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Synlett 2009(16): 2633-2636  
DOI: 10.1055/s-0029-1217972
LETTER
© Georg Thieme Verlag Stuttgart ˙ New York

Syntheses of Novel Sulfated Glycans for Cell-Adhesion Interaction Studies

Vipin Kumar, Robert D. Locke, Khushi L. Matta*
Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
Fax: +1(716)8458768; e-Mail: khushi.matta@roswellpark.org;

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Publication History

Received 23 April 2009
Publication Date:
09 September 2009 (online)

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Abstract

Stereoselective syntheses of two 3-O-Gal sulfated trisaccharides GalNAcβ(1-4)[(3-SE)-Gal]β(1-3)GalNAcα-O-All and GalNAcβ(1-4)[(3-SE)-Gal]β(1-4)Glcβ-O-All were accomplished through the use of three novel glycosyl acceptors, namely, allyl 4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-α-d-galactopyranoside, methyl 2,6-di-O-benzoyl-3-O-naphthylmethyl-α-d-galactopyranoside and allyl 6-O-acetyl-2-O-benzoyl-3-O-naphthylmethyl-β-d-galactopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-β-d-glucopyranoside. These sulfated trisaccharides were expected to act as potential reference compounds for human β4GalNAc transferase and can be effectively used as antigens when linked to KLH.

Key words

glycosylations - oligosaccharides - stereoselective synthesis - sulfated glycans - sulfotransferases

Biosynthesis of oligosaccharides, polysaccharides, and glycoconjugates is mainly carried out by a large family of enzymes known as glycosyltransferases (Glycosyl-Ts). [¹] Aberrant glycosylation and overexpression of carbohydrate structures leads to the growth of tumor cells with different adhesion properties. [²] It is a well-established fact that the changes in the structures of cancer-associated glycans are driven by the expression and activity alterations of Glycosyl-Ts involved in their biosynthesis. [³] Since, sulfotransferases (Sulf-Ts) and sialyltransferases (STs) can compete with GTs for the same sites during an assembly of glycans, [4] it became important to study the role of both the enzyme families in the development of malignant cells. Based on this principal, over the years, the prime objective of our laboratory has been to develop the chemistry for understanding the enzymic machinery of glycans, especially O-glycans. [4] [5] It is evident from the literature reports [6] and our previous biochemical investigations [5] that Sulf-Ts are highly specific enzymes which incorporate a sulfate ester (SE) to a specific position of a specific oligosaccharide acceptor. For instance, two distinct types of Gal:3-O-sulfotransferases (Gal3Sulf-Ts) in tumor tissues and cancer cells demonstrated distinctive acceptor preferences. Enzymes from breast cancer cells prefer to sulfate 3-O position of Gal in the Galβ1→3GalNAcα moiety of the mucin core 2 structure. In contrast, enzymes from colon cancer cell lines and colon tumor tissues prefer to act on the Galβ1→4GlcNAcβ moiety. [5b] [c] These studies have led us to identify whether human β4GalNAc-transferase has the capability to generate GalNAcβ(1-4)[(3-SE)-Gal]β sequence. Herein, we report the syntheses of ­GalNAcβ(1-4)-[(3-SE)-Gal]β(1-3)GalNAcα(1-O)-All (1) and GalNAcβ(1-4)-[(3-SE)-Gal]β(1-4)Glcβ(1-O)-All (6) as potential reference compounds that can be effectively used as antigens when linked to keyhole limpet hemocyanin (KLH) under conventional conditions. [7]

As envisaged from the retrosynthetic scheme (Figure  [¹] ), coupling between 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-galactopyranosyl-(1→4)-2,6-di-O-benzoyl-3-O-naphthylmethyl-d-galactopyranosyl trichloroacetimidate (2) and allyl 4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)­-α-d-galactopyranoside (3) would yield the desired sulfated trisaccharide 1. The disaccharide donor 2 could in turn be obtained by the reaction of known 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-d-galacto­pyranosyl trichloroacetimidate (4) [8] with methyl 2,6-di-O-benzoyl-3-O-naphthylmethyl-α-d-galactopyranoside (5). Similarly, synthesis of second target molecule 6 necessitates the use of N-trichloroethoxycarbonyl (N-Troc) Gal imidate 4 and suitably protected lactose acceptor 7 having free 4′-OH accessible for glycosylation reaction. In order to introduce acetamido groups in the final trisaccharides 1 and 6 (examples of β-glycosides), N-Troc-protected glycosyl donor 4 was preferred as it is known to furnish β-glycosides in high yield with good β-stereoselectivity. [8] [9] Also their transformation into acetamido group is straightforward. [¹0] To accomplish the introduction of sulfate group at the 3′-OH of Gal unit of trisaccharides 1 and 6, protection with 2-naphthylmethyl (NAP) group proved to be highly suitable for our purpose owing to its stability under various reaction conditions, viz. acetal hydrolysis, glycosylation, and ester deprotection. Also, its chemoselective removal under DDQ oxidative conditions as required in the later part of our synthetic strategy is an added advantage. [¹¹] We opted to make the allyl glycoside derivatives 1 and 6 because of their ease in attachment to gold nanoparticles and KLH following standard protocols. [7] [¹²] We intend to utilize these compounds for developing monoclonal antibodies and for discovery of carbohydrate-binding aptamers.

Figure 1 Retrosynthetic analysis of target sulfated trisaccharides 1 and 6

Syntheses of starting materials required for the construction of sulfated trisaccharide 1 is depicted in Scheme  [¹] . 4,6-O-Benzylidene protection of allyl α-d-GalNAc (8) followed by hydrolysis of its acetamido group provided amine 9 in 70% yield over two steps. [¹³] Troc protection of 9 using TrocCl and NaHCO3 afforded the required acceptor 3 in reasonable yield. The poor yield of 3 may be attributed to the low solubility of free amine 9 in the reaction medium (water). Attempts to improve the yield of 3 in the above reaction are ongoing. Similarly, 3-O-NAP galactoside 5 was prepared from commercially available methyl α-d-galactoside (10). Reaction of 10 with NAPBr in presence of Bu4NI and Bu2SnO brought about the regioselective introduction of NAP to afford methyl 3-O-NAP-α-d-galactoside (11) in a very high yield (96%). [¹¹] It is worth mentioning here that the 3-O-NAP group could be chemoselectively removed at the ­final stage of the synthetic strategy to endure sulfation. Selective benzoylation at 2- and 6-OH of 11 was successfully achieved using BzCl (2 equiv) in pyridine at -30 ˚C to provide the desired acceptor 5 in 80% yield (Scheme  [¹] ).

Scheme 1 Preparation of the key building blocks 3 and 5. Reagents and conditions: a) i) PhCH(OMe)2, PTSA, DMF, r.t., overnight; ii) 30% KOH solution, 1,4-dioxane-EtOMe (5:3 v/v), refluxed at 120 ˚C, 18 h (70%, 2 steps); b) TrocCl, NaHCO3, Et2O-H2O (1:1 ­v/v), r.t., 1.5 h (40%); c) Bu2SnO-dry benzene, refluxing, 4 h, then NAPBr, n-Bu4NI, 80-85 ˚C, 18 h (96%). d) BzCl, pyridine, -30 ˚C, 6 h (80%).

With the required starting materials in hand, we turned our attention on the synthesis of one of the target molecules 1. Condensation of alcohol 5 with imidate 4 was performed under standard glycosylation conditions using TMSOTf as the catalyst [¹4] to provide the desired β-(1→4) linked disaccharide 12 in 86% yield (Scheme  [²] ). In order to synthesize disaccharide donor 2, compound 12 was first subjected to acetolysis [¹5] with AcOH-Ac2O-H2SO4 followed by removal of the resulting anomeric acetate by hydrazinium acetate [¹6] to afford the 1-hydroxy compound 13 in a good yield of 75% over two steps. Compound 13 upon further treatment with trichloroacetonitrile in the presence of DBU [8] gave the desired trichloroacetimidate 2 in a 78% yield. Under the similar conditions as mentioned for the synthesis of 12, coupling between 2 and 3 was carried out successfully to obtain the β-(1→3) linked trisaccharide 14 in moderate yield. Subsequent removal of 4,6-O-benzylidene ring of 14 followed by conversion of NHTroc into NHAc [¹0] afforded 15 in 69% over three steps (Scheme  [²] ). Chemoselective removal of NAP from 15 was effected using DDQ to afford 16 in 75% yield. [¹¹] Finally, sulfation using SO3-pyridine complex in DMF [¹7] followed by complete deprotection using NaOMe/MeOH, neutralization with IR-120 (H+) resin, and passage through IR-120 (Na+) resin afforded the title compound 1 [¹8] as a white solid in 95% yield (over two steps, Scheme  [²] ).

Scheme 2 Synthesis of trisaccharide 1. Reagents and conditions: a) TMSOTf, 4 Å MS, dry CH2Cl2, N2, -50 ˚C, 40 min (86%); b) i) Ac2O-AcOH (11:9 v/v), H2SO4, 0 ˚C to -4 ˚C, overnight; ii) N2H2˙HOAc, DMF, 50 ˚C, 2 h (75%, 2 steps); c) CCl3CN, DBU, CH2Cl2, 0 ˚C, 30 min (78%); d) TMSOTf, 4 Å MS, dry CH2Cl2, N2, -50 ˚C to 0 ˚C, 30 min (55%); e) i) 80% aq AcOH, 55 ˚C, 30 min; ii) Cd powder, DMF-AcOH (2:1 v/v), r.t., overnight; iii) Ac2O-pyridine (1:2 v/v), CH2Cl2, r.t., 70 h (69%, 3 steps); f) DDQ, CH2Cl2-MeOH (4:1 v/v), overnight (75%). g) i) SO3-pyridine, dry DMF, 50 ˚C, 2 h; ii) NaOMe/MeOH, r.t., 24 h (95%, 2 steps).

Scheme  [³] outlines the synthesis of 3′-O-SO3Na trisaccharide 6. 3′-O-NAP protection of 17 [¹9] as described before provided 18 in a reasonable yield. 4,6-O-Benzylidene protection of 18 followed by conventional benzoylation furnished 19 in 70% yield over two steps. Finally, treatment of 19 with 80% AcOH cleaved the benzylidene ring to afford diol which was selectively acetylated at the 6′-OH (primary hydroxyl group) using 1 equivalent of Ac2O in pyridine at low temperature to get the desired lactose acceptor 7 in 81% yield. TMSOTf-mediated coupling of 7 with imidate 4 provided the desired β-(1→4) linked trisaccharide 20 in a good yield (Scheme  [³] ). Following the same strategy as described for 1; conversion of NHTroc into NHAc, removal of NAP, sulfation, and ­finally complete deprotection and treatment with IR-120 (Na+) resin, afforded the final sulfated trisaccharide 6 [²0] as a white solid in an overall yield of 58% over five steps.

Scheme 3 Synthesis of trisaccharide 6. Reagents and conditions: a) i) Bu2SnO-dry toluene, refluxing, 4 h, then NAPBr, n-Bu4NI, 110-115 ˚C, 48 h (60%); b) i) PhCH(OMe)2, PTSA, MeCN, r.t., 1 h; ii) BzCl, pyridine, r.t., overnight (70%, 2 steps); c) i) 80% AcOH, 75-80 ˚C, 6 h; ii) Ac2O, DMAP, pyridine, -30 ˚C, 7 h (81%, 2 steps); d) TMSOTf, 4 Å MS, dry CH2Cl2, N2, -50 ˚C, 3 h (76%); e) i) Cd powder, DMF-AcOH (2:1 v/v), r.t., overnight; ii) Ac2O-py (1:2 v/v), CH2Cl2, r.t., 20 h (80%, 2 steps); f) DDQ, CH2Cl2-MeOH (4:1 v/v), overnight (76%); g) i) SO3-pyridine, dry DMF, 50 ˚C, 4 h; ii) NaOMe/MeOH, r.t., 48 h (95%, 2 steps).

In summary, we have developed a concise and practical synthesis of two 3-O-Gal-sulfated trisaccharides 1 and 6 based on the rationally designed synthetic strategy. We argue that the sulfated compounds prepared here will be useful for cell-adhesion interaction studies which recognize sulfated glycans. Furthermore, attempts to explore the synthetic utility of intermediates 2, 3, and 7 for obtaining higher oligosaccharides with similar structures are currently being undertaken. These compounds will be further employed for examining the specificity of glycosyltransferases and sulfotransferases.

Acknowledgment

We acknowledge grant support from DOD (W81XWH-06-1-0013) and support, in part, by the NCI Cancer Center Support Grant to the Roswell Park Cancer Institute (P30 -CA016056).

18

Analytical Data for Compound 1
¹H NMR (400 MHz, CD3OD): δ = 1.97 (s, 3 H, NHCOCH 3), 2.03 (s, 3 H, NHCOCH 3), 3.93-3.41 (m, 16 H), 4.03-3.98 (m, 2 H), 4.11-4.09 (m, 1 H), 4.23-4.17 (m, 1 H), 4.39 (d, J = 7.6 Hz, 1 H, H-1′), 4.46-4.41 (m, 1 H), 4.63 (d, J = 8.4 Hz, 1 H, H-1′′), 5.17 (dd, ³ J = 10.4 Hz, ² J = 1.6 Hz, 1 H, OCH2CH=CH cis H trans ), 5.32 (dd, ³ J = 17.2 Hz, ² J = 1.6 Hz, 1 H, OCH2CH=CH cis H trans ), 5.99-5.89 (m, 1 H, OCH2CH=CH2) ppm. ¹³C NMR (100 MHz, CD3OD): δ = 22.9 (COCH3), 23.2 (COCH3), 55.5, 61.8, 62.8, 62.9, 69.4, 70.3, 72.4, 72.8, 72.9, 74.7, 74.8, 75.8, 77.0, 77.9, 78.5 (C-3), 78.9 (C-3′), 98.2, 104.6, 106.5 (C-1, C-1′, C-1′′), 117.8 (CH=CH2), 135.7 (CH=CH2), 174.2 (COCH3), 175.4 (COCH3) ppm. ESI-MS: m/z calcd for C25H41N2NaO19S: 728.2; found: 751.2 [M + Na]+.

20

Analytical Data for Compound 6
¹H NMR (400 MHz, CD3OD): δ = 2.06 (s, 3 H, NHCOCH 3), 3.26 (m, 1 H), 3.91-3.46 (m, 14 H), 4.16-4.01 (m, 2 H), 4.36-4.28 (m, 4 H, H-1, J = 8.0 Hz, incorporated with other protons), 4.44 (d, J = 7.6 Hz, 1 H, H-1′), 4.61 (d, J = 8.4 Hz, 1 H, H-1′′), 5.16 (dd, ³ J = 10.8 Hz, ² J = 1.2 Hz, 1 H, OCH2CH=CH cis H trans ), 5.32 (dd, ³ J = 17.6 Hz, ² J = 1.6 Hz,
1 H, OCH2CH=CH cis H trans ), 5.99-5.93 (m, 1 H, OCH2CH=CH2) ppm. ¹³C NMR (100 MHz, CD3OD): δ = 23.5 (COCH3), 54.2, 61.4, 61.9, 62.7, 69.8, 70.9, 71.3, 73.8, 74.9, 75.7, 76.4, 76.6, 76.7, 76.8, 81.0 (C-3), 81.1 (C-3′), 103.4, 104.9, 105.0 (C-1, C-1′, C-1′′), 117.6 (CH=CH2), 135.8 (CH=CH2), 174.9 (COCH3) ppm. ESI-MS: m/z calcd for C23H38NNaO19S: 687.2; found: 710.2 [M + Na]+.

18

Analytical Data for Compound 1
¹H NMR (400 MHz, CD3OD): δ = 1.97 (s, 3 H, NHCOCH 3), 2.03 (s, 3 H, NHCOCH 3), 3.93-3.41 (m, 16 H), 4.03-3.98 (m, 2 H), 4.11-4.09 (m, 1 H), 4.23-4.17 (m, 1 H), 4.39 (d, J = 7.6 Hz, 1 H, H-1′), 4.46-4.41 (m, 1 H), 4.63 (d, J = 8.4 Hz, 1 H, H-1′′), 5.17 (dd, ³ J = 10.4 Hz, ² J = 1.6 Hz, 1 H, OCH2CH=CH cis H trans ), 5.32 (dd, ³ J = 17.2 Hz, ² J = 1.6 Hz, 1 H, OCH2CH=CH cis H trans ), 5.99-5.89 (m, 1 H, OCH2CH=CH2) ppm. ¹³C NMR (100 MHz, CD3OD): δ = 22.9 (COCH3), 23.2 (COCH3), 55.5, 61.8, 62.8, 62.9, 69.4, 70.3, 72.4, 72.8, 72.9, 74.7, 74.8, 75.8, 77.0, 77.9, 78.5 (C-3), 78.9 (C-3′), 98.2, 104.6, 106.5 (C-1, C-1′, C-1′′), 117.8 (CH=CH2), 135.7 (CH=CH2), 174.2 (COCH3), 175.4 (COCH3) ppm. ESI-MS: m/z calcd for C25H41N2NaO19S: 728.2; found: 751.2 [M + Na]+.

20

Analytical Data for Compound 6
¹H NMR (400 MHz, CD3OD): δ = 2.06 (s, 3 H, NHCOCH 3), 3.26 (m, 1 H), 3.91-3.46 (m, 14 H), 4.16-4.01 (m, 2 H), 4.36-4.28 (m, 4 H, H-1, J = 8.0 Hz, incorporated with other protons), 4.44 (d, J = 7.6 Hz, 1 H, H-1′), 4.61 (d, J = 8.4 Hz, 1 H, H-1′′), 5.16 (dd, ³ J = 10.8 Hz, ² J = 1.2 Hz, 1 H, OCH2CH=CH cis H trans ), 5.32 (dd, ³ J = 17.6 Hz, ² J = 1.6 Hz,
1 H, OCH2CH=CH cis H trans ), 5.99-5.93 (m, 1 H, OCH2CH=CH2) ppm. ¹³C NMR (100 MHz, CD3OD): δ = 23.5 (COCH3), 54.2, 61.4, 61.9, 62.7, 69.8, 70.9, 71.3, 73.8, 74.9, 75.7, 76.4, 76.6, 76.7, 76.8, 81.0 (C-3), 81.1 (C-3′), 103.4, 104.9, 105.0 (C-1, C-1′, C-1′′), 117.6 (CH=CH2), 135.8 (CH=CH2), 174.9 (COCH3) ppm. ESI-MS: m/z calcd for C23H38NNaO19S: 687.2; found: 710.2 [M + Na]+.

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Figure 1 Retrosynthetic analysis of target sulfated trisaccharides 1 and 6

Scheme 1 Preparation of the key building blocks 3 and 5. Reagents and conditions: a) i) PhCH(OMe)2, PTSA, DMF, r.t., overnight; ii) 30% KOH solution, 1,4-dioxane-EtOMe (5:3 v/v), refluxed at 120 ˚C, 18 h (70%, 2 steps); b) TrocCl, NaHCO3, Et2O-H2O (1:1 ­v/v), r.t., 1.5 h (40%); c) Bu2SnO-dry benzene, refluxing, 4 h, then NAPBr, n-Bu4NI, 80-85 ˚C, 18 h (96%). d) BzCl, pyridine, -30 ˚C, 6 h (80%).

Scheme 2 Synthesis of trisaccharide 1. Reagents and conditions: a) TMSOTf, 4 Å MS, dry CH2Cl2, N2, -50 ˚C, 40 min (86%); b) i) Ac2O-AcOH (11:9 v/v), H2SO4, 0 ˚C to -4 ˚C, overnight; ii) N2H2˙HOAc, DMF, 50 ˚C, 2 h (75%, 2 steps); c) CCl3CN, DBU, CH2Cl2, 0 ˚C, 30 min (78%); d) TMSOTf, 4 Å MS, dry CH2Cl2, N2, -50 ˚C to 0 ˚C, 30 min (55%); e) i) 80% aq AcOH, 55 ˚C, 30 min; ii) Cd powder, DMF-AcOH (2:1 v/v), r.t., overnight; iii) Ac2O-pyridine (1:2 v/v), CH2Cl2, r.t., 70 h (69%, 3 steps); f) DDQ, CH2Cl2-MeOH (4:1 v/v), overnight (75%). g) i) SO3-pyridine, dry DMF, 50 ˚C, 2 h; ii) NaOMe/MeOH, r.t., 24 h (95%, 2 steps).

Scheme 3 Synthesis of trisaccharide 6. Reagents and conditions: a) i) Bu2SnO-dry toluene, refluxing, 4 h, then NAPBr, n-Bu4NI, 110-115 ˚C, 48 h (60%); b) i) PhCH(OMe)2, PTSA, MeCN, r.t., 1 h; ii) BzCl, pyridine, r.t., overnight (70%, 2 steps); c) i) 80% AcOH, 75-80 ˚C, 6 h; ii) Ac2O, DMAP, pyridine, -30 ˚C, 7 h (81%, 2 steps); d) TMSOTf, 4 Å MS, dry CH2Cl2, N2, -50 ˚C, 3 h (76%); e) i) Cd powder, DMF-AcOH (2:1 v/v), r.t., overnight; ii) Ac2O-py (1:2 v/v), CH2Cl2, r.t., 20 h (80%, 2 steps); f) DDQ, CH2Cl2-MeOH (4:1 v/v), overnight (76%); g) i) SO3-pyridine, dry DMF, 50 ˚C, 4 h; ii) NaOMe/MeOH, r.t., 48 h (95%, 2 steps).