1

Supplementary Methods

Discovery of a new peptide natural product by Streptomyces coelicolor genome mining

S. Lautru, R. J. Deeth, L. M. Bailey and G. L. Challis Structure determination of coelichelin

Accurate mass measurements were made using a 9.4 Tesla Bruker BioAPEX II FTICR mass

spectrometer equipped with a Bruker/Analytica Bramford nanospray source in positive ion mode or

a Bruker MicroTOF mass spectrometer equipped with a electrospray source in positive ion mode.

ESI-MS-MS spectra were measured using a Bruker Esquire HCT mass spectrometer equipped with

an electrospray source in positive ion mode. NMR spectra were measured on an approximately

1mM d6-DMSO solution of Ga-coelichelin in a 3mm NMR tube at 320K using a Bruker Avance

700MHz NMR spectrometer equipped with a TCI cryoprobe. 1H, DQF-COSY, TOCSY (mixing

time of 60 ms), HMBC and ROESY (mixing time of 500 ms) spectra were measured. Inter-residue

αCH-NH distances (r) were calculated from the ROESY data using the equation: signal intensity =

A/r6; where the value of A was calculated from the intensity of the strongest geminal correlation

observed assuming a value of r = 1.75 Å. The αH-NH and αH-βH dihedral angles were calculated

from the 3J coupling constants using appropriate Karplus relationships.

ESI-FTICR-MS analysis of purified desferri-coelichelin indicated a molecular formula of

C21H39N7O11 (measured mass = 566.27864; mass calculated for C21H40N7O11+

= 566.27866;

Supplementary Figure 1). Analysis of the y fragments in the ESI-MS-MS spectrum of

desferricoelichelin, produced by collision-induced dissociation of the C21H40N7O11+ (m/z = 566.2),

suggested the presence of two hfOrn residues, one Thr residue and one hOrn residue. This data also

indicated that the hOrn residue was acylated on one amino group with the dipeptide hfOrn-Thr and

on the other amino group with the second hfOrn residue (Figure 2). Due to the instability of

desferri-coelichelin in aqueous solution, it was converted to the gallium complex by reaction with

gallium sulfate prior to further analysis by NMR spectroscopy. The Ga-coelichelin was purified by

semi-preparative HPLC and formation of the complex was confirmed by ESI-TOF-MS (measured

masses = 632.1818, 634.1814; mass calculated for C21H37Ga69N7O11+ = 632.1807; mass calculated

for C21H37Ga71N7O11+ = 634.1798; Supplementary Figures 3 and 4). The 1H NMR spectrum of Ga-

coelichelin in DMSO confirmed the presence of two amide groups and two formyl groups in the

2

complex. COSY and TOCSY experiments identified the 1H spin systems for the Thr residue and

three ornithine-containing residues and allowed complete assignment of the 1H NMR spectrum

(Table 1). Inter-residue correlations in the HMBC spectrum of Ga-coelichelin from the hfOrn1

carbonyl carbon to the Thr NH, the hfOrn1 carbonyl carbon to the Thr α-H, and the hfOrn2

carbonyl carbon to the hOrn NH allowed the sequence of residues in coelichelin to be

unambiguously assigned. The carbonyl carbon signals for each residue were assigned on the basis

of intra-residue correlations to the α-H and the characteristic chemical shift for the carbonyl carbon

of a threonine-derived hydroxamic acid (Figure 5). The sequence and connectivity of Ga-

coelichelin were confirmed by inter-residue correlations in the ROESY spectrum of the complex

between the hfOrn1 α-H and the Thr N-H, the Thr α-H and one of the hOrn δ-Hs, the hOrn NH and

the hfOrn2 α-H, and intra-residue correlations between the each of the formyl Hs and one of the δ-

Hs in hfOrn1 and hfOrn2, respectively (Figure 6).

The relative stereochemistry of the α-carbons in Ga-coelichelin was elucidated theoretically.

The need for extensive conformational searching precluded a quantum chemical approach.

However, the absence of suitable force field parameters for metal complexes in general and Ga-

hydroxamate complexes in particular necessitated development of a novel set of parameters. The

inter-residue α-H to NH distances in the lowest energy conformers calculated by stochastic searches

for each of the eight possible diastereomers of Ga-coelichelin were compared to the corresponding

distances calculated from the ROESY data. Good agreement between theory and experiment was

observed for only one of the eight diastereomers and then only when the carboxylate of hOrn3 and

the free amine of hfOrn4 were in their ionised forms. The dihedral angles between α-Hs and NHs

and the α- and β-Hs in the model of this diastereomer also agreed well with those calculated from

the 1H NMR spectrum of Ga-coelichelin, whereas those in the models of the other seven

diastereomers did not. The calculations appear able to discriminate both the stereochemistry and

charge states of the ligand’s ionisable groups.

The Ligand Field Molecular Mechanics (LFMM) model1 was specially parameterised for

Ga3+-hydroxamate complexes via density functional theory (DFT) calculations on the model

complex [Ga(N-methylhydroxamate)3]. The chosen DFT method gave excellent reproduction of the

3

molecular structure and was further used to estimate the energetic cost of shortening the optimal

Ga-L bond lengths by 0.1 Å (12 kcal mol-1), lengthening them by up to 0.2 Å (26 kcal mol-1), and

twisting the structure from the approximately octahedral minimum to a trigonal prism (12 kcal mol-

1). The LFMM parameters for Ga-L bond stretching and L-L repulsion (there is no ligand field

contribution for formally d0 Ga3+) were then optimised to reproduce these energies, paying special

attention to the octahedral-trigonal prism twisting energy. All LFMM calculations employed the

DommiMOE2 program and the supplied MMFF94 force field including electrostatic contributions

as described in the supporting information. Metric data from the lowest energy conformers from

three 500-step stochastic searches are given in the table in the supplementary information.

A search of the Cambridge Structural Database on ferric hydroxamate complexes suggests

the Fe-O distances are insensitive to the details of the ligand coordination. Only one Ga

hydroxamate complex was located but this was virtually isostructural with its Fe(3+) analogue.

Density Functional Theory (DFT) calculations on [Ga(N-methylhydroxamate)3] (Amsterdam

Density Functional program version 2003, TZP basis on Ga, DZP on C, N and O, DZ on H) were

employed to calibrate the MM structures and energies. The DFT optimisations (at the local density

approximation level) give excellent reproduction of the experimental M-L bond lengths and further

suggest that fac and mer isomers are virtually equienergetic. A trigonal prismatic model was

derived from the fac isomer by enforcing D3 symmetry. The FF parameters were designed to

reproduce the DFT trigonal prism/octahedral energy difference of 12 kcal mol-1. All MM

computations used our DommiMOE package. The FF parameters were derived from the MMFF94

parameters supplied with MOE version 2004.03. Standard MMFF94 partial atomic charges were

used apart from the oxygen donor atoms. Based on DFT estimates of the changes in partial atomic

charges between coordinated and free hydroxamates, the carbonyl oxygen charge was reduced from

–0.57 to –0.47 and the other oxygen from –0.712 to –0.512 resulting in a partial charge on the Ga of

1.35. In the absence of ligand field effects for Ga3+, all the Angular Overlap Model parameters

were set to zero. The additional LFMM parameters required are thus r0, D and α for the Morse

function and the ligand-ligand repulsion parameters ALL/dn:

4

Bond r0 D α

Ga+3 O= 1.93 60 1.00

Ga+3 OM 1.87 60 1.00

Donor ALL n

O=/OM 1100 6

Conformational searches were carried out using the stochastic search algorithm implemented in

DommiMOE. Three 500-step searches were run, the second and third starting from the minimum

energy structure from the previous run. To prevent the program sampling irrelevant regions of

conformation space, chirality was enforced as was the trans arrangement of the amide bonds in the

ligand backbone. The final structures were re-optimised with all restraints removed. Three sets of

calculations were undertaken. In the first, Set 1, all ionisable groups (the two free amines and the

carboxylic acid moiety) were assumed to be unionised. Set 2 assumed all groups were ionised

(giving an overall molecular charge of +1). Set 3 had the carboxylic acid group and the amine group

of hfOrn4 ionised and the amine group of hfOrn1 unionised (giving a molecular charge of zero).

No calculations from Set 1 were able to satisfy all the observed constraints suggesting a strong salt

bridge between hfOrn4 and fOrn3 is required. Although all the relevant groups are expected to be

ionised in aqueous solution, the NMR experiments were conducted in DMSO. The close physical

approach of the carboxylic acid group and its neigbouring amine would facilitate an internal proton

transfer suggesting Set 3 as a better representation than Set 2. (See Table 2 for comparison of αH-

NH and dihedral angles for each model of Set 3 with experimentally derived data).

The relative stereochemistry of the α- and β- carbons of the Thr residue was elucidated by

analysis of N-trifluroacetyl isopropyl ester derivative prepared from the acid hydrolysate of Ga-

coelichelin. Ga-Coelichelin (~ 50 �g) was hydrolysed in 100 µl of concentrated HI at 100°C for 24 h

and excess HI was removed under vacuum. Coelichelin hydrolysate, L-, D-, L-allo and D-allo

threonine were treated with 3 M HCl in propan-2-ol for 30 min at 100°C and excess reagents were

removed under vacuum. Samples were next treated with trifluoroacetic anhydride in

dichloromethane (1:1 v/v) for 10 min at 100°C and excess reagents removed under a stream of

argon. 0.1µl of the N-trifluoroacetyl propan-2-yl ester derivatives in ethylacetate were injected on a

5

BPX5 30m x 0.32 mm x 0.5µm g.c. column connected to a Shimadzu 17A instrument (flame

ionisation detector and splitting ratio of 1:40). The injector and the detector temperatures were

maintained at 250°C and helium was used as carrier gas. A gradient of temperature of 4°C/ min

from 80°C to 170°C was used. Comparison with the standards indicated that the Thr residue has

allo relative stereochemistry.

1. Deeth R. J., Coord. Chem. Rev. 212, 11- (2001).

2. Deeth R. J., Fey N., Williams-Hubbard, B. J. J. Comp. Chem. 26, 123- (2005).

6

Figure 1: High resolution ESI-FTICR mass spectrum of desferricoelichelin. Top trace: simulated spectrum for C21H40N7O11

+. Bottom trace: measured spectrum.

Figure 2: ESI-MS-MS spectrum of desferricoelichelin. Major y fragment ions are assigned. The peak at m/z = 249.9 corresponds to the y ion resulting from loss of both hfOrn residues.

566.278644

7

Figure 3: HPLC purification of Ga-coelichelin. A Phenomenex Synergy 4µ Fusion RP 250 x 10 mm, (4 µm) column was used under the conditions detailed in the methods section.

Figure 4: ESI-TOF Mass spectrum of Ga-coelichelin. Top trace: measured spectrum of Ga-coelichelin. Bottom trace: simulated spectrum for C21H37N7O11Ga+.

-10

190

390

590

790

990

0 5 10 15 20 25 30

Time (min)

Abso

rban

ce a

t2

00

nm

(m

AU

)

-10

190

390

590

790

990

0 5 10 15 20 25 30

Time (min)

Abso

rban

ce a

t2

00

nm

(m

AU

)

Ga-coelichelin

8

Table 1: 1H and 13C NMR assignments for Ga-coelichelin Assignment δH δC

hfOrn1-C1 - 168.5 hfOrn1-C2 3.97, dd, 3.0 Hz, 3.0 Hz hfOrn1-C3 1.91, m; 1.50, m hfOrn1-C4 1.42, m; 1.50, m hfOrn1-C5 3.41, m; 3.51, m hfOrn1-NH2 - - hfOrn1-C(O)H 8.17, s 153.5 Thr2-C1 - 162.5 Thr2-C2 4.59, dd, 9.0 Hz, 9.0 Hz 58.0 Thr2-C3 3.82, m 66.0 Thr2-C4 1.14, d, 5.5 Hz 21.0 Thr2-NH 8.75, d, 9.0 Hz - Thr2-OH 5.08, d, 6.2 Hz - hOrn3-C1 - 173 hOrn3-C2 4.24, dd, 8.5 Hz, 11.0 Hz hOrn3-C3a 1.76, m hOrn3-C4 1.83, m; 1.83, m hOrn3-C5 3.45, m; 3.93, m hOrn3-NH 7.57, d, 8.5 Hz - hfOrn4-C1 - 170 hfOrn4-C2 4.15, dd, 11.0 Hz, 1.9 Hz hfOrn4-C3 1.64, m; 2.29, m hfOrn4-C4 1.96, m, 1.76, m hfOrn4-C5a 3.76, m hfOrn4-C(O)H 8.22, s 153.5 hfOrn4-NH2 - - a The signal for the other proton attached to these carbon atoms was masked by a solvent signal.

H2NN

OOH

N

O

O

N

ONH2

NO

O

NO

O

Ga

O OH

H

H

HH

HH

H H

12

3

4

5

12

34

1

23

4

5

12

3

4

5

hfOrn1

Thr2

hOrn3

hfOrn4

9

Figure 5: Region of the HMBC spectrum of Ga-coelichelin showing correlations between αH and C=O

H2NN

O

N

O

O

N

ONH2

NO

O

NO

O

Ga

O OH

H

H

HH

HH

H H

H

HOH

H

1

2

3

H

4

5

6

Figure 6: Region of the ROESY spectrum of Ga-coelichelin showing important correlations

10

Table 2: Comparison of key inter-proton distances and dihedral angles from models of each α-C diastereomer of Ga-coelichelin with values calculated from experiment. Conformer experimental RRSR RRRR RRSS RSSR SRSR RRRS RSRR RSSS

E - -166.0 -173.6 -165.0 -168.2 -167.9 -167.7 -163.3 -169.3 �E(next) - 3.5 7.8 5.4 2.2 1.9 0.0* 3.2 4.8

tor1 60 63 -57 -50 -63 (-49)

69 (49)

-58 (99)

-69 -49

tor2 60 52 174 65 -178 (66)

174 (66)

-174 (144)

175 66

tor3 140-220 153 159 149 152 (-147)

147 (-147)

160 (-165)

-158 17

tor4 160 179 37 -37 -44 (41)

54 (41)

39 (48)

155 42

tor5 80 64 -76 77 70 (-74)

-62 (-74)

-74 (-67)

41 -72

tor6 140-220 126 168 -141 -163 (-143)

-129 (-143)

159 (138)

130 -167

tor7 180 176 177 170 -179 (-161)

162 (161)

155 (-178)

178 -166

tor8 60 60 64 55 65 (83)

-82 (83)

-89 (65)

66 -52

dis1 2.59 2.40 2.67 2.40 2.39 (2.55)

2.40 (2.55)

2.72 (2.39)

2.40 2.46

dis2 2.58 2.52 2.39 2.53 3.66 (3.42)

3.54 (3.42)

2.36 (2.81)

3.69 2.29

dis3 1.83 2.06 2.36 2.35 4.14 (2.13)

2.15 (2.13)

2.38 (2.54)

2.11 1.95

dis4 2.46 2.45 3.77 3.79 2.21 (2.32)

2.42 (2.31)

2.24 (2.38)

3.81 3.77

dis5 2.32 2.36 2.51 2.54 2.42 (2.58)

2.37 (2.58)

2.59 (2.33)

2.56 2.32

* Next confomer energy -165.2 (+2.5) Torsions more than 30 degrees from experiment, or outside experimental range, are in bold. Distances > 0.4 Å in bold and > 1.0 Å in bold and underlined. Data for conformers within 2.2 kcal mol-1 of lowest energy structure in parentheses. 2 kcal/mol cutoff implies >= 97% lowest energy conformer 3 kcal/mol cutoff implies >= 99% lowest energy conformer Order of chiral centres is hfOrn1-Thr2-hOrn3-hfOrn4

H2NN

O

N

O

O

N

ONH2

NO

O

NO

O

Ga

O OH

H

H

HH

HH

H H

H

HOH

H

HH

tor1, tor2

tor3

HH

tor4, tor5

HH

tor7, tor8

tor6

dis1

dis2

dis3

dis4

dis5

11

HPLC analysis of coelichelin production HPLC analysis of S. coelicolor M145, W5, W6, wild type S. fungicidicus B-5477 and S. fungicidicus B-5477 containing the cch cluster were carried out as described in the methods section (Figure 7).

-50

350

750

1150

1550

-50

350

750

1150

1550

-50

350

750

1150

1550

-50

350

750

1150

1550

-50

350

750

1150

1550

0 2 4 6 8 10

Retention time/min

A435

/mA

U

a

b

c

d

e

Coelichelin

Figure 7: HPLC analysis of coelichelin production in iron deficient media: (a) by S. coelicolor M145; (b) by S. coelicolor W5; (c) S. coelicolor W6; (d) by S. fungicidicus B-5477; (e) by one transfromant of S. fungicidicus B-5477 containing the cch cluster. The retention times of coelichelin in culture supernatants of S. coleicolor and the three independent transformants of S. fungicidicus B-5477 containing the cch cluster are 2.78 min, 2.76 min, 2.79 min and 2.76 min, respectively.