Synthesis of the Non-Reducing Hexasaccharide Fragment of Saccharomicin B

Abstract

A synthesis of the nonreducing end hexasaccharide of saccharomicin B, α-_L-Eva-(1→4)-α-_L-Eva-(1→4)-α-_L-Dig- (1→4)-α-_L-Eva-(1→4)-α-_L-Dig-(1→4)-β-_D-Fuc, has been developed. Selective glycosylations of _L-digitoxose (_L-Dig) using AgPF₆/TTBP-mediated thioether activation and _L-4-epi-vancosamine (_L-Eva) using Tf₂O/DTBMP-mediated sulfoxide activation produced the hexasaccharide as a single diastereomer in very good yield. This hexasaccharide is properly functionalized to serve as a glycosyl donor for the total synthesis of saccharomicin B.

Graphical Abstract

The heptadecasaccharide antibiotics saccharomicins A and B were first isolated from the rare actinomycete Saccharothrix espanaensis bacteria by Kong and co-workers.¹ These molecules possess significant antibacterial activity against Gram-positive and Gram-negative bacteria.² While an unknown mode of toxicity precluded their further development as antibiotics, it is possible that saccharomicin analogues could have potential antimicrobial activity without undesirable side effects. In order to understand the antibacterial activity, mechanism of toxicity, and structure−activity relationships of the saccharomicins, it is essential to access a significant quantity of the material, which is technically challenging to isolate from natural sources with adequate purity, as fermentation produces 5.6 mg/L of saccharomicin B.¹ Since the biosynthesis of saccharomicin B is unknown,³ chemical synthesis remains the best option for obtaining this material for answering questions about the saccharomicins.

Saccharomicin B consists five different deoxy sugars that include _L-rhamnose (Rha) and the rare monosaccharides _D- fucose (Fuc), _L-4-epi-vancosamine (Eva), _L-digitoxose (Dig), and _D-saccharosamine (Sac). To date, the synthesis of four fragments of the saccharomicins have been reported. McDonald et al. have reported the construction of the Fuc- aglycone moiety and a Dig-10 to Fuc-12 fragment of the molecule in high yield and excellent selectivity.⁴ Very recently, our laboratory has reported efficient and stereoselective routes to the Fuc-8 to Dig-10 branching point and the Fuc-5 to Sac-7 fragment of saccharomicin B (Figure 1).⁵ Herein, we report the first synthetic route to the nonreducing hexasaccharide fragment of saccharomicin B which corresponds to the Fuc- 12 to Eva-17 sector, which would be the largest fragment of the molecule so far synthesized.

Figure 1.

Retrosynthetic outline of hexasaccharide fragment in saccharomicin B. Fragments circled in green have been previously synthesized by our group. The fragment in red is this work.

Retrosynthetically, we envisioned that the hexasaccharide fragment could be obtained from a 4 + 2 coupling between the tetrasaccharide 3 and disaccharide 4.⁶ Tetrasaccharide 3 could ultimately be traced back to three suitably functionalized monosaccharides: _L-digitoxose 6, _L-4-epi-vancosamine 8, and _D- fucose 10. The _L-4-epi-vancosamine⁷ can be synthesized from vancomycin−HCl using our group’s modification^5a to the procedure described by Kahne and Walsh.⁸ Similarly, _L- digitoxose can be obtained from _L-rhamnal as most recently described by our group^5a (for the preparation of the monosaccharides, see Supporting Information). Meanwhile, disaccharide 4 could be obtained through glycosylation between _L-4-epi-vancosamines 8 and 9.^6,8

Our initial studies focused on construction of the disaccharide 7. Initial attempts to couple donor 6 and acceptor 10 using AgPF₆ in the presence of 2,6-di-tert-butyl-4- methylpyridine (DTBMP)⁹ afforded disaccharide 11 in moderate yield as a mixture of anomers (Table 1, entries 1 and 2). After several unfruitful approaches, both the yield and α-selectivity were recovered by replacing DTBMP with less basic 2,4,6-tri-tert-butylpyrimidine (TTBP) at 0 °C.^10,11 Under these conditions, 11 was produced in 72−85% yield as a single α-anomer (Table 1, entries 3 and 4). Interestingly, we found that running the reaction for too long (5.5 vs 4 h) resulted in lower yield, presumably due to product decomposition. Treating 11 with DDQ to unmask the Nap protecting group afforded the disaccharide acceptor 7 without anomerization in 93% yield (Scheme 1).¹²

Table 1.

Optimization of Disaccharide 11 Synthesis^a

entry	D/A	base	temp (°C)	time (h)	yield % (α:β)b
1c	1.1/1	DTBMP	−10	10	41 (4:1)
2c	1/1.5	DTBMP	−10 to 0	22	43 (3:1)
3d	1/1.5	TTBP	0	5.5	72 (1:0)
4d	1/1.5	TTBP	0	4	85 (1:0)

^aAgPF₆ (3.0 equiv), 4 Å molecular sieves unless otherwise noted.

^bYield of isolated product after purification.

^cDTBMP (5.0 equiv).

^dTTBP (4.0 equiv).

Scheme 1.

Synthesis of Disaccharide Acceptor 7

We initially envisioned that the _L-4-epi-vancosamine could be installed on this disaccharide 7 using our previously described cyclopropenium cation mediated dehydrative glycosylation.¹³ All attempts to carry out this coupling failed, however, which led us to reconsider our strategy. After examining several approaches, we ultimately drew inspiration from the Kahne group, who reported the construction of the vancomycin disaccharide through activating a _L-vancosamine sulfoxide donor with Tf₂O.⁸

To this end, in a two step activation, first the thioglycoside donor 8 was converted to sulfoxide using m-CPBA, which was activated using Tf₂O and glycosylated with acceptor 7 to produce the trisaccharide 12 in moderate yield (38%) as a single α-isomer.⁸ After optimization, we found that using a 2:1 donor/acceptor ratio, the addition of molecular sieves, and a shorter reaction time provided an optimum yield (61%) of the desired trisaccharide (Table 2, entry 5). In addition, we also found that addition of a proton scavenger, such as β-pinene (A)¹⁴ or 4-allyl-1,2-dimethoxybenzene (B),¹⁵ minimized unwanted side reactions (Table 2, entries 4−6).¹⁶ Finally, changing the order of addition of donor did not have any additional impact on the yield (Table 2, entry 6).¹⁷ The coupling constant of the anomeric proton of the _L-Eva residue at δ 4.90 ppm (J_H1 = 4.5 Hz) and _L-Dig residue at δ 4.84 ppm (J_H1 = 3.5 Hz) confirmed the α-stereochemistry of the newly formed linkage. Finally, removal of the Nap protecting group on 12 using DDQ afforded trisaccharide acceptor 5 in 90% yield (Scheme 2).¹²

Table 2.

Optimization of Trisaccharide Fragment 12 Synthesis^a

entry	donor (equiv)	scavenger	time (h)	yieldb (%)
1c	1.5		3	38
2c,d	2		2	51
3	2		1	59
4	2	A	1	56
5	2	B	1	61
6e	2	B	1	59

^am-CPBA (1.0 equiv), acceptor (1.0 equiv), DTBMP (4.0 equiv), Tf₂O (2.0 equiv), reaction temp −78 °C, 4 Å molecular sieves unless otherwise noted.

^bYield of isolated product after purification.

^cWithout molecular sieves.

^dDonor was preactivated over 5 min.

^eDonor was added dropwise to the solution of acceptor, DTBMP, scavenger, and Tf₂O in DCM at −78 °C. A: β-pinene, B: 4-allyl-1,2- dimethoxybenzene.

Scheme 2.

Successful Synthesis of Trisaccharide Acceptor 5

With the trisaccharide acceptor 5 in hand, we examined its coupling to _L-digitoxose 6 using AgPF₆ and DTBMP⁹ in DCM at −5 °C. Under these conditions, we were able to obtain tetrasaccharide 13 in 66% yield as a single α-isomer. Shortening the reaction time and increasing the amount of donor in the reaction helped to improve the yield to 76% without compromising selectivity (Table 3, entry 3). The stereochemistry of the glycosidic linkages in the tetrasaccharide fragment 13 was deduced from the anomeric ¹H−C coupling constants in the proton-coupled ¹³C NMR spectrum.¹⁸ The anomeric carbons of the two _L-Dig residues show J_C1−H1 coupling constants of 167.5 and 170.0 Hz, while J_C1−H1 for L-Eva was 166.2 Hz, which confirmed the α-stereochemistry of those linkages. The coupling constant of the anomeric proton of the two _L-Dig residues at δ 5.16 ppm (J_H1 = 4.0 Hz), 4.85 ppm (J_H1 = 4.5 Hz) and _L-Eva residue at δ 4.90 ppm (J_H1 = 4.0 Hz) further confirmed the α-stereochemistry. Finally, Nap removal under standard conditions (DDQ) gave tetrasacchar- ide acceptor 3 in 88% yield (Scheme 3).¹²

Table 3.

Optimization of Tetrasaccharide Fragment 13 Synthesis^a

entry	donor (equiv)	time (h)	yieldb (%)
1	2	2.5	66
2	2	1	70
3	2.5	1	76

^aAcceptor (1.0 equiv), DTBPM (5.0 equiv), AgPF₆ (3.0 equiv), reaction temp −5 °C, 4 Å molecular sieves unless otherwise noted.

^bYield of isolated product after purification.

Scheme 3.

Glycosylation Attempt To Synthesize Pentasaccharide 14

With 3 in hand, we initially chose to examine a linear synthesis of the molecule. To this end, we turned our attention to the synthesis of pentasaccharide 14. Coupling 8 and 3 using a two-step activation method led to an inseparable mixture of the pentasaccharide 14 and what appeared to be a trehalose derivative 15 in a modest 33% yield (Scheme 3). Attempts to improve upon this result failed. We therefore chose to examine a more convergent 4 + 2 route to the molecule.

This approach began with construction of the disaccharide 16. To this end, sequential oxidation and Tf₂O-mediated coupling of donor 8 with acceptor 9 produced disaccharide 16 in a good yield of 58% and moderate selectivity (Table 4 entry 1). With further optimization, we found that using a 2:1 donor/acceptor ratio slightly improved the yield (Table 4, entry 2). Finally, a shorter reaction time and addition of the scavenger 4-allyl-1,2-dimethoxybenzene (Table 4, entries 3 and 4) improved the disaccharide and afforded the product in 77% yield as an inseparable 3:1 (α/β) mixture of isomers.^15,17 Following Nap removal, it was possible to isolate the desired α- anomer 18, which was converted to hemiacetal donor 19 under standard conditions. Next, we converted the hemiacetal donor 19 into the corresponding acetate 20 in anticipation of converting this species to the requisite thioglycoside.¹⁹ Attempts to synthesize thioglycoside 4 using BF₃·OEt₂ failed, instead affording a mixture of Eva thioglycosides 21 and 22 (Scheme 4).¹⁹ Given the apparent sensitivity of this compound to Lewis acids, we chose to directly convert the hemiacetal 19 into the corresponding thioglycoside 4 using our p- toluenesulfonyl chloride (TsCl) mediated dehydrative glyco- sylation methodology.²⁰ Under these conditions, we were able to obtain thioglycoside 4 in a moderate 37% yield as 2:1 (α:β) mixture of anomers.²¹

Table 4.

Optimization of Eva-Eva Disaccharide 16^a

entry	donor (equiv)	scavenger	time (h)	yieldb (%)
1c,d	1.1		2	58
2e,f	2		2	61
3d,e	2	B	1	72
4d,e	2	B	1	77

^am-CPBA (1.0 equiv), acceptor (1.0 equiv), DTBMP (4.0 equiv), reaction temp −78 °C, 4 Å molecular sieves unless otherwise noted.

^bYield of isolated product after purification.

^cTf₂O (1.2 equiv)

^dDonor was added dropwise to the solution of acceptor, DTBMP, scavenger, and Tf₂O in DCM at −78 °C.

^eTf₂O (2.2 equiv).

^fDonor was coactivated with acceptor, DTBMP, and scavenger (B). B: 4-allyl- 1,2-dimethoxybenzene.

Scheme 4.

Optimization of Eva-Eva Disaccharide Thioglycoside 4

With both coupling partners in hand, we set about examining the 4 + 2 glycosylation for the construction of the hexasaccharide target. Pleasingly, subsequent oxidation of the thiol 4 by m-CPBA^8a,b and activation with Tf₂O in the presence of the proton scavenger 4-allyl-1,2-dimethoxybenzene in DCM at −78 °C furnished the target hexasaccharide 2 in excellent yield 50% and α-selectivity (Scheme 5).^8,15,17 All of the anomeric carbon linkages were confirmed by the proton- coupled HSQC NMR spectrum. The J_C1−H1 coupling constant for the anomeric carbons in the proton-coupled HSQC NMR spectrum are 172.5 Hz (C-1_Eva), 171.1 Hz (C-1_Eva), 172.3 Hz (C-1_Dig), 172.2 Hz (C-1_Eva), 170.7 Hz (C-1_Dig), and 155.7 Hz (C-1_Fuc), respectively. The coupling constants of the anomeric proton of the hexasaccharide are δ 5.14 ppm (two protons overlapped, H-1, H-1 ), 4.90 ppm (J = 4.5 Hz, H-1 ), 4.86 ppm (J_H1 = 5.0 Hz, H-1_Dig), 4.79 ppm (J_H1 = 4.5 Hz, H-1_Eva), and 4.66 (J_H1 = 10.0 Hz, H-1_Fuc), further confirming the stereochemistry of the linkages.

Scheme 5.

Successful Attempt for the Synthesis of Hexasaccharide Fragment 2

In summary, we have developed an expedient chemical synthesis of the hexasaccharide 2, which corresponds to the nonreducing end of saccharomicin B. This compound, which is the largest fragment of saccharomicin synthesized to date, bears a thioglycoside aglycone, which will allow it to directly serve as a donor for elaboration to the natural product. The chemistry reported above also probes the efficiency of different glycosylations using deoxy-sugar donors in a challenging setting. In particular, it demonstrates the utility of both the Kahne sulfoxide glycosylation reaction and AgPF₆-mediated thioglycoside activation in the construction of highly complex deoxy-sugar oligosaccharides. Studies to elaborate 2 into saccharomicin B are currently underway in our laboratory.