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Kyle W. Knouse Baran Group Meeting 2/10/18 Starch Cellulose

Kyle W. Knouse Baran Group Meeting 2/10/18 Starch Cellulose

Baran Group Meeting Kyle W. Knouse Carbs 2/10/18 6 OH C-5 Contents: Not included in this group meeting: 6 OH 4 What are ?, Roles Biomass to feedstock, Enzymatic glycosylation, 4 β 5 O Reference 5 O HO O OH in biological systems/metabolism, Synthetic polymers, Carbohydrates HO 1 HO OH HO 2 Glycoconjugates, Synthetic as chirons (Martinez, 2014), Nucleoside Chemistry HO 2 3 OH α 3 OH 1 OH challenges/highlights, (O’Hara, 2012; Gianatassio, 2013) HO OH History: α-Pyranose OH -10,000 BC: Primitive sugarcane processing in New Guinea β- OH 62% 37% D- -1500 BC: Cotton cloth from India speards to Persia and China OH HO O -1891: Fischer reports the structure of glucose, , , and HO -1929: Haworth proposes ring structures for pyranose/furanose OH CHO OHC H -1940’s: produce protective immune respons H HO H HO HO OH -1956-1963: OH OH HO H H OH -Bacterial peptidoglycan backbone structure HO HO OH O O HO H -major natural polysaccharides (, , peptidoglycan) are β(1→4) linked H OH H OH HO H -mechanism of peptidoglycan biosynthesis and penicillin action OH -1972: high field NMR for glycan structure analysis CH OH HOH2C α-Furanose OH β-Furanose OH 2 0.5% 0.5% D-Glucose L-Glucose What is a Carb? - Carbohydrates are defined as “hydrates of carbon” polyhydroxyaldehydes, polyhydroxyketones and their derivatives Alpha vs. Beta ( vs. Cellulose): HO OH HO O Drugs: -Heparin OH HO -erythropoietin O O O Branching HO α(1→6) OH O O OH HO Starch OH O Linear O α(1→4) HO OH O Class Monomers Subclass Examples Starch - most common carbohydrate in human diets 6 Major classes - (1-2) Mono/, Polyols Glucose, , Sorbitol (potatoes, wheat, maize (corn), rice, and cassava) - (3-9) Malto-oligosaccharides -water soluble Polysaccharides (>9) , , Cellulose -Hexosamines -highly branched polymer of glucose -Deoxyhexoses -linear glucose with α(1→4) glycosidic bonds. -Uronic Acids Other definitions: -branching α(1→6) bonds occurring every 24 to 30 glucose units -Sialic Acids - = carbs that cannot be hydrolyzed into a simpler form - Polysaccharide = repeating oligosaccharides Amylose Cellulose - Most abundant biomass on Earth. Main component of the primary cell wall of - Glycan = general term for any of these… (left-haded helix) green plants, algae and some bacteria (biofilms) - Often encountered conjugated, >50% of all proteins are glycosylated - Glycoconjugate = mono/ attatched to -Cotton, wood, hemp, etc. a non-carbohydrate moiety (aglycone) -linear glucose with β(1→4)-glycosidic bonds. -no coiling or branching OH “Reducing Linear Cellulose End” Capable of reducing OH β(1→4) OH O O HO OH H2O2, Ferricyanide, O O HO Cu2+, Ag+ O O OH HO HO OH OH Baran Group Meeting Kyle W. Knouse Carbs 2/10/18 Monomeric Building Blocks Oligomeric Biomolecules Central Dogma of Molecular Biology: Letters of the alphabet OR Written Novel O Transcription Translation B B HO O O B DNA RNA Proteins O HO 5’ O P X O O OH B Replication HO B O O RO P DNA DNA Duplication DNA Linear O 4 canonical -displace X nucleobases O “DNA Chemistry” -oxidation O 3’ Information Modification results in (Peters, 2017) similar stereochemical complexity

O Simple PG strategy O RHN N NH O 3’→5’ or C→N H N OA* 2 OH R R R HN R O NH Peptides H N Achiral link O Transcription 2 O RNA Information 20 proteogenic AA’s -activate RNA Synthesis R R -displace OA* C mRNA Often Post-Translationaly Nuclear Envelope Modified ( complexity) OR HO O O Linear and HO OH X Branched HO α RO OH Ribosome O RO OR OH O Information HO OH OH 6 HO O Protein HO 4 O Translation OH O HO β O Protein Synthesis “Highlights in Peptide and Carbohydrates RO HO 2 OH 3 OH 1 reducing Protein Synthesis” RO OR O R Only 36 building end (Mallins, 2016) H 5 potential sites Chiral link N OH blocks are needed to for reaction N construct 75% of the >50% of all proteins are glycosylated H 3299 mammalian Diversity: Hexamer R O oligosaccharides. Nucleotide* = 46 = 4096 Mammalian glycan Cell Surface Peptide* = 206 = 64 million structures comprise 78% of all the Motility/membrane eukaryotic glycans Carbohydrate** = 192 billion Signaling movement (charge distribution) ACS Chem. Biol., 2007, 2, 685. Chem. Sci. 2011, 2, 337. Provide structure (peptidoglycan) and preservation () *Assuming natural NH2 phosphodiester/ CO H amide linkages Glycans are abundantly expressed on the 2 **based on the “ten outer-surface of prokaryoate and common” mammal eukaryote cells, creating a landscape of monosaccharides recognition sites, barriers and transporters Baran Group Meeting Kyle W. Knouse Carbs 2/10/18

Glycosyl Donors: O O Donor (D) Acceptor (A) (D-A) RO O Y Y = potential leaving group O Z Z O O Promotor (P) O Y O Y O Promotor β-linkage RO X HO RO O + Solvent (Ingnored) Z HO Y Z Z Z RO X Solvent O or Z Z RO α -linkage Two ways to set up reaction: X = Leaving Group Glycosyl Acceptor: O Z 1) Normal Procedure (disarmed donors) 2) Inverse Procedure (armed donors) R = Protecting Group O Y *Bulky groups at C-6 diminish 1→4 Z = participating or Z P D non-participating group (TBDPS, TBDMS, Piv) D + A D-A D + P + A P + A D-A Glycosyl Donors: For armed donors . O NH D P will decompose O O R O O O O . X P R Decomp. D.P A P A + P X CCl3 3 P P

Glycosyl halides Glycosyl phosphates Trichloroacetimidates Pentenyl glycosides A D (TMSOTf) OBn X = Cl, Br (AgOTf) (TMSOTf) (NIS/Et3SiOTf D-A or F (SnCl /AgClO ) or Br ) 2 4 R=Alkyl, O-Alkyl 2 AcO HO O X = O, S, lone pair AcO O OTBS O O O AcO N O O OAc 3 S(O)Ar S BnO XR SEt O Et2O, OBn + TMSOTf (0.01 eq.) OBn S BnO O OBn NP: 43% Me O OBn Glycosyl sulfide Glycosyl sulfoxides Glycals IP: 78% Xanthathes OBn AcO (NIS/TfOH (Tf2O) (TMSOTf) (ZnCl2) Me O O O AcO O or DMTST) O OTBS O CCl O O O O 3 AcO N O R OAc 3 O R NH N O Tetrahedron Lett. 1991, 32, 3353 N O O R = H, Me PG Strategies: R R = OR’, SR’, CN Selective 1° protection (Bulky, TBS, Trt, 0 °C, limiting reagents, etc.) 2° equitorial. > 2° axial. OH Armed/Disarmed logic: cis/trans vicinal diols (acetal formation) O Highly dependent Slow HO OH O O on stereochem HO Selective 2° protection, X OH hemiacetal Bu2SnO, R-X + OBz OBz Esters induce slight Two step monomer: (H , ROH) Armed: C-2 = Ether positive charge, oxonium Disarmed C-2 - Ester, Amide, etc. formation is slower OH OAc OAc O Ac O O BF OEt O O O 2 3 Fast HO OH AcO OAc AcO X X HO AcO - AcO OH Pyridine OAc X OAc OBn OBn 100 °C, 1 hr. Baran Group Meeting Kyle W. Knouse Carbs 2/10/18

Not always the case! Kiliani Fischer Synthesis: 1. Acetone, H SO 1. NaH, CS , MeI 2 4 2 CN CN CHO CHO 2. TsCl, pyridine 2. Bu3SnH, AIBN CHO OH OTBS + O OH 3. NaBH4, DMSO 3. H OBn HO OH O HO OH + OH NaCN Heat [H] O 4. H , DMSO O 4. Ac2O, pyridine O OH OH OH OH CH2OH CH OH CH OH OH CH OH CH OH HO 5. Bu SnO HO 5. PhSH, BF OEt H2O 2 2 H2O 2 2 OH 2 O 3 BnO HO 6. BnBr, NaH OH O 6. NaOMe Br D- D- D- 7. TBSCl, Pyridine 7. BnBr, NaH Cycle is repeated until desired chain length (requires separation of epimers) 8. H2, Pd/C Me Me 8. Br2 Wohl Degradation: PG Migration: OH OR CHO N CN O SPh OR OR OH NH2OH OH Ac2O OH NaOMe CHO O SPh BzO O SPh OH OH OH OH Tf2O TfO N H2O AcOH MeOH O 3 HO N CH2OH CH2OH CH2OH CH2OH 3 Pyr. HO N3 Chem. Ber., 1893, 730 BzO O N Ph Modern Approach: Total Synthesis of the L-Hexoses - K. B. Sharpless Access to all 8 hexoses, D and L Epimerizations: Eliminations: OH OH SPh RO RO TsO N CHO i. [O], [H] 3 O O O NaN O CHO OR * OR OR ii. Mitsonobu OR O 3 OR O OR R-M AE * Payne OR Pummerer; OR DMF O OR CH2OR OR OR OR OR HO OR HO OR TsO CH2OR CH2OR CH2OR CH2OR TBSO TBSO Interesting transformations: *(+) or (–) tartrate *DIBAL = Retention O O K CO /MeOH = Inversion O PhMgBr O 2 3 OBz OBz “Since the mirror image of every compound in Fig. 2 can be prepared by simple O OMe O F TBSO exchange of the chiral ligand in the AE reaction, the formal synthesis of the D- Me DAST Me hexoses has also been achieved” Science, 1983, 220, 949 N 76% N 3 OH 3 OMe Selective 1° protection Ph J. Carb. Chem., 1985, 627 (Bulky, TBS, Trt, 0 °C, limiting reagents, etc.) Works well for: Ph Mannose BzO H N O BzO TBSO OH Arabinose O 1. H5IO6 O O O O BzO OH O Highly dependent O H H O 2. TEA, MeOH OH HO hemiacetal OMe on stereochem OMe (H+, ROH) HO R HO H OH R O O O BzO OH BzO OH Me Ph NH H O H WO 2010/015637 A1 90:10:-, 77% Ph O -:100:-, 97% DMTrO Proposed glucose ii. 1° protection N O N O R O H-bonding in CHCl3 Good for functionalizing RO N H O iPr N O OMe 2’-ribofuranoses O + O O i. acetalization E = TESCl, AcCl, MsCl H OH X NMI = bad selectivity and/or yield HO OTBS MeN N iii. oxidation R= H, OMe O O 2:98, 93% X= iPr, c-pentyl Nature Chem. 2013, 790. Tet. Lett. 2003, 1731 Baran Group Meeting Kyle W. Knouse Carbs 2/10/18

Generalizations made independent of subtitution and stereochem @ C-(3 to 6) Solvent Effects: O Et Z Z “Reverse Anomeric Effect” O O O O O O R Et OR OR Et2O OR R α-glycosidation Z Z OR O OR OR X Promoter or R 1,2-cis 1,2-trans 1,2-cis 1,2-trans OR O O MeCN β-glycosidation 2-D-glycero: 2-L-glycero: R X R allo-, gluco-, gulo-, altro-, manno-, ido-, OR OR and galactopyranosides and talopyranosides O N Low selectivities R Cl for mannosides H “The Nitrile Effect” OR (see below) H O 3 MeCN molecules form N Ar O O Ac 2-deoxy- 2-keto-3-deoxy- a β-nitrilium-nitrile CO2H glycosides OH ulosonic acids O J. Chem. Soc., Perkin Trans I, 1990, 747 H H OR OR Non-participating group at C-2: Glycosyl Halides - α-selective [major] Participating Group at C-2? 1,2 - trans rule! O [Lemieux] [minor] ORʹ O ROH O O Slow O O O O OR ʹR-OH OR X BnO Et4NBr BnO BnO O Br OR Br BnO O O HO O (good for , galactose, fructose) Blocks bottom OR Fast R O R O face ORʹ Br Participating group β-Mannosides: R OR Et NBr O (Ester or Amide) O O 4 i. R-X, insoluble Ag Blocks top O O Chem. Ber. 1981, 114, 3102 R ii. Priveledged donors J. Org. Chem. 1996, 61, 4506 O O face ORʹ R R O iii. Epimerization Carbohydr. Res. 1980, 79, C13 iv. IAD J. Am. Chem. Soc. 1992, 114, 1087 O O O O OR OBn OBn ʹR-OH X O i. [Paulsen] BnO BnO BnO O O O Insoluble silver Ag ROH ORsalts, silver silicate ORʹ or silver oxide work Non-Participating Group at C-2? 1,2 - cis rule? (try again) MeCN Br Br Shields α face OBn (2-D)-1,2-cis O . -1,2-trans ii. iii. BnO BnO Ag (2-L)-1,2-cis TBSO TBSO OH O O * O O O O . -1,2-trans Tf2O, ROH OH- X OBn OR OR OR OR O OTf OR S 1:1 to > 20:1 β/α D O Et “Can always just epimerize” In the absence of a (2- )-1,2-cis . -1,2-trans MsO MsO Non-Participating group participating group, O O O OH (Ether or Azide) (2-L)-1,2-cis ROH O O you must fight the OR * OR [H] anomeric effect. . -1,2-trans OR OR BnO MeCN, 48 hrs OTs O O O 78%, 4.9:1 β/α X O *Varying C-2 PG gives access to all but iv. Y Y R’ OBn OBn HO X-Y-X, O OR' O O HO BnO (2-L)-1,2-cis O R’OH O O O OR (mainly β-Mannosides) L L OR’ Baran Group Meeting Kyle W. Knouse Carbs 2/10/18

“ There are no universal reaction conditions UDP: O Uses DMTr for colorimetric loading analysis for oligosaccharides synthesis” - Hans Paulsen Cleavage: (Cross-Metathesis) O O NH BnO BnO HO O O Angew. Chem. Int. Ed. Engl. 1982, 21, 155 O P O P O 3-steps O O O OH OH O Cl O BnO BnO Enzymatic: OPiv PivO - uses unprotected donor/acceptors Longer chain length for higher loading O - high d.r, 105-108 rate acelerations - No linkers, immobilized enzymes OH OH OH BnO OAc BnO OAc O O Limited by number of available n O BnO OH O BnO BnO O P HO glycosyltransferases (GT) BnO BnO TBSO O OBu HO OH OH BnO OBu O OH BnO O O CCl3 PivO HO OH UDP O O HO O BnO HO O NH OH β1-4-glycosyltransferase HO OH BnO HO n = 6, heptasaccharide, largest to date on resin aqueous buffer, M2+ OH OH O Org. Lett. 1999, 1, 211 New linker = high loading = hundreds of mg’s For greater detail, see Blixt O., Razi N. (2008) Enzymatic Glycosylation by Transferases. In: Fraser-Reid B.O., Tatsuta K., Thiem J. (eds) Glycoscience. Springer, Berlin, Heidelberg OBz OH Solid-Phase: FmocO HO Based on pionerring work of Merrifield O O General Requirements: HO <1 week 3-steps BnO HO 28 -C-1 reactive LG BnO OH 1% yield, average 96% each Cl -temporary vs. persistent PG’s O SEt O OH ONBz Standard SPPS Cleavage: (O ) ACIE. 2013, 52, 5862. HO 3 ONBz HO O OH O BnO OH O ONBz BnO HO O BnO Br O HO BnO BnO HO O BnO O HO BnO HO 48 BnO OH O 1) 0.2 M NaOEt, EtOH, 30 min O O BnO O BnO O 2) 1:4 resin/monomer, PhH, BnO HO 60 hr in the dark BnO HO 5% yield, 102 steps - average O OBz OH FmocO O 97.1% each JACS, 1971, 492. 75% O O BnO HO BnO HO 100 Benzyl Ethers - several - RO HO treatments (72-96 hr) H Pd/C Polystyrene Cleavage: (F ) Cl SEt O 2 BuLi; O O Chem. Commun., 2017, 53, 9085 Si(R) R O R3O SiR2Cl2 2 1 R2O R4O O A R=Ph, stable, R=iPr, minor cleavage 8 Solvents 16 reagents O Free hydroxyl generated -40 to +80°C O 1) DMDO, Acetone, 0°C (Capping = double rxn time) O 90 min, repeat 40 min R1O O R O O R2O 1 OH R2O 2) A, ZnCl2, THF, 0°C - rt O 8 hrs R3O R4O Homemade Synthesizer The Glyconeer ® Long times / cold temperatures Science, 2001, 1523 JACS, 1995, 5712 (Max Planck) Nature, 2002, 785 (Glycouniverse) Baran Group Meeting Kyle W. Knouse Carbs 2/10/18

OLev Solid-Phase (contd): Solution-Phase One Pot: Armed/Disarmed Theory: OR OBn O Fraser-Reid, B. J. Am. Chem. Soc. 1988, 110, 2662 O HO L BnO O P(OBu) OR O 2 O O Armed O O BnO OPiv O Promoter RO BnO HO O L O O RO OR OBn O RO Anomeric Control OR O HO L Protecting Group Coupling: TMSOTf OLev Control (Constant L) (L1>L2) O Disarmed Deprotection: H2NNH2 OH OH RO O OBn RO BnO O P(OBu)2 O O OR BnO Select Examples: O OPiv O O EDG L L1 BnO STol OR Homogeneous PG’s = ease of synthesis BnO O OH OH RO 3 hours per full cycle NHTroc O OBn O RRV = 6500 O O RO OR EWG L L RO O O 2 OR HO STol O AcO RO O NHTroc OH Releative Reactivity Value (RRV) RO RRV = 302 O O OR O BnO STol R STol R OMe BnO Automated 10 Donor 80% yield Science 2001, 291, 1523 OH N3 NIS, MeOH HPLC ratio (Seeberger) machine RRV = 23 hours gives RRV O O O BnO OR R STol R OMe Manual 122 20% yield BnO “Reducing End” (Nicolaou) reaction J. Am. Chem. Soc. 1997, 119, 449 N Reference hours RRV = 0 3 unreactive

Wong, C.-H. J. Am. Chem. Soc. 1999, 121, 734 OH OBn OBn O OR Me O STol Estimated OptiMer database would only OH OBn a O O 1 need ~500 building blocks analyzed to 2nd gen route HO OBn prepare most bioactive saccharides. BnO STol OH O OH OBn O OLev 83% yield OH O HO RRV = 72,000 RRV = 4000 OH O O HO OH OBn OBn OBz OBz NBzO ONBz HO OBz OBz NHAc O O O OptiMer STol b O OH BnO O O O O OptiMer STol 2 OH TrocHN BnO HO HO TrocHN Globo H RRV = 6 RRV = 850 O 1) Mix 1 + 2 + 3 1) Mix a + b + c HO OH OBn 2) Deprotect OH OBn 2) PG Manipulations NBzO ONBz O O 63% for couplings 3 O 67% for couplings O OH 20% overall O c Me BnO OR HO STol OH OBn BnO Minimizes PG manipulations OBn Suitable for 3-6 length O(ClBn) saccharides per one-pot reaction RRV = 13 Baran Group Meeting Kyle W. Knouse Carbs 2/10/18

OH β-cyclodextrin Entraps hydrophilic compounds = Febreeze HO O O O Solublizes hydrophobic compounds OH HO OH O OH OH Hydrophillic HO OH O HO OH O O O HO HO OH H OH HO H R O OH galactose OH H Hydrophobic OH isomerase, 2 Mg (D-Gal) Hydrogen Bonding: hydrophobic packing O required for catalytic activity OH H-Bond residues: Aspartic acid, Asparginine, OH Glutamate, Glutamine, Arganine OH Negative Free Energy of Binding Glycan Code: Increses diversity in the proteome O OH OHO O O i.Hydrogen Bonding i. (site of linkage, stereo) OOH ii. Association with Metals ii. Type HO iii. Branched/Linear O HO OH NH3 iii. Hydrophobic packing Hoogstein face iv. Ionic interactions iv. Length OH O

O N NH Glucose Metabolism: Aminoglycoside After digestion of food (poly to monosaccharides): P Antigen: OH Normal glucose level = O O N NH2 O N (I) Gucose conversion to oxalate, NADH, ATP. O O i. peptide O O HO H ii. oligosaccharide HO OH O Antibody: N H iii. lipid HO O High glucose levels (poor diet, diabetes)= O OH O H2 (II) Saturates glycolysis pathway, glucose HO stored as HO Glycoconjugates: O (III) Activates Polyol Pathway, (rapidly (II) Glycogen-isis/lysis i. N-Linked - asparganine, arginine consuming NADPH) leads to cell damage. ii. O-Linked - serine, threonine, tyrosine + NADH (I) Glycolysis Blood types: OH O + ATP -antigens are determined by GT’s = present, Glucose (80%) O NADP+ OH - creates a barrier to zoonotic transmision of Foods HO HO Acetyl CoA viruses Immune System: HO HO O -Cell to cell adhesion Not Advanced Glycation End glucose OH Product (AGE): -Monoclonal antibodies O O Liver CHO Lysine OH OH tyrosine HO Arginine OH OH OH H O CH OH HO O OH H2N NH2 2 HO OH N N HO HO NH OH + NADP+ HO Glucosepane H (III) Polyol Pathway HO N Sorbitol Glucose Other monosaccharides: H H O HO (Fructose, Galactose) go to liver; Over 40 glycosylation disorders identified in humans converted to glyceraldehyde NH2 -No treatment for any Converted to glucose, then -80% in CNS metabolized N Handbook of Clinical Neurology, 2013, 1737. R NADH Baran Group Meeting Kyle W. Knouse Carbs 2/10/18

Mammalian OH Hexoses(8): OH OH OH Carbohydrates: HO2C OH OH HO O O OH OH OH OH HO HO OH O O O O O HO HO HO HO NH2 HO OH OH HO HO OH mannose OH OH OH OH (D-Man) * OH OH OH OH OH OH glucose OH *derivatives = sialic acids (D-Sia) glucose galactose (D-Glc) OH OH OH OH OH OH OH OH OH OH OH OH O O O HO O O O HO HO HO HO OH HO HO OH NHAc galactose OH OH OH OH OH OH OH (D-Gal) N-Acetylglucosamine mannose (D-GlcNAc) "All Altruists Gladly Make Gum in Gallon Tanks": OH allose, altrose, glucose, mannose, gulose, idose, galactose, talose. OH O HO HO OH Pentoses(5): O OH OH OH Me O O O O O HO OH OH OH OH OH NHAc fucose HO HO HO HO N-Acetylgalactosesamine (L-Fuc) HO OH HO OH HO OH HO OH HO (D-GalNAc) HO C 2 O xylose arabinose HO OH HO : OH O OH O OH O glucuronic acidOH OH HO OH HO OH (D-GlcA) HO O O OH OH O OH OH OH OH HO OH HO D-erythropentulose D-threopentulose HO OH D-fructose OH HO HO HO2C OH OH O OH O OH O iduronic acid xylose OH OH HO OH OH HO OH (L-IdoA) (D-Xyl) D- HO OH OH OH OH L-erythropentulose D- L-threopentulose

OH Other sugars OH of interest: O Me O OH HO HO OH 18F 18 HO OH OSO3H 2-[ F]-Fluoro-2-deoxy-D-glucose Most successful PET tracer in history OH L- O avian influenza virus particles HO OH O HO rhamnose OH OH (L-Rha) Me OH Sulfoquinovose plant cellulose OH

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