wenlond, Y., Cao, H, et. al. Carbohyr. Res. 2015, 401, 5.

Chemical and Enzymatic Combined Syntheses Wenlond, Y., Cao, H, et. al. Carbohyr. Res. 2015, 401, 5.

Chemical and Enzymatic Combined Syntheses Wenlond, Y., Cao, H, et. al. Carbohyr. Res. 2015, 401, 5.

Chemical and Enzymatic Combined Syntheses Wenlond, Y., Cao, H, et. al. Carbohyr. Res. 2015, 401, 5.

One-Pot Multienzyme (OMPE) Systems Glycosyltransferase and sugar nucleotide biosynthetic enzymes used in same pot Must optimize reaction conditions (ex. pH) for proper functioning of all enzymes Allows for sugar nucleotide regeneration Avoids tedious sugar nucleotide purification Glycosyltransferases less subject to feedback inhibition Avoids instability issues of some sugar nucleotides

Enzymatic Generation of Sugar Nucleotides GlyK: glycokinase NucT: nucleotidyltransferase NTP: nucleoside triphosphate PPi: pyrophosphate PpA: inorganic pyrophosphatase Pi: inorganic phosphate CTP: cytidine triphosphate CCS: CMP-sialic acid synthetase

One-Pot Multienzyme (OMPE) Systems Yu, H. and Chen, X. Org. Biomol. Chem. 2016, 14, 2809.

One-Pot Multienzyme (OMPE) Systems Yu, H. and Chen, X. Org. Biomol. Chem. 2016, 14, 2809.

Enzymatic Regeneration of Sugar Nucleotides Wong, C.H, Haynie, S.L., and Whitesides, G.M. J. Org. Chem. 1982, 47, Ichikawa, Y., Look G.C., and Wong, C.H. Anal. Biochem. 1992, 202, 215. One-pot synthesis of N-acetyllactosamine

Enzymatic Regeneration of Sugar Nucleotides Schmaltz, R.M., Hanson, S.R., and Wong, C.H. Chem. Rev. 2011, 111, 4259.

Enzymatic Regeneration of Sugar Nucleotides Křen V. and Thiem J. Angew. Chem. Int. Ed. Engl. 1995, 34, 893. One-pot synthesis of sialyl T-antigen

Enzymatic Regeneration of Sugar Nucleotides Chen, X., Wang, P.G. et. al. J. Am. Chem. Soc. 2001, 123, Sugar nucleotide regeneration beads

Enzymatic Regeneration of Sugar Nucleotides Chen, X., Wang, P.G. et. al. J. Am. Chem. Soc. 2001, 123, 2081.

Sequential One-Pot Multienzyme (OMPE) Systems

Yu, H. Chen, X., et. al. Angew. Chem. Int. Ed , Enzymatic synthesis of disialyl lacto-N-neotetraose OP4E system OP2E system

Sequential One-Pot Multienzyme (OMPE) Systems Tsai, T., Wong, C.H., et. al. J. Am. Chem. Soc. 2013, 135, Large-scale enzymatic synthesis of Globo H and SSEA4

Sequential One-Pot Multienzyme (OMPE) Systems Tsai, T., Wong, C.H., et. al. J. Am. Chem. Soc. 2013, 135,

Sequential One-Pot Multienzyme (OMPE) Systems Tsai, T., Wong, C.H., et. al. J. Am. Chem. Soc. 2013, 135,

Sequential One-Pot Multienzyme (OMPE) Systems Tsai, T., Wong, C.H., et. al. J. Am. Chem. Soc. 2013, 135,