Investigations of desymmetric glucosamine acceptors

Reagents and conditions: (a) Ac2O, NaHCO3, MeOH, rt, 30%. (b) Boc2O, NaHCO3, MeOH, rt, 76%.

(c) HO(CH2)6Cl 7, NIS, cat. TMSOTf, 4A MS, -40 ^oC; (d) Ac2O, pyr., rt; 50% (2 steps). (e) CbzCl, NaHCO3, MeOH, rt, 82%. (f) HO(CH2)6Cl 7, NIS, cat. TMSOTf, 4A MS, -65 ^oC, 88%. (g) PdCl2,

Et3SiH, Et3N, CH2Cl2, rt, 45%.

arbonyl group for its versatile deprotection methods. Treatment of 20 with NaHCO3

in MeOH, followed by di-tert-butyl dicarbonate gave desymmetric N-benzyl-N-tert- butoxy protected glucosamine thioglycoside 22 in 76% yield. Unfortunately, subsequent glycosylation of 22 with acceptor 7 was characterized by the cleavage of the N-substituent, and the main compound recovered was the amine 23. The amine 23 was further acylated to 24 for characterizations. After searching literatures, we found that the experimental result was similar with Boullanger’s work, and the formation of 23 could be due to removal of one of the carbamate protons from the intermediate A (Scheme 20) to form isobutylene together with 23. Boullanger et al. conducted a study on the glycosylation of simple acceptor alcohols with various N-alkoxycarbonyl derivatives of glucosamine. Their investigation inspired us to adopt the benzyloxycarbonyl group to protect the secondary amine in glucosamine derivative 20.

Again, treatment of 20 with NaHCO3 in MeOH, followed by benzyloxycarbonyl chloroformate gave desymmetric N-benzyl-N-benzyloxycarbonyl (N-BnCbz) protected glucosamine thioglycoside 25 in high yield of 82%. Subsequent glycosylation of 25 with primary alcohol 7 gave the -glycoside 26 in 88% yield (Scheme 19). Although the yields of 25 and 26 were satisfactory and their preliminary

BnO O HO

OBn

Bn O

O S

 BnO O

OBn

Bn N O

O Nu

BnO O HO

OBn

Bn N O

intermediate A S Tol



identifications were evidenced by HRMS, the assignments of NMR spectra were difficult due to the peak broadening of the resonance signals. After searching literatures, we found that the peak broadening phenomenon was quite similar with Lafont’s research.⁹¹ The ¹H NMR spectrum of N-acetyl-N-allyloxycarbonyl protected glucosamine derivative A (Figure 6) revealed the presence of two conformers (45:55).

They pointed that this observation could be due to the restricted rotations around the amide bond, and the coalescence of signals appeared when the temperature was increased. Compared to our study, the ¹H NMR spectrum was unclear at room temperature. When the temperature was raised, the broadened peaks became sharp at 100 ^oC as shown in Figure 7 (ca VT-NMR from 30 to 100 ^oC in deuterated DMSO solvent). This phenomenon could also be attributed to the restricted rotations around the amide bond based on our VT-NMR spectra and Lafont’s research. Although we could get clear spectrum at elevated temperature of 100 ^oC, it was unpractical since the operation of NMR machine at such elevated temperature were quite time-spending and laborious. We predicted that the broadening of resonance signal was resulted from the presence of the carbamate function. In order to prove our predictions, the Cbz protecting group was removed under mild conditions (Scheme 19).⁹² Consistent with our reasoning, the broadening phenomenon was disappeared, and the clear NMR spectrum of -glycoside 27 (data were shown in experimental section) could be obtained at room temperature.

AcO O

OAc

AcO N

OAc Ac Alloc compound A

Figure 6. ¹H NMR spectra of compound A at different temperatures

BnO O

OBn HO N(Cbz)Bn

OR 26, R = (CH₂)₆Cl

2.7 Glycosylation studies of the glucosamine acceptor 25

After characterizations of the desymmetric N-BnCbz protected glucosamine 25, the next step was glycosylation studies of the acceptor 26 with thioglycosides 11, 12, 28, and 29 (Table 11). Glycosylations of 26 with thioglactopyranosides 11, 28, and 29 furnished the expected Gal--(1→3)-GlcNAc (type 2 LacNAc) disaccharides 3032 in high (73-80%) yields (Table 11, entries 13). The coupling of thiofucopyranoside

Table 11. Glycosylation studies of the desymmetric glucosamine acceptor 26

thioglycoside donor glucosamine acceptor disaccharide +

11, 12, 28, or 29 26 30, 31, 32, or 33 NIS, cat. TMSOTf, 4A MS

CH2Cl2, T

Entry Thioglycoside donor T (^oC)

Disaccharide product Yield (%)

1 O

BnO OBn BnO OBzSTol

65

BnO O

OBn O N(Cbz)BnOR BnOBnOO

AllO OBzSTol 28

65

BnO OBnO O N(Cbz)BnOR BnOBnOO

PMBO OBzSTol 29

65

O BnO OBn

O N(Cbz)BnOR BnO O O N(Cbz)BnOR

33, R = (CH₂)₆Cl O

BnOOBn

OBn 93

12 and acceptor 26 led to the Fuc--(1→3)-GlcNAc disaccharide 33 which is the backbone of type 1 Lewis antigens in high yield of 93% (Table 11, entry 4). All the glycosylations proceeded smoothly and the yields were satisfactory. The peak broadening was also occurred in the NMR spectroscopy of disaccharides 3033. We tried in vain to remove the Cbz protecting group in disaccharide 30 using the same condition which was feasible for monosaccharide 26 (Scheme 21). The reason is not clear at this stage.

PdCl2, Et3SiH, NEt3

CH2Cl2, rt, 45%

BnO O

OBn HO N(Cbz)Bn

OR 26, R = (CH₂)₆Cl

BnO O

OBn

HO NHBn

OR 27, R = (CH₂)₆Cl

BnO O

OBn O N(Cbz)Bn O OR

BnO OBn

BnO OBz 30, R = (CH₂)₆Cl

PdCl₂, Et₃SiH, NEt₃ CH₂Cl₂, rt

BnO O

OBn

O NHBn

O OR BnO OBn

BnO OBz R = (CH₂)₆Cl

Scmeme 21. Selective deprotection studies of the Cbz protecting group

2.8 Hydrogenolysis of oxazolidinone protected disaccharides

Hence, the Pd-catalyzed hydrogenolysis method may be another choice for the removal of Cbz protecting groups. At the same time, O-benzyl ethers and N-benzyl groups could be removed simultaneously during hydrogenolysis reaction.

Consequently, the conditions for the total debenzylations in the presence of Cbz protecting groups were optimized. We used glucosamine derivative 26 as a model to study hydrogenolysis reactions and the results were shown in Table 12.

Table 12. Deprotection of desymmetric glucosamine acceptor 26

catalyst, H₂ (1 atm) solvent, T, 12h BnO O

OBn HO N(Cbz)Bn

OR 26, R = (CH₂)₆Cl

AcO O

OAc AcO NHAc

OR 34, R = (CH₂)₆Cl

Entry Catalyst Solvent T (^oC) Results

1 Pd MeOH:AcOH = 10:1 25 26^a

2 Pd MeOH:HCOOH = 10:1 25 not fully deprotected^a 3 Pd EtOH:HCOOH = 10:1 25 not fully deprotected^a

4 Pd THF:H2O = 4:1 25 26^a

5 Pd THF:H2O:AcOH = 8:2:1 25 26^a

6 Pd THF:H2O:HCOOH = 8:2:1 25 not fully deprotected^a

7 Pd(OH)2 AcOH 25 26^a

8 Pd(OH)2 MeOH:AcOH = 2:1 25 26^a

9 Pd(OH)2 EtOAc:H2O:AcOH = 2:1:4 25 26^a 10 Pd(OH)2 EtOAc:H2O:AcOH = 2:1:4 60 34 (70%)^b

a Judged from TLC plates. ^b After acetylation, we got the low mass data.

At first, hydrogenolysis of monosaccharide 26 did notproceed over 10% Pd/C in solvent mixtures of MeOH/AcOH (10:1), THF/H2O (4:1), and THF/H2O/AcOH (8:2:1) at room temperature (Table 12, entries 1, 4 and 5).⁷⁰ Incomplete deprotections were observed and a series of spots were noted on the TLC plate, and changing the solvent mixture to MeOH/HCOOH (10:1), EtOH/HCOOH (10:1), and THF/H2O/HCOOH (8:2:1) did not give the desired product either. After these failures, we took a step back to do a search hoping for finding the solutions to overcome this problem. We found that 20 % Pd(OH)2 catalyst was used for the removal of N-benzyl groups, maybe it would fulfill all our requirements for total debenzylations along with the removal of Cbz protecting group. Hydrogenolysis reactions of monosaccharide 26 were not successful even over 20 % Pd(OH)2 in solvent system of AcOH, MeOH/AcOH (10:1), EtOAc/H2O/AcOH (2:1:4) at room temperature (Table 12, entries 79).^93,94 Eventually, the hydrogenolysis temperature was best performed at 60

oC.⁷⁴ After raising the temperature to 60 ^oC, the deprotection was achieved (Table 12, entry 10). To our delight, we applied the optimized condition to disaccharides 30 and 33. As shown in Scheme 22, both of their hydrogenolysis reactions worked well in solvent mixture of EtOAc/H2O/AcOH (the ratio was based on the solubility of disaccharide) over 20% Pd(OH)2 under 1 atm H2 at 60 ^oC. For NMR characterization, the resulting debenzylated products were further acetylated to produce the type 1 LacNAc glycoside 35 and the peracetyl Fuc--(1→3)-GlcNAc disaccharide 36 which was similar to part of Lewis Y structure.

BnO O

OBn O

N(Cbz)Bn O OR

BnO OBn

BnO OBz

30, R = (CH₂)₆Cl BnO O

OBn O N(Cbz)Bn

33, R = (CH₂)₆Cl O

BnOOBn OBn

1. Pd(OH)2, H2, EtOAc:H₂O:AcOH = 2:1:4 for 30,

5:1:4 for 33 60 ^oC, overnight 2. Ac₂O, pyr., rt

AcO O

OAc

O NHAc

O OR AcO OAc

AcO OBz

From 30

35, R = (CH₂)₆Cl, 58%

AcO O

OAc O

NHAc OR

From 33

36, R = (CH2)6Cl, 50%

O AcOOAc OAc

Scmeme 22. Hydrogenolysis reactions of disaccharides 30 and 33

2.9 Synthesis of disaccharides using desymmetric aminoprotecting strategy

At present, we had developed the desymmetric aminoprotecting strategy. The scope of investigation included the installation, deprotection, and application to glycosylations. At this stage, we focused on the removal of the oxazolidinone ring in disaccharide 16. It seemed that basic condition (t-BuOK/DMSO) at room temperature would be practical (Table 10, entry 5), but it didn’t proceed. The reaction outcome was not reproducible, and more intriguingly, the installation of Cbz protecting group failed when the residue was treated with NaHCO3 in MeOH, followed by benzyloxycarbonyl chloroformate (Scheme 23). What we could do was using the other basic conditions (1,4-dioxane/1M NaOH, v/v = 1:1) which was also feasible for the monosaccharide 19 (Table 10, entry 4). Fortunately, the removal of the oxazolidinone ring was successful and further installation of Cbz protecting group furnished desymmetric protected disaccharide 38 in high yield.

CbzCl, NaHCO₃ MeOH, rt, 82%

Scmeme 23. Removal studies of the N-benzyl oxazolidinone ring in disaccharides 16

2.10 Synthesis of trisaccharide 41 via one-pot glycosylation and deprotection

After some experimentation, the key component of disaccharide 38 was finally obtained. To investigate its chemical properties, 38 was coupled with primary alcohol 7 using NIS/Lewis acid promoter system (Table 13). At first, coupling of 38 and 7 gave desired product 39 in 86% yield (Table 13, entry 1). However, further glycosylation of 39 with fucosyl thioglycoside 12 gave undesired N-succinimide side product. Then we decided to change the Lewis acid to TfOH as the promoter, most disaccharides 38 were not activated at 60 ^oC, but we got the desired product 39 in 88% yield after raising the temperature to 50 ^oC (Table 13, entries 2 and 3).

According to the experimental results in Table 13, the NIS/TfOH promoter system was employed in further glycosylation with fucosyl thioglycosides 12. According to our retrosynthetic analysis, glycosylation of 39 with 12 should give the Lewis Y tetrasaccharide; however, we obtained the trisaccharide glycosylation product 40 even using 4 equiv of fucosyl thioglycoside and 1.2 equiv of TfOH.

Table 13. Glycosylation studies of donor 38 and acceptor 7

O BnO OBn BnO

OH O O

OBn HO N(Cbz)Bn

STol

HO(CH₂)₆Cl 7, NIS, Lewis acid

DCM, T

O BnO OBn BnO

OH O O

OBn HO N(Cbz)Bn

38 39, R = (CH₂)₆Cl

Entry Lewis acid promoter (equiv.) T (^oC) Yield (%)

1 TMSOTf (0.3) 65 39 (86%)

2 TfOH (0.3) 60 38 (80%)

3 TfOH (0.3) 50 39 (88%)

Owing to the phenomenon of resonance peak broadening for 40, its preliminary identification was evidenced by mass spectroscopy. In addition, the one-pot deprotection by hydrogenolysis reaction was useful for the trisaccharide 40 by using the Pd(OH)2 catalyst (Degussa type) at room temperature instead of 60 ^oC. For further characterizations of the structure, the resulting debenzylated product was acetylated to produce the peracetyl Fuc--(1→3)-Gal--(1→4)-GlcNAc trisaccharide 41 which constitutes part of the H blood group substrate. In this stage, we have synthesized the trisaccharide glycosylation product 40 by stepwise glycosylation. Subsequently, synthesis of 40 from 38 was attempted. Fortunately, we obtained the trisaccharide 40 in 55% yield by one-pot glycosylation reaction (Scheme 24). It was a pity that the 3-OH group in the GlcNAc could not be glycosylated with the fucosyl thioglycoside to give Lewis Y oligosaccharide probably because of the steric hindrance.

O BnO OBn BnO

OH O O

OBn HO N(Cbz)Bn

STol 38

O BnO OBn BnO

O O O

OBn HO N(Cbz)Bn

O(CH₂)₆Cl

O BnOOBn OBn

40 O

AcO OAc AcO

O O O

OAc AcO NHAc

O(CH2)6Cl

O AcOOAc OAc

one-pot glycosylation

one-pot deprotection&

acetylation

HO(CH₂)₆Cl 7

BnOOBn OBnSTol 12

Scmeme 24. Synthesis of trisaccharide 41 via one-pot glycosylation and deprotection strategy

3. Conclusion

In summary, the joined use of N-benzyl oxazolidinoe and N-BnCbz desymmetric amino protection is a versatile strategy for protection of 2-amino sugars. We have used oxazolidinone protected thioglucosamines which have lower anomeric reactivity as acceptors in reactivity-based chemoselective glycosylation. In addition, we have developed the desymmetric N-BnCbz protecting group including its installation, deprotection, and application in oligosaccharides synthesis. Disaccharides such as type 1 LacNAc, type 2 LacNAc, Fuc-a-(1→3)-GlcNAc, and Fuc-a-(1→4)-GlcNAc and trisaccharides H blood group substrate were synthesized based on this strategy.

Moreover, one-pot global deprotection by hydrogenolysis reaction was also developed for both disaccharide and trisaccharide. Further application of this strategy such as Lewis Y tetrasaccharide synthesis is under investigations.

4. Experimental 4.1 General procedures

Chemicals were purchased as reagent grade from commercial venders and used without further purification. All of solvents were dried and distilled by standard techniques unless mentioned.Optical rotations were measured with the JASCO DIP-1000 polarimeter at 30˚C. Flash column chromatography was performed over silica gel 60 (70230 mesh, E. Merck). NMR spectra were recorded with the Brüker console, Varian Unity-300 and Varian Unity-500. Chemical shifts are reported in ppm relative to internal tetramethylsilane (δ = 0.00 ppm) for ¹H and ¹³C resonance signal of CDCl3 (δ = 77.00 ppm) for ¹³C NMR spectra. Coupling constant(s) in hertz (Hz) were derived from ¹H NMR spectra.

4.2 General procedure for glycosylations

Glycosyl donor (8, 9, 10, 11, 12, 27 or 28), glycosyl acceptor (4 or 26), and activated 4 Å MS (100mg/1mL DCM, AW300) in CH2Cl2 were stirred at room temperature under nitrogen for 20 min. The mixture was then cooled in a cooling bath followed by addition of TMSOTf and NIS. After disappearance of acceptor detected by TLC, the mixture was diluted with CH2Cl2, quenched by Et3N, and few droplets of sat. NaHCO3 and excess of Na2S2O3(s) were added, removed from cooling bath, and then stirred at room temperature for 2h. The quenching mixture was then stirred at room temperature for 2h, filtered and finally concentrated. The residue was purified by column chromatography over silica gel to give 13, 14, 15, 16, 17, 30, 31, 32 or 33.

The stoichiometric amounts of substrates and reagents were listed below (Table 13).

Table 14. The amounts of glycosyl donor, glycosyl acceptor, NIS, and TMSOTf used in glycosylation Glycosyl donor

(mg, mmol)

Glycosyl acceptor (mg, mmol)

NIS (mg, mmol)

TMSOTf (uL, mmol, mM)

DCM (mL)

Temp.

(^oC)

Product

8, 160, 0.244 4, 100, 0.204 55, 0.244 9, 0.049, 0.016 3 70 13 9, 159, 0.265 4, 100, 0.204 60, 0.265 10, 0.053, 0.013 4 60 14 10, 173, 0.265 4, 100, 0.204 60, 0.265 10, 0.053, 0.013 4 65 15 11, 175, 0.265 4, 100, 0.204 60, 0.265 10, 0.053, 0.013 4 70 16 12, 165, 0.306 4, 100, 0.204 69, 0.306 11, 0.061, 0.015 4 60 17 11, 734, 1.112 26, 600, 0.856 252, 1.112 31, 0.171, 0.007 24 70 30 28, 780, 1.275 26, 597, 0.850 289, 1.275 31, 0.170, 0.007 25 65 31 29, 198, 0.293 26, 158, 0.225 67, 0.293 8, 0.045, 0.008 6 65 32 12, 486, 0.899 26, 420, 0.599 204, 0.899 22, 0.120, 0.007 17 70 33

4.3 Procedures and experimental data

p-Tolyl N-Benzyl-2-amino-4,6-O-benzylidene-2,3-N,O-carbonyl-2-deoxy-1-thio--

D-glucopyranoside (3):

O STol HO

OH HO

NHTroc

C₆H₅CH(OMe)₂, cat. TsOH CH₃CN, rt 1

O STol NHTroc OO

Ph HO

BnBr, NaH DMF, 0^oCrt

O STol OO

O NBn O

To a solution of compound 1 (2.0 g, 4.36 mmol) and TsOH (67 mg, 0.35 mmol) in CH3CN (15 mL) at room temperature, was added C6H5CH(OMe)2 (0.78 mL, 5.23 mmol). After stirring for 4 h, the mixture was neutralized with NEt3 and then concent- rated under reduced pressure. The residue was crystallized from (EtOAc/hexane) to give compound 2 (2.2 g, 92%) as white solid. Counpound 2 (2.2 g, 4.02 mmol) was dissolved in dry DMF (20 mL) and stirred in an ice bath under nitrogen for 20 min.

Then NaH (241 mg, 6.03 mmol, 60% in mineral oil) was added, followed by addition of benzyl bromide (0.58 mL, 4.82 mmol). After stirring for 30 min, the reaction mixture was warmed to room temperature and stirred for 2h. The reaction was concentrated under reduced pressure and purified by column chromatography over silica gel (EtOAc/CH2Cl2/hexane, 1:1:3) to give 3 (1.57 g, 80%) as white solid. Rf = 0.45 (EtOAc/CH2Cl2/hexane, 1:1:3); []³⁰D -66 (c 1, CHCl3); ¹H NMR (300 MHz, CDCl3) δ: 7.46–7.29 (m, 10H; Ar-H), 7.17–7.07 (m, 4H; Ar-H), 5.56 (s, 1H;

4.75 (d, J= 15.9 Hz, 1H; PhCH2), 4.31–4.24 (m, 2H; H-5, H-6), 3.99 (t, J= 9.3 Hz, 1H; H-4), 3.86 (t, J= 10.3 Hz, 1H; H-6), 3.54–3.45 (m, 2H; H-3, H-2), 2.33 (s, 3H, Ar-CH3); ¹³C NMR (75 MHz, CDCl3) δ: 158.9 (C=O), 139.1, 136.4, 133.2, 129.9, 129.3, 128.7, 128.3, 128.0, 127.9, 127.6, and 126.1 (Ar-C), 101.4 (benzylidene-C), 88.0 (C-1), 78.9 (C-5), 78.4 (C-4), 72.7 (C-3), 68.3 (C-6), 61.5(C-2) , 47.7 (PhCH2), 21.1 (Ar-CH3).

p-Tolyl N-Benzyl-2-amino-6-O-benzyl-2,3-N,O-carbonyl-2-deoxy-1-thio--D- glucopyranoside (4)

O STol HO

O NBn O

OBn

A solution of compound 3 (200 mg, 0.41 mmol), triethylsilane (322 L, 2.05 mmol), and activated 4 Å MS (200 mg, AW300) in CH2Cl2 (2 mL) was stirred at room temperature under nitrogen atmosphere for 20 min. The mixture was then cooled to 20 ^oC in a cooling bath followed by addition of dry trifluoroacetic acid (177L, 2.5 mmol). After 1h, the mixture was diluted with CH2Cl2 (6 mL), and then quenched with Et3N (0.4 mL, 3.3 mmol) at 20 ^oC. MS in the mixture was filtered off and the filtrate was concentrated. The residue was purified by column chromatogram- phy on silica gel (EtOAc/CH2Cl2/hexane, 1:1:8→1:1:3) to give 4 (161 mg, 80%) as white solid. Rf = 0.09 (EtOAc/ CH2Cl2/hexane, 1:1:3); []³⁰D -54 (c 2.38, CHCl3); ¹H NMR (300 MHz, CDCl3) δ: 7.40–7.21 (m, 12H; Ar-H), 7.03–7.01 (d, 2H; Ar-H), 4.73 (s, 1H; PhCH2), 4.69 (d, J= 9.3 Hz, 1H; H-1), 4.57 (d, J= 12.3 Hz, 1H; PhCH2), 4.53 (d, J= 12.0 Hz, 1H; PhCH2), 4.04 (t, J= 10.3 Hz, 1H; H-3), 3.96 (t, J= 9.2 Hz, 1H;

H-4), 3.76 (d, J= 4.5 Hz, 2H; H-6), 3.54–3.48 (m, 1H; H-5), 3.37 (dd, J= 10.7 Hz, J

= 9.6 Hz, 1H; H-2), 2.30 (s, 3H, Ar-CH3); ¹³C NMR (75 MHz, CDCl3) δ: 159.5 (C=O), 138.6, 137.7, 136.3, 132.9, 129.8, 128.6, 128.4, 128.1, 127.74, 127.69, and 127.5 (Ar-C), 86.7 (C-1), 82.5 (C-3), 79.9 (C-5), 73.5 (PhCH2), 69.5 (C-6), 68.5 (C-4), 60.1(C-2) , 47.4 (PhCH2), 21.1 (Ar-CH3); HRMS (ESI) calcd for C28H29NO5S [M+Na]⁺: 514.1664, found: 514.1659.

p-Tolyl N-Benzyl-2-amino-4-acetyl-6-O-benzyl-2,3-N,O-carbonyl-2-deoxy-1-thio-

-D-glucopyranoside (5)

O STol AcO

O NBn O

OBn

To a solution of compound 4 (500 mg, 1.02 mmol) in pyridine (5 mL) at room temperature, was added acetic anhydride (145 μL, 1.53 mmol). After stirring for 2h, the mixture was directly concentrated under reduced pressure, and the residue was purified by column chromatography over silica gel (EtOAc/hexane, 1:7→1:4) to give 5 (516 mg, 95%) as colorless foam. Rf = 0.30 (EtOAc/hexane, 1:2); ¹H NMR (300 MHz, CDCl3) δ: 7.33 (m, 12H; Ar-H), 6.94 (d, J = 7.8 Hz, 2H; Ar-H), 5.20 (dd, J = 10.4 Hz, J = 8.4 Hz, 1H), 4.674.64 (m, 3H; H-1, PhCH2), 4.47 (d, J = 11.7 Hz, 1H), 4.40(d, J = 11.7 Hz, 1H), 4.094.02 (m, 1H), 3.633.51 (m, 3H), 3.44 (dd, J = 11.3 Hz, J = 9.3 Hz, 1H), 2.23 (s, 3H; Ar-CH3), 1.93 (s, 3H; CH3CO);¹³C NMR (75 MHz, CDCl3) δ: 138.7, 137.6, 136.0, 132.9, 129.8, 128.6, 128.3, 128.12, 128.08, 127.7, 127.63, 127.56, 86.7 (C-1), 79.9, 78.7, 73.4, 68.6, 67.8, 60.1, 47.4, 21.0, 20.6.

p-Tolyl N-Benzyl-2-amino-6-O-benzyl-2,3-N,O-carbonyl-2-deoxy-1-thio--D- glucopyranoside (6)

HOO O O NBn

OBn

6 STol

Based on literature stoichiometric amounts of reagents and the same experimental procedure as in the synthesis 4, a mixture of 4 and 6 was obtained, and the ratio was determined by integration of the ¹H NMR spectrum (4:6 = 1:6, 65%).

We separated the -glycoside 6 and obtained its characterization data. Rf = 0.15 (EtOAc/CH2Cl2/hexane, 1:1:3); []³⁰D 168 (c 0.24, CHCl3); ¹H NMR (300 MHz, CDCl3) δ: 7.36–7.23 (m, 12H; Ar-H), 7.09 (d, 2H; Ar-H), 5.32 (d, J= 4.5 Hz, 1H;

H-1), 4.78 (d, J= 14.7 Hz, 1H; PhCH2), 4.59 (d, J= 12.0 Hz, 1H; PhCH2), 4.49 (d, J

= 12.0 Hz, 1H; PhCH2), 4.36 (dd, J= 11.9 Hz, J= 9.8 Hz, 1H; H-3), 4.19–4.11 (m, 2H; H-5, PhCH2), 4.00 (td, J= 9.3 Hz, J= 3.0 Hz, 1H; H-4), 3.78 (dd, J= 10.5 Hz, J

= 4.5 Hz, 1H; H-6), 3.71 (dd, J= 10.5 Hz, J= 4.5 Hz, 1H; H-6), 3.49 (dd, J= 12 Hz, J= 4.5 Hz, 1H; H-2), 2.99 (d, 1H, J= 3.3 Hz, 4-OH), 2.34 (s, 3H, Ar-CH3); ¹³C NMR (75 MHz, CDCl3) δ: 158.6 (C=O), 138.3, 137.5, 134.4, 132.4, 129.94, 129.0, 128.9, 128.5, 128.4, 127.9, and 127.8 (Ar-C), 85.2 (C-1), 78.4 (C-3), 73.6 (PhCH2), 72.9 (C-5), 69.4 (C-4), 68.7 (C-6), 59.2(C-2) , 47.4 (PhCH2), 21.1 (Ar-CH3).

p-Tolyl 2,3,4-Tri-O-benzyl-6-O-levulinoyl--D-galactopyranosyl-(1→4)-N-benzyl- 2-amino-6-O-benzyl-2,3-N,O-carbonyl-2-deoxy-1-thio--D-glucopyranoside (13)

O STol O

O NBn O

OBn O

BnO OLev BnO

BnO

Compound 13 was prepared from 8 and 4, and the stoichiometric amounts were referred to Table 13. The residue was purified by column chromatography over silica gel (EtOAc/CH2Cl2/hexane, 1:1:7→1:1:3) to give 13 (146 mg, 70%) as amorphous white solid. Rf = 0.21 (EtOAc/CH2Cl2/hexane, 1:1:3); []³⁰D +9 (c 2.67, CHCl3); ¹H NMR (300 MHz, CDCl3) δ: 7.42–7.22 (m, 27H, Ar-H), 6.99 (d, J = 11.1 Hz, 2H;

Ar-H), 5.44 (d, J = 3.6 Hz, 1H; H-1), 4.94 (d, J= 11.1 Hz, 1H; PhCH2), 4.87–4.64 (m, 7H), 4.62–4.55 (m, 2H), 4.47 (d, J= 12.0 Hz, 1H; PhCH2), 4.21–4.06 (m, 4H), 3.94 (dd, J= 11.1 Hz J= 5.7 Hz, 1H), 3.763.65 (m, 6H), 3.45 (t, J= 10.1 Hz, 1H; H-2), 2.672.62 (m, 2H; CH2), 2.452.40 (m, 2H; CH2), 2.29 (s, 3H; Ar-CH3), 2.12 (s, 3H;

CH3); ¹³C NMR (75 MHz, CDCl3) δ: 206.2 (C=O), 172.1 (C=O), 158.7 (C=O), 138.6, 138.5, 138.1, 138.0, 136.2, 132.8, 129.8, 128.6, 128.32, 128.26, 128.22, 128.14, 128.06, 127.7, 127.6, 127.5, and 127.3 (Ar-C), 96.3 (C’-1), 86.4 (C-1), 82.7, 79.1, 78.2, 75.8, 74.4 (PhCH2), 74.1, 73.34 (PhCH2), 73.29 (PhCH2), 73.1 (PhCH2), 71.0, 69.2, 69.0, 63.4, 60.0 (C-2), 47.4 (PhCH2), 37.7 (CH2), 29.8 (CH3), 27.6 (CH2), 21.1 (Ar-CH3). HRMS (ESI) calcd for C60H63NO12S [M+Na]⁺: 1044.3969, found:

1044.3962.

p-Tolyl 3,4,6-Tri-O-benzyl-2-O-levulinoyl--D-galactopyranosyl-(1→4)-N-benzyl- 2-amino-6-O-benzyl-2,3-N,O-carbonyl-2-deoxy-1-thio--D-glucopyranoside (15)

O STol O

O NBn O O OBn BnO OBn

BnO

OLev 15

Compound 15 was prepared from 10 and 4, and the stoichiometric amounts were

referred to Table 13. The residue was purified by column chromatography over silica gel (EtOAc/CH2Cl2/hexane, 1:1:8→1:1:3) to give 15 (135 mg, 65%) as amorphous white solid. Rf = 0.19 (EtOAc/CH2Cl2/hexane, 1:1:3); []³⁰D -21 (c 1, CHCl3); ¹H NMR (500 MHz, CDCl3) δ: 7.42–7.26 (m, 27H; Ar-H), 7.05–7.02 (m, 2H, Ar-H), 5.37 (t, J= 9.0 Hz, 1H), 4.93 (dd, J = 11.5 Hz, J = 3.5 Hz, 1H), 4.794.74 (m, 2H), 4.694.60 (m, 4H), 4.574.46 (m, 5H), 4.21 (td, J = 10.5 Hz, J = 4.5 Hz, 1H), 4.104.06 (m, 1H), 4.003.97 (m, 1H), 3.823.71 (m, 4H), 3.663.63 (m, 1H), 3.613.59 (m, 1H), 3.49 (dd, J = 10.3 Hz, J = 2.75 Hz, 1H), 3.423.37 (m, 1H), 2.682.62 (m, 2H; CH2), 2.532.46 (m, 2H; CH2), 2.32 (s, 3H; Ar-CH3), 2.11 (s, 3H;

CH3); ¹³C NMR (125 MHz, CDCl3) δ: 206.1 (C=O), 171.2 (C=O), 158.9 (C=O), 138.4, 138.2, 138.1, 137.9, 137.7, 136.3, 132.8, 129.7, 128.4, 128.24, 128.23, 128.14, 128.10, 128.05, 128.00, 127.95, 127.7, 127.60, 127.56, 127.48, 127.44, 127.37, and 127.33 (Ar-C), 100.3 (C’-1), 86.2 (C-1), 81.0, 80.0, 79.6, 74.31 (PhCH2), 74.28, 73.5, 73.4 (PhCH2), 73.3 (PhCH2), 72.3, 71.8 (PhCH2), 71.6, 68.2, 68.1, 60.2, 47.3 (PhCH2), 37.5 (CH2), 29.6 (CH2), 27.7 (CH2), 21.0 (Ar-CH3); HRMS (ESI) calcd for C60H63NO12S [M+Na]⁺: 1044.3969, found: 1044.4080.

p-Tolyl 2-O-Benzoyl-3,4,6-tri-O-benzyl--D-galactopyranosyl-(1→4)-N-benzyl-2- amino-6-O-benzyl-2,3-N,O-carbonyl-2-deoxy-1-thio--D-glucopyranoside (16)

O STol O

O NBn O O OBn BnO OBn

BnO

OBz 16

Compound 16 was prepared from 11 and 4, and the stoichiometric amounts were

referred to Table 13. The residue was purified by column chromatography over silica gel (EtOAc/CH2Cl2/hexane, 1:1:7→1:1:4) to give 16 (167 mg, 80%) as amorphous white solid. Rf = 0.20 (EtOAc/CH2Cl2/hexane, 1:1:4); []³⁰D -12 (c 2.19, CHCl3); ¹H NMR (300 MHz, CDCl

), 47.3

3) δ: 7.97 (d, J = 8.4 Hz, 2H; Ar-H), 6.91 (t, J = 8.1 Hz, 1H;

Ar-H), 7.417.07 (m, 29H; Ar-H), 6.91 (d, J = 8.1 Hz, 2H; Ar-H), 5.76 (dd, J = 10.1 Hz, J = 8.0 Hz, 1H), 4.94 (d, J = 11.4 Hz, 1H), 4.694.52 (m, 7H), 4.474.35 (m, 3H), 4.183.94 (m, 4H), 3.753.66 (m, 3H), 3.463.39 (m, 3H), 3.40 (dd, J = 10.2 Hz, J = 2.7 Hz, 1H), 2.22 (s, 3H, Ar-CH3); ¹³C NMR (75 MHz, CDCl3) δ: 165.6 (C=O), 158.9 (C=O), 138.4, 138.2, 138.0, 137.9, 137.6, 137.4, 136.2, 128.5, 128.3, 128.2, 128.18, 128.13, 128.06, 128.01, 127.8, 127.6, 127.3 (Ar-C), 100.8 (C’-1), 86.2 (C-1 81.3, 79.4, 79.0, 74.7, 74.4, 73.6, 73.5, 72.7, 72.0, 71.9, 71.3, 68.1, 67.7, 60.0,

(PhCH2), 21.0 (Ar-CH3); HRMS (ESI) calcd for C62H61NO11S [M+Na]⁺: 1050.3863, found: 1050.3860.

p-Tolyl 2,3,4-Tri-O-benzyl--L-fucopyranosyl-(1→4)-N-benzyl-2-amino-6-O- benzyl-2,3-N,O-carbonyl-2-deoxy-1-thio-D-glucopyranoside (17)

O BnO OBn

OBn O O STol

O NBn O

OBn

17 (:= 1:3.5)

Compound 17 was prepared from 12 and 4, and the stoichiometric amounts were referred to Table 13. The residue was purified by column chromatography over silica gel (EtOAc/CH2Cl2/hexane, 1:1:9→1:1:5) to give 17 (157 mg, 85%) as an

Ar-CH3 1H resonance signals at ca. 2.3 ppm of the reaction mixture. Rf = 0.28 (EtOAc/ CH2Cl2/hexane, 1:1:5); HRMS (ESI) calcd for C55H57NO9S [M+Na]⁺: 930.3652, found: 930.3723.

p-Tolyl N-Benzyl-2-amino-4,6-di-O-benzyl-2,3-N,O-carbonyl-2-deoxy-1-thio--D- glucopyranoside (19)

O STol BnO

O NBn O

OBn

Counpound 1 (600 mg, 1.31 mmol) was dissolved in dry DMF (10 mL) and stirred at 15 ^oC under nitrogen for 20 min. Then NaH (314 mg, 7.84 mmol, 60% in mineral oil) was added, followed by addition of benzyl bromide (0.7 mL, 5.88 mmol).

After stirring in a cooling bath for 30 min, the reaction mixture was warmed to room temperature and stirred for 2h. The reaction was quenched with crushed ice, stirred at room tempeture for 10 min, and the whole was extracted with EtOAc (30 mL × 3).

The organic layer was washed with brine (30 mL), dried over MgSO4, filtered, and finally concentrated. The residue was purified by column chromatography over silica gel (EtOAc/hexane, 1:3) and then crystallized from (EtOAc/hexane) to give 19 (608 mg, 80%) as white amorphous solid. Rf = 0.43 (EtOAc/hexane, 1:3); []³⁰D -32 (c 0.87, CHCl3); ¹H NMR (300 MHz, CDCl3) δ: 7.43–7.23 (m, 17H, Ar-H), 7.00 (d, J= 8.1 Hz, 2H; Ar-H), 4.88 (d, J= 11.1 Hz, 1H; PhCH2), 4.86 (s, 2H; PhCH2), 4.69 (d, J

= 9.3 Hz, 1H; H-1), 4.56 (d, J = 12.0 Hz, 1H; PhCH2), 4.54 (d, J = 11.4 Hz, 1H;

PhCH2), 4.48 (d, J= 12.0 Hz, 1H; PhCH2), 4.16 (dd, J= 11.1 Hz, J= 9.9 Hz, 1H;

H-3), 3.86 (dd, J= 9.6 Hz, J=8.7 Hz, 1H; H-4), 3.75 (dd, J= 10.8 Hz, J= 2.1 Hz, 1H;

H-6), 3.67 (dd, J= 10.8 Hz, J= 4.5 Hz, 1H; H-6), 3.58–3.53 (m, 1H; H-5), 3.44 (dd, J

= 11.1 Hz, J= 9.6 Hz, 1H; H-2), 2.29 (s, 3H; Ar-CH3); ¹³C NMR (75 MHz, CDCl3) δ:

159.2 (C=O), 138.6, 138.0, 137.2, 136.3, 128.6, 128.4, 128.35, 128.29, 128.11, 127.96, 127.89, 127.81, 127.7, 127.6 and 127.5 (Ar-C), 86.6 (C-1), 83.4 (C-3), 79.8 (C-5), 73.6 (C-4), 73.3 and 73.1 (PhCH2), 68.4 (C-6), 60.2 (C-2), 47.4 (PhCH2), 21.1 (Ar-CH3); HRMS (ESI) calcd for C28H29NO5S [M+Na]⁺: 514.1664, found: 514.1659.

p-Tolyl N-Benzyl-2-amino-4,6-di-O-benzyl-2-deoxy-1-thio--D-glucopyranoside (20)

O STol BnO

HO NHBn OBn

To a solution of compound 19 (190 mg, 0.33 mmol) in DMSO (5 mL) at room temperature, was added t-BuOK (183 mg, 1.64 mmol). After stirring for l h, the reaction mixture was diluted with EtOAC, and then washed with water. The aqueous layer was washed with EtOAc three times. The combined organic phase was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography over silica gel (EtOAc/hexane, 1:3) to give 20 (136 mg, 75%) as yellow oil. Rf = 0.18 (EtOAc/hexane, 1:3); []³⁰D -9 (c 2.83, CHCl3); ¹H NMR (300 MHz, CDCl3) δ: 7.47 (d, J= 8.1 Hz, 2H; Ar-H), 7.33–7.20 (m, 15H; Ar-H), 7.01 (d, J= 8.1 Hz, 2H; Ar-H), 4.86 (d, J= 11.1 Hz, 1H; PhCH2), 4.62 (d, J = 10.2 Hz, 1H; H-1), 4.59 (d, J= 11.1 Hz, 1H; PhCH2), 4.58 (d, J= 11.1 Hz, 1H;

PhCH2), 4.51 (d, J= 12.0 Hz, 1H; PhCH2), 3.94 (d, J= 12.3 Hz, 1H; PhCH2), 3.84 (d, J= 12.3 Hz, 1H; PhCH2), 3.81–3.62 (m, 3H; H-3, H-6), 3.52–3.43 (m, 2H; H-4, H-5), 2.63 (t, J= 9.9 Hz, 1H; H-2), 2.28 (s, 3H, Ar-CH3); ¹³C NMR (75 MHz, CDCl3) δ:

140.0, 138.3, 137.6, 132.2, 129.6, 129.4, 128.4, 128.3, 128.19, 128.18, 127.8, 127.6,

127.5, 127.4, and 127.1 (Ar-C), 87.3 (C-1), 78.8 (C-4), 77.9 (C-5), 76.0 (C-3), 74.2 and 73.2 (PhCH2), 69.2 (C-6), 61.7 (C-2), 49.7 (PhCH2), 21.0 (Ar-CH3); HRMS (ESI) calcd for C34H37NO4S [M+H]⁺: 556.2522, found: 556.2516.

p-Tolyl N-Benzyl-N-tert-butoxycarbonyl-2-amino-4,6-di-O-benzyl-2-deoxy-1-thio-

-D-glucopyranoside (22)

O STol BnO

N(Boc)Bn HO

OBn

To a solution of compound 20 (340 mg, 0.61 mmol) and NaHCO3 (410 mg, 4.88 mmol) in non-dried MeOH (6 mL) at room temperature, was added (Boc)2O chloroformate (0.26 mL, 1.22 mmol). After stirring for 2h, MeOH was removed under reduced pressure. The residue was diluted with CH2Cl2 (5 mL), poured into a two-layer mixture of CH2Cl2 (10 mL) and brine (10 mL), and the whole was extracted with CH2Cl2 (20 mL × 3). The combined organic phase was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography over silica gel (EtOAc/CH2Cl2/ hexane, 1:1:5) to give 22 (304 mg, 76%) as yellow oil. Rf = 0.49 (EtOAc/CH2Cl2/ hexane, 1:1:3); HRMS (ESI) calcd for C39H45NO6S [M+Na]⁺: 678.2865, found: 678.2860.

Standard ¹H and ¹³C NMR spectra were obscured by peak broadening.

N-Benzyl-2-amino-3-O-acetyl-4,6-di-O-benzyl-1,2-cis-O,N-carbonyl-2-deoxy-D- glucopyranoside (24)

BnO O

OBn AcO

BnN O O 24

Rf = 0.47 (EtOAc/CH2Cl2/ hexane, 2:2:3); ¹H NMR (300 MHz, CDCl3) δ: 7.30–7.15

3 3

30 31 7

-Tolyl N-Benzyl-N-benzyloxycarbonyl-2-amino-4,6-di-O-benzyl-2-deoxy-1-thio- (m, 15H; Ar-H), 5.76 (d, J = 7.2 Hz, 1H; H-1), 5.24–5.23 (m, 1H), 4.88 (d, J = 15.3 Hz, 1H), 4.63 (d, J = 11.7 Hz, 1H), 4.43 (dd, J = 11.7 Hz, J = 4.5 Hz, 2H), 4.27 (d, J

= 11.7 Hz, 1H), 3.89 (d, J = 15.3 Hz 1H), 3.77–3.72 (m, 1H), 3.66 (d, J = 8.4 Hz, 1H), 3.61–3.56 (m, 1H) 3.52 (dd, J = 15.6 Hz, J = 2.4 Hz, 1H), 3.38 (dd, J = 11.0 Hz, J = 3.6 Hz, 1H), 1.92 (s, 3H; CH CO); ¹³C NMR (75 MHz, CDCl ) δ: 169.6, 156.9, 137.7, 137.1, 134.5, 129.0, 128.50, 128.46, 128.29, 128.27, 128.19, 127.8, 127.7, 93.7 (C-1), 73.3, 72.9, 72.0, 69.6, 68.7, 65.9, 52.7, 46.0, 20.8 (CH CO); HRMS (ESI) calcd for C H NO [M+H]⁺: 518.2179, found: 518.2180.

-D-glucopyranoside (25)

O STol BnO

N(Cbz)Bn HO

OBn

To a solution of compound 20 (870 mg, 1.57 mmol) and NaHCO3 (1.32g, 15.68 mmol) in non-dried MeOH (10 mL) at ro

2 2

4 centr

om temperature, was added benzyl chloroformate (0.8 mL, 2.36 mmol, 50 wt% solution in toluene). After stirring for 2h, MeOH was removed under reduced pressure. The residue was diluted with CH Cl (10 mL), poured into a two-layer mixture of CH Cl (20 mL) and brine (20 mL), and the whole was extracted with CH Cl (40 mL × 3). The combined organic phase was separated, dried over MgSO , filtered, and con ated under reduced pressure. The

residue was purified by column chromatography over silica gel (EtOAc/CH2Cl2/ hexane, 1:1:4) to give 25 (886 mg, 82%) as yellow oil. Rf = 0.31 (EtOAc/CH2Cl2/ hexane, 1:1:4); []³⁰D -6 (c 4, CHCl3); HRMS (ESI) calcd for C42H43NO6S [M+H]⁺: 690.2889, found: 690.2894. Standard ¹H and ¹³C NMR spectra were obscured by peak broadening.

6-Chlorohexyl N-Benzyl-N-benzyloxycarbonyl-2-amino-4,6-di-O-benzyl-2-deoxy-

-D-glucopyranoside (26)

O OR BnO

N(Cbz)Bn HO

OBn

26, R = (CH₂)₆Cl

Compound 25 (1.60 g, 2.32 mmol), 6-chlorohexanol 7 (0.46 mL, 3.48 mmol), and activated 4 Å MS (6.5 g, 100mg/1mL CH2Cl2, AW300) in CH2Cl2 (65 mL) were stirred at room temperature under nitrogen for 20 min. The mixture was then cooled in a cooling bath at 65 ^oC followed by addition of TMSOTf (84 L, 7 mM) and NIS (526 mg, 2.32 mmol). After disappearance of acceptor detected by TLC, the mixture was diluted with CH2Cl2 (65 mL), quenched by Et3N, and few droplets of sat.

NaHCO3 and pieces of Na2S2O3(s) were added. The quenching mixture was then stirred at room temperature for 2h, filtered and finally concentrated. The residue was

在文檔中環胺基酸酯結合非對稱胺基保護在寡糖合成上的應用 (頁 53-0)