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Synlett 2008(14): 2122-2126  
DOI: 10.1055/s-2008-1077978
LETTER
© Georg Thieme Verlag Stuttgart ˙ New York

Synthesis of a Biotin-Labeled Quorum-Sensing Molecule: Towards a General Method for Target Identification

Richard J. Spandla, Rebecca L. Nicholsona, David M. Marsdena, James T. Hodgkinsona, Xianbin Sua, Gemma L. Thomasa, George P. C. Salmondb, Martin Welchb, David R. Spring*a
a Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, UK
Fax: +44(1223)336362; e-Mail: drspring@ch.cam.ac.uk;
b Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, UK
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Publication History

Received 23 May 2008
Publication Date:
15 July 2008 (online)

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Abstract

The synthesis of bacterial quorum-sensing regulator N-(3-oxohexanoyl)-l-homoserine lactone (OHHL) and biotin-tagged OHHL is reported. The latter will be applied to developing a general method to address the ‘target identification problem’ in chemical genetics.

Key words

drugs - high-throughput screening - proteins - OHHL - quorum sensing

    References and Notes

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2

The complementary approach, reverse chemical genetics, involves modulating a known protein and analyzing the resulting phenotype.¹b

11

N-(3-Oxohexanoyl)-l-homoserine lactone(2): R f = 0.23 (SiO2; EtOAc-PE, 8:2). IR (neat): νmax = 3301 (w, br), 2965 (w), 2878 (w), 1774 (s), 1716 (m), 1649 (s), 1535 (m), 1379 (m), 1221 (m), 1169 (s), 1021 (m) cm-¹. ¹H NMR (400 MHz, CDCl3): δ = 7.73 (1 H, br s, CONH), 4.63-4.51 [1 H, br m, C(2)H], 4.43 [1 H, br t, J = 9.1 Hz, C(4)HaHb], 4.27-4.18 [1 H, br m, C(4)HaHb], 3.42 (2 H, s, COCH2CO), 2.68-2.58 [1 H, br m, C(3)HaHb], 2.47 (2 H, t, J = 7.3 Hz, CH3CH2CH2), 2.30-2.16 [1 H, br m, C(3)HaHb], 1.54 (2 H, sext, J = 7.3 Hz, CH3CH2CH2), 0.86 (2 H, t, J = 7.5, CH3CH2CH2). ¹³C NMR (100 MHz, CDCl3): δ = 206.1 (C), 175.2 (C), 166.9 (C), 65.9 (CH2), 48.9 (CH), 48.7 (CH2), 45.4 (CH2), 29.2 (CH2), 16.8 (CH2), 13.4 (CH3). HRMS: m/z calcd for C10H15NO4Na+: 236.0899; found [ESI - Na+]: 236.0892; Δppm = -1.5; mp 80-81 ˚C (EtOAc-PE). [α]D ²5 +7.36 (c 0.95, CHCl3).

13

Compound 2: [α]D ²5 +7.36 (c 0.95, CHCl3). Sigma OHHL [α]D ²5 +8.5 (c 0.12, CHCl3). These specific rotation values are slightly lower than those reported by Blackwell and co-workers,¹²a that is, [α]D ²5 +12.2 (c 2.7, CHCl3). Although some racemization may have occurred during the synthesis reported here, this did not affect binding of CarR. In our hands coupling with HOBt was less successful.

14

Polymer-bound DMAP was required in the final EDC-mediated coupling to aid purification. The reaction products and DMAP had very similar R f values.
Synthesis of 18
A round-bottom flask, equipped with a magnetic stirrer, containing the ester 17 (529 mg, 1.02 mmol), LiOH˙H2O (98 mg, 2.33 mmol) and 66% aq MeOH (25 mL) was stirred at r.t. for 16 h. The solvent was removed in vacuo to give the lithium salt of the corresponding acid (structure not shown) as a white solid (550 mg). The salt was used in subsequent reactions without further purification. A round-bottom flask, equipped with a magnetic stirrer, containing the lithium salt (0.55 g, 1.09 mmol), EDC (0.27 g, 1.42 mmol), polymer-bound DMAP (5 mmol/g, 1.1 g, 5.46 mmol), and DMF (40 mL) was stirred at r.t. for 15 min before being charged with l-homoserine lactone hydrobromide (1.02 g, 5.6 mmol) and stirred at r.t. for 16 h. The crude reaction mixture was filtered and solvent removed in vacuo. The crude product was purified by column chromatography to give 18 as a colorless oil (0.43 g, 68% over 2 steps).
R f = 0.36 (SiO2; CH2Cl2-MeOH, 85:15). IR (neat): νmax = 3391 (s, br), 2932 (w, br), 1766 (m), 1645 (s) br, 1549 (s), 1474 (m), 1355 (m), 1063 (s) cm-¹. ¹H NMR (400 MHz, CD3OD): δ = 4.69-4.52 [2 H, m, NHCHCH2S and C(2)H], 4.48 [1 H, t, J = 9.2 Hz, C(4)HaHb], 4.41-4.29 [2 H, m, NHCHCH2S and C(4)HaHb], 4.13-3.94 [4 H, m, C(OCH2CH2O)CH2], 3.67-3.54 (6 H, m, OCH2CH2O and NHCH2CH2O), 3.54 (2 H, t, J = 6.1 Hz, OCH2CH2CH2C), 3.35-3.47 (2 H, br m, NHCH2CH2O), 3.30-3.22 (1 H, m, SCH), 2.97 (1 H, dd, J = 12.7, 5.1 Hz, SCHaHb), 2.75 (1 H, d, J = 12.7 Hz, SCHaHb), 2.66-2.49 [3 H, m, C(OCH2CH2O)CH2CO and C(3)HaHb)], 2.43-2.30 [1 H, m, C(3)HaHb], 2.26 (2 H, t, J = 7.1 Hz, CH2CH2CH2CH2CONH], 1.88-1.55 (8 H, CH2CH2CH2CH2CONH and OCH2CH2CH2C), 1.53-1.41 (2 H, m, CH2CH2CH2CH2CONH). ¹³C NMR (100 MHz, CD3OD): δ = 174.5 (C), 173.3 (C), 168.9 (C), 163.1 (C), 107.7 (C), 69.3 (CH2), 68.4 (CH2), 68.2 (CH2), 67.7 (CH2), 64.5 (CH2), 63.4 (CH2), 60.5 (CH), 58.8 (CH), 54.1 (CH), 47.0 (CH), 42.2 (CH2), 38.3 (CH2), 37.5 (CH2), 33.9 (CH2), 32.5 (CH2), 26.9 (CH2), 26.7 (CH2), 26.5 (CH2), 24.0 (CH2), 22.0 (CH2). LCMS (MeCN): 587 [MH]. HRMS: m/z calcd for C26H43N4O9S1 +: 587.2746; found [ESI - H+]: 587.2746; Δppm = +0.1.
Synthesis of 1
A round-bottom flask, equipped with a magnetic stirrer and open to air, containing the acetal 18 (140 mg, 0.23 mmol), CH2Cl2 (2.5 ml), and TFA (0.25 mL, 33.6 mmol) at r.t. was stirred for 2.5 h and the solvent was removed in vacuo. The crude product was purified by column chromatography to give the title compound 1 (66 mg, 52%) as a colorless oil.
R f = 0.21 (SiO2; CH2Cl2-MeOH, 9:1). IR (neat): νmax = 3292 (w, br), 2926 (w, br), 1774 (m), 1671 (s, br), 1541 (m), 1469 (m), 1332 (m), 1200(s), 1175 (s), 1127 (s), 1020 (m) cm-¹. ¹H NMR (400 MHz, CD3OD): δ = 4.58 [1 H, dd, J = 10.8 9.3, C(2)H], 4.53-4.40 [2 H, m, NHCHCH2S and C(4)HaHb], 4.38-4.23 [2 H, m, NHCHCH2S and C(4)HaHb], 3.62-3.50 (6 H, m, OCH2CH2O and NHCH2CH2O), 3.46 (2 H, t, J = 6.3 Hz, OCH2CH2CH2), 3.39-3.32 [4 H, m, NHCH2CH2O and C(O)CH2C(O)], 3.24-3.16 (1 H, m, SCH), 2.93 (1 H, dd, J = 5.1, 12.7 Hz, SCHaHb), 2.74-2.63 (3 H, m, SCHaHb and OCH2CH2CH2), 2.62-2.52 [1 H, m, C(3)HaHb], 2.37-2.17 [3 H, m, C(3)HaHb, CH2CH2CONH], 1.84 [2 H, quin, J = 6.9 Hz, OCH2CH2CH2C(O)], 1.77-1.53 (4 H, m, CH2CH2CH2CH2), 1.44 (2 H, quin, J = 3.9 Hz, CH2CH2CH2CH2). ¹³C NMR (100 MHz, CD3OD): δ = 197.5 (C), 175.7 (C), 174.8 (C), 167.9 (C), 164.7 (C), 69.8 (CH2), 69.8 (CH2), 69.7 (CH2), 69.2 (CH2), 65.9 (CH2), 61.9 (CH), 60.2 (CH), 55.6 (CH), 48.7 (CH), 48.5 (CH2), 39.6 (CH2), 39.0 (CH2), 38.9 (CH2), 35.4 (CH2), 28.3 (CH2), 28.2 (CH2), 28.1 (CH2), 25.4 (CH2), 23.3 (CH2). HRMS: m/z calcd for C24H39N4O8S+: 543.2489; found [ESI - H+]: 543.2497; Δppm = +0.7. [α]D ²5 +11.7 (c 0.54, CHCl3).