Design and Synthesis of Peptide Mimetics of GDP-Fucose: Targeting Inhibitors of Fucosyltransferases

Toru Tanaka; Chihiro Tsuda; Tsuyoshi Miura; Toshiyuki Inazu; Shuichi Tsuji; Shoko Nishihara; Mitsuko Hisamatsu; Tetsuya Kajimoto

doi:10.1055/s-2003-44984

LETTER

Design and Synthesis of Peptide Mimetics of GDP-Fucose: Targeting Inhibitors of Fucosyltransferases

Toru Tanaka^a, Chihiro Tsuda^a, Tsuyoshi Miura^b, Toshiyuki Inazu^b, Shuichi Tsuji^c, Shoko Nishihara^d, Mitsuko Hisamatsu^d, Tetsuya Kajimoto*^a
^a Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan
Fax: +81(42)3778170; e-Mail: kajimoto@cc.ocha.ac.jp;
^b The Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo 173-0003, Japan
^c The Glycoscience Institute, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8555, Japan
^d Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji-shi, Tokyo 192-8577, Japan

Abstract

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Abstract

Novel peptide mimetics of GDP-fucose were designed and synthesized targeting inhibitors of the fucosyltransferases that transfer l-fucose from GDP-fucose to oligosaccharides, on the basis of the background that nikkomycin Z, a peptide mimetic of UDP-N-acetylglucosamine, shows potent inhibitory activity toward an N-acetylglucosamine transfer enzyme. The synthetic routes of the GDP-fucose mimetics take advantage of an enzymatic aldol reaction catalyzed by l-threonine aldolase to prepare the guanine carrying β-hydroxy-α-l-amino acid, a key synthetic intermediate.

Key words

aldol reactions - amino acids - enzymes - inhibitors - nucleotides

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References

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<A NAME="RU21603ST-8A">8a</A> The absolute stereochemistry of the a-carbon was determined to be l-configuration by the observation that 5a and 5b were not substrates of the d-amino acid oxidase but those of the l-amino acid oxidase. The erythro- and threo-configuration was determined by converting to the corresponding oxazolidones treating with ethyl chlorocarbonate in 1 M aq NaOH. See also: Saeed A. Yong DW. Tetrahedron 1992, 48: 2507

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Compound 13a: ¹H NMR (CDCl₃): δ = 1.14 (d, 3 H, J = 6.5 Hz, Me-6), 1.69 (m, 2 H), 2.00, 2.08, 2.17 (s, each 3 H, 3 × Ac), 2.13 (m, 2 H), 3.41 (dt, 1 H, J = 10.0, 6.5 Hz, A part of AB type), 3.69 (dt, 1 H, J = 10.0 Hz, B part of AB type), 4.16 (br q, 1 H, J = 6.5 Hz, H-5), 4.95-5.02 (m, 2 H), 5.05 (d, 1 H, J = 4.0 Hz, H-1), 5.11 (dd, 1 H, J = 4.0, 10.5 Hz, H-2), 5.30 (dd, 1 H, J = 1.0, 3.5 Hz, H-4), 5.35 (dd, 1 H, J = 3.5, 10.5 Hz, H-3), 5.81 (ddt, 1 H, J = 10.5, 17.0, 7.5 Hz). ¹³C NMR (CDCl₃): δ = 15.8, 20.6 × 2, 20.7, 28.4, 30.0, 64.2, 67.5, 68.0, 68.2, 71.1, 96.0, 115.0, 137.7, 170.0, 170.4, 170.6.

Compound 13b: ¹H NMR (CDCl₃): δ = 1.23 (d, 3 H, J = 6.5 Hz, Me-6), 1.70 (m, 2 H), 2.00, 2.06, 2.18 (s, each 3 H, 3 × Ac), 2.11 (m, 2 H), 3.49 (m, 1 H), 3.81 (br q, 1 H, J = 6.5 Hz, H-5), 3.92 (dt, 1 H, J = 9.5, 6.0 Hz), 4.43 (d, 1 H, J = 7.5, H-1), 4.95-5.05 (m, 2 H), 5.02 (dd, 1 H, J = 3.0, 10.5 Hz, H-3), 5.20 (dd, 1 H, J = 7.0, 10.5 Hz, H-2), 5.24 (br d, 1 H, J = 3.0 Hz, H-4), 5.80 (ddt, 1 H, J = 10.0, 17.0, 6.5 Hz).

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Compound 20a: ¹H NMR (CD₃OD): δ = 0.99 (d, 3 H, J = 6.5 Hz, Me-6), 1.80 (m, 2 H), 1.85, 1.92, 2.04 (s, each 3 H, 3 × Ac), 2.30 (t, 2 H, J = 7.5 Hz), 3.33 (dt, 1 H, J = 10.0, 6.0 Hz), 3.60 (dt, 1 H, J = 10.0, 6.0 Hz), 4.01 (dd, 1 H, J = 9.0, 15.0 Hz, H-γ), 4.07 (br q, 1 H, J = 6.5 Hz, H-5), 4.14 (dd, 1 H, J = 3.0, 15.0 Hz, H-γ′), 4.16 (m, 1 H, H-β), 4.53 (d, 1 H, J = 5.5 Hz, H-α), 4.88 (d, 1 H, J = 3.5 Hz, H-1), 4.94 (dd, 1 H, J = 3.5, 11.0 Hz, H-2 or H-3), 5.05, 5.08 (d, each 1 H, AB type, J = 13.0 Hz, CH ₂Ph), 5.15 (dd, 1 H, J = 1.0, 3.5 Hz, H-4), 5.20 (dd, 1 H, J = 3.5, 11.0 Hz, H-2 or H-3), 7.25 (m, 5 H, Ph), 7.53 (s, 1 H, guanine H-8). FAB MS: Calcd for C₃₂H₄₀N₆O₁₃: 716.3. Found: 717.4.

Compound 20b: ¹H NMR (CD₃OD): δ = 1.06 (d, 3 H, J = 6.5 Hz, Me-6), 1.76 (m, 2 H), 1.84, 1.94, 2.04 (s, each 3 H, 3 × Ac), 2.25 (t, 2 H, J = 8.0 Hz), 3.45 (dt, 1 H, J = 10.0, 6.0 Hz), 3.74 (dt, 1 H, J = 10.0, 6.0 Hz), 3.81 (br q, 1 H, J = 6.5 Hz, H-5), 4.03 (dd, 1 H, J = 8.5, 14.0 Hz, H-γ), 4.16 (dd, 1 H, J = 4.0, 14.0 Hz, H-γ′), 4.19 (m, 1 H, H-β), 4.44 (d, 1 H, J = 7.5 Hz, H-1), 4.51 (d, 1 H, J = 5.5 Hz, H-α), 4.94 (dd, 1 H, J = 7.5, 10.5 Hz, H-2), 4.98 (dd, 1 H, J = 3.5, 10.5 Hz, H-3), 5.06, 5.09 (d, each 1 H, AB type, J = 12.5 Hz, CH ₂Ph), 5.11 (dd, 1 H, J = 1.0, 3.5 Hz, H-4), 7.30 (m, 5 H, Ph), 7.56 (s, 1 H, guanine H-8). FAB MS: Calcd for C₃₂H₄₀N₆O₁₃: 716.3. Found: 717.4.

Compound 2a: ¹H NMR (D₂O): δ = 1.01 (d, 3 H, J = 6.5 Hz, Me-6), 1.73 (m, 2 H), 2.24 (m, 2 H), 3.34 (m, 1 H), 3.54 (m, 1 H), 3.59 (dd, 1 H, J = 4.0, 10.0 Hz, H-2), 3.62 (br d, 1 H, J = 3.5 Hz, H-4), 3.70 (dd, 1 H, J = 3.5, 10.0 Hz, H-3), 3.88 (br q, 1 H, J = 6.5 Hz, H-5), 3.91 (dd, 1 H, J = 8.0, 14.5 Hz, H-γ), 4.04 (dd, 1 H, J = 6.0, 14.5 Hz, H-γ′), 4.18 (d, 1 H, J = 3.0 Hz, H-α), 4.37 (m, 1 H, H-β), 4.71 (d, 1 H, J = 4.0 Hz, H-1), 7.63 (s, 1 H, guanine H-8). MALDI-TOF MS: Calcd for C₁₉H₂₈N₆O₁₀ + Na⁺: 523.2. Found: 523.1.

Compound 2b: ¹H NMR (D₂O): δ = 1.01 (d, 1 H, J = 6.5 Hz, Me-6), 1.72 (m, 2 H), 2.23 (m, 2 H), 3.24 (dd, 1 H, J = 7.5, 10.0 Hz, H-2), 3.38 (dd, 1 H, J = 3.5, 10.0 Hz, H-3), 3.46 (dt, 1 H, J = 10.5, 6.5 Hz, A part of AB type), 3.48-3.55 (m, 2 H, H-4 and H-5), 3.72 (dt, 1 H, J = 10.5, 6.5 Hz, B part of AB type), 3.95-4.12 (m, 3 H, H-β and H₂-γ), 4.13 (d, 1 H, J = 7.5 Hz, H-1), 4.19 (d, 1 H, J = 5.5 Hz, H-α), 7.62 (s, 1 H, guanine H-8). FAB MS: Calcd for C₁₉H₂₈N₆O₁₀ + Na⁺: 523.2. Found: 523.2.

<A NAME="RU21603ST-19">19</A> Nishihara S. Nakazato M. Kudo T. Kimura H. Ando T. Narimatsu H. Biochem. Biophys. Res. Commun. 1993, 190: 42

The assay was performed in 50 mM cacodylate buffer (pH 6.8) containing 5 mM ATP, 10 mM l-Fuc, 25 mM MnCl₂, 15 mM acceptor substrate, Galβ1-3GlcNAcβ1-3Galβ1-4Glc-2-aminobenzamide (for FUT 3) or Galβ1-4GlcNAcβ1-3Galβ1-4Glc-2-aminobenzamide (for FUT 6), 75 µM donor substrate GDP-Fuc, and 2a or 2b (0 mM for the positive control; 0.75 mM, and 7.5 mM respectively for the inhibitory assay). After incubation at 37 °C for 2 h in the presence of the fucosyltransferases (FUT 3 or FUT 6), the enzyme reaction was terminated by heating at 97 °C for 5 min followed by adding H₂O. After centrifugation of the reaction mixture, in order to detect the fucosylated products and estimate their amounts, each supernatant was filtered and subjected to reverse-phase HPLC analysis on TSK-gel ODS-80Ts QA column (4.6 × 250 mm; Tosoh, Tokyo, Japan) and eluted with 20 mM ammonium acetate buffer (pH 4.0) containing 7% MeOH at flow rate of 1.0 mL/min at
50 °C, with monitoring by a fluorescence spectrophotometer (JASCO FP-920; Nihon Bunkoh, Tokyo, Japan).