1. 25.17 Carbohydrate Structure Determination

2. Carbohydrate Structure Determination Spectroscopy X-Ray Crystallography Chemical Tests once used extensively; now superceded by spectroscopic methods and x-ray crystallography reactions of carbohydrates can involve either open-chain form, furanose, or pyranose form

3. 25.18 Reduction of Carbohydrates

4. Reduction of Carbohydrates Carbonyl group of open-chain form is reduced to an alcohol. Product is called an alditol. Alditol lacks a carbonyl group so cannot cyclize to a hemiacetal.

5. Reduction of D -Galactose  - D -galactofuranose  - D -galactofuranose  - D -galactopyranose  - D -galactopyranose CH 2 OH H OH H HO H HO H OHCHO H OH H HO H HO H OH D -Galactitol (90%) reducing agent: NaBH 4, H 2 O (catalytic hydrogenation can also be used)

6. 25.19 Oxidation of Carbohydrates

7. Benedict's Reagent Benedict's reagent is a solution of the citrate complex of CuSO 4 in water. It is used as a test for "reducing sugars." Cu 2+ is a weak oxidizing agent. A reducing sugar is one which has an aldehyde function, or is in equilibrium with one that does. A positive test is the formation of a red precipitate of Cu 2 O. + 2Cu 2+ RCHO 5HO – + + Cu 2 O RCO – O 3H 2 O +

8. Examples of Reducing Sugars Aldoses: because they have an aldehyde function in their open-chain form. Ketoses: because enolization establishes an equilibrium with an aldose. CH 2 OH C O RCHOHC OH R CH CHOH RO oxidized by Cu 2+

9. Examples of Reducing Sugars Disaccharides that have a free hemiacetal function. O HOCH 2 OH OH HO OH HO HO O O Maltose

10. Examples of Reducing Sugars Disaccharides that have a free hemiacetal function. oxidized by Cu 2+ O HOCH 2 OH HO OH HO HO OOH CH O Maltose

11. Glycosides are not reducing sugars O OCH 3 OH HO HO HOCH 2 Methyl  - D -glucopyranoside lacks a free hemiacetal function; cannot be in equilibrium with a species having an aldehyde function

12. Oxidation of Reducing Sugars The compounds formed on oxidation of reducing sugars are called aldonic acids. Aldonic acids exist as lactones when 5- or 6- membered rings can form. A standard method for preparing aldonic acids uses Br 2 as the oxidizing agent.

13. Oxidation of D -Xylose HO H OH H OH HCHO CH 2 OH Br 2 H2OH2OH2OH2O HO H OH H OH H CH 2 OH CO 2 H D -Xylose D -Xylonic acid (90%)

14. Oxidation of D -Xylose HO H OHOHOHOH H OH H CH 2 OH CO 2 H D -Xylonic acid (90%) O O OH OH HOCH 2 OOOH HO HOHOHOHO +

15. Nitric Acid Oxidation Nitric acid oxidizes both the aldehyde function and the terminal CH 2 OH of an aldose to CO 2 H. The products of such oxidations are called aldaric acids.

16. Nitric Acid Oxidation CHO CH 2 OH H OH H OH H H OH HO HNO 3 60°C CO 2 H H OH H OH H H OH HO D -Glucaric acid (41%) D -Glucose

17. Uronic Acids CHO CO 2 H H OH H OH H H OH HO D -Glucuronic acid HO HO OH OH HO 2 C O Uronic acids contain both an aldehyde and a terminal CO 2 H function.

18. 25.20 Cyanohydrin Formation and Carbohydrate Chain Extension

19. Extending the Carbohydrate Chain Carbohydrate chains can be extended by using cyanohydrin formation as the key step in C—C bond-making. The classical version of this method is called the Kiliani-Fischer synthesis. The following example is a more modern modification.

20.  - L -arabinofuranose  - L -arabinofuranose  - L -arabinopyranose  - L -arabinopyranose CH 2 OH H HO H HO H OHCHO Extending the Carbohydrate Chain HCN CH 2 OH HO H H HO OH HCN CHOH the resulting cyanohydrin is a mixture of two stereoisomers that differ in configuration at C-2; these two diastereomers are separated in the next step

21. Extending the Carbohydrate Chain CH 2 OH HO H H HO OH HCN CHOH CH 2 OH HO H H HO OH H H OHOHOHOHCN HO H H HO OH H HOHOHOHO HCN+ separate L -Mannononitrile L -Gluconononitrile

22. Extending the Carbohydrate Chain CH 2 OH HO H H HO OH H H OHOHOHOHCN L -Mannononitrile H 2, H 2 O Pd, BaSO 4 L -Mannose (56% from L -arabinose) CH 2 OH HO H H HO OH H H OHOHOHOH CHCHCHCHO

23. Likewise... HO H H HO OH H HOHOHOHO HCN L -Gluconononitrile H 2, H 2 O Pd, BaSO 4 L -Glucose (26% from L -arabinose) CH 2 OH HO H H HO OH H HOHOHOHO H CHCHCHCHO

24. 25.21 Epimerization, Isomerization, and Retro-Aldol Reactions of Carbohydrates

25. Enol Forms of Carbohydrates Enolization of an aldose scrambles the stereochemistry at C-2. This process is called epimerization. Diastereomers that differ in stereochemistry at only one of their stereogenic centers are called epimers. D -Glucose and D -mannose, for example, are epimers.

26. Epimerization CHO CH 2 OH H OH H OH H H OH HO D -Mannose D -Glucose Enediol CHO CH 2 OH H OH H OH H HO H HO H OH H OH H OH HOCHOHC This equilibration can be catalyzed by hydroxide ion.

27. Enol Forms of Carbohydrates The enediol intermediate on the preceding slide can undergo a second reaction. It can lead to the conversion of D -glucose or D -mannose (aldoses) to D -fructose (ketose).

28. Isomerization D -Fructose D -Glucose or D -Mannose Enediol CH 2 OH H OH H OH H HO H OH H OH H OH HOCHOHC CHO H OH H OH H HO CHOH CO

29. Retro-Aldol Cleavage The D -fructose 6-phosphate formed according to the preceding slide undergoes phosphorylation of its free CH 2 OH group to give D -fructose 1,6-diphosphate. D -Fructose 1,6-diphosphate is cleaved to two 3- carbon products by a reverse aldol reaction. This retro-aldol cleavage is catalyzed by the enzyme aldolase.

30. Isomerization aldolase D -Fructose 1,6-phosphate CH 2 OP(O)(OH) 2 H OH H OH H HO CO H OH CHO CO CH 2 OH

31. 25.22 Acylation and Alkylation of Hydroxyl Groups in Carbohydrates

32. Reactivity of Hydroxyl Groups in Carbohydrates acylation alkylation Hydroxyl groups in carbohydrates undergo reactions typical of alcohols.

33. O OH OH HO HO HOCH 2 + CH 3 COCCH 3 OO 5 Example: Acylation of  - D -glucopyranose

34. O OH OH HO HO HOCH 2 + CH 3 COCCH 3 OO 5 O O CH 3 COCH 2 O CH 3 CO O O O OCCH 3 pyridine (88%)

35. Example: Alkylation of methyl  - D -glucopyranoside O OCH 3 OH HO HO HOCH 2 + 4CH 3 I Ag 2 O, CH 3 OH

36. Example: Alkylation of methyl  - D -glucopyranoside O OCH 3 OH HO HO HOCH 2 + 4CH 3 I Ag 2 O, CH 3 OH O OCH 3 CH 3 O CH 3 OCH 2 (97%)

37. Classical Method for Ring Size Ring sizes (furanose or pyranose) have been determined using alkylation as a key step. O OCH 3 OH HO HO HOCH 2 O OCH 3 CH 3 O CH 3 OCH 2

38. Classical Method for Ring Size Ring sizes (furanose or pyranose) have been determined using alkylation as a key step. O OCH 3 CH 3 O CH 3 OCH 2 H2OH2OH2OH2O H+H+H+H+ (mixture of  +  ) O OH CH 3 O CH 3 OCH 2

39. Classical Method for Ring Size Ring sizes (furanose or pyranose) have been determined using alkylation as a key step. (mixture of  +  ) O OH CH 3 O CH 3 OCH 2 CH 2 OCH 3 H OHOHOHOH OCH 3 H H CH 3 O H OCH 3 CHO

40. Classical Method for Ring Size Ring sizes (furanose or pyranose) have been determined using alkylation as a key step. CH 2 OCH 3 H OHOHOHOH OCH 3 H H CH 3 O H OCH 3 CHO This carbon has OH instead of OCH 3. Therefore,its O was the oxygen in the ring.

41. 25.23 Periodic Acid Oxidation of Carbohydrates

42. Recall Periodic Acid Oxidation Cleavage of a vicinal diol consumes 1 mol of HIO 4. CC HO OH HIO 4 C O O C + Section 15.12: Vicinal diols are cleaved by HIO 4.

43. Also Cleaved by HIO 4 Cleavage of an  -hydroxy carbonyl compound consumes 1 mol of HIO 4. One of the products is a carboxylic acid. C RCRCRCRC OH HIO 4 C O O C +  -Hydroxy carbonyl compounds O R HO

44. Also Cleaved by HIO 4 2 mol of HIO 4 are consumed. 1 mole of formic acid is produced. HIO 4 R2CR2CR2CR2CO R' 2 C O+ Compounds that contain three contiguous carbons bearing OH groups HCOHO OH R2CR2CR2CR2CCH CR' 2 OHHO +

45. O HOCH 2 HO OH OCH 3 Structure Determination Using HIO 4 Distinguish between furanose and pyranose forms of methyl arabinoside HO HO OOH OCH 3 2 vicinal OH groups; consumes 1 mol of HIO 4 3 vicinal OH groups; consumes 2 mol of HIO 4