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Dec 18, 2017 - Yuta Maki, Takanori Mima, Ryo Okamoto, Masayuki Izumi, and Yasuhiro Kajihara*. Department of Chemistry, Graduate School of Science, ...
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Cite This: J. Org. Chem. 2018, 83, 443−451

Semisynthesis of Complex-Type Biantennary Oligosaccharides Containing Lactosamine Repeating Units from a Biantennary Oligosaccharide Isolated from a Natural Source Yuta Maki, Takanori Mima, Ryo Okamoto, Masayuki Izumi, and Yasuhiro Kajihara* Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan S Supporting Information *

ABSTRACT: Poly-N-acetyllactosamine (poly-LacNAc) structures on glycoproteins play important roles in essential biological events such as cell−cell adhesion. Here, we report a new strategy for the semisynthesis of LacNAc-extended complex-type biantennary oligosaccharides. We found an efficient isopropylidenation reaction that selectively protects the terminal Gal-3,4OH of a biantennary complex-type nonasaccharide isolated from a natural source. This finding enabled the conversion of the nonasaccharide into the two types of oligosaccharides containing di-LacNAc units at one or two antennae via ten-step chemical sequences.

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tion steps12,13 are usually required, resulting in the scarcity of the final compounds. Here, we report the semisynthesis of complex-type oligosaccharide 1 containing two LacNAc units with only 10 conversion steps from biantennary asialo-nonasaccharide 3, which can be isolated from hen egg yolk in multigram quantities.14,15 Previously, we reported a novel semisynthetic strategy using nonasaccharide 3 to obtain triantennary oligosaccharides within 10 conversion steps.16 The structure of nonasaccharide 3 was efficiently used as a scaffold for the desired undecasaccharides. This new method dramatically decreased the number of synthetic steps in comparison to that of conventional oligosaccharide synthesis. To expand this method, we envisaged that various poly-LacNAc units could be incorporated into nonasaccharide 3 by the selective protection of the 24 hydroxy groups (Scheme 1). First, we studied the isopropylidenation of Galf,i-3,4-OH of biantennary nonasaccharide 3 (Scheme 2). The Galf,i-3,4-OH shows a specific 1,2-cis configuration, and therefore we examined isopropylidenation conditions using acetone/DMF and TsOH. After optimization, we obtained the desired product 6 in 14% yield (conversion: 22%) by reversed-phase HPLC (RP-HPLC) purification, along with mono-O-isopropylidenated products 4 (12%, conversion: 23%), 5 (13%, conversion: 25%), and starting material 3 (8%), as shown in Supporting Information (SI), Figure S1. The isopropylidena-

epetition of a Gal-β1,4-GlcNAc moiety (poly-LacNAc) at the nonreducing terminals of oligosaccharides is a typical structural feature observed in complex-type oligosaccharides. Oligosaccharides of glycoproteins play important roles in many biological events.1 In particular, poly-LacNAc structures of glycoproteins on cell surfaces are often involved in direct cell− cell adhesion.2,3 S-Type lectins (galectins) recognize βgalactoside units of glycoconjugates.4,5 Multivalent-oligomeric galectins support cell−cell adhesion through binding between poly-LacNAc and galectins. In addition, Rudd et al. reported the structural analyses of four types of erythropoietin drugs, revealing that over 30% of complex-type oligosaccharides contain poly-LacNAc structures.6 However, why erythropoietin drugs need to have much poly-LacNAc structures in oligosaccharides is still unclear. Although LacNAc-extended complex-type oligosaccharides have been synthesized by sophisticated techniques, strategies for a facile synthesis are still required. The Ogawa and Nakahara groups independently synthesized four timesrepeated LacNAc structures,7,8 and the Boons group chemoenzymatically synthesized asymmetric complex-type oligosaccharides having LacNAc repeating units.9,10 Moreover, the Paulson group enzymatically extended poly-LacNAc units at the two terminals of complex-type biantennary oligosaccharides.11 In general, chemical synthesis allows to insert any sugar analogues and unnatural glycosidic linkages, which is useful for studying the relationship between the oligosaccharide structure and its bioactivity. However, repetitive protection/deprotec© 2017 American Chemical Society

Received: September 30, 2017 Published: December 18, 2017 443

DOI: 10.1021/acs.joc.7b02485 J. Org. Chem. 2018, 83, 443−451

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The Journal of Organic Chemistry

disaccharides were not applicable to nonasaccharide 3. In most cases, selective protections of nonasaccharide 3 resulted in the multiple products due to a large number of hydroxy groups and low solubility in organic solvents. We concluded that mild isopropylidenation using nonasaccharide 3 (6.7 mM) and acetone (670 equiv) was the best condition for not only obtaining the desired product 6 but also recovering the starting material and the mono-O-isopropylidenated isomers 4 and 5, which can be reused in the isopropylidenation reaction and can be used to asymmetrically extend LacNAc units. Purification of oligosaccharides using RP-HPLC resulted in lower isolated yields than conversion yields estimated by the peak areas of the RP-HPLC chromatograms. This is potentially due to the nature of the reversed-phase columns we used. After the protection of other functional groups of the isopropylidenated oligosaccharide 6, we performed NMR structural analysis to precisely determine the isopropylidenated hydroxy groups. First, the carboxylic acid of oligosaccharyl asparagine 6 was protected as the phenacyl ester, followed by per-acetylation of the remaining hydroxy groups (Scheme 3).

Scheme 1. Semisynthetic Strategy of Complex-Type Oligosaccharide 1 Containing LacNAc Repeating Units from Isolated Nonasaccharide 3

Scheme 3. Synthesis of Glycosyl Acceptor 2a

tion of Gal-3,4-OH was not completed, potentially due to the multiple hydroxy groups and the low solubility of nonasaccharide 3 in organic solvents. Using alternative reagents such as 2,2-dimethoxypropane did not improve the yield of the desired product 6 and resulted in the formation of undesired multiple products such as tri- and tetra-O-isopropylidenated products. Typical protection protocols toward mono- and

a

Reagents and conditions: (i) 2-bromoacetophenone, iPr2NEt, DMF, 2 h; (ii) Ac2O/pyridine (1:1), DMAP, 2 h, 53% (2 steps); (iii) 50% aq TFA, 0 °C, 5 min, 58%; (iv) CH3C(OCH3)3, TsOH, CH2Cl2, 20 min; (v) 80% aq AcOH, 20 min, 55% (2 steps).

Scheme 2. Isopropylidenation Reaction of Asialo-nonasaccharide 3a

Reagents and conditions: (i) TsOH, acetone/DMF (1:2), 37 °C, 29 h, 12% 4, 13% 5, 14% 6, 8% 3.

a

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DOI: 10.1021/acs.joc.7b02485 J. Org. Chem. 2018, 83, 443−451

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The Journal of Organic Chemistry

twice to enhance the attachment of isopropylidene groups, and the di-O-isopropylidenated product 6 was roughly purified by silica gel column chromatography (SI, Figure S6). The resulting oligosaccharide was then protected by phenacyl ester and acetyl groups as previously described. After the acidic hydrolysis of isopropylidene groups and the formation of orthoester, followed by the acidic opening reaction, diol 2 was obtained in 6% yield (6 steps from 3) after HPLC purification (SI, Figure S6). Glycosylation of acceptor 2 with lactosamine donor 9 was then examined (Scheme 4). After extensive optimization, the best conditions were found to be as follows: acceptor 2 (8 mM), donor 9 (10 equiv), NIS (17 equiv), and TfOH (0.3 equiv). On the basis of the RP-HPLC peak areas, we obtained the desired diglycosylated product 10 in 34% conversion yield along with two monoglycosylated products (10% and 6% conversion yields) and acceptor 2 (3% recovery yield) as shown in SI, Figure S7. The use of additional donor 9 did not improve the conversion yield. We also examined glycosylation reactions using an imidate donor; however, the attempts were not successful, and LC-MS analysis showed unknown multiple peaks. A previous study also reported the difficulty to complete simultaneous glycosylations at multiple hydroxy groups of an N-linked oligosaccharide.18 The desired product 10 was, however, successfully isolated by RP-HPLC purification. NMR analyses displayed the low-field (glycosyl) shift of Galf,i-C3 and HMBC correlation signals between Galf,i-C3 and GlcNj,j-H1 (SI, Figure S8), indicating the success of the glycosylation reaction. Stepwise deprotections of the protected oligosaccharide 10 were then performed (Scheme 4). The Troc group of protected oligosaccharide 10 was reduced and simultaneous acetamidation was performed by zinc and Ac2O. Subsequent saponification of acetyl groups and reintroduction of the cleaved Fmoc group to the asparagine moiety yielded desired product 1 (20% over 3 steps). HRMS and NMR analyses indicated that we successfully synthesized oligosaccharide 1 containing di-LacNAc repeating units. To demonstrate the efficient chemical modification of nonasaccharide 3, we examined synthesis of an asymmetric LacNAc-extended biantennary oligosaccharide because natural N-linked oligosaccharides usually have asymmetric antennae. We used mono-O-isopropylidenated oligosaccharide 5 and performed protection/deprotection manipulation, similar to the previous improved protocol (Scheme 5). As a result, we obtained glycosyl acceptor 11 in 31% yield (5 steps), which was then glycosylated with thioglycoside 9 to yield biantennary oligosaccharide 12 that asymmetrically has an additional LacNAc unit in 21% yield (conversion: 45%, SI, Figure S11). Moreover, sequential deprotection steps afforded asymmetric LacNAc-extended oligosaccharide 13 in 30% yield (3 steps). In summary, we have established a facile semisynthetic strategy to obtain not only symmetric LacNAc-extended tridecasaccharide 1 but also asymmetric LacNAc-extended undecasaccharide 13. A successful isopropylidenation reaction enabled the conversion of nonasaccharide 3, isolated from hen egg yolk, into two LacNAc-extended oligosaccharides via a tenstep chemical sequences. This strategy would allow for the preparation of a library of LacNAc-extended oligosaccharides by using various glycosyl donors.

The resulting fully protected oligosaccharide 7 was purified by RP-HPLC and obtained in 53% yield (2 steps). NMR analysis showed HMBC correlation signals between the acetal carbons of two isopropylidene groups and Galf,i-H3,4 (SI, Figure S3), indicating that Galf,i-3,4-OH was successfully protected with isopropylidene groups. Next, acetate 7 was briefly treated with 50% TFA at 0 °C to obtain tetraol 8 in 58% yield. Extensive and careful NMR analyses indicated that tetraol 8 was the major product and byproduct due to acetyl migration was not observed. De-Oacetylated products, which were barely detected by LC/MS analyses, could be separated from the desired tetraol 8 by HPLC purification. Selective glycosylation of Galf,i-3-OH of tetraol 7 was examined using disaccharyl thioglycoside donor 9 (Scheme 4), Scheme 4. Synthesis of Complex-Type Oligosaccharide 1 Containing LacNAc Repeating Unitsa

a Reagents and conditions: (i) donor 9, NIS, TfOH, CH2Cl2, 0 °C, molecular sieves 4A, 1 h, 9% (conversion: 34%); (ii) Zn, AcOH/ Ac2O (1:1), 1 h; (iii) 5 M NaOH/MeOH (3:5), 15 min, then NaHCO3, FmocOSu, CH3CN, 2 h, 20% (3 steps).

which was prepared as previously described.16 Unfortunately, no selectivity was found in the nucleophilicity between the 3OH and 4-OH of Galf,i; thus, the glycosylation reactions using tetraol 8 resulted in mono- to tetra-glycosylated products. We hypothesized that the reactivity of the equatorial 3-OH was not sufficiently greater than that of the axial 4-OH because the glycosyl acceptor used here was a large oligosaccharide. To overcome this problem, tetraol 8 was converted into diol 2 having free Galf,i-3-OH (Scheme 3). Tetraol 8 was treated with trimethyl orthoacetate, and then the formed orthoester was subjected to 80% AcOH treatment. Acid-promoted ringopening reactions of orthoesters are known to form axial acetyl groups and equatorial hydroxy groups.17 This protocol afforded suitably protected acceptor 2 in 55% yield (2 steps). We also examined the selective protection of nonasaccharide 3 using trimethyl orthoacetate; however, we have not observed appropriate selectivity toward Gal-3,4-OH. To synthesize acceptor 2 from nonasaccharide 3 promptly and efficiently, we examined an improved protocol to omit HPLC purification in each synthetic step. Treatment of nonasaccharide 3 with acetone and TsOH was repeated 445

DOI: 10.1021/acs.joc.7b02485 J. Org. Chem. 2018, 83, 443−451

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The Journal of Organic Chemistry

mm) column, and a Proteonavi (Shiseido, 2.0 mm × 150 mm) were also used for analytical HPLC using 0.1% aq HCOOH and 90% aq CH3CN containing 0.1% HCOOH. An XBridge prep column (Waters, 10 mm × 250 mm) was used for preparative HPLC using 50 mM aq NH4OAc and CH3CN or 0.1% aq TFA and 90% aq CH3CN containing 0.1% TFA. A Proteonavi (Shiseido, 10 mm × 250 mm) used for preparative HPLC using 0.1% aq TFA and 90% aq CH3CN containing 0.1% TFA. N2-(9-Fluorenylmethyloxycarbonyl)-N4-{O-(3,4-O-isopropylidene-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-2-deoxyβ-D-glucopyranosyl)-(1→2)-O-α-D-mannopyranosyl-(1→3)-O[β-D -galactopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-β- D glucopyranosyl)-(1→2)-O-α-D-mannopyranosyl-(1→6)]-O-β-Dmannopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-(2-acetamido-2-deoxy-β- D -glucopyranosyl)}-L-asparagine (4). N2-(9-Fluorenylmethyloxycarbonyl)-N4{O-β-D-galactopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-β-Dglucopyranosyl)-(1→2)-O-α- D -mannopyranosyl-(1→3)-O[(3,4-O-isopropylidene-β- D -galactopyranosyl)-(1→4)-O-(2acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-O-α-D-mannopyranosyl-(1→6)]-O-β- D-mannopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-(2-acetamido-2deoxy-β-D-glucopyranosyl)}-L-asparagine (5). N2-(9-Fluorenylmethyloxycarbonyl)-N4-{O-(3,4-O-isopropylidene-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-O-α-D-mannopyranosyl-(1→3)-O-[(3,4-O-isopropylidene-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-2-deoxyβ-D-glucopyranosyl)-(1→2)-O-α-D-mannopyranosyl-(1→6)]-Oβ-D-mannopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-(2-acetamido-2-deoxy-β-D-glucopyranosyl)}-L-asparagine (6). To a solution of nonasaccharide 3 (200 mg, 101 μmol) in DMF/acetone (10.0 mL/5.0 mL), p-toluenesulfonic acid monohydrate (101 mg, 0.531 mmol) was added, and this mixture was kept at 37 °C using a dry bath incubator (major science). After 29 h, N,N′-diisopropylethylamine (52.0 μL) was added to neutralize the reaction mixture. Furthermore, ice-cold Et2O was added to the mixture to give a precipitate, which was purified by RP-HPLC (YMC packed column D-ODS-5A, ϕ20 mm × 250 mm, 50 mM NH4OAc/ CH3CN = 78:22 to 70:30 over 60 min at 7.5 mL/min). Fractions containing the desired products were collected and applied to ODS column chromatography (ϕ25 mm × 150 mm) to remove ammonium acetate, respectively. Fractions containing the desired products were collected and lyophilized to give mono-O-isopropylidenated oligosaccharide 4 (24.4 mg, 12.1 μmol, 12%), mono-Oisopropylidenated oligosaccharide 5 (27.4 mg, 13.6 μmol, 13%), and di-O-isopropylidenated oligosaccharide 6 (28.5 mg, 13.8 μmol, 14%) as white solids. The starting material 3 was also collected (16.7 mg, 8.44 μmol) and used for isopropylidenation reaction again to accumulate isopropylidenated oligosaccharides 4, 5, and 6. Mono-O-isopropylidenated Oligosaccharide 4. [α]D25 −1.2 (c 1.5, H2O). HRMS (ESI/LIT-Orbitrap) m/z: [M + 2H]2+ calcd for C84H126N6O50 1009.3745, found 1009.3748. 1H NMR (700 MHz, D2O, 4.79 ppm) δ 7.93−7.82 (m, 2H), 7.73−7.61 (m, 2H), 7.54− 7.44 (m, 2H), 7.44−7.33 (m, 2H), 5.11 (s, 1H), 5.00 (d, J = 9.65 Hz, 1H), 4.92 (s, 1H), 4.75 (s, 1H), 4.59−4.52 (m, 3H), 4.52−4.48 (m, 2H), 4.46 (d, J = 8.10 Hz, 1H), 4.39−4.31 (m, 2H), 4.31−4.26 (m, 1H), 4.24 (brd, 1H), 4.22−4.16 (m, 2H), 4.11 (brd, 1H), 4.08 (brdd, 1H), 4.02−3.42 (m), 2.76−2.56 (m, 2H), 2.07 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.89 (s, 3H), 1.53 (s, 3H), 1.38 (s, 3H). 13C NMR (175 MHz, D2O) δ 176.6, 175.7, 175.5, 173.9, 158.5, 144.7, 144.6, 141.9, 129.0, 128.5, 128.4, 126.1, 126.0, 125.7, 121.1, 112.0, 103.9, 103.1, 102.2, 101.4, 100.5, 100.4, 98.0, 81.4, 80.4, 79.8, 79.5, 79.4, 79.1, 77.3, 77.2, 77.1, 76.3, 75.6(1), 75.5(8), 75.3, 74.7, 74.5, 74.3, 73.8, 73.7, 73.6, 73.4, 73.0, 72.9, 72.8, 71.9, 71.1, 70.4, 70.3, 69.5, 68.3, 68.2, 67.4, 66.7, 66.6, 62.6(2), 62.5(6), 61.9, 61.7, 60.8(9), 60.8(5), 60.7, 55.9, 55.8, 54.7, 52.4, 49.8, 47.9, 38.7, 31.2, 28.0, 26.2, 23.3, 23.2, 22.9. Mono-O-isopropylidenated Oligosaccharide 5. [α]D25 −0.8 (c 1.5, H2O). HRMS (ESI/LIT-Orbitrap) m/z: [M + 2H]2+ calcd for C84H126N6O50 1009.3745, found 1009.3749. 1H NMR (700 MHz, D2O, 4.79 ppm) δ 7.72−7.51 (m, 2H), 7.51−7.38 (m, 2H), 7.38− 7.06 (m, 4H), 5.12 (s, 1H), 4.98 (brd, 1H), 4.91 (s, 1H), 4.73 (s,

Scheme 5. Synthesis of Complex-Type Biantennary Oligosaccharide 13 Containing a LacNAc Repeating Unita

a

Reagents and conditions: (i) 2-bromoacetophenone, iPr2NEt, DMF, 2 h; (ii) Ac2O/pyridine (1:1), DMAP, 2 h; (iii) 50% aq TFA, 0 °C, 5 min; (iv) CH3C(OCH3)3, TsOH, CH2Cl2, 2 h; (v) 60% aq AcOH, 5 min, 31% (5 steps); (vi) donor 9, NIS, TfOH, CH2Cl2, 0 °C to rt, molecular sieves 4A, 6 h, 21%; (vii) Zn, THF/AcOH/Ac2O (3:2:1), 23 h; (viii) 5 M NaOH/MeOH (1:1), 1 h; (ix) NaHCO3, FmocOSu, CH3CN/H2O, 4 h, twice, 30% (3 steps).

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EXPERIMENTAL SECTION

General Experimental Methods. 1H and 13C NMR spectra were recorded on a 400 MHz spectrometer (Bruker Avance III) or a 700 MHz spectrometer (Bruker Avance I) and are reported as follows: chemical shift (δ), multiplicity (s = singlet, d = doublet, dd = double doublet, br = broad, m = multiplet), coupling constants (Hz), and integration. All 1H chemical shifts are reported in parts per million (ppm) relative to CD3CN (1.94 ppm), D2O (4.79 ppm), or H2O in CD3CN (2.13 ppm). All 13C chemical shifts are assigned according to CD3CN (118.3 ppm) or 1,4-dioxane in D2O as an external standard (67.2 ppm). All NMR signals were assigned on the basis of 1H and 13 C NMR, DQF-COSY, TOCSY, HSQC, HMBC, and HSQCTOCSY. High-resolution mass spectrometry was performed by ESI/ LIT-Orbitrap (Thermo Fisher Scinentific, LTQ Orbitrap XL), and other mass spectra were recorded on a Bruker Esquire 3000plus (ion trap), a Bruker Compact (Q-TOF), or a Bruker Amazon ETD (ion trap) mass spectrometers. TLC-analysis was conducted on Silica Gel 60 F254 (Merck TLC plates), and visualizations were performed with UV light (254 nm) and sulfuric acid stain (5% H2SO4 in methanol). Analytical RP-HPLC analyses were performed on a Waters 2996 HPLC system equipped with a multiwavelength detector on a Thermo Ultimate 3000 HPLC system equipped with a variable wavelength detector. Preparative RP-HPLC separation was performed with a Waters 2487 HPLC system equipped with a variable wavelength detector. A Cadenza CD-C18 (Imtakt, 4.6 mm × 75 mm) column was used for analytical HPLC using 0.1% aq TFA and 90% aq CH3CN containing 0.1% TFA. A Capcell Pack C18 (Shiseido, 2.0 mm × 50 mm) column, a Cadenza CD-C18 (Imtakt, 2 mm × 100 446

DOI: 10.1021/acs.joc.7b02485 J. Org. Chem. 2018, 83, 443−451

Note

The Journal of Organic Chemistry

C NMR (175 MHz, CD3CN, 118.3 ppm) δ 193.3, 172.5, 171.9, 171.8(3), 171.7(6), 171.7, 171.6, 171.5(4), 171.4(9), 171.4(7), 171.4, 171.2(0), 171.1(5), 171.1, 171.0, 170.9(2), 170.8(9), 170.8, 170.7, 170.6, 170.5, 156.9, 145.0, 142.1, 135.0, 129.9, 128.7(3), 128.6(6), 128.1, 126.1, 120.9, 110.9(9), 110.9(5), 101.9, 101.3, 101.2(3), 101.1(9), 100.2, 100.1, 99.3, 99.2, 79.5, 77.8(2), 77.7(7), 77.6, 77.5, 77.4, 77.1, 76.0, 75.3(8), 75.3(6), 75.0, 74.5(5), 74.4(5), 74.3, 74.0(0), 73.9(7), 73.9, 73.8, 73.1, 73.0, 72.6, 71.6(4), 71.5(9), 71.5, 71.0, 70.5, 70.2, 69.5, 68.2, 67.9, 67.4, 65.9, 65.7, 63.7(4), 63.6(6), 63.4, 63.1(9), 63.1(7), 63.0, 62.9, 62.6, 55.0, 54.7, 53.7, 51.5, 47.9, 38.2, 28.2, 27.9, 26.4(3), 26.4(1), 23.1(1), 23.0(6), 23.0, 21.6, 21.5, 21.2, 21.1(2), 21.1(0), 21.0(7), 21.0(3), 21.0(1), 20.9(8), 20.9(6), 20.9(3), 20.9(0), 20.6. N2-(9-Fluorenylmethyloxycarbonyl)-N4-{O-(2,6-di-O-acetylβ-D-galactopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-acetyl2-deoxy-β-D-glucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-Dmannopyranosyl)-(1→3)-O-[(2,6-di-O-acetyl-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β- Dglucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→6)]-O-(2,4-di-O-acetyl-β-D-mannopyranosyl)-(1→4)O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)(1→4)-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)}-L-asparagine Phenacyl Ester (8). Di-O-isopropylidenated product 7 (15.0 mg, 4.97 μmol) was dissolved in ice-cooled 50% aq TFA (1 mL), which was stirred at 0 °C for 5 min. The resulting mixture was immediately evaporated and co-evaporated with toluene, and the residue was dissolved in aq CH3CN and purified by RPHPLC (XBridge, ϕ10 mm × 250 mm, 50 mM NH4OAc/CH3CN = 60:40 to 40:60 over 90 min at 4 mL/min). During HPLC purification, fractions were kept on an ice bath to prevent aspartimide formation.16,19 Fractions containing the desired product were collected, treated with Dowex to remove ammonium salt, and lyophilized to give tetraol 8 (8.4 mg, 2.9 μmol, 58%) as a white solid. HRMS (ESI/LIT-Orbitrap) m/z: [M + 2H] 2+ calcd for C129H168N6O71 1468.4854, found 1468.4864. 1H NMR (700 MHz, CD3CN, HDO: 2.13 ppm) δ 7.92−7.82 (m, 2H), 7.80−7.72 (m, 2H), 7.64−7.54 (m, 3H), 7.50−7.43 (m, 2H), 7.40−7.32 (m, 2H), 7.30− 7.24 (m, 2H), 7.21 (brd, 1H), 6.78 (d, J = 9.77 Hz, 1H), 6.74 (d, J = 9.77 Hz, 1H), 6.67 (brd, 1H), 6.34 (d, J = 8.75 Hz, 1H), 6.24 (brd, 1H), 5.40−5.27 (m, 3H), 5.26−5.16 (m, 2H), 5.09 (dd, J = 10.03 Hz, 1H), 5.05−4.95 (m, 5H), 4.93 (dd, J = 11.01, 9.30 Hz, 1H), 4.85 (dd, J = 8.79 Hz, 1H), 4.77 (brd, 1H), 4.74 (s, 1H), 4.71 (dd, J = 10.50, 3.24, 1H), 4.69 (brd, 1H), 4.68−4.60 (m, 2H), 4.48 (d, J = 8.38 Hz, 1H), 4.44 (d, J = 8.49 Hz, 1H), 4.40−4.19 (m, 10H), 4.19−4.05 (m, 8H), 4.05−3.75 (m, 15H), 3.74−3.55 (m, 8H), 3.55−3.44 (m, 5H), 3.36−3.17 (m, 2H), 2.76−2.59 (m, 2H), 2.08 (s, 3H), 2.04 (s, 3H), 2.03 (s, 6H), 2.01 (s, 3H × 2), 2.00 (s, 3H), 1.99 (s, 3H), 1.98 (s, 3H × 2), 1.97 (s, 6H), 1.94 (s, 6H), 1.93 (s, 3H), 1.92 (s, 3H), 1.91 (s, 3H), 1.89 (s, 3H), 1.86 (s, 3H), 1.79 (s, 3H), 1.78 (s, 3H), 1.74 (s, 3H), 1.73 (s, 3H). 13C NMR (125 MHz, CD3CN, 118.3 ppm) δ 193.5, 172.4, 172.3, 172.2, 171.9, 171.8, 171.7, 171.6, 171.5(4), 171.5(2), 171.4(9), 171.4, 171.3, 171.2, 171.1(3), 171.0(9), 171.0(2), 170.9(4), 170.8(7), 170.7, 170.4, 156.9, 144.9(9), 144.9(5), 142.1, 135.0, 129.9, 128.7(3), 128.6(5), 128.1, 126.1(4), 126.1(1), 120.9, 102.0, 101.9, 101.8, 101.2, 100.3, 100.0, 79.1, 78.1, 77.6, 77.4, 77.1, 76.7, 76.2, 75.8, 75.0, 74.6, 74.4, 73.9, 73.8, 73.2(8), 73.2(5), 73.2, 73.1, 72.7, 72.2, 72.1(4), 72.0(6), 70.6, 70.5, 70.2, 69.7, 69.4, 68.0, 67.5, 67.4, 66.3, 65.6, 64.0, 63.4, 63.3, 63.2, 62.5, 62.2, 54.9, 54.5, 54.0, 53.1, 51.5, 47.9, 38.2, 23.1, 22.9, 21.8, 21.4(4), 21.3(5), 21.3, 21.2(1), 21.1(5), 21.1(3), 21.1(0), 21.0(6), 21.0(1), 20.9(8), 20.9(4), 20.9(3), 20.9(0), 20.8(8), 20.8(7). N2-(9-Fluorenylmethyloxycarbonyl)-N4-{O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetylα-D-mannopyranosyl)-(1→3)-O-[(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxyβ-D-glucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→6)]-O-(2,4-di-O-acetyl-β-D-mannopyranosyl)(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→4)-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)}-L-asparagine Phenacyl Ester (2). To a solution of tetraol 8 (8.8 mg, 3.0 μmol) in CH2Cl2 (400 μL), trimethyl 13

1H), 4.64−4.51 (m, 3H), 4.51−4.39 (m, 2H), 4.39−3.33 (m), 2.81− 2.26 (m, 2H), 2.04 (s, 9H), 1.88 (s, 3H), 1.47 (s, 3H), 1.31 (s, 3H). 13 C NMR (175 MHz, D2O) δ 176.8, 175.6(2), 175.5(7), 175.4, 173.9, 158.3, 144.6, 144.4, 141.7, 128.8, 128.3, 126.0, 121.0,111.9, 103.9, 103.2, 102.2, 101.3, 100.5, 100.4, 98.0, 81.4, 80.3, 79.9, 79.6, 79.5, 79.4, 79.1, 77.3(2), 77.2(7), 77.1, 76.3, 75.7, 75.5, 75.3(3), 75.2(6), 74.6, 74.5, 74.3, 73.8, 73.7, 73.6, 73.4, 73.0, 72.9, 71.9, 71.1, 70.4, 70.3, 69.5, 68.3, 68.2, 67.5, 66.5, 62.6(3), 62.5(7), 61.9, 61.7, 60.9(1), 60.8(6), 60.7, 55.9, 55.8, 54.7, 47.6, 38.9, 28.1, 26.3, 23.3, 23.2, 23.0. Di-O-isopropylidenated Oligosaccharide 6. [α]D25 −3.2 (c 1.5, H2O). HRMS (ESI/LIT-Orbitrap) m/z: [M + 2H]2+ calcd for C87H130N6O50 1029.3902, found 1029.3904. 1H NMR (700 MHz, D2O, 4.79 ppm) δ 7.96−7.81 (m, 2H), 7.75−7.60 (m, 2H), 7.55− 7.34 (m, 4H), 5.12 (s, 1H), 4.99 (d, J = 9.56 Hz, 1H), 4.92 (s, 1H), 4.74 (s, 1H), 4.63−4.54 (m, 3H), 4.54−4.40 (m, 4H), 4.38−4.29 (m, 3H), 4.28−4.15 (m, 5H), 4.15−4.05 (m, 3H), 4.00−3.40 (m, 44H), 2.76−2.66 (m, 1H), 2.63−2.52 (m, 1H), 2.06 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.89 (s, 3H), 1.53 (s, 3H), 1.51 (s, 3H), 1.37 (s, 3H), 1.36 (s, 3H). 13C NMR (175 MHz, D2O) δ 175.7, 175.5, 174.1, 158.5, 144.8, 144.6, 141.8, 129.0, 128.5, 128.4, 126.1(3), 126.0(6), 121.1, 112.0, 103.1(4), 103.1(0), 102.2, 101.3, 100.5, 100.4, 98.0, 81.4, 80.3, 79.8(3), 79.7(7), 79.5(4), 79.5(0), 79.4(8), 79.1, 77.3, 77.2, 77.1, 75.6, 75.5, 75.3(2), 75.2(6), 74.7, 74.5, 74.3, 73.8, 73.7(3), 73.7(1), 73.6, 73.0, 72.9, 72.8, 71.1, 70.4, 70.3, 68.3, 68.2, 67.4, 66.6(3), 66.5(5), 62.6(2), 62.5(6), 61.7, 60.8, 60.7, 55.9, 55.8, 54.7, 53.0, 47.9, 39.1, 28.0(3), 28.0(2), 26.2, 23.3, 23.2, 22.9. N2-(9-Fluorenylmethyloxycarbonyl)-N4-{O-(2,6-di-O-acetyl3,4-O-isopropylidene-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-O(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→3)-O-[(2,6-di-Oacetyl-3,4-O-isopropylidene-β-D-galactopyranosyl)-(1→4)-O(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)(1→2)-O-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→6)]-O(2,4-di-O-acetyl-β-D-mannopyranosyl)-(1→4)-O-(2-acetamido3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→4)-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)}-L-asparagine Phenacyl Ester (7). To a solution of di-O-isopropylidenated oligosaccharide 6 (18.9 mg, 9.18 μmol) in DMF (1.2 mL), 2bromoacetophenone (5.5 mg, 28 μmol) and N,N′-diisopropylethylamine (8.0 μL, 46 μmol) were added, and this mixture was stirred at room temperature. After 2 h, ice-cold ether (13 mL) was added to give a precipitate. The precipitate was collected by centrifugation and then dried. Ac2O/pyridine (1:1, 920 μL) was added to the resulting residue, followed by addition of DMAP (1.1 mg, 9.0 μmol). The resulting mixture was then stirred at room temperature. After 2 h, MeOH (2 mL) was added to the mixture on an ice bath, and the temperature was elevated to room temperature. The mixture was evaporated and co-evaporated with toluene. The residue was dissolved in aq CH3CN and purified by RP-HPLC (XBridge, ϕ10 mm × 250 mm, 40 mM NH4OAc/CH3CN = 50:50 to 30:70 over 90 min at 4.0 mL/min). During HPLC purification, fractions were kept on an ice bath to prevent aspartimide formation.16,19 Fractions containing the desired product were collected, treated with Dowex to remove ammonium salt, and lyophilized to give acetate 7 (14.6 mg, 4.84 μmol, 53%) as a white solid. [α]D24 −4.1 (c 0.55, CHCl3). HRMS (ESI/LIT-Orbitrap) m/z: [M + 2H] 2+ calcd for C135H176N6O71 1508.5167, found 1508.5162. 1H NMR (700 MHz, CD3CN, HDO: 2.13 ppm) δ 7.93−7.83 (m, 2H), 7.83−7.73 (m, 2H), 7.66−7.54 (m, 3H), 7.53−7.43 (m, 2H), 7.41−7.33 (m, 2H), 7.33− 7.23 (m, 2H), 7.18 (d, J = 8.45 Hz, 1H), 6.50 (d, J = 9.49 Hz, 2H), 6.40−6.26 (m, 2H), 6.19 (d, J = 8.73 Hz, 1H), 5.43−5.25 (m, 3H), 5.15 (dd, J = 10.01 Hz, 1H), 5.09 (dd, J = 10.33 Hz, 1H), 5.07−4.85 (m, 7H), 4.78 (s, 1H), 4.75−4.60 (m, 6H), 4.50 (d, J = 8.31 Hz, 1H), 4.44 (d, J = 8.45 Hz, 1H), 4.41 (d, J = 11.72 Hz, 1H), 4.37−4.04 (m, 22H), 4.04−3.88 (m, 7H), 3.87 (dd, J = 2.80, 1.61 Hz, 1H), 3.85− 3.57 (m, 11H), 3.57−3.26 (m, 6H), 2.79−2.61 (m, 2H), 2.10 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 2.00 (s, 6H), 1.99 (s, 3H), 1.98 (s, 3H), 1.97 (s, 3H), 1.97 (s, 3H), 1.96 (s, 3H), 1.93 (s, 6H), 1.91 (s, 3H), 1.91 (s, 3H), 1.90 (s, 3H), 1.88 (s, 3H), 1.79 (s, 3H), 1.79 (s, 3H), 1.76 (s, 3H), 1.71 (s, 3H), 1.41 (s, 3H), 1.38 (s, 3H), 1.24 (s, 3H), 1.23 (s, 3H). 447

DOI: 10.1021/acs.joc.7b02485 J. Org. Chem. 2018, 83, 443−451

Note

The Journal of Organic Chemistry orthoacetate (160 μL, 1.3 mmol) and p-toluenesulfonic acid monohydrate (10 mg, 58 μmol) were added, and then the reaction mixture was stirred at room temperature for 20 min. N,N′Diisopropylethylamine (15 uL, 88 μmol) was added to the mixture to quench the reaction. Subsequently, AcOH (15 μL, 0.25 μmol) was added to the mixture to avoid aspartimide formation under basic condition, and then the mixture was evaporated. To the resulting residue, 80% aq AcOH (2.0 mL) was added, and the mixture was stirred at room temperature for 20 min and then evaporated. The obtained residue was applied to silica gel column chromatography (EtOAc, and then EtOAc:MeOH = 20:1 to EtOAc:MeOH = 5:1) to remove the reagents. The resulting residue was further purified by RPHPLC (XBridge, ϕ10 mm × 250 mm, 0.1% aq TFA:90% aq CH3CN containing 0.1% TFA = 50:50 to 30:70 over 90 min at 2.5 mL/min). Fractions containing the desired product were collected and lyophilized to give diol 2 (5.0 mg, 1.7 μmol, 55%) as a white solid. [α]D22 −19.3 (c 0.45, CHCl3). HRMS (ESI/LIT-Orbitrap) m/z: [M + 2H]2+ calcd for C133H172N6O73 1510.4960, found 1510.4964. 1H NMR (400 MHz, CD3CN, HDO: 2.13 ppm) δ 7.90−7.82 (m, 2H), 7.81−7.71 (m, 2H), 7.63−7.54 (m, 3H), 7.50−7.42 (m, 2H), 7.38− 7.30 (m, 2H), 7.30−7.20 (m, 3H), 6.62−6.51 (m, 2H), 6.42 (d, J = 8.59 Hz, 1H), 6.37 (d, J = 9.07 Hz, 1H), 6.21 (d, J = 8.47 Hz, 1H), 5.41−5.24 (m, 3H), 5.21−4.86 (m, 11H), 4.77 (s, 1H), 4.75−4.58 (m, 6H), 4.48 (d, J = 8.38 Hz, 1H), 4.45−4.38 (m, 2H), 4.38−4.12 (m, 10H), 4.12−3.60 (m, 29H), 3.59−3.34 (m, 6H), 2.77−2.59 (m, 2H), 2.07 (s, 3H), 2.01 (s, 12H), 1.99 (s, 15H), 1.97 (s, 3H), 1.96 (s, 6H), 1.94 (s, 12H), 1.93 (s, 6H), 1.90 (s, 6H), 1.77 (s, 3H), 1.76 (s, 3H), 1.73 (s, 3H), 1.67 (s, 3H). 13C NMR (100 MHz, CD3CN, 118.3 ppm) δ 193.4, 172.3, 171.9, 171.8, 171.6(4), 171.5(5), 171.5, 171.4, 171.3, 171.2(4), 171.1(7), 171.0, 170.9(4), 170.9(0), 170.8, 170.6, 170.5, 159.6, 159.2, 156.9, 145.0, 142.1, 135.0, 129.9, 128.7(4), 128.6(6), 128.1, 126.1, 120.9, 101.9, 101.8(0), 101.7(6), 101.3, 100.2, 100.1, 99.7, 99.4, 79.4, 77.5, 77.4, 77.2, 77.0, 76.4, 75.6, 75.4, 75.0, 74.7, 74.1, 73.9, 73.8, 73.6, 73.4, 73.2, 73.1, 73.0, 72.7, 72.1, 71.9, 71.7, 70.9, 70.7, 70.6, 70.5, 70.2, 69.5, 68.1, 67.9, 67.5, 67.4, 66.1, 65.7, 63.6, 63.3, 63.1(4), 63.0(5), 62.8, 62.6, 62.5, 55.1, 54.6, 53.8, 53.7, 51.5, 47.9, 38.2, 23.0(8), 23.0(5), 23.0, 21.7, 21.5, 21.2, 21.1(1), 21.0(5), 21.0, 20.9. N2-(9-Fluorenylmethyloxycarbonyl)-N4-{O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetylα-D-mannopyranosyl)-(1→3)-O-[(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxyβ-D-glucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→6)]-O-(2,4-di-O-acetyl-β-D-mannopyranosyl)(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→4)-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)}-L-asparagine Phenacyl Ester (2). Improved protocol: To a solution of nonasaccharide 3 (113 mg, 57.1 μmol) in DMF/acetone (9.3 mL/3.0 mL), p-toluenesulfonic acid monohydrate (55.1 mg, 290 μmol) was added, and this mixture was kept at 37 °C using a dry bath incubator (major science). After 20 h, N,N′diisopropylethylamine (55.0 μL) was added to neutralize the reaction mixture. Furthermore, ice-cold Et2O was added to the mixture to give a precipitate, which was dissolved in distilled water and applied to ODS column chromatography (Cosmosil 75C18−OPN, ϕ25 mm × 150 mm, H2O then MeOH). Fractions containing oligosaccharides were collected, evaporated, dissolved in distilled water, and lyophilized. To the resulting residue in DMF/acetone (9.3 mL/3.0 mL), p-toluenesulfonic acid monohydrate (54.8 mg, 288 μmol) was added, and this mixture was kept at 37 °C using a dry bath incubator (major science). After 20 h, N,N′-diisopropylethylamine (55.0 μL) was added to neutralize the reaction mixture. Furthermore, ice-cold Et2O was added to the mixture to give a precipitate, which was dissolved in EtOAc/MeOH/H2O (4:2:2) and applied to silica gel column chromatography (ϕ10 mm × 80 mm, EtOAc/MeOH/H2O (4:2:2), then EtOAc/MeOH/H2O (2:2:1)) to roughly separate the di-O-isopropylidenated product from mono-O-isopropylidenated products and the starting material. Fractions containing the di-Oisopropylidenated product were collected and used for the next reaction without further purification (36.6 mg). To the resulting

residue in DMF (2.0 mL), 2-bromoacetophenone (10.6 mg, 53.3 μmol) and N,N′-diisopropylethylamine (15.5 μL, 89 μmol) were added, and this mixture was stirred at room temperature. After 1 h, ice-cold ether (13 mL) was added to give a precipitate. The precipitate was collected by centrifugation and then dried. Ac2O/ pyridine (1:1, 2.2 mL) was added to the resulting residue, followed by addition of DMAP (2.2 mg, 18 μmol). The resulting mixture was then stirred at room temperature. After 3 h, MeOH (5 mL) was added to the mixture on an ice bath, and the mixture was warmed to room temperature. The mixture was evaporated and co-evaporated with toluene. The residue was dissolved in MeOH and applied to gel filtration column chromatography (LH-20, ϕ18 mm × 320 mm, MeOH). Fractions containing the desired product were collected and evaporated. The resulting residue was dissolved in ice-cooled 50% aq TFA (1.0 mL), and the mixture was stirred at 0 °C for 5 min. The resulting mixture was immediately evaporated and coevaporated with toluene, To the resulting residue in CH2Cl2 (2.5 mL), trimethyl orthoacetate (624 μL, 5.0 mmol), and p-toluenesulfonic acid monohydrate (60.9 mg, 320 μmol) were added, and then the reaction mixture was stirred at room temperature for 2 h. N,N′Diisopropylethylamine (58.0 μL, 333 μmol) was added to the mixture to quench the reaction. Subsequently AcOH (57.0 μL, 997 μmol) was added to the mixture to avoid aspartimide formation under basic condition,16,19 and then the mixture was evaporated. To the resulting residue, 60% aq AcOH (2.0 mL) was added, and the mixture was stirred at room temperature for 1 h, evaporated, and co-evaporated with toluene. The resulting mixture was dissolved in aq CH3CN and purified by RP-HPLC (XBridge, ϕ10 mm × 250 mm, 0.1% aq TFA:90% aq CH3CN containing 0.1% TFA = 60:40 to 40:60 over 90 min at 4.0 mL/min). Fractions containing the desired product were collected and lyophilized to give diol 2 (9.7 mg, 3.2 μmol, 6% over 6 steps) as a white solid. HRMS (ESI/LIT-Orbitrap) m/z: [M + 2H]2+ calcd for C133H172N6O73 1510.4960, found 1510.4949. 1H NMR (700 MHz, CD3CN, 1.94 ppm) δ 8.00−7.89 (m, 2H), 7.89−7.79 (m, 2H), 7.73−7.60 (m, 3H), 7.60−7.50 (m, 2H), 7.48−7.38 (m, 2H), 7.38− 7.30 (m, 2H), 7.26 (d, J = 8.47 Hz, 1H), 6.68−6.58 (m, 2H), 6.47− 6.37 (m, 2H), 6.25 (d, J = 8.58 Hz, 1H), 5.46−5.34 (m, 3H), 5.29− 5.17 (m, 3H), 5.17−4.93 (8H), 4.84 (s, 1H), 4.80−4.64 (6H), 4.55 (d, J = 8.68 Hz, 1H), 4.53−4.46 (m, 2H), 4.46−4.20 (m, 10H), 4.18− 3.95 (m, 11H), 3.95−3.69 (m, 16H), 3.62−3.41 (m, 6H), 2.84−2.69 (m, 2H), 2.15 (s, 3H), 2.15 (s, 3H), 2.09 (s, 6H), 2.09 (s, 3H), 2.07 (s, 9H), 2.06 (s, 3H), 2.05 (s, 3H), 2.04 (s, 6H), 2.02 (s, 9H), 2,01 (s, 6H), 1.98 (s, 3H), 1.96 (s, 3H), 1.95 (s, 3H), 1.93 (s, 3H), 1.85 (s, 3H), 1.84 (s, 3H), 1.81 (s, 3H), 1.75 (s, 3H). 13C NMR (175 MHz, CD3CN, 118.3 ppm) δ 193.3, 172.0, 171.9, 171.8, 171.6(3), 171.5(5), 171.5(0), 171.4(7), 171.4, 171.2(2), 171.1(8), 171.1(6), 171. 0(2), 170.9(9), 170.9(2), 170.8(9), 170.8(0), 170.7(9), 170.6, 170.5, 156.9, 145.0, 142.1, 135.0, 129.9, 128.7(3), 128.6(5), 128.1, 126.1, 120.9, 101.9, 101.8, 101.3, 100.2, 100.1, 99.6, 99.4, 79.5, 77.5, 77.4, 77.2, 77.0, 76.4, 75.6, 75.4, 75.0, 74.6, 74.1, 73.9, 73.7, 73.6, 73.4, 73.2, 73.0, 72.7, 72.1, 71.9, 71.7, 70.9, 70.7, 70.6(4), 70.5(7), 70.5, 70.2, 69.5, 68.1, 67.9, 67.5, 67.4, 66.1, 65.7, 63.6, 63.2, 63.1(0), 63.0(5), 62.8, 62.6, 62.5, 55.0, 54.6, 53.8, 53.6, 51.4, 47.9, 38.2, 23.1(2), 23.0(7), 23.0, 21.7, 21.5, 21.2, 21.1(4), 21.1(1), 21.0(9), 21.0(5), 21.0(1), 20.9(9), 20.9(8), 20.9(6), 20.9(4), 20.9(3), 20.9(1). N2-(9-Fluorenylmethyloxycarbonyl)-N4-{O-(2,3,4,6-tetra-Oacetyl-β- D -galactopyranosyl)-(1→4)-O-(3,6-di-O-acetyl-2deoxy-2-(2,2,2-trichloroethoxy)-carbonylamino-β-D-glucopyranosyl)-(1→3)-O-(2,4,6-tri-O-acetyl-β- D-galactopyranosyl)(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→ 3)-O-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-O(3,6-di-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxy)-carbonylamino-β-D-glucopyranosyl)-(1→3)-O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2deoxy-β-D-glucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-Dmannopyranosyl)-(1→6)]-O-(2,4-di-O-acetyl-β-D-mannopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β- Dglucopyranosyl)-(1→4)-(2-acetamido-3,6-di-O-acetyl-2deoxy-β-D-glucopyranosyl)}-L-asparagine Phenacyl Ester (10). A mixture of glycosyl donor 9 (28.5 mg, 33.1 μmol) and 448

DOI: 10.1021/acs.joc.7b02485 J. Org. Chem. 2018, 83, 443−451

Note

The Journal of Organic Chemistry oligosaccharyl acceptor 2 (10 mg, 3.3 μmol) was dried in vacuo for 2 h. Distilled CH2Cl2 (400 μL) and freshly activated 4 A molecular sieves (40 mg) were added to the mixture at room temperature. After addition of NIS (12.5 mg, 55.6 μmol), the reaction temperature was allowed to cool to 0 °C, and then TfOH (0.99 μmol) was added to the mixture. The resulting mixture was stirred at 0 °C. After 30 min, additional glycosyl donor (66 μmol), NIS (111 μmol), and TfOH (0.99 μmol) were added to the mixture. The reaction mixture was stirred at 0 °C for another 30 min. Then, the mixture was applied to silica gel chromatography (ϕ10 mm × 100 mm, EtOAc to EtOAc:MeOH = 20:1) to remove donor derivatives and furthermore applied to gel permeation chromatography (Sephadex LH-20, MeOH). The resulting residue was purified by RP-HPLC (XBridge, 0.1% aq TFA:90% aq CH3CN containing 0.1% TFA = 45:55 to 25:75 over 90 min at 2.5 mL/min). Fractions containing the desired product were collected and lyophilized to give diglycosylated oligosaccharide 10 (1.3 mg, 0.29 μmol, 9%) as a white solid. HRMS (ESI/LITOrbitrap) m/z: [M + 3H]3+ calcd for C187H241Cl6N8O107 1506.7259, found 1506.7260. 1H NMR (400 MHz, CD3CN, HDO: 2.13 ppm) δ 7.97−7.85 (m, 2H), 7.85−7.75 (m, 2H), 7.70−7.56 (m, 3H), 7.56− 7.45 (m, 2H), 7.43−7.35 (m, 2H), 7.35−7.24 (m, 3H), 6.70−6.55 (m, 2H), 6.34 (d, J = 8.87 Hz, 1H × 2), 6.24 (d, J = 8.47 Hz, 1H), 6.09 (d, J = 9.39 Hz, 1H), 5.98 (d, J = 9.53 Hz, 1H), 5.41−5.29 (m, 4H), 5.29−5.23 (m, 2H), 5.20 (dd, J = 9.67 Hz, 1H), 5.15−4.61 (m, 23H), 4.61−4.17 (m), 4.17−3.22 (m), 2.80−2.70 (m, 2H), 2.07 (s, 3H), 2.05 (s, 9H), 2.04 (s, 12H), 2.01 (s, 12H), 1.99 (s, 3H), 1.98 (s, 3H), 1.97 (s, 3H), 1.96 (s, 6H), 1.95 (s, 6H), 1.94 (s), 1.86 (s, 6H), 1.82 (s, 3H), 1.82 (s, 3H), 1.77 (s, 3H), 1.71 (s, 3H). 13C NMR (100 MHz, CD3CN, 118.3 ppm) δ 193.3, 172.1, 172.0, 171.8, 171.6, 171.5, 171.4, 171.3, 171.2, 171.1, 171.0(2), 170.9(9), 170.9, 170.8(3), 170.7(6), 170.7, 170.6, 170.5, 170.4, 156.8, 155.1, 145.1, 142.1, 135.0, 129.9, 128.8, 128.7, 128.1, 126.2, 121.0, 102.2, 102.0, 101.6, 101.5, 101.4, 100.3, 100.1, 99.7, 96.6(9), 96.6(6), 79.4, 79.1, 79.0, 77.6, 77.5, 77.2, 77.0, 76.9, 75.7, 75.6, 75.1(4), 75.0(7), 75.0, 74.9, 74.2, 74.1, 73.7(9), 73.7(5), 73.3, 73.2, 72.7, 72.5, 72.2, 71.8, 71.6, 71.5, 71.4, 71.1, 71.0, 70.9, 70.6, 70.5, 70.3, 70.0, 69.5, 68.1, 68.0(2), 67.9(9), 67.8, 67.4, 66.2, 65.7, 63.7, 63.3, 63.1, 62.6, 62.0, 61.8, 57.1, 54.9, 54.6, 54.1, 53.4, 51.5, 47.9, 38.2, 23.1(4), 23.0(9), 21.7, 21.5, 21.2, 21.1(2), 21.0(9), 21.0, 20.9(1), 20.8(5), 20.7. N2-(9-Fluorenylmethyloxycarbonyl)-N4-{O-β-D-galactopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)(1→3)-O-β- D -galactopyranosyl-(1→4)-O-(2-acetamido-2deoxy-β- D-glucopyranosyl)-(1→2)-O-α- D -mannopyranosyl(1→3)-O-[β- D -galactopyranosyl-(1→4)-O-(2-acetamido-2deoxy-β- D-glucopyranosyl)-(1→3)-O-β-D -galactopyranosyl(1→4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)-Oα-D-mannopyranosyl-(1→6)]-O-β-D-mannopyranosyl-(1→4)O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-(2-acetamido-2-deoxy-β-D-glucopyranosyl)}-L-asparagine (1). To a solution of diglycosylated product 10 (2.8 mg, 0.62 μmol) in AcOH/Ac2O (1 mL/1 mL), activated Zn (228 mg) was added. The resulting mixture was stirred at room temperature. After 1 h, MeOH was added to the mixture, and the resulting mixture was evaporated. The resulting residue was applied to silica gel column chromatography (ϕ10 mm × 100 mm, EtOAc, EtOAc:MeOH = 10:1, 5:1, and 3:1) and moreover applied to gel permeation chromatography (Sephadex LH-20, CH3CN). A solution of MeOH/5 M NaOH (400 μL/240 μL) was added to the resulting residue, and the mixture was stirred at room temperature. After 15 min, 5 M HCl (240 μL) was added to the reaction mixture, and then NaHCO3 (2.2 mg, 26 μmol), Fmoc-OSu (7.5 mg, 22 μmol), and CH3CN (400 μL) were added to the reaction mixture, which was stirred at room temperature for 2 h. The mixture was applied to ODS column chromatography (H2O, then CH3CN/H2O (3:7)) and gel permeation chromatography (Sephadex G25, H2O). Additional RP-HPLC purification (Cadenza Imtact, 0.1% aq TFA/90% aq CH3CN containing 0.1% TFA = 80:20 to 70:30 over 30 min at 1 mL/min) afforded the desired product 1 (0.3 mg, 0.1 μmol, 20% over 3 steps) as a white solid. HRMS (ESI/LIT-Orbitrap) m/z: [M + 2H] 2+ calcd for C109H168N8O70 1354.4911, found 1354.4905. 1H NMR (700 MHz, D2O, 4.79 ppm) δ 7.96−7.90 (m, 2H), 7.76−7.68 (m, 2H), 7.54−

7.48 (m, 2H), 7.48−7.41 (m, 2H), 5.12 (s, 1H), 5.00 (d, J = 9.45 Hz, 1H), 4.93 (s, 1H), 4.77 (s, 1H), 4.73−4.67 (m), 4.61−4.55 (m), 4.52−4.43 (m), 4.39−4.33 (m, 1H), 4.28−4.23 (m, 2H), 4.19 (brs, 1H), 4.16 (brs, 2H), 4.11 (brs, 1H), 4.03−3.41 (m), 2.77−2.68 (m, 1H), 2.58−2.48 (m, 1H), 2.07 (s, 3H), 2.05 (s, 3H), 2.05 (s, 3H), 2.03 (s, 6H), 1.89 (s, 3H). Chemical shift of 13C (175 MHz, D2O, Chemical shift was picked up from HSQC except for quaternary carbon, FID resolution 68.8 Hz) δ 129.0, 128.4, 126.1, 121.1, 103.8, 103.7, 102.2, 101.0, 100.5, 100.4, 97.9, 83.0, 81.4, 80.3, 79.5, 79.0, 72.3, 71.2, 77.1, 76.3, 75.8, 75.5, 74.5, 73.8, 73.7, 73.4, 73.0, 71.9, 71.1, 70.9, 70.3, 69.5, 69.2, 68.2, 67.3, 62.6, 61.9, 60.8, 60.6, 56.1, 55.7, 54.7, 53.7, 47.8, 39.5, 23.3, 23.0. N2-(9-Fluorenylmethyloxycarbonyl)-N4-{O-(2,3,4,6-tetra-Oacetyl-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-Oacetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→3)-O-[(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2deoxy-β-D-glucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-Dmannopyranosyl)-(1→6)]-O-(2,4-di-O-acetyl-β-D-mannopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β- Dglucopyranosyl)-(1→4)-(2-acetamido-3,6-di-O-acetyl-2deoxy-β-D-glucopyranosyl)}-L-asparagine Phenacyl Ester (11). To a solution of mono-O-isopropylidenated oligosaccharide 5 (25.0 mg, 12.4 μmol) in DMF (2.5 mL), 2-bromoacetophenone (7.9 mg, 40 μmol) and N,N′-diisopropylethylamine (10.8 μL, 62 μmol) were added, and the mixture was stirred at room temperature. After 2 h, ice-cold ether (13 mL) was added to give a precipitate. The precipitate was collected by centrifugation and then dried. Ac2O/ pyridine (1:1, 1.2 mL) was added to the resulting residue, followed by addition of DMAP (1.5 mg, 12 μmol). The resulting mixture was then stirred at room temperature. After 2 h, MeOH (5 mL) was added to the mixture on an ice bath, and the mixture was warmed to room temperature. The mixture was evaporated and coevaporated with toluene. The residue was dissolved in MeOH and applied to gel filtration column chromatography (LH-20, ϕ18 mm × 320 mm, MeOH). Fractions containing the desired product were collected and evaporated. The resulting residue was dissolved in ice-cooled 50% aq TFA (1.0 mL), and the mixture was stirred at 0 °C for 5 min. The resulting mixture was immediately evaporated and co-evaporated with toluene. To the resulting residue in CH2Cl2 (1.5 mL), trimethyl orthoacetate (368 μL, 2.9 mmol), and p-toluenesulfonic acid monohydrate (35.9 mg, 189 μmol) were added, and then the reaction mixture was stirred at room temperature for 2 h. N,N′Diisopropylethylamine (33.0 μL, 189 μmol) was added to the mixture to quench the reaction. Subsequently, AcOH (32.0 μL, 560 μmol) was added to the mixture to avoid aspartimide formation under basic condition, and then the mixture was evaporated. To the resulting residue, 60% aq AcOH (1.0 mL) was added, and the mixture was stirred at room temperature for 5 min, evaporated, and co-evaporated with toluene. The resulting mixture was dissolved in aq CH3CN and purified by RP-HPLC (XBridge, ϕ10 mm × 250 mm, 0.1% aq TFA:90% aq CH3CN containing 0.1% TFA = 60:40 to 40:60 over 90 min at 4.0 mL/min). Fractions containing the desired product were collected and lyophilized to give diol 11 (11.7 mg, 3.82 μmol, 31% over 5 steps) as a white solid. [α]D22 −18.8 (c 0.50, H2O). HRMS (ESI/LIT-Orbitrap) m/z: [M + 2H]2+ calcd for C135H174N6O74 1531.5013, found 1531.5005. 1H NMR (700 MHz, CD3CN, 1.94 ppm) δ 7.98−7.88 (m, 2H), 7.88−7.79 (m, 2H), 7.71−7.60 (m, 2H), 7.58−7.48 (m, 2H), 7.46−7.38 (m, 2H), 7.36−7.26 (m, 3H), 6.75− 6.63 (m, 2H), 6.51 (d, J = 9.45 Hz, 1H), 6.46 (d, J = 8.83 Hz, 1H), 6.27 (d, J = 8.99 Hz), 5.46−5.34 (m, 3H), 5.30 (dd, J = 3.41 Hz, 1H), 5.24 (dd, J = 3.21 Hz, 1H), 5.21 (dd, J = 10.04, 10.04 Hz, 1H), 5.16− 4.94 (m, 9H), 4.90 (dd, J = 10.47, 8.19 Hz, 1H), 4.83 (s, 1H), 4.78 (d, J = 10.47, 3.30 Hz, 1H), 4.76−4.72 (m, 3H), 4.72−4.67 (m, 1H), 4.59−4.54 (m, 2H), 4.52−4.46 (m, 2H), 4.43 (d, J = 8.08 Hz, 1H), 4.42−4.21 (m, 8H), 4.18−3.94 (m, 14H), 3.94−3.70 (m, 14H), 3.61−3.49 (m, 4H), 3.49−3.39 (m, 2H), 2.81−2.69 (m, 2H), 2.15 (s, 3H), 2.09 (s, 6H), 2.09 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.04 (s, 3H), 2.02 (s, 6H), 2.01 (s, 6H), 2.00 (s, 9H), 1.98 (s, 3H), 1.96 (s, 3H), 1.95 (s, 3H), 1.93 (s, 3H), 1.89 (s, 3H), 1.86 (s, 3H), 1.85 (s, 3H), 1.82 (s, 3H), 1.76 (s, 449

DOI: 10.1021/acs.joc.7b02485 J. Org. Chem. 2018, 83, 443−451

Note

The Journal of Organic Chemistry 3H). 13C NMR (175 MHz, CD3CN, 118.3 ppm) δ 193.3, 172.3, 171.9, 171.8, 171.6(4), 171.5(6), 171.5(4), 171.4(9), 171.4(7), 171.4(3), 171.3(5), 171.2(3), 171.1(5), 171.1(2), 171.0(7), 171.0(0), 170.9(7), 170.9(3), 170.8(9), 170.8, 170.7, 170.6, 170.5, 170.4, 156.9, 145.0, 144.9, 142.0, 135.0, 129.8, 128.7, 128.6, 128.1, 126.1, 120.9, 101.9, 101.7, 101.5, 101.2, 100.1(4), 100.0(6), 99.6, 99.4, 79.4, 77.5, 77.4, 77.2, 77.0, 76.4, 75.5, 75.4, 75.0, 74.6, 74.1, 73.9, 73.6, 73.4, 73.1, 73.0, 72.7, 72.0, 71.7, 71.6, 71.4, 70.9, 70.6, 70.5, 70.2, 70.0, 69.5, 68.1, 67.9, 67.5, 67.4, 66.0, 65.7, 63.6, 63.2, 63.1, 63.0, 62.8, 62.6, 62.5, 61.9, 55.1, 54.6, 53.8, 53.7, 51.4, 47.9, 38.1, 23.1, 23.0, 22.9, 21.6, 21.5, 21.2, 21.1(2), 21.1(0), 21.0(3), 21.0(0), 20.9(8), 20.9(6), 20.9(2), 20.8(9), 20.8(5), 20.8, 20.6(9), 20.6(8). N2-(9-Fluorenylmethyloxycarbonyl)-N4-{O-(2,3,4,6-tetra-Oacetyl-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-Oacetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→3)-O-[(2,3,4,6-tetra-O-acetyl-β-Dgalactopyranosyl)-(1→4)-O-(3,6-di-O-acetyl-2-deoxy-2-(2,2,2trichloroethoxy)-carbonylamino-β-D-glucopyranosyl)-(1→3)O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1→2)-O(3,4,6-tri-O-acetyl-α-D-mannopyranosyl)-(1→6)]-O-(2,4-di-Oacetyl-β-D-mannopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-Oacetyl-2-deoxy-β-D-glucopyranosyl)-(1→4)-(2-acetamido-3,6di-O-acetyl-2-deoxy-β-D-glucopyranosyl)}-L-asparagine Phenacyl Ester (12). A mixture of glycosyl donor 9 (59.3 mg, 68.9 μmol) and oligosaccharyl acceptor 11 (10.5 mg, 3.43 μmol) was dried in vacuo for 2 h. Distilled CH2Cl2 (425 μL) and freshly activated 4 A molecular sieves (48.0 mg) were added to the mixture at room temperature. After addition of NIS (12.2 mg, 54.2 μmol), the reaction mixture was allowed to cool to 0 °C, and then TfOH (2.5 μmol) was added to the mixture, and the mixture was stirred at 0 °C. After 3 h, the mixture was warmed to room temperature and stirred for another 3 h. Then, the mixture was applied to silica gel chromatography (ϕ10 mm × 100 mm, EtOAc:hexane = 1:1, EtOAc, then EtOAc:MeOH = 5:1) to remove donor derivatives. Fractions containing the desired product and starting material were collected and evaporated. The resulting residue was purified by RP-HPLC (XBridge, 0.1% aq TFA:90% aq CH3CN containing 0.1% TFA = 50:50 to 30:70 over 90 min at 4.0 mL/min). Fractions containing the desired product and starting material were collected, respectively, and lyophilized to give the desired product 12 (2.8 mg, 0.73 μmol, 21%) and starting material 11 (1.7 mg, 0.56 μmol, 16%) as white solids. HRMS (ESI/LITOrbitrap) m/z: [M + 2H]2+ calcd for C162H208Cl3N7O91 1906.0459, found 1906.0441. 1H NMR (700 MHz, CD3CN, 1.94 ppm) δ 7.99− 7.88 (m, 2H), 7.88−7.79 (m, 2H), 7.72−7.61 (m, 3H), 7.58−7.50 (m, 2H), 7.47−7.36 (m, 2H), 7.36−7.27 (m, 2H), 6.72−6.63 (m, 2H), 6.46 (d, J = 8.96 Hz, 1H), 6.43 (d, J = 8.78 Hz, 1H), 6.29 (d, J = 8.60 Hz, 1H), 6.13 (d, J = 9.31 Hz, 1H), 5.46−5.32 (m, 4H), 5.32− 5.28 (m, 2H), 5.27 (dd, J = 2.52 Hz, 1H), 5.22 (dd, J = 9.82, 9.82 Hz, 1H), 5.17−4.93 (m, 12H), 4.93−4.87 (m, 2H), 4.85 (s, 1H), 4.82 (dd, J = 9.13 Hz, 1H), 4.77 (dd, J = 10.42, 3.25 Hz, 1H), 4.75 (s, 1H), 4.72−4.66 (m, 1H), 4.64−4.48 (m, 6H), 4.47−4.20 (m, 11 H), 4.20− 3.94 (m, 16H), 3.94−3.69 (m, 16H), 3.65 (ddd, J = 7.55 Hz, 1H), 3.63−3.48 (m, 5H), 3.48−3.34 (m, 3H), 2.82−2.74 (m, 2H), 2.15 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 2.04 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 2.00 (s, 3H), 1.98 (s, 6H), 1.97 (s, 3H), 1.96 (s, 3H), 1.93 (s, 3H), 1.89 (s, 3H), 1.89 (s, 3H), 1.85 (s, 3H), 1.84 (s, 3H), 1.80 (s, 3H), 1.74 (s, 3H). 13C NMR (175 MHz, CD3CN, 118.3 ppm) δ 193.3, 172.0, 171.9, 171.7(7), 171.7(6), 171.6(1), 171.5(6), 171.5(1), 171.4(8), 171.4, 171.3(4), 171.2(6), 171.2(3), 181.1(9), 171.1(4), 171.1(2), 171.0(7), 171.0(6), 170.9(9), 170.9(6), 170.9(3), 170.8(9), 170.8(6), 170.8(3), 170.7(7), 170.7(2), 170.7(0), 170.6, 170.5, 170.4(1), 170.3(9), 170.3(7), 156.9, 155.4, 145.0(3), 144.9(6), 142.1, 135.0, 129.0, 129.8, 128.7, 128.1, 126.1, 120.9, 102.2, 101.9, 101.5, 101.4, 100.3, 100.1, 99.9, 99.6, 96.7, 79.4, 79.1, 77.6, 77.5, 77.2, 77.1, 76.9, 76.7, 75.7, 75.6, 75.1, 74.9, 74.1, 74.0, 73.8, 73.6, 73.3, 73.1, 72.8, 72.4, 71.8, 71.6, 71.4(3), 71.3(5), 71.1, 70.9, 70.5(3), 70.4(7), 70.2, 70.0, 69.9, 69.5, 68.1, 68.0(0), 67.9(6), 67.9, 67.4, 66.1, 65.7, 63.6, 63.2, 63.1(2), 63.09,

63.0, 62.8, 62.6(2), 62.5(5), 61.9, 61.8, 57.2, 55.0, 54.5, 54.0, 53.5, 51.5, 47.9, 38.1, 23.1(3), 23.1(2), 23.0(7), 23.0(6), 21.7, 21.5, 21.2(1), 21.1(8), 21.1(3), 21.1(0), 21.0(5), 21.0(2), 20.9(8), 20.9(7), 20.9(3), 20.9(1), 20.8(9), 20.8. N2-(9-Fluorenylmethyloxycarbonyl)-N4-{O-β-D-galactopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)(1→2)-O-α-D-mannopyranosyl-(1→3)-O-[β-D-galactopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→ 3)-O-β-D-galactopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-βD-glucopyranosyl)-(1→2)-O-α-D-mannopyranosyl-(1→6)]-O-βD-mannopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-(2-acetamido-2-deoxy-β- D -glucopyranosyl)}-L-asparagine (13). To a solution of glycosylated product 12 (3.9 mg, 1.0 μmol) in THF/AcOH/Ac2O (480 μL/360 μL/240 μL), activated Zn (266 mg) was added on an ice bath. The resulting mixture was stirred, and the reaction mixture was warmed to room temperature. After 23 h, MeOH (1 mL) was added to the reaction mixture, which was further diluted with CH3CN. The resulting mixture was filtered through a pad of Celite and evaporated. To the resulting residue, a solution of MeOH/5 M NaOH (500 μL/500 μL) was added, and the mixture was stirred at 0 °C. After 1 h, 5 M HCl (500 μL) was added to the reaction mixture, and then NaHCO3 (11.0 mg, 131 μmol) and Fmoc-OSu (3.4 mg, 10 μmol) were added to the reaction mixture, which was stirred at room temperature for 2 h. The mixture was applied to gel permeation chromatography (Sephadex LH-20, ϕ10 mm × 180 mm, H2O). Fractions containing the desired product was collected and lyophilized. To the resulting residue, a solution of NaHCO3 (11.4 mg, 136 μmol) in H2O (500 μL) and a solution of Fmoc-OSu (3.9 mg, 12 μmol) in CH3CN (500 μL) were added, and the mixture was stirred at room temperature for 4 h to ensure the completion of the introduction of Fmoc group. The resulting mixture was diluted with H2O (2.0 mL), filtered, and purified by RP-HPLC purification (Proteonavi, 0.1% aq TFA:90% aq CH3CN containing 0.1% TFA = 80:20 to 60:40 over 30 min at 4.0 mL/min) afforded the desired product 13 (0.7 mg, 0.3 μmol, 30% over 3 steps) as a white solid. HRMS (ESI/LIT-Orbitrap) m/z: [M + H]+ calcd for C95H144N7O60 2342.8426, found 2342.8416. 1H NMR (400 MHz, D2O, 4.79 ppm) δ 7.99−7.86 (m, 2H), 7.78−7.65 (m, 2H), 7.57−7.48 (m, 2H), 7.48−7.38 (m, 2H), 5.13 (s, 1H), 5.01 (d, J = 9.64 Hz, 1H), 4.94 (s, 1H), 4.77 (s, 1H), 4.71 (d, J = 8.14 Hz, 1H), 4.68−4.51 (m), 4.51−4.42 (m), 4.42−4.30 (m), 4.26 (brs, 1H), 4.20 (brd, J = 2.95 Hz, 1H), 4.17 (brd, J = 2.79 Hz, 1H), 4.11 (brd, J = 2.48 Hz, 1H), 4.07−3.34 (m), 2.84−2.52 (m, 2H), 2.08 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.90 (s, 3H). Chemical shift of 13C (175 MHz, D2O, Chemical shift was picked up from HSQC except for quaternary carbon, FID resolution 25.6 Hz) δ 129.0, 128.5, 126.1, 121.2, 103.9, 103.7, 102.2, 101.4, 100.5, 100.4, 98.0, 83.0, 81.4, 80.4, 79.5, 79.4, 79.1, 77.3, 77.2, 77.1, 76.3, 75.8, 75.7, 75.5, 75.3, 74.5, 73.8, 73.6, 73.5, 73.1, 73.0, 72.9, 71.9, 71.1, 70.9, 70.3, 69.5, 69.2, 68.3, 67.8, 67.4, 66.7, 66.6, 62.6, 61.9, 60.9, 60.7, 56.1, 55.9, 55.8, 54.7, 52.6, 47.9, 38.9, 23.3.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.7b02485.

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HPLC, MS, 1H, 13C, and HSQC data of the compounds (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] ORCID

Yuta Maki: 0000-0002-5838-302X Ryo Okamoto: 0000-0001-9529-2525 Masayuki Izumi: 0000-0001-6486-9678 Yasuhiro Kajihara: 0000-0002-6656-2394 450

DOI: 10.1021/acs.joc.7b02485 J. Org. Chem. 2018, 83, 443−451

Note

The Journal of Organic Chemistry Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Japan Society for the Promotion of Science (Grants-in-Aid for Creative Scientific Research: 26248040, 17H01214 to Y.K.) is acknowledged and appreciated. We thank GlyTech, Inc., for preparing a biantennary oligosaccharide.

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REFERENCES

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DOI: 10.1021/acs.joc.7b02485 J. Org. Chem. 2018, 83, 443−451