Introduction

The mucins, a family of highly O-glycosylated glycoproteins that are found in mucus and on the surface of epithelial cells, play an important role in various biological processes^{1, 2, 3, 4, 5, 6, 7}. The glycan core structures of mucins possess at the reducing end an N-acetyl galactosamine residue that is -glycosidically linked to the hydroxy groups of L-Ser or L-Thr^{1, 2, 3, 4, 5, 6, 7, 8}. Eight core structures and the T_N and ST_N antigens have been identified to date (Fig. 1). These basic structures are constituents of various other glycoproteins, for instance, cell membrane glycoproteins, blood group determinants, immunoglobulins and anti-freeze glycoproteins. Because of the importance of these glycoproteins, several methods for the synthesis of O-linked glycosyl amino acids and glycopeptides have been introduced. For example, for the stereoselective construction of -glycosidic linkages between 2-acetamido-2-deoxy-D-galactopyranose residues and the side chain hydroxy group of L-Ser or L-Thr, the nonparticipating azido group was selected as latent amino functionality at position-2 (refs. 9–21).

Figure 1: T_N-, ST_N-antigens and the mucin core structures.

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We have demonstrated that concatenation of 2-nitrogalactal and O-glycosylated derivatives is a useful tool for forming -glycosidic bonds to L-Ser or L-Thr^{22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33}. With this methodology in hand, practically all core structures of the mucin family^{22, 23, 25}, as well as various O-glycosides²⁶, thioglycosides²⁷, glycosyl phosphonates²⁹, C-glycosides³² and nucleosides³³ have been obtained. This methodology was also successfully applied to the synthesis of hybrid sugars³⁴ and C-glycosyl amino acids (Fig. 2)³⁵.

Figure 2: Michael-type addition of O-, N-, C-, S- and P-nucleophiles to 2-nitrogalactal as donor.

Some typical examples.

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Experimental design

Synthesis of the core structures of the mucin family is reviewed in refs. 23 and 25 and the synthesis of the most prominent building block, namely the core 1 structure, is covered in this protocol. A schematic representation of the synthetic route adopted here is shown in Figure 3. In our approach, the formation of a (1-3)-glycosidic bond between galactose and N-acetyl galactosamine is required. For this purpose, galactal derivative 3 is obtained by glycosylation of 3,4-O-unprotected galactal 1 (ref. 36) with glycosyl donor 2 (ref. 37) in the presence of catalytic amounts of Sn(OTf)₂ as mild catalyst in dichloromethane at 0 °C. Removal of the O-acetyl group at 2b and the following benzylation of 2b-OH and 4a-OH in the presence of BnBr/sodium hydride (NaH) subsequently furnish the galactal derivative 4 in good yield. Nitration of galactal derivative 4 with acetyl nitrate, generated in situ by adding nitric acid to acetic anhydride under strict temperature control, leads to the formation of 5. Addition of triethylamine (Et₃N) to a solution of 5 in dichloromethane gives 6 in 68% yield. Michael-type addition of Ser or Thr derivatives 7a and 7b to nitrogalactal 6, in the presence of catalytic amounts of potassium tert butoxide in toluene leads to the highly stereocontrolled formation of adducts 8a or 8b, which contain two stereogenic centers. The reduction of the nitro groups with zinc/hydrochloric acid (Zn/HCl)³⁸ and the acetylation of the resulting amines with an acetic anhydride/pyridine mixture afford the ultimate target molecules 9a, 9b in good yields. The Zn/HCl procedure worked well for the reduction of one nitro group. Other Zn-based reduction methods, as for instance Zn/hydrazinium formate³⁹, were not investigated. Also nitro group reduction with platinized Raney-Ni (T₄) as catalyst and hydrogen worked well in small-scale applications⁴⁰. Reduction of the nitro group with excess of samarium (III) iodide²⁵ in tetrahydrofuran/methyl alcohol (THF/MeOH) leads to hydroxylamines; subsequent N-acetylation using pyruvic acid in aqueous N,N-dimethyl formamide (DMF) at 50 °C furnished the acetylamino derivatives. This method worked well for the concomitant reduction of two nitro groups also.

Figure 3: Synthesis of core 1 building blocks 9a,b.

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2-Nitroglycal concatenation has been mainly investigated with 2-nitrogalactal derivatives. Using alcohols as nucleophiles and strong bases as catalysts, mainly or exclusively -galacto-configurated adducts are obtained. For instance, with 3,4,6-tri-O-benzyl-2-nitrogalactal and 7 as acceptor in the presence of tert-butoxide as base, a 3:1 mixture of the / anomers is formed⁴¹. On the other hand, introducing bulky protecting groups such as triisopropylsilyl (TIPS) or tert-butyldimethylsilyl (TBDPS) at the 6-position affords, as shown for 6 as glycosyl donor, the exclusive formation of -glycosidic bonds with Ser/Thr²⁵. Hence, the high stereocontrol associated with the Michael-type addition is based on stereoelectronic and/or steric effects. Some studies using weaker bases such as Et₃N proved that also products in the -configuration can be preferentially obtained²⁶. Highly stereoselective Michael-type additions to 2-nitroglucal have been also carried out²⁵. All these examples demonstrate that the choice of the base and the selection of the protecting group pattern play significant roles in the stereoselection^{25, 26, 40}.

Materials

Reagents

• Unprotected galactal derivative 1, obtained as reported in ref. 36
• Isomeric galactal derivative 2, obtained as reported in ref. 37
• Tin (II) trifluoromethanesulfonate (Aldrich, cat. no. 388122)
  Caution Irritant and corrosive.
• Benzyl bromide (Fluka, cat. no. 13250)
  Caution Irritant and toxic.
• NaH (Aldrich, cat. no. 388122)
  Caution Highly flammable.
• Sodium methoxide (Fluka, cat. no. 71750)
• Concentrated nitric acid (65–70%)
  Caution Corrosive and harmful.
• HCl (1 N solution)
• Acetic anhydride
• Acetic acid (AcOH)
• Et₃N
• Acetonitrile
• Dichloromethane
• Toluene
• Ethyl acetate
• Petroleum ether
  Caution Highly flammable.
• Diethyl ether
  Caution Highly flammable.
• Pyridine
  Caution Toxic and harmful.
• Methanol (Fluka, cat. no. 65543)
  Caution Toxic.
• THF
  Caution Toxic and highly flammable.
• DMF (Fluka, cat. no. 40248)
  Caution Toxic.
• Silica gel (Silica gel 60, 0.04–0.063 mm; Macherey-Nagel)
• Thin-layer chromatography (TLC) aluminum sheets (Silica gel 60F254; Merck, cat. no. 1.05554.009)
• L-Ser (Novabiochem, cat. no. 04-13-0065)
• L-Thr (Novabiochem, cat. no. 04-12-0015)
• Potassium tert-butoxide (Fluka, cat no. 60098)
• Zn dust (Aldrich, cat. no. 206032)
• Magnesium sulfate, anhydrous
• Sodium hydrogen carbonate (NaHCO₃)

Equipment

• Round-bottomed flasks, one- and two-necked (50 and 100 ml)
• Dual argon vacuum manifold with vacuum line
• Cooling apparatus for - 35 °C (can be used dry ice/acetone bath)
• Rubber septums
• Disposable syringes and injection needles
• Filter papers
• Rotary evaporator
• Chromatographic columns
• ¹H NMR and ¹³C NMR spectrometers

Reagent setup

• Dry reagents To obtain dry toluene and dry THF, distill from the solvent still over sodium wire under argon atmosphere.
• To obtain dry dichloromethane and dry acetonitrile, distill from the solvent still over CaCl₂ under argon atmosphere.
  Caution All chemical operations and experiments should be performed in a fume hood and standard laboratory apparel must be worn.

Procedure

1. Points from here (point 1) up to and including point 19 are related to Synthesis of disaccharide (4)Timing: 30 hWeigh 3.0 g (4.66 mmol) of racemic galactal derivative 2, obtained as reported in ref. 37, and 1.09 g (4.0 mmol) of 3,4-unprotected galactal derivative 1, obtained as reported in ref. 36, into 100-ml round-bottomed flask.
2. Dissolve in toluene (10 ml), and evaporate two times with toluene (10 ml 2) on rotary evaporator at 40 °C. It takes 10 min. Add a Teflon-coated magnetic stir bar.
3. Dry under vacuum for 2 h, and then fill the flask with argon.
4. Add dry dichloromethane (20 ml) using a syringe, and turn the magnetic stirrer on.
5. Cool the flask to 0 °C, and stir for 10 min. Add dropwise 47 mg (0.119 mmol) of tin (II) trifluoromethanesulfonate in 1-ml dry acetonitrile.
6. Stir the reaction mixture for 0.5–1 h. Monitor the progress of the reaction by TLC (R_f, of 3 = 0.44 in petroleum ether/ethyl acetate, 6:1). Terminate the reaction by adding few drops of Et₃N using a syringe.
7. Remove the volatile materials by rotary evaporation at 40 °C (it takes 15 min), and purify the desired product 3 by flash chromatography on silica gel ( 80 g, 5.2 cm internal diameter (i.d.) 40 cm length column) using 3:1 petroleum ether/ethyl acetate as eluent.
8. Evaporate solvents from the eluate using a rotary evaporator at 40 °C. It takes 1 h. Dry under vacuum ( 3, 2.73 g).Pause Point Product 3 can be dried overnight under vacuum.
9. Dissolve 1.0 g (1.28 mmol) of 3 in dry methanol (20 ml), and add a Teflon-coated magnetic stir bar. Add 10 mg (0.18 mmol) of sodium methoxide and stir the resulting mixture for 30 min.
10. Evaporate the solvent on a rotary evaporator at 40 °C. It takes 15 min. Dry the residue under vacuum for 1 h and fill the reaction flask with argon.
11. Dissolve the residue from Step 10 in dry dimethylformamide (15 ml) and add a Teflon-coated magnetic stir bar.
12. Cool the flask to 0 °C in an ice/water bath, and turn the magnetic stirrer on. Add NaH (60% in mineral oil) 0.154 g (6.24 mmol) and stir for 30 min.
    Caution NaH is highly flammable. Special care should be taken when handling it.
    Caution H₂ gas evolves from the solution. H₂ gas is extremely flammable, it can form explosive mixtures with air, and it may react violently with oxidants.
13. Add dropwise 0.38 ml benzyl bromide (3.19 mmol), and stir for 1 h. Remove the reaction flask from the ice/water bath, and stir the reaction mixture at room temperature (25 °C) for 16 h.
14. Terminate the reaction by adding 50 ml water, and transfer the resulting solution to a separatory funnel. Extract then the organic materials with ethyl acetate (20 ml 2) and combine the organic phase.
15. Wash the organic phase with water (100 ml) and then with 20 ml of an NaCl-saturated aqueous solution (brine).
16. Dry the organic phase over anhydrous magnesium sulfate (add 2–3 g, and stir the mixture vigorously for 3 min).
17. Filter the inorganic materials through filter paper, and remove volatiles by rotary evaporation at 40 °C. It takes 20 min.
18. Purify the desired product 4 (R_f of 4 = 0.79 in petroleum ether/ethyl acetate, 4:1) by flash column chromatography on silica gel ( 50 g, 4.5 cm i.d. 45 cm length column) using 10:1 toluene/ethyl acetate as eluent.
19. Evaporate solvents from the eluate using a rotary evaporator at 40 °C. It takes 1 h. Dry under vacuum. ( 4, 0.915 g).Pause Point Compound 4 can be dried overnight under vacuum.
20. Points from here (point 20) up to and including point 38 are related to Synthesis of 2-deoxy-2-nitrogalactal derivative (6)Timing: 8 hFit a 50-ml round-bottomed flask containing a Teflon-coated magnetic stir bar with a rubber septum and connect the flask to an argon line.
21. Add 5 ml acetic anhydride using a syringe.
22. Cool the flask to - 10 °C, and turn the magnetic stirrer on.
23. Add concentrated nitric acid (3.71 mmol) dropwise (2–3 min). For the researcher's safety (see CAUTION below) make sure that during the addition of nitric acid the internal temperature does not exceed 10–20 °C, and that the external temperature is maintained at - 10 °C.
    Caution Concentrated nitric acid is corrosive and harmful. Special care should be taken when handling it.
    Caution Addition of nitric acid to acetic anhydride known to generate acetyl nitrate, which is known to be an explosive material. Strict temperature control should be maintained.
24. Cool the reaction mixture to - 30 to - 35 °C and add to it a solution of galactal derivative (4) (0.900 g, 0.983 mmol) in acetic anhydride (5 ml).
25. Stir the reaction mixture for 30 min at - 30 to - 35 °C, and then allow it to warm to 0 °C.
26. Pour the reaction mixture into an ice/water mixture (10 ml), and then add brine (10 ml).
27. Transfer the mixture to a separatory funnel and extract the aqueous layer with diethyl ether (15 ml 2). Combine the organic phase and dry it with anhydrous magnesium sulfate (add 1–2 g, and stir the mixture vigorously for 3 min).
28. Filter the inorganic materials through filter paper.
29. Remove the solvents by co-evaporation with toluene using rotary evaporator at 40 °C. It takes 30 min. Dry under vacuum for 1 h ( 5, yellow oil).
    Critical step Make sure that acetic acid is evaporated completely. The next step of the PROCEDURE calls, in fact, for the Et₃N-promoted elimination from compound 5 of acetate in the form of acetic acid. If residual acetic acid is not thoroughly evaporated at this point, Et₃N may be inactivated to triethylammonium acetate and the elimination reaction may be incomplete. The addition of excess of Et₃N may therefore become necessary.Pause Point Compound 5 can be dried overnight under vacuum.
30. Fit a 50-ml round-bottomed flask containing a Teflon-coated magnetic stir bar with a rubber septum and connect the flask to the argon line.
31. To the flask, add Et₃N (4.91 mmol) and dichloromethane (5 ml) using a syringe. Cool the flask to 0 °C in an ice/water bath and turn the magnetic stirrer on.
32. Add dropwise compound 5 in dichloromethane (5 ml) using a syringe or a dropping funnel.
33. Remove the flask from the ice/water bath, and stir the reaction mixture at room temperature for 20–30 min.
34. Transfer the reaction mixture to a separatory funnel, and add 1 N hydrochloric acid (10 ml).
35. Extract the organic materials with dichloromethane (10 ml 2) and combine the organic phase. Dry the organic phase over anhydrous magnesium sulfate (add 1–2 g, and stir the resulting mixture vigorously for 3 min).
36. Filter the inorganic materials through filter paper, and remove the volatile materials by rotary evaporation at 40 °C. It takes 15 min.
37. Purify the desired product 6 (R_f of 6 = 0.50 in toluene/ethyl acetate, 4:1) by flash column chromatography on silica gel (50 g, 4 cm i.d. 40 cm length column) using 40:1 toluene/ethyl acetate as eluent.
38. Evaporate solvents from the eluate using a rotary evaporator at 40 °C. It takes 1 h. Dry under vacuum ( 6, 0.642 g).Pause Point 6 can be stored at room temperature for 2 d.
39. Points from here (point 39) up to and including point 55 are related to Synthesis of mucin core 1 building blocks (9a,b)Timing: 12 hWeigh 0.200 g (0.21 mmol) of 2-nitrogalactal derivative (6), and L-Ser or L-Thr (0.25 mmol) into 50-ml round-bottomed flask.
40. Dissolve in toluene (5 ml), and evaporate two times with toluene (5 ml 2) using a rotary evaporator at 40 °C. It takes 10 min. Add a Teflon-coated magnetic stir bar.
41. Dry under vacuum for 1 h, and then fill the flask with argon.
42. Add dry toluene (5 ml) using a syringe, and turn the magnetic stirrer on.
43. Add potassium tert-butoxide (1 M solution in dry THF, 27 l) using a micro syringe. Stir the reaction mixture at room temperature for 2 h.
44. Terminate the reaction by adding few drops of acetic acid using a syringe, and remove the volatile materials by rotary evaporation at 40 °C. It takes 10 min.
45. Purify the desired product (R_f of 8a = 0.53 in petroleum ether/ethyl acetate, 5:1; R_f of 8b = 0.50 in petroleum ether/ethyl acetate, 5:1) by flash chromatography on silica gel (50 g, 2.8 cm i.d. 45 cm length column) using 40:1 toluene/ethyl acetate as eluent.
46. Evaporate solvents from the eluate using a rotary evaporator at 40 °C. It takes 50 min. Dry under vacuum for 2 h ( 8a,b, 0.177 g).Pause Point 8a and 8b can be stored at room temperature for 2 d.
47. Weigh 0.150 g (0.122 mmol) 2-nitro compound (8a, or 8b) into 50-ml round-bottomed flask equipped with a Teflon-coated magnetic stir bar.
48. Dissolve the compound from Step 47 in 10 ml of the following mixed solvent: THF/concentrated HCl/AcOH/H₂O (25:1:6:10). Cool the flask to 0 °C in ice/water bath and turn the magnetic stirrer on.
49. Add 190 mg (2.93 mmol) of Zn dust and stir for 2 h at 0 °C. Filter (to remove excess Zn dust) and transfer the reaction solution to a separatory funnel. Extract it then with dichloromethane (10 ml 2).
50. Extract the dichloromethane solution with water (10 ml) and then with 1 M NaHCO₃ solution (10 ml). Dry the organic phase over anhydrous magnesium sulfate (add 1–2 g and stir the resulting mixture vigorously for 3 min).
51. Filter the inorganic materials through filter paper, and remove the volatile materials by rotary evaporation at 40 °C. It takes 15 min. Dry under vacuum for 1 h.Pause Point The impure desired products 9a and 9b can be dried overnight under vacuum.
52. Dissolve impure residues from Step 51 in acetic anhydride/pyridine mixture (1:2, 5 ml), and add a Teflon-coated magnetic stir bar.
53. Stir for 2 h and remove the volatile materials by rotary evaporation at 40 °C.
54. Purify the desired product (9a or 9b) by flash chromatography on silica gel (50 g, 2.8 cm i.d. 45 cm length column) using 2:1 petroleum ether/ethyl acetate as eluent (R_f of 9a = 0.25; R_f of 9b = 0.23).
55. Evaporate solvents from the eluate using a rotary evaporator at 40 °C. It takes 1 h. Dry under vacuum ( 9a, b, 0.108 g).

Timing

Steps 1–19: 30 h; Steps 20–38: 8 h; Steps 39–55: 12 h

Anticipated results