1

Supplementary data

Chemical mechanism of petal color development of Nemophila menziesii by a

metalloanthocyanin, nemophilin

Kumi Yoshida*,†, Kensuke Tojo†, Mihoko Mori‡,**, Keiko Yamashita†, Sayoko Kitahara† Masanori

Noda††, Susumu Uchiyama††, †††

†Graduate School of Information Science, ‡Graduate School of Human Informatics, Nagoya University,

Chikusa, Nagoya 464-8601, Japan ††Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka,

Suita 565-0871, Osaka, Japan †††Department of Bioorganization Research, Okazaki Institute for Integrative Bioscience, 5-1,

Higashiyama, Myodaiji-cho, Okazaki 444-8787, Japan

*Corresponding Author: Phone +81-52-789-5638; Fax: +81-52-789-5638

E-mail: yoshidak@is.nagoya-u.ac.jp

**Present address: Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato

Ward, Tokyo 108-8641, Japan

2

Isolation and structure identification of petal components

3-O-(6-O-p-coumaroyl-β-glucopyranosyl)-5-O-(6-O-malonyl-β-glucopyranosyl)petunidin (2,

NM) .

Red amorphous powder of TFA salt, 1H NMR (600 MHz, 10% TFA-d-CD3OD, 24 °C) δ ppm

(multiplicity, J in Hz): 3.48 (1H, t, 9.5), 3.51 (1H, dd, 9.2, 8.8), 3.60 (1H, dd, 9.2, 8.8), 3.65 (1H, dd,

9.2, 8.8), 3.77 (1H, dd, 8.8, 8.4), 3.80 (1H, dd, 8.8, 8.4), 3.82 (1H, m), 3.96 (1H, m), 3.98 (3H, s), 4.26

(1H, dd, 12.0, 6.6), 4.46 (1H dd, 12.0, 8.0), 4.53 (1H, dd, 12.5, 3.0), 4.56 (1H, dd, 12.0, 2.2), 5.21 (1H,

d, 8.0), 5.49 (1H, d, 8.0), 6.22 (1H, d, 16.0), 6.73 (1H, d, 16.0), 6.73 (2H, d, 8.8), 6.93 (1H, s), 7.00 (1H,

s), 7.22 (2H, d, 8.8), 7.74 (1H, d, 1.5), 7.90 (1H, d, 1.5), 8.92 (1H, s); FABMS: m/z = 873 [M]+.

7-O-β-glucopyranosyl-4’-O-(6-O-malonyl-β-glucopyranosyl)apigenin (3, MP).

Amorphous powder, [α]D24 -58 (c 0.1, MeOH); IR (neat) 3362, 1736, 1657, 1606, 1497, 1347, 1239,

1176, 1070 cm-1; UV (MeOH) λmax nm (ε) 318 (19,400), 269 (20,900); 1H NMR (500 MHz, CD3OD,

50°C) δ ppm (multiplicity, J in Hz): 3.37 (1H, t, 8.5, G4’-4), 3.41 (1H, t, 8.5, G7-4), 3.47-3.55 (3H, m,

G7-2, G7-3, G7-5), 3.49 (2H, m, G4’-2, G4’-3), 3.71 (1H, ddd, 8.5, 7.0, 2.0, G4’-5), 3.72 (1H, dd, 12.0,

6.5, G7-6a), 3.93 (1H, dd, 12.0, 2.0, G7-6b), 4.27 (1H, dd, 12.0, 7.0, G4’-6a), 4.59 (1H, dd, 12.0, 2.0,

G4’-6b), 5.01 (1H, d, 7.5, G4’-1), 5.06 (1H, d, 7.5, G7-1), 6.50 (1H, d, 1.5, H-6), 6.68 (1H, s, H-3),

6.80 (1H, d, 1.5, H-8), 7.22 (2H, d, 8.5, H-3’,5’), 7.95 (2H, d, 8.5, H-2’,6’). 13C NMR (125 MHz,

CD3OD, 20°C) δ ppm: 61.1, 64.2, 69.9, 70.2, 73.4, 74.2, 76.4, 76.5, 77.0, 94.8, 99.9, 100.2, 103.8,

105.8, 116.7, 124.7, 128.1, 157.6, 160.7, 161.5, 163.5, 164.7, 167.1, 168.8, 182.7. HRMS: calcd. for

C30H31O18 [M-H]- 679.1516, found, 679.1505.

3-O-(6-O-α-rhamnopyranosyl-β-glucopyranosyl)-7-O-β-glucopyranosylkaempferol (5) and

3-O-(6-O-α-rhamnopyranosyl-β-glucopyranosyl)-7-O-α-rhamnopyranosylkaempferol (6).

A fraction of the Amberlite XAD-7 column chromatography (0.5% TFA-15% MeCN aq.) product,

which was equivalent to ca. 500 g of petals, was purified with preparative ODS-HPLC eluted with

0.5% TFA-12% to 20% MeCN aq. to yield pure 5 (79 mg) and 6 (5.0 mg), respectively. 5: 1H NMR

(600 MHz, C5D5N, 24 °C) δ ppm (multiplicity, J in Hz): 1.48 (3H, d, 3.7), 3.95 (1H, dd, 11.0, 5.0),

4.01 (1H, t, 9.5), 4.09 (1H, t, 8.0), 4.14 (2H, d, 7.5), 4.18 (1H, m), 4.33 (9H, m), 4.53 (1H, d, 11.0),

4.57 (1H, d, 12.5), 5.27 (1H, s), 5.81 (1H, d, 7.5), 6.05 (1H, d, 6.5), 6.75 (1H, brs), 6.99 (1H, brs), 7.23

(2H, d, 6.5), 8.41 (2H, d, 6.5); FABMS: m/z = 757 [M+H]+, 6: 1H NMR (600 MHz, C5D5N, 24 °C) δ

3

ppm (multiplicity, J in Hz): 1.41 (3H, d, 3.7), 1.42 (3H, d, 3.7), 3.89 (1H, dd, 11.0, 6.0), 3.92 (1H, dd,

9.5, 9.0), 3.99 (1H, brt, 7.5), 4.07 (2H, d, 7.5), 4.27 (1H, d, 6.5), 4.28 (1H, s), 4.31 (1H, t, 9.5), 4.31

(1H, dd, 9.5, 9.0), 4.38 (1H, d, 11.0), 4.52 (1H, dd, 9.5, 9.0), 4.83 (1H, dd, 9.5, 3.0), 4.85 (1H, d, 3.5),

4.99 (1H, dd, 9.0, 6.5), 5.19 (1H, s), 6.39 (1H, d, 7.5), 6.43 (1H, s), 6.65 (1H, brs), 6.69 (1H, brs), 7.31

(2H, d, 9.0), 8.51 (2H, d, 9.0); FABMS: m/z = 741 [M+H]+.

Assignment of 1H NMR signals and NOE observations of nemophilin for analysis of

intermolecular stackings.

Analysis of 1a

Using various 1D and 2D NMR with ambient temperature, aromatic protons of 1a were assigned. For

assignment, the NMR analysis of commelinin, cyanosalvianin and protocyanin were also referred.

Broad singlet signals observed at 8.00 and 8.22 ppm had a correlation each other in COSY (Figure

S13) and the former signal had a NOE correlation with OMe (3.41 ppm, s) in NOESY (Figure S14).

Therefore the signal at 8.00 was assigned to be H-2’ of 2 and that of 8.22 ppm as H-6’ of 2,

respectively. Signals at 6.40 and 6.46 ppm had a COSY correlation and the area of each signal seemed

to be 2H by integration. Protons at 7.46, 6.78 and 6.58 ppm also had COSY correlations each other. In

commelinin and cyanosalvianin, protons of B-ring of flavone components were observed separately,

but the protons of p-coumaroyl residue did not. Thus, signals at 6.40 ppm and 6.46 ppm were assigned

to be H-2” and H-6”, and H-3” and H-5” of 2, respectively. The signals of 7.46, 6.78 and 6.58 ppm

were deduced to be signals at B-ring of 3. Signals at 5.99 and 5.58 ppm, and those at 5.50 ppm and

4.96 ppm showed a correlation in a 1D-HOHAHA and decoupling experiments (1H: 800 MHz, D2O, rt,

40°C), respectively indicating that each of them might be a pair of H-6 and H-8 of 2 and 3.

Comparison of signals of Zn-Zn protocyanin, the protons at 5.99 ppm, and 5.50 ppm, 5.58 ppm and

4.96 ppm were deduced to be H-6 of 3, H-8 of 3, H-6 of 2 and H-8 of 2, respectively. In addition H-3

of 3 was overlapped at 5.58 ppm. Correlated signals of broad doublet at 5.84 and 6.78 ppm by COSY

experiment were determined to be α and β protons of p-coumaroyl residue of 2. Remaining singlet

4

signal at 6.73 ppm was determined to be H-4 of 3. After those assignment of aromatic signals of 1a,

long-range NOEs observed between protons of 2 and 3 were analyzed using (1H: 500 MHz and 800

MHz, D2O, rt and 40°C). From H-3 of 3 intermolecular NOEs were observed with H-8, H-2’, H-2”,

H-3” and β of 2. Between H-8 of 2 and H-6 of 3, and H-2” of 2 and H-8 of 3, long-range NOEs were

also observed. Combining with those NOESY correlations and the analogy of previously determined

stacking structure that anthocyanin and flavone, the molecular stacking of 2 and 3 with a clockwise

manner was proposed.

Analysis of 1f

The 1H NMR spectrum of 1b was very broad and unanalyzable because of paramagnetic ferric ion.

Therefore, we choose diamagnetic Ga3+ complex, Mg-Ga nemophilin (1f), for conformational analysis

according to the previously reported study on Al-Mg protocyanin by Ueda et al (Tetrahedron Lett.

1998, 39, 8307-8310). We carried out various 1D and 2D NMR experiments with anbient temperature.

In 1f, signals attributable to protons at B-ring of 2 observed two species each. This is the same

phenomenon as was observed in the case of protocyanin, therefore, formation of a binuclear complex

arising from two different metal ions was confirmed. The signals observed around 8.1 to 8.6 ppm were

assigned to be H-2’ and H-6’ of 2. Using NOE correlation between OMe signals at 3.42 and 3.38 ppm,

it was deduced that the protons at 8.24 and 8.15 ppm were H-2’ and those at 8.54 and 8.33 ppm to be

H-6’. Ga3+ ion has stronger acidity than that of Mg2+, suggesting that 2 coordinating to Ga3+ ion may be

more deshielded than that of Mg2+, then, observed at the lower field. The signals at 6.45 and 6.41 ppm

and those at 6.52 and 6.50 ppm were determined to be H-2” and H-6”, and H-3” and H-5”, respectively

by COSY correlation and comparison of the assignment of 1a. Using the COSY and NOESY

correlations and comparison with the signals of 1a, protons at 5.78 ppm and that of 6.84 ppm were

assigned to be α and β protons of 2. Protons of B-ring of 3 were estimated to be the signals at 7.46,

7.38, 6.84 and 7.17 ppm by COSY experiments with different temperature and magnetic field. Results

5

of integration suggested that one more proton might exist in the overlapped signals around 5.78 ppm.

This signal showed only the correlation with H-2” and 6”, thus this might be H-3 of 3. Remaining

signals were assigned using COSY and spectrum comparison with 1a and Al-Mg protocyanin and

proposed that the signals at 5.60 and 5.59 ppm to be H-6 of 2, that of 4.99 ppm to be H-8 of 2, 6.01

ppm to be H-6 of 3, and 5.53 and 5.48 ppm to be H-8 of 3, and signal at 6.72 ppm to be H-4 of 2,

respectively. The stacking manner of two 2s in 1f was studied. From CD experiment, a strong negative

Cotton effect around λVISmax indicates anticlockwise stacking manner of 2s, therefore, long-range

NOEs were analyzed using this information. NOE between different signal of H-6’ of 2 coordinating to

Mg2+ and Ga3+ ions, respectively was observed. In Al-Mg protocyanin NOEs between H-2’ protons of

B-ring of anthocyanin, however, in 1f, the chromophore is petunidin containing OMe group at

2’-position of B-ring, then, the geometry of B-ring might rotate. Long-range NOEs between the protons

of 2 and 3 were almost the same as those observed in 1a. Therefore, the stack manner of 2 and 3 may

be the same as shown in Figure 6A in the main text.

Legends of supporting figures.

Figure S1. 1H NMR spectrum of MP (3) (500 MHz, CD3OD, rt).

Figure S2. 13C NMR spectrum of MP (3) (125 MHz, CD3OD, rt).

Figure S3. 1D-HOHAHA of MP (3) (irr. arrowed signal: H-6 of glucose attached at 7-OH, 500 MHz,

CD3OD, 50°C).

Figure S4. 1D-HOHAHA of MP (3) (irr. arrowed signal: H-6 of glucose attached at 4’-OH, 500 MHz,

6

CD3OD, 50°C).

Figure S5. NOESY of MP (3) (1H: 500 MHz, CD3OD, 50°C).

Figure S6. HSQC of MP (3) (1H: 500 MHz, 13C: 125 MHz, CD3OD, rt).

Figure S7. HMBC of MP (3) (1H: 500 MHz, 13C: 125 MHz, CD3OD, rt).

Figure S8. HRMS of MP (3).

Figure S9. UV-VIS spectra and CD of metal-substituted nemophilins of 1a type. (0.05 mM, H2O,

pathlength: 1.0 mm, rt)

Figure S10. UV-VIS spectra and CD of trivalent metal-substituted nemophilins of 1b type. (0.05 mM,

H2O, pathlength: 1.0 mm, rt)

Figure S11. UV-VIS spectra and CD of divalent metal-substituted nemophilins of 1b type. (0.05 mM,

H2O, pathlength: 1.0 mm, rt)

Figure S12. Results of analytical ultracentrifugation (AUC) analyses for 1a. (A-D: Concentration

gradient of AUC sedimentation equilibrium experiments at 40,000 r.p.m., E: Extraplotting graph)

Figure S13. COSY of Mg-Mg nemophilin (1a) (500 MHz, D2O, rt).

Figure S14. NOESY of Mg-Mg nemophilin (1a) (500 MHz, D2O, rt).

7

Figure S15. COSY of Mg-Ga nemophilin (1f) (500 MHz, D2O, rt).

Figure S16. NOESY of Mg-Ga nemophilin (1f) (500 MHz, D2O, rt).

Figure S17. Petal color variation of blue Nemophila menziesii.

Figu

re S

1. 1 H

NM

R sp

ectru

m o

f MP

(3) (

500

MH

z, C

D3O

D, r

t).

8

Figu

re S

2. 13

C N

MR

spec

trum

of M

P (3

) (12

5 M

Hz,

CD

3OD

, rt).

9

Figu

re S

3. 1

D-H

OH

AH

A o

f MP

(3) (

irr. a

rrow

ed si

gnal

: H-6

of g

luco

se a

ttach

ed a

t 7-O

H, 5

00 M

Hz,

CD

3OD

, 50°

C).

(thousandths)

!10.0010.020.030.040.0

X :

part

s p

er M

illi

on

: 1

H

5.5

5.4

5.3

5.2

5.1

5.0

4.9

4.8

4.7

4.6

4.5

4.4

4.3

4.2

4.1

4.0

3.9

3.8

3.7

3.6

3.5

3.4

3.3

3.2

3.1

10

Figu

re S

4. 1

D-H

OH

AH

A o

f MP

(3) (

irr. a

rrow

ed si

gnal

: H-6

of g

luco

se a

ttach

ed a

t 4’-

OH

, 500

MH

z, C

D3O

D, 5

0°C

).

(thousandths)

!10.0010.020.030.040.050.0

X :

part

s p

er M

illi

on

: 1

H

5.5

5.4

5.3

5.2

5.1

5.0

4.9

4.8

4.7

4.6

4.5

4.4

4.3

4.2

4.1

4.0

3.9

3.8

3.7

3.6

3.5

3.4

3.3

3.2

3.1

11

Figu

re S

5. N

OES

Y o

f MP

(3) (

1 H: 5

00 M

Hz,

CD

3OD

, 50°

C).

X :

part

s p

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illi

on

: 1

H

8.0

7.0

6.0

5.0

4.0

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9.08.07.06.05.04.0

ab

un

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abundance

00.20.40.6

Filename = tko20150109 ydk noesy

Author = yoshida

Experiment = noesy_wgs_phase.

Sample_id = 00

Solvent = METHANOL!D3

Creation_time = 10!JAN!2015 04:01:41

Revision_time = 12!JAN!2015 16:01:34

Current_time = 12!JAN!2015 16:05:01

Comment = Api!diGlc mal phase s

Data_format = 2D COMPLEX COMPLEX

Dim_size = 819, 512

Dim_title = 1H 1H

Dim_units = [ppm] [ppm]

Dimensions = X Y

Site = ECA500

Spectrometer = JNM!ECA500

Field_strength = 11.7473579[T] (500[MH

X_acq_duration = 0.10911744[s]

X_domain = 1H

X_freq = 500.15991521[MHz]

X_offset = 5.0[ppm]

X_points = 1024

X_prescans = 4

X_resolution = 9.16443788[Hz]

X_sweep = 9.38438438[kHz]

Y_domain = 1H

Y_freq = 500.15991521[MHz]

Y_offset = 5.0[ppm]

Y_points = 256

Y_prescans = 0

Y_resolution = 29.30859844[Hz]

Y_sweep = 7.5030012[kHz]

Irr_domain = 1H

Irr_freq = 500.15991521[MHz]

Irr_offset = 5.0[ppm]

Tri_domain = 1H

Tri_freq = 500.15991521[MHz]

Tri_offset = 5.0[ppm]

Clipped = FALSE

Mod_return = 1

Scans = 32

Total_scans = 8192

X_acq_time = 0.10911744[s]

X_atn = 3.2[dB]

X_pulse = 12.7[us]

Y_acq_time = 34.11968[ms]

Y_p0_correction = 0

Y_p1_correction = 180

Irr_mode = Off

Tri_mode = Off

Dante_presat = FALSE

Grad_1 = 1[ms]

Grad_1_amp = 45[mT/m]

Initial_wait = 1[s]

Mix_time = 0.15[s]

Raw_suppression = Off

Recvr_gain = 50

Relaxation_delay = 1.5[s]

Repetition_time = 1.60911744[s]

Scramble = 1[ms]

T1 = 50.384[us]

T1_cal1 = 50.384[us]

T1_cal2 = 0.117024[ms]

T1_cal3 = 0.183664[ms]

T1_cal4 = 0.250304[ms]

12

Figu

re S

6. H

SQC

of M

P (3

) (1 H

: 500

MH

z, 13

C: 1

25 M

Hz,

CD

3OD

, rt).

13

Figu

re S

7. H

MB

C o

f MP

(3) (

1 H: 5

00 M

Hz,

13C

: 125

MH

z, C

D3O

D, r

t).

14

Figu

re S

8. H

RM

S of

MP

(3).

15

200 400 600 800

Wavelength (nm)

mdeg

Abs

30

-30

0

CD

UV-VIS

0

1

Figure S9. UV-VIS spectra and CD of metal-substituted nemophilins of 1a type. (0.05 mM, H2O, path lenth: 1.0 mm, rt)

: Zn-Zn nemophilin (1c)

: Mn-Mn nemophilin (1d)

16

200 400 600 800

mdeg

Abs

30

-30

0

CD

UV-VIS

0

1

Figure S10. UV-VIS spectra and CD of trivalent metal-substituted nemophilins of 1b type. (0.05 mM, H2O, path lenth: 1.0 mm, rt)

: Mg-Al nemophilin (1e)

: Mg-Ga nemophilin (1f)

: Mg-In nemophilin (1g)

17

200  400  600  800 

mdeg

Abs

30

-30

0

CD

UV-VIS

0

1

Figure S11. UV-VIS spectra and CD of divalent metal-substituted nemophilins of 1b type. (0.05 mM, H2O, path lenth: 1.0 mm, rt)

: Zn-Fe nemophilin (1h)

: Mn-Fe nemophilin (1i)

: Cd-Fe nemophilin (1j)

18

A: 0.1 mg/ml, 430 nm B: 0.5 mg/ml, 430 nm

C: 0.75 mg/ml, 430 nm D: 1.0 mg/ml, 480 nm

Figure S12. Results of analytical ultracentrifugation (AUC) analyses for 1a. (A-D: Concentration gradient of AUC sedimentation equilibrium experiments at 40,000 rpm, E: Extrapolating graph)

E

19

Figure S13. COSY of Mg-Mg nemophilin (1a) (500 MHz, D2O, rt).

a: signals of anthocyanin component (NM, 2), f: signals of flavone component (MP, 3).

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Y :

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7.0

6.0

5.0

4.0

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abundance

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20

Figure S14. NOESY of Mg-Mg nemophilin (1a) (500 MHz, D2O, rt).

a: signals of anthocyanin component (NM, 2), f: signals of flavone component (MP, 3).

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21

Figure S15. COSY of Mg-Ga nemophilin (1f) (500 MHz, D2O, rt).

a: signals of anthocyanin component (NM, 2), f: signals of flavone component (MP, 3).

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Figure S16. NOESY of Mg-Ga nemophilin (1f) (500 MHz, D2O, rt).

a: signals of anthocyanin component (NM, 2), f: signals of flavone component (MP, 3).

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a-OMe

a-4

a-4

a-6 a-8

a-6

G-1

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Figure S17. Petal color variation of blue Nemophila menziesii.

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