The Ralstonia solanacearum pathogenicity regulator HrpB induces 3-hydroxy-oxindole synthesis -- Delaspre et al. 104 (40): 15870 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Delaspre et al. 10.1073/pnas.0700782104.

Supporting Information

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SI Figure 5
SI Figure 6
SI Table 1
SI Figure 7
SI Figure 8
SI Table 2

SI Figure 5

Fig. 5. HMBC spectrum of U-[¹⁵N,¹³C] HDF at 18°C in acetonitrile-d₄. Assignment of ¹³C resonances is illustrated by horizontal lines. In addition to HSQC and H2BC spectra, the HMBC spectrum revealed the signals arising from quaternary carbons. Correlation peaks with quaternary carbons are labeled according to their respective ¹H and ¹³C resonances associated with the atom numbering.

SI Figure 6

Fig. 6. ¹H-decoupled ¹³C NMR spectrum of U-[¹⁵N,¹³C] HDF performed in acetonitrile-d₄. Based on ¹³C-¹³C scalar coupling analysis and in addition to HMBC, the ¹³C NMR spectrum permitted the unambiguous assignment of the quaternary carbons; C4 at 129.8 ppm gave a quartet due to its homonuclear scalar coupling with C3, C5, and C9, whereas C5 at 143.2 ppm gave a triplet due to its homonuclear scalar coupling with only C6 and C4. The intense solvent peak is labeled as CD₃CN.

SI Figure 7

Fig. 7. ¹H-¹⁵N and ¹H-¹³C two dimensional HN(CO)CA spectra of U-[¹⁵N,¹³C] HDF was performed in acetonitrile at 18°C. This experiment confirmed the presence of an indole ring, because amide proton magnetization could be transferred via the C2 carbonyl carbon to carbon C3. The coherence pathway between the two spectra is the same, with the exception that ¹⁵N coherence evolved during t1 for the ¹H-¹⁵N spectrum whereas ¹³C coherence evolved in the ¹H-¹³C spectrum.

SI Figure 8

Fig. 8. LC-MS analysis of HDF extracted from a Ralstonia solanacearum culture (A) and from chemically synthesized HDF (B).

Table 1. ¹H and ¹³C chemical shifts and corresponding scalar homonuclear ¹H and ¹³C spin couplings of HDF in acetonitrile measured at 18°C

Atom No	δ _1H, ppm	³J_HH(multiplicity)	δ _13C, ppm	¹J_CC (multiplicity)
1	8.22	(broad)
2			178.6	54.6 Hz (d)
3	4.88	7.0 Hz (d)	70.7	51.3 Hz (t)
4			129.8	57.2 Hz (q)
5			143.2	62.0 Hz (t)
6	6.85	7.7 Hz (d)	111.0	61.5 Hz (t)
7	7.24	7.7 Hz (t)	130.7	57.2 Hz (t)
8	7.01	7.5 Hz (t)	123.4	56.6 Hz (t)
9	7.33	7.5 Hz (d)	70.7	57.8 Hz (t)
10	4.02	7.0 Hz (d)

Table 2. LC-MS analysis of HDF, 3-OH-PAME, and N-hexanoyl-HSL (C₆-HSL) and N-octanoyl-HSL (C₈-HSL): applied parameters

		HDF	3-OH-PAME	C₆-/C₈-HSL
HPLC	Flow rate, ml •min-¹	200	200	200
	Mobile phase	50% methanol in water; isocratic	Acetonitrile; isocratic	Solvent A: water, solvent B: acetonitrile; Gradient: 0-1 min: 20% B, 10 min: 80% B, 10-12 min: 80% B
	Injection volume, ml	10	10	10
	Standard conc., mM	0.1, 1, 10, 50	0.05, 0.1, 0.5, 2	0.1, 0.5, 1
MS	Vaporizer temp., °C	200	250	230
	Sheath gas flow, arb.	50	40	36
	Aux gas flow, arb.	10	20	7
	Sweep gas flow, arb.	0	5	0
	Discharge current, mA	5	10	5
	Capillary temp., °C	275	275	275
	Capillary voltage, V	7	6	22.5
	Tube lense, V	38	40	48