Synthesis of Partially N-Acetylated Chitooligosaccharides and Muropeptides

Emiliano Bedini; Luigi Cirillo; Roberta Marchetti; Sara Basso; Diego Tufano; Antonio Molinaro; Michelangelo Parrilli

doi:10.1055/s-0033-1340323

Synlett, Table of Contents

Synlett 2014; 25(3): 365-370
DOI: 10.1055/s-0033-1340323

letter

Synthesis of Partially N-Acetylated Chitooligosaccharides and Muropeptides

Emiliano Bedini*

Dipartimento di Scienze Chimiche, Università degli Studi di Napoli ‘Federico II’, Complesso Universitario Monte Sant’Angelo, Via Cintia 4,80126 Napoli, Italy Fax: +39(81)674393 Email: ebedini@unina.it

,

Luigi Cirillo

Dipartimento di Scienze Chimiche, Università degli Studi di Napoli ‘Federico II’, Complesso Universitario Monte Sant’Angelo, Via Cintia 4,80126 Napoli, Italy Fax: +39(81)674393 Email: ebedini@unina.it

,

Roberta Marchetti

Dipartimento di Scienze Chimiche, Università degli Studi di Napoli ‘Federico II’, Complesso Universitario Monte Sant’Angelo, Via Cintia 4,80126 Napoli, Italy Fax: +39(81)674393 Email: ebedini@unina.it

,

Sara Basso

Dipartimento di Scienze Chimiche, Università degli Studi di Napoli ‘Federico II’, Complesso Universitario Monte Sant’Angelo, Via Cintia 4,80126 Napoli, Italy Fax: +39(81)674393 Email: ebedini@unina.it

,

Diego Tufano

Dipartimento di Scienze Chimiche, Università degli Studi di Napoli ‘Federico II’, Complesso Universitario Monte Sant’Angelo, Via Cintia 4,80126 Napoli, Italy Fax: +39(81)674393 Email: ebedini@unina.it

,

Antonio Molinaro

Dipartimento di Scienze Chimiche, Università degli Studi di Napoli ‘Federico II’, Complesso Universitario Monte Sant’Angelo, Via Cintia 4,80126 Napoli, Italy Fax: +39(81)674393 Email: ebedini@unina.it

,

Michelangelo Parrilli

Dipartimento di Scienze Chimiche, Università degli Studi di Napoli ‘Federico II’, Complesso Universitario Monte Sant’Angelo, Via Cintia 4,80126 Napoli, Italy Fax: +39(81)674393 Email: ebedini@unina.it

› Author Affiliations

Abstract

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Abstract

Partially N-acetylated chitooligosaccharides and muropeptides are referred to as microbial associated molecular patterns (MAMPs). To shed light on the molecular basis of their recognition by the innate immunity system of living organisms, their production in pure form is necessary. To this end, we present here the first synthetic strategy for the obtainment of a series of partially N-acetylated muropeptides as well as of a chitodisaccharide and a chitotetrasaccharide, all possessing a well-defined N-acetylation pattern.

Key words

bioorganic chemistry - oligosaccharides - glycopeptides - glycosylation - chitosan - peptidoglycan

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References

References and Notes

For two comprehensive reviews, see:

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22 A solution of 10 (10.7 mg, 13.2 μmol) in CH₂Cl₂ (500 μL) was diluted with MeOH (1.0 mL), AcOH (600 μL) and H₂O (500 μL). The resulting monophasic, clear solution was treated with Pearlman’s catalyst (16.0 mg, 22.8 μmol). The mixture was stirred under a H₂ atmosphere at r.t. overnight, then filtered on a Celite pad. Volatiles were removed by rotoevaporation. The residue was freeze-dried to give a yellowish powder that was purified by HPLC (0.05% v/v TFA–H₂O to 0.05% v/v TFA–MeOH). Pure 1 (3.7 mg, 66%) was obtained as a white powder: [α]_D ²² +33 (c = 0.2, H₂O); ¹H NMR (500 MHz, D₂O): δ = 4.89 (d, J = 3.0 Hz, 1 H), 4.88 (d, J = 8.5 Hz, 1 H), 3.95–3.85 (m, 6 H), 3.80–3.75 (m, 2 H), 3.71–3.63 (m, 2 H), 3.55–3.43 (m, 3 H), 3.14 (dd, J = 10.5, 8.5 Hz, 1 H), 2.04 (s, 3 H), 1.60 (sext., J = 7.0 Hz, 2 H), 0.91 (t, J = 7.0 Hz, 3 H); ¹³C NMR (100 MHz, D₂O): δ = 175.1, 98.1, 97.2, 77.8, 77.0, 72.6, 70.8, 70.7, 70.2, 69.7, 61.0, 60.9, 56.5, 54.4, 22.5, 10.5; MS (MALDI TOF): m/z = 425.08 [M + H]⁺.
23 A mixture of 12 (62.0 mg, 62.9 μmol) and 15 (152 mg, 126 μmol) was coevaporated three times with anhydrous toluene (3 mL). The mixture was dried and then mixed with AW-300 4 Å molecular sieves under Ar. The mixture was then suspended in CH₂Cl₂ (7.0 mL) and cooled to –30 °C. After a few minutes stirring at –30 °C, the mixture was treated with a 0.13 M solution of TMSOTf in CH₂Cl₂ (39 μL, 5.07 μmol). After 1 h stirring at –30 °C, two drops of Et₃N were added. The mixture was then filtered over a Celite pad and concentrated. The residue was subjected to column chromatography (14:1 to 8:1 v/v toluene–EtOAc) to afford 16 (91.7 mg, 72%) as a white foam: [α]_D ²² –20.6 (c = 1.0, CH₂Cl₂); ¹H NMR (600 MHz, CDCl₃): δ = 7.37–7.25 (m, 40 H, H-Ar), 7.07 (d, J = 8.6 Hz, 2 H), 6.80 (d, J = 8.6 Hz, 2 H), 5.87 (m, 1 H), 5.31–5.27 (m, 2 H), 5.22 (dd, J = 10.4, 1.2 Hz, 1 H), 5.16 (d, J = 12.5 Hz, 1 H), 5.11–4.98 (m, 5 H), 4.93 (d, J = 3.6 Hz, 1 H), 4.82 (d, J = 10.9 Hz, 1 H), 4.78 (d, J = 10.9 Hz, 1 H), 4.70–4.57 (m, 8 H), 4.51–4.43 (m, 5 H), 4.37 (d, J = 9.4 Hz, 1 H), 4.34 (d, J = 12.2 Hz, 1 H), 4.30–4.23 (m, 3 H), 4.19 (dd, J = 14.1, 6.6 Hz, 1 H), 4.17 (d, J = 9.4 Hz, 1 H), 4.06 (dt, J = 10.3, 3.7 Hz, 1 H), 4.01–3.93 (m, 5 H), 3.87–3.80 (m, 2 H), 3.79 (s, 3 H), 3.72–3.60 (m, 5 H), 3.54 (d, J = 8.5 Hz, 1 H), 3.44–3.39 (m, 3 H), 3.29 (d, J = 7.9 Hz, 1 H), 3.25–3.08 (m, 5 H), 2.96 (d, J = 9.7 Hz, 1 H); ¹³C NMR (50 MHz, CDCl₃): δ = 159.3, 155.2, 154.9, 154.1, 153.9, 138.6, 138.4, 138.0, 137.9, 137.7, 137.3, 135.4, 133.1, 130.1, 129.4-127.5, 118.4, 113.8, 100.9, 100.8, 100.3, 96.3, 95.6, 95.4, 83.1, 81.2, 77.6, 77.3, 75.6, 75.3, 75.0, 74.8, 74.4, 73.5, 73.1, 70.3, 69.6, 68.7, 68.6, 67.9, 67.7, 67.6, 66.4, 66.0, 56.5, 55.2, 54.2; MS (MALDI TOF): m/z = 2053.40 [M + Na]⁺.
24 A solution of 18 (16.3 mg, 10.9 μmol) in CH₂Cl₂ (400 μL) was diluted with MeOH (2.0 mL), AcOH (800 μL) and H₂O (400 μL), and the resulting monophasic, clear solution was treated with Pearlman’s catalyst (16.0 mg, 22.8 μmol). The mixture was stirred under a H₂ atmosphere at r.t. overnight, then filtered on a Celite pad. Volatiles were removed by rotoevaporation. The residue was freeze-dried to give a white powder that was purified by HPLC (0.05% v/v TFA–H₂O to 0.05% v/v TFA–MeOH). Pure 2 (4.7 mg, 55%) was obtained as a white powder: [α]_D ²² +2.3 (c = 0.4, H₂O); ¹H NMR (600 MHz, D₂O): δ = 4.88 (d, J = 4.6 Hz, 1 H), 4.82 (d, J = 8.3 Hz, 2 H), 4.58 (d, J = 7.8 Hz, 1 H), 3.94–3.84 (m, 10 H), 3.79–3.75 (m, 5 H), 3.70–3.64 (m, 5 H), 3.57 (m, 2 H), 3.48 (m, 2 H), 3.16 (t, J = 8.3 Hz, 1 H), 3.14 (t, J = 8.3 Hz, 1 H), 2.06 (s, 3 H), 2.04 (s, 3 H), 1.60 (sext., J = 7.0 Hz, 2 H), 0.91 (t, J = 7.0 Hz, 3 H); ¹³C NMR (150 MHz, D₂O): δ = 175.4, 175.1, 102.2, 99.0, 97.4, 79.3, 77.7, 77.1, 75.8, 75.1, 73.1, 72.1, 70.9, 70.4, 69.8, 61.1, 61.0, 60.4, 56.5, 56.2, 54.5, 22.9, 22.7, 22.6, 10.6; MS (MALDI TOF): m/z = 789.13 [M + H]⁺.
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26 A solution of 22 (26.6 mg, 27.1 μmol) in CH₂Cl₂ (300 μL) was diluted with MeOH (900 μL), AcOH (600 μL) and H₂O (600 μL), and the resulting monophasic, clear solution was treated with Pearlman’s catalyst (26.6 mg, 37.9 μmol). The mixture was stirred under a H₂ atmosphere at r.t. overnight, then filtered on a Celite pad. Volatiles were removed by rotoevaporation and the residue was freeze-dried to give a yellowish powder that was purified by HPLC (0.05% v/v TFA–H₂O to 0.05% v/v TFA–MeOH). Pure 4 (7.5 mg, 53%) was obtained as a white powder: [α]_D ²² +44 (c = 0.2, H₂O); ¹H NMR (600 MHz, D₂O): δ = 4.93 (d, J = 3.6 Hz, 1 H), 4.82 (d, J = 8.2 Hz, 1 H), 4.40 (q, J = 7.1 Hz, 1 H), 4.37 (q, J = 8.2 Hz, 1 H), 4.06 (t, J = 9.3 Hz, 1 H), 3.96 (m, 2 H), 3.87 (m, 2 H), 3.82 (t, J = 10.2 Hz, 1 H), 3.76 (m, 2 H), 3.69 (t, J = 9.6 Hz, 1 H), 3.64 (m, 1 H), 3.51 (m, 1 H), 3.44 (m, 2 H), 3.14 (dd, J = 9.6, 8.2 Hz, 1 H), 1.99 (s, 3 H), 1.59 (sext, J = 7.4 Hz, 2 H), 1.45 (d, J = 8.2 Hz, 3 H), 1.38 (d, J = 7.1 Hz, 3 H), 0.90 (t, J = 7.4 Hz, 3 H); ¹³C NMR (150 MHz, D₂O): δ = 177.0, 175.7, 174.5, 98.1, 97.4, 78.5, 77.4, 77.3, 75.1, 72.6, 71.3, 70.4, 61.7, 60.9, 57.2, 53.9, 49.2, 22.8, 22.5, 19.1, 17.3, 10.4; MS (MALDI TOF): m/z = 568.16 [M + H]⁺.
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28 A solution of 20 (30.9 mg, 33.9 μmol) and 23 (133 mg, 305 μmol) in DMF (1.25 mL) was treated with a 0.27 M solution of PyBOP in DMF (250 μL), then with a 0.27 M solution of HOBt in DMF (250 μL) and finally with DIPEA (65 μL, 280 μmol). After 3 h stirring at r.t., the reaction mixture was diluted with CH₂Cl₂ (50 mL) and washed with 0.1 M HCl (50 mL). The organic phase was collected, dried over anhydrous Na₂SO₄, filtered, and concentrated. After column chromatography (3:1 to 1:3 v/v n-hexane–EtOAc), pure 27 (32.4 mg, 79%) was obtained as a colorless oil: [α]_D ²² +13.7 (c = 2.0, CH₂Cl₂); ¹H NMR (600 MHz, CDCl₃): δ = 7.33–7.26 (m, 20 H), 7.02 (d, J = 8.6 Hz, 2 H), 6.79 (d, J = 8.6 Hz, 2 H), 5.82 (m, 1 H), 5.23 (dd, J = 17.2, 1.5 Hz, 1 H), 5.16 (dd, J = 10.4, 1.5 Hz, 1 H), 5.11 (d, J = 12.3 Hz, 1 H), 5.08 (d, J = 12.3 Hz, 1 H), 5.07 (d, J = 3.9 Hz, 1 H), 4.80 (s, 2 H), 4.69 (d, J = 12.0 Hz, 1 H), 4.65 (d, J = 10.5 Hz, 1 H), 4.57 (dq, J = 7.9, 5.0 Hz, 1 H), 4.51 (d, J = 11.8 Hz, 1 H), 4.46–4.41 (m, 4 H), 4.36 (app. quint., J = 7.0 Hz, 1 H), 4.21 (d, J = 8.2 Hz, 1 H), 4.08 (dd, J = 13.0, 5.2 Hz, 1 H), 4.03–3.93 (m, 4 H), 3.76 (s, 3 H), 3.74 (d, J = 9.9 Hz, 1 H), 3.70–3.56 (m, 8 H), 3.29 (dd, J = 9.8, 8.3 Hz, 1 H), 3.22 (t, J = 9.0 Hz, 1 H), 3.19 (m, 1 H), 2.38–2.29 (m, 2 H), 2.23–2.15 (m, 1 H), 2.05–1.98 (m, 1 H), 1.98 (s, 3 H), 1.35 (d, J = 7.4 Hz, 3 H), 1.33 (d, J = 7.5 Hz, 3 H); ¹³C NMR (100 MHz, CDCl₃): δ = 174.7, 173.3, 172.1, 171.5, 171.4, 159.2, 137.9, 137.7, 137.5, 137.4, 133.7, 130.0, 128.7-127.6, 117.6, 113.9, 100.8, 96.1, 83.2, 76.7, 76.4, 75.3, 74.4, 74.3, 73.4, 73.1, 71.8, 70.6, 68.5, 68.3, 67.8, 67.3, 66.4, 55.2, 53.9, 53.8, 51.9, 51.7, 49.2, 29.8, 26.8, 23.1, 19.3, 17.8; MS (MALDI TOF): m/z = 1238.19 [M + Na]⁺.
29 Adinolfi M, Iadonisi A, Ravidà A, Valerio S. Tetrahedron Lett. 2006; 47: 2595

Supplementary Material

Supporting Information