Synthesis of Fluorinated Analogues of Tumor-Associated Carbohydrate and Glycopeptide Antigens

Abstract

Partial structures of tumor-associated mucin glycoproteins are interesting target structures for the development of selective anticancer vaccines. To probe the effect of fluorination on the immunological and metabolic properties of mucin glycopeptides, six novel fluorinated glycosyl-threonine conjugates have been synthesized. The synthesis of the orthogonally protected glycosyl amino acids was achieved using microwave irradiation in key fluorination and glycosylation steps. The 2′-deoxy-2′-fluoro- and 6′-deoxy-6′-fluoro-T antigen building blocks were applied to the synthesis of analogues of MUC1 tandem repeat-glycopeptide antigens via SPPS.

Key words

fluorinated carbohydrates - glycopeptide antigens - MUC1 - solid-phase synthesis - microwave-assisted glycosylations

References and Notes

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12

CEM Discover microwave reactor.

19

Typical Experimental Procedure
A solution of acceptor 7 (150 mg, 0.25 mmol) in anhyd MeNO₂-CH₂Cl₂ (3:2, 4 mL) was stirred with Hg(CN)₂ (125 mg, 0.49 mmol) and activated pulverized MS 4 Å (200 mg) for 30 min under argon. A solution of donor 3 (185 mg, 0.49 mmol) in anhyd MeNO₂-CH₂Cl₂ (3:2, 4 mL) was added, and the reaction mixture was irradiated in a microwave reactor for 5 h (80 ˚C, 100 W), diluted with CH₂Cl₂ (20 mL), and filtered through Hyflo Supercel. The filtrate was washed with sat. aq NaHCO₃ (10 mL) and brine (10 mL), dried (MgSO₄), and concentrated in vacuo. Flash chromatography on silica gel (cyclohexane-EtOAc, 5:1) afforded 9 as a colorless, amorphous solid (162 mg, 73%); R _f = 0.53 (cyclohexane-EtOAc, 5:1); [α]_D ^²³ 39.42 (c 1, CHCl₃).

25

Compound β-20
[α]_D ^²³ = 55.74 (c 1, CHCl₃); t _R = 14.6 min [Perfectsil C18, grad.: MeCN-H₂O, (50:50) → (90:10), 30 min → (100:0), 10 min]. ^¹H NMR (400 MHz, CDCl₃, COSY): δ = 5.43 (d, 1 H, 4-H, J _3,4 = 2.0 Hz), 5.36-5.32 (m, 1 H, 4′-H), 5.10-5.00 (m, 1 H, 3′-H), 4.87 (d, 1 H, 1-H, J _1,2 = 3.5 Hz), 4.65 (dt, 1 H, 2-H, J _2,1 = 3.6 Hz, J _2,3 = 10.6 Hz), 4.60-4.37 (m, 4 H, 1′-H {4.57}, 2′-H, CH₂ (Fmoc) {4.52}), 4.29-4.08 (m, 6 H, T^α {4.23}, T^β {4.16}, 6a-H {4.11}, 5′-H {4.13}, 6a′-H {4.21}, 9-H (Fmoc) {4.22}), 4.03-3.84 (m, 3 H, 5-H, 6b-H, 6b′-H), 4.03-3.94 (m, 1 H, 3′-H), 3.79 (dd, 1 H, 3-H, J _3,4 = 3.2 Hz, J _2,3 = 11.0 Hz), 2.12 [s, 3 H, CH₃(Ac)], 2.10 [s, 3 H, CH₃(Ac)], 2.03 [s, 9 H, 3 × CH₃(Ac)], 1.97 [s, 3 H, CH₃(NHAc)], 1.44 [s, 9 H, CH₃(t-Bu)], 1.28 (d, 3 H, T^γ, J _{T γ ,Tβ} = 6.1 Hz) ppm. ^¹³C NMR (100.6 MHz, CDCl₃, BB, HMQC): δ = 101.9 (d, C1′, J _{C1 ′ ,F} = 23.5 Hz), 100.3 (C1), 87.8 (d, C2′, J _{C2 ′ ,F} = 186.2 Hz), 83.2 [Cq(t-Bu)], 77.5 (C3), 77.2 (T^β), 70.8 (d, C3′, J _{C3 ′ ,F} = 19.1 Hz), 70.5 (C5), 69.0 (C4), 68.1 (C5′), 67.2 (d, C4′, J _{C4 ′ ,F} = 8.0 Hz,), 66.8 (CH₂-Fmoc), 63.2 (C6′), 60.8 (C6) 59.0 (T^α), 48.0 (C2), 47.2 (CH-Fmoc), 28.0 [CH₃(t-Bu)], 23.0 [CH₃(NHAc)], 20.8, 20.7, 20.6, 20.5, 20.5 [CH₃ (Ac)], 18.5 (T^γ) ppm. ESI-MS (pos. ion mode): m/z calcd for C₄₇H₅₉FN₂NaO₁₉: 997.36; found: 997.34 [M + Na]⁺.

27

Applied Biosystems 433A peptide synthesizer.

29

Rapp Polymere GmbH.

32

Compound 22
[α]_D ^²5 = -97.61 (c 1, CHCl₃); t _R = 21.2 min [Luna C18, grad.: MeCN-H₂O + 0.1% TFA, (5:95) → (50:50), 80 min → (100:0), 20 min]. ^¹H NMR (400 MHz, DMSO-d ₆, COSY): δ = 8.25 (d, 1 H, R^NH, J _{NH, α} = 7.2 Hz), 8.21 (d, 1 H, D^NH, J _{NH, α} = 8.7 Hz), 8.10 (d, 1 H, A^NH, J _{NH, α} = 7.3 Hz) 8.00 (d, 1 H, A^NH, J _{NH, α} = 7.3 Hz), 7.92 (d, 1 H, S^NH, J _{NH, α} = 8.0 Hz), 7.61-7.53 (m, 2 H, T^NH {7.56}, R^Gua {7.58}), 6.93 (d, 1 H, NH-GalNAc, J _NH,2 = 8.7 Hz), 4.69-4.62 (m, 2 H, 1-H {4.67}, D^α {4.65}), 4.58 (m, 1 H, 6′a-H), 4.54-4.43 (m, 3 H, A^α {4.51}, R^α {4.47}, 6′b-H {4.47}), 4.43-4.36 (m, 2 H, A^α {4.40}, T^α {4.39}), 4.36-4.29 (m, 2 H, 2 × P^α {4.33}, {4.31}), 4.28-4.23 (m, 2 H, S^α {4.26}, 1′-H {4,24}), 4.20 (dd, 1 H, P^α, J _{α , β a} = 4.4 Hz, J _{α , β b} = 8.7 Hz), 4.17-4.12 (m, 1 H, 2-H), 4.11-4.08 (m, 1 H, T^β), 3.89-3.85 (m, 1 H, 4-H), 3.76-3.60 (m, 3 H, 5′-H {3.69}, 5-H {3.65}, 4′-H {3.63}), 3.61-3.38 (m, 11 H, S^β {3.52}, 3 × P^δ {3.54}, 6a-H {3.43}, 6b-H {3.41}, 3-H {3.57}), 3.35-3.28 (m, 2 H, 2′-H {3.52}, 4′-H {3.30}), 3.15-3.01 (m, 2 H, R^δ), 2.75 (dd, 1 H, D^{β a}, J _{β a , α} = 6.4 Hz, J _{β a , β b} = 16.5 Hz), 2.49 (m, 1 H, D^{β b}, under DMSO-d ₆), 2.18-2.09 (m, 1 H, P^{β a}), 2.07-1.97 (m, 2 H, 2 × P^{β a}, {2.06}, {2.01}), 1.94-1.74 [m, 15 H, 6 × P^γ {1.91}, {1.88}, {1.83}, 3 × P^{β b} {1.84}, {1.81}, {1.78}, 2 × CH₃ (NHAc)], 1.73-1.65 (m, 1 H, R^{β a}), 1.59-1.42 (m, 3 H, R^β, {1.52}, R^{β b} {1.49}), 1.19 (d, 3 H, A^β, J _{α , β} = 7.4 Hz), 1.17 (d, 3 H, A^β, J _{α , β} = 7.4 Hz), 1.10 (d, 3 H, T^γ, J _{β , γ} = 6.4 Hz) ppm. ^¹³C NMR (100.6 MHz, CDCl₃, BB, HMQC): δ = 173.3, 171.9, 171.8, 171.0, 170.8, 170.5, 170.5, 169.7, 169.7, 169.6, 169.4 (12 × C=O), 156.8 (C=N), 104.8 (C1′), 98.6 (C1), 83.3 (d, C6′, J _{C6 ′ ,F} = 166.9 Hz), 78.8 (C3), 75.0 (T^β), 73.1 (d, C5′, J _{C5 ′ ,F} = 19.1 Hz), 72.5 (C3′), 71.3 (C5), 70.5 (C2′), 68.0 (d, C4′, J _{C4 ′ ,F} = 6.5 Hz), 67.8 (C4), 61.8 (S^β), 60.6 (C6), 59.5, 59.0, 58.5 (3 × P^α), 55.8 (T^α), 55.0 (S^α), 50.1 (R^α), 49.3 (D^α), 48.0 (C2), 46.7, 46.5, 46.3, 46.3 (2 × A^α, 3 × P^δ), 40.5 (R^δ), 35.9 (D^β), 29.1, 28.8, 28.6 (3 × P^β), 28.3 (R^β), 24.8 (R^γ), 24.6, 24.4, 24.3 (3 × P^γ), 23.1, 22.6 [2 × CH₃(NHAc)], 18.3 (T^γ) 17.1, 16.6 (2 × A^β) ppm. ESI-MS (pos. ion mode): m/z calcd for C₅₄H₈₇FN₁₃O₂₄: 1320.59; found: 1320.60 [M + H]⁺.