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

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

• For reviews on chemical genetics, see:
• 1a MacBeath G. Genome Biol.  2001,  2:  2005.1 
• 1b Spring DR. Chem. Soc. Rev.  2005,  34:  472 
• 1c Walsh DP. Chang Y.-T. Chem. Rev.  2006,  106:  2476 
• For reviews on DOS, see:
• 3a Spandl RJ. Thomas GL. Diaz-Gavilan M. O’Connell KMG. Spring DR. Chem. Rec.  2008,  129 
• 3b Nielsen TE. Schreiber SL. Angew. Chem. Int. Ed.  2008,  47:  48 
• 3c Spandl RJ. Spring DR. Bender A. Org. Biomol. Chem.  2008,  6:  1149 
• 3d Tan DS. Nat. Chem. Biol.  2005,  1:  74 
• 3e Thomas GL. Wyatt EE. Spring DR. Curr. Opin. Drug Discovery Dev.  2006,  9:  700 
• For reviews and approaches to solving the target identification problem, see:
• 4a Ahn YH. Chang YT. Acc. Chem. Res.  2007,  40:  1025 
• 4b Wong CC. Cheng KW. He QY. Chen F. Proteomics: Clin. Appl.  2008,  2:  338 
• 4c Zheng XFS. Chan TF. Zhou HH. Chem. Biol.  2004,  11:  609 
• 5 Burdine L. Kodadek T. Chem. Biol.  2004,  11:  593 
• For reviews of quorum sensing involving N-acylated homoserine lactones, see:
• 6a Hodgkinson JT. Welch M. Spring DR. ACS Chem. Biol.  2007,  2:  715 
• 6b Geske GD. Oneill JC. Miller DM. Wezeman RJ. Mattmann ME. Lin Q. Blackwell HE. ChemBioChem  2008,  9:  389 
• For selected recent examples, see:
• 7a Thomas GL. Bohner CM. Williams HE. Walsh CM. Ladlow M. Welch M. Bryant CE. Spring DR. Mol. BioSyst.  2006,  2:  132 
• 7b Welch M. Mikkelsen H. Swatton JE. Smith D. Thomas GL. Glansdorp FG. Spring DR. Mol. BioSyst.  2005,  1:  196 
• 7c Welch M. Dutton JM. Glansdorp FG. Thomas GL. Smith DS. Coulthurst SJ. Barnard AML. Salmond GPC. Spring DR. Bioorg. Med. Chem. Lett.  2005,  15:  4235 
• 7d Glansdorp FG. Thomas GL. Lee JJK. Dutton JM. Salmond GPC. Welch M. Spring DR. Org. Biomol. Chem.  2004,  2:  3329 
• 8 A solution-phase route to OHHL(2): Dekhane M. Douglas KT. Gilbert P. Tetrahedron Lett.  1996,  37:  1883 
• The synthesis of native N-acylated homoserine lactones [including OHHL(2)] and non-natural analogues on solid support:
• 9a Geske GD. O’Neill JC. Blackwell HE. ACS Chem. Biol.  2007,  2:  426 
• 9b Geske GD. O’Neill JC. Miller DM. Mattmann ME. Blackwell HE.
  J. Am. Chem. Soc.  2007,  129:  13613 
• 9c Geske GD. Wezeman RJ. Siegel AP. Blackwell HE. J. Am. Chem. Soc.  2005,  127:  12762 
• 10 Analogues of the related signaling molecule N-3-(oxododecanoyl)-l-homoserine lactone (OdDHL), used in Pseudomonas aeruginosa, have been synthesized by coupling using the acid, Meldrum’s acid, and the amine in one pot: Chhabra SR. Harty C. Hooi DSW. Daykin M. Williams P. Telford G. Pritchard DI. Bycroft BW. J. Med. Chem.  2003,  46:  97 ; in our hands this method proved less fruitful than the stepwise method employed therein
• 12 Blackwell HE, Geske GD, and Wezeman RJ. inventors; WO 2006/084056  A2. 

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): ν_max = 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)H_aH_b], 4.27-4.18 [1 H, br m, C(4)H_aH_b], 3.42 (2 H, s, COCH₂CO), 2.68-2.58 [1 H, br m, C(3)H_aH_b], 2.47 (2 H, t, J = 7.3 Hz, CH₃CH₂CH₂), 2.30-2.16 [1 H, br m, C(3)H_aH_b], 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): ν_max = 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)H_aH_b], 4.41-4.29 [2 H, m, NHCHCH₂S and C(4)H_aH_b], 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, SCH_aH_b), 2.75 (1 H, d, J = 12.7 Hz, SCH_aH_b), 2.66-2.49 [3 H, m, C(OCH₂CH₂O)CH₂CO and C(3)H_aH_b)], 2.43-2.30 [1 H, m, C(3)H_aH_b], 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): ν_max = 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)H_aH_b], 4.38-4.23 [2 H, m, NHCHCH₂S and C(4)H_aH_b], 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, SCH_aH_b), 2.74-2.63 (3 H, m, SCH_aH_b and OCH₂CH₂CH₂), 2.62-2.52 [1 H, m, C(3)H_aH_b], 2.37-2.17 [3 H, m, C(3)H_aH_b, 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).