Semisynthesis of Complex-Type Biantennary Oligosaccharides

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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 J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02485 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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The Journal of Organic Chemistry

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 e-mail: [email protected]

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-3,4-OH 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.

Repetition of a Gal-β1,4-GlcNAc moiety (poly-LacNAc) at the non-reducing 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 β-galactoside 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

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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 times-repeated 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/deprotection 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 ten conversion steps from biantennary asialo-nonasaccharide 3, which can be isolated from hen egg yolk in multi-gram quantities.14,15 Previously, we reported a novel semisynthetic strategy using nonasaccharide 3 to obtain triantennary oligosaccharides within ten 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).


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Scheme 1. Semisynthetic strategy of complex-type oligosaccharide 1 containing LacNAc repeating units from isolated nonasaccharide 3.

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 Figure S1. The isopropylidenation 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 towards mono- and di-saccharides 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.

Scheme 2. Isopropylidenation reaction of asialo-nonasaccharide 3. Reagents and condition; (i) TsOH, acetone/DMF (1:2), 37 oC, 29 h, 12% 4, 13% 5, 14% 6, 8% 3.

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-phased 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). 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 (Figure S3), indicating that Galf,i-3,4-OH was successfully protected with isopropylidene groups.


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Scheme 3. Synthesis of glycosyl acceptor 2. 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 oC, 5 min, 58%. (iv) CH3C(OCH3)3, TsOH, CH2Cl2, 20 min. (v) 80% aq. AcOH, 20 min, 55% (2 steps).

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-O-acetylated 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), which was prepared as previously described.16 Unfortunately, no selectivity was found in the nucleophilicity between the 3-OH 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 ring-opening 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 twice to enhance the attachment of isopropylidene groups, and the di-O-isopropylidenated product 6 was roughly purified by silica gel column chromatography (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 (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). Based on the RP-HPLC peak areas, we obtained the desired di-glycosylated product 10 in 34% conversion yield along with two mono-glycosylated products (10% and 6% conversion yields) and acceptor 2 (3% recovery yield) as shown in 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,l-H1 (Figure S8), indicating the success of the glycosylation reaction.


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Scheme 4. Synthesis of complex-type oligosaccharide 1 containing LacNAc repeating units. Reagents and conditions; (i) donor 9, NIS, TfOH, CH2Cl2, 0 oC, 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).

Stepwise deprotections of the protected oligosaccharide 10 were then performed (Scheme 4). The Troc group of protected oligosaccharide 10 was reduced and simultaneously acetylated by zinc and Ac2O. Subsequent saponification of acetyl groups and re-introduction 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

mono-O-isopropylidenated

usually

have

oligosaccharide

5

asymmetric and

performed

antennae.

We

used

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%, Figure S11). Moreover, sequential deprotection steps afforded asymmetric LacNAc-extended oligosaccharide 13 in 30% yield (3 steps).



Scheme 5. Synthesis of complex-type biantennary oligosaccharide 13 containing a LacNAc repeating unit. Reagents and conditions; (i) 2-bromoacetophenone, iPr2NEt, DMF, 2 h. (ii) Ac2O/pyridine (1:1), DMAP, 2 h. (iii) 50% aq. TFA, 0 oC, 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 oC 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).

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 ten-step chemical sequences. This strategy would allow for the preparation of a library of LacNAc-extended oligosaccharides by using various glycosyl donors.


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Experimental section General Experimental Methods 1

H 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 HSQC-TOCSY. 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 multi wavelength 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 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 XBridgeTM prep column (Waters, 10 × 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 × 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-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 → 2)-O-α-D-mannopyranosyl-(1

→

6)]-O-β-D-mannopyranosyl-(1

4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1


→ →


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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-D-glucopyranosyl)-(1 → 2)-O-α-D-mannopyranosyl-(1 → →

3)-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 → 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 (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-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-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 oC 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, φ20×250 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 × 150 mm) to remove ammonium acetate, respectively. Fractions containing

the

desired

mono-O-isopropylidenated mono-O-isopropylidenated

products

were

oligosaccharide oligosaccharide

4 5

collected

and

lyophilized

(24.4

mg,

12.1

(27.4

mg,

13.6

to

give

µmol,

µmol,

12%),

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


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(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, 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). NMR

(175

MHz,

13

C

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-acetyl-3,4-O-isopropylidene-β-D-



Page 12 of 25

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-O-acetyl-3,4-O-isopropylidene-β-D-galactopyranosyl)-(1

→

4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(1

→


→

6)]-O-(2,4-di-O-acetyl-β-D-mannopyranosyl)-(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), 2-bromoacetophenone (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 ×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).

13

C NMR (175 MHz, CD3CN, 118.3 ppm) δ 193.3, 172.5, 171.9,


Page 13 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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-acetyl-β-D-galactopyranosyl)-(1

→


→


→

3)-O-[(2,6-di-O-acetyl-β-D-galactopyranosyl)-(1

→


→


→


→


→


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 oC 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 RP-HPLC (XBridge, φ10×250 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),



Page 14 of 25

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

→


→

3)-O-[(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1

→


→


→


→


→


phenacyl

ester (2). To a solution of tetraol 8 (8.8 mg, 3.0 µmol) in CH2Cl2 (400 µL), trimethyl 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 RP-HPLC (XBridge, φ10×250 mm, 0.1% aq. TFA:


Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 = 8.38Hz, 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

→


→


→


→


→


→


→


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 oC 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× 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

o

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 (φ10×80 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-O-isopropylidenated 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, φ18×320 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 oC for 5 min. The resulting mixture was immediately evaporated and co-evaporated 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, φ10×250 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


Page 16 of 25

Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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).

13

C 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), 1 71.1(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, 9 9.6, 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, 7 3.2, 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, 6 7.4, 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, 3 8.2, 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), 2 0.9(8), 20.9(6), 20.9(4), 20.9(3), 20.9(1).

N2-(9-Fluorenylmethyloxycarbonyl)-N4-{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-glucopyranosy l)-(1

→

3)-O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1

→


→


→

3)-O-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1

→


→


→


→


→


→


→



phenacyl


Page 18 of 25

ester (10). A mixture of glycosyl donor 9 (28.5 mg, 33.1 µmol) and oligosaccharyl acceptor 2 (10 mg, 3.3 µmol) was dried in vacuo for 2 h. Distilled CH2Cl2 (400 µL) and freshly activated 4A 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 oC. And then TfOH (0.99 µmol) was added to the mixture. The resulting mixture was stirred at 0 oC. 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 oC for another 30 min. Then, the mixture was applied to silica gel chromatography (φ10×100 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 di-glycosylated oligosaccharide 10 (1.3 mg, 0.29 µmol, 9%) as a white solid. HRMS (ESI/LIT-Orbitrap) 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).

13

C 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-2-deoxy-β-D-glucopyranosyl)-(1 → 2)-O-α-D-mannopyranosyl-(1 →


Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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-β-D-glucopyranosyl)-(1 → 2)-O-α-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

di-glycosylated 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 (φ10×100 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-O-acetyl-β-D-galactopyranosyl)-( 1

→




→ →


Page 20 of 25


→


→


→


→


→


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 co-evaporated with toluene. The residue was dissolved in MeOH and applied to gel filtration column chromatography (LH-20, φ18×320 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 oC 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, φ10×250 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


Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(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,

3H).

13

C

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, 1 70.6, 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, 1 01.9, 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-O-acetyl-β-D-galactopyranosyl)-( 1

→


→


→

3)-O-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1

→


→


→


→


→


→


→


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 4A 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 oC. And



then TfOH (2.5 µmol) was added to the mixture, and the mixture was stirred at 0 oC. 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 (φ10×100 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/LIT-Orbitrap) 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.


Page 22 of 25

Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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-β-D-glucopyranosyl)-(1 → 2)-O-α-D-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 oC. 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× 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.

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

Author Information Corresponding Author *E-mail: [email protected]

Acknowledgements 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. The authors thank GlyTech, Inc., for preparing a biantennary oligosaccharide.

References 1. Essentials of glycobiology; Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze, H. H.; Stanley, P.; Bertozzi, C. R.; Hart, G. W.; Etzler, M. E., Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2009. 2. Thiemann, S.; Baum, L. G., Annu. Rev. Immunol. 2016, 34, 243. 3. Liu, F.-T.; Rabinovich, G. A., Nat. Rev. Cancer 2005, 5, 29. 4. Barondes, S. H.; Castronovo, V.; Cooper, D. N. W.; Cummings, R. D.; Drickamer, K.; Felzi, T.; Gitt, M. A.; Hirabayashi, J.; Hughes, C.; Kasai, K.-i.; Leffler, H.; Liu, F.-T.; Lotan, R.; Mercurio, A. M.; Monsigny, M.; Pillai, S.; Poirer, F.; Raz, A.; Rigby, P. W. J.; Rini, J. M.; Wang, J. L., Cell 1994. 76, 597. 5. Hirabayashi, J.; Hashidate, T.; Arata, Y.; Nishi, N.; Nakamura, T.; Hirashima, M.; Urashima, T.; Oka, T.; Futai, M.; Muller, W. E. G.; Yagi, F.; Kasai, K.-i., Biochim. Biophys. Acta 2002, 1572, 232. 6. Shahrokh, Z.; Royle, L.; Saldova, R.; Bones, J.; Abrahams, J. L.; Artemenko, N. V.; Flatman, S.; Davies, M.; Baycroft, A.; Sehgal, S.; Heartlein, M. W.; Harvey, D. J.; Rudd, P. M., Mol. Pharm. 2011, 8, 286. 7. Shimizu, H.; Ito, Y.; Kanie, O.; Ogawa, T., Bioorg. Med. Chem. Lett. 1996, 6, 2841.


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8. Ueki, A.; Takano, Y.; Kobayashi, A.; Nakahara, Y.; Hojo, H.; Nakahara, Y., Tetrahedron 2010, 66, 1742. 9. Wang, Z.; Chinoy, Z. S.; Ambre, S. G.; Peng, W.; McBride, R.; de Vries, R. P.; Glushka, J.; Paulson, J. C.; Boons, G. J., Science 2013, 341, 379. 10. Gagarinov, I. A.; Li, T.; Toraño, J. S.; Caval, T.; Srivastava, A. D.; Kruijtzer, J. A. W.; Heck, A. J. R.; Boons, G.-J., J. Am. Chem. Soc. 2017, 139, 1011. 11. Nycholat, C. M.; Peng, W.; McBride, R.; Antonopoulos, A.; de Vries, R. P.; Polonskaya, Z.; Finn, M. G.; Dell, A.; Haslam, S. M.; Paulson, J. C., J. Am. Chem. Soc. 2013, 135, 18280. 12. Zhu, X.; Schmidt, R. R., Angew. Chem. Int. Ed. 2009, 48, 1900. 13. Kaeothip, S.; Demchenko, A. V., Carbohydr. Res. 2011, 346, 1371. 14. Seko, A.; Koketsu, M.; Nishizono, M.; Enoki, Y.; Ibrahim, H. R.; Juneja, L. R.; Kim, M.; Yamamoto, T., Biochim. Biophys. Acta 1997, 1335, 23. 15. Kajihara, Y.; Suzuki, Y.; Yamamoto, N.; Sasaki, K.; Sakakibara, T.; Juneja, L. R., Chem. – Eur. J. 2004, 10, 971. 16. Maki, Y.; Okamoto, R.; Izumi, M.; Murase, T.; Kajihara, Y., J. Am. Chem. Soc. 2016, 138, 3461. 17. King, J. F.; Allbutt, A. D., Can. J. Chem. 1970, 48, 1754. 18. Walczak, M. A.; Danishefsky, S. J. J. Am. Chem. Soc. 2012, 134, 16430. 19. Yamamoto, N.; Takayanagi, A.; Yoshino, A.; Sakakibara, T.; Dawson, P. E.; Kajihara, Y., Tetrahedron Lett. 2006, 47, 1341.


Semisynthesis of Complex-Type Biantennary Oligosaccharides

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