Supplementary Table and Figures

Supplementary Table 1Expected HMBC correlation patterns for hop bitter acid acyl side chain signals

CompoundMe group

(1H signal multiplicity)2J & 3J HMBC to C

Humolone,

Lupolone

4’ (d) 2’ (CH2); 3’ (CH); 5’ (CH3)

5’ (d) 2’ (CH2); 3’ (CH); 4’ (CH3)

Cohumulone,

Colupolone

3’ (d) 1’ (C=O); 2’ (CH2); 4’ (CH3)

4’ (d) 1’ (C=O); 2’ (CH2); 3’ (CH3)

Adhumulone,

Adlupolone

4’ (t) 2’ (CH); 3’(CH2)

5’ (d) 1’ (C=O); 2’ (CH); 3’(CH2)

Supplementary Figure 1

Traces parallel to the 1H axis of different HMBC spectra of HHS resin under variation of

number of scans and number of increments. Normalized signal intensity of the minor cross

peak at 1H/13C of 3.24/108.5 ppm assigned to C-7 of the enol tautomer of lupulones is

displayed (indicated by an arrow). HMBC spectra were recorded at three different acquisition

parameters: (a) 2 scans and 256 increments; (b) 8scans and 256 increments and (c) 16 scans

and 192 increments for total acquisition times of 28, 89 and 146 min, respectively

Supplementary Figure 2HMBC spectrum of HHE hop resin acquired in CD3OD. As outlined in the text, the spectrum

can be roughly divided into 4 main regions along the F2 proton dimension labelled as:

I: 1H 5.5 - 8.5 ppm;

II: 1H 4.0 - 5.5 ppm;

III: 1H 1.8 - 4.0 and

IV: 1H 0.0 - 1.8 ppm

Supplementary Figure 3(a) General structure of α-acids and iso α-acids showing the change in the hybridization status

of C-5 from sp2 to sp3 and its associated 13C upfield chemcal shift (in ppm). Arrows

represent HMBC correlations that were readily observed in reference HMBC spectra of

α-acids

(b) Partial HMBC spectrum of aged solution of hops α acids showing different marker cross

peaks for α-bitter acids and their isomerization products labelled as follows:

1, H2-12 C-6 of α-bitter acids (humulones)

2, H2-7 C-4 of cis-isohumulones

3, H2-7 C-5 of cis-isohumulones

4, H2-7 C-5 of trans-isohumulones

5, H2-7 C-5 of dihydro trans-isohumulones

Other signals could not be assigned due to lack of literature NMR data

OH

R

HO OHO

12

34

56

O

R

HO OH

O

1234

5

Humulones trans-Isohumulones

6

7O

R

HO OH

O

12

345

6

7

cis-Isohumulones

7

12

C-5: 172-173 ppm C-5: 89-95 ppm C-5: 85-89 ppm

a

Supplementary Figure 4

Flowchart showing the path of data extraction from a HMBC spectrum to principal component analysis. Step (1): acquisition of HMBC spectra of hop samples using Agilent (Varian) (CHEMPACK 5.1) spectrometer software. Step (2): Integration of HMBC cross peaks using ACD/NMR Manager lab version 10.0 software to reduce the complex HMBC profile into pixels (i.e. constant small area2D buckets) that cover the 1H and 13C dimensional grid. Step (3): Integration volumes are then plotted against different pixels to generate a comprehensive 2D NMR chemomatrix. Step (4): The generated chemomatrix is fed into statistic software R (2.9.2) using Bioconductor package pcaMethods together with custom-written procedures (Supplementary Word File, or Download from ipb-halle.de) to produce scores and loading plots for classification of samples.

-100 -50 0 50

-40

-20

020

40

PC1 (67.5%)

ATHM ATPE

CZAG EHM

ENB HHE

HHM ∆

HHS ▪HHT

HPE ○

HSE

HTU

TPE

PC

2 (1

8.1%

)

A

1

2

34

H0.74/C208.3 0,741 208,30850,155 0,145 H0.74/C208.3 0,741 208,30850,155 0,145Pixel position Intensity

Supplementary Figure 5

PCA scoring plots of PC1 and PC2 scores using different pixel sizes in both F1 and F2

dimensions (F1xF2): a, (85x128); b, (64x256); c, (85x256) and d, (128x256). HMBC cross

peaks intensities were recorded in the F1 range ( -6.0-231) ppm and F2 range (0.4–8.5)

ppm. Triplicates of both CZAG and HSE cultivars are displayed individually as an indicator

of extraction & spectral reproducibility, respectively.

Supplementary Figure 6

Loading plot for PC1 components of the region from 1H 0.4-3.6 ppm and 13C 0-230

ppm showing cross peaks contributing to PC1 variance. Signals with positive contribution to

PC1 are displayed in green and marked with dashed arrows, while those with a negative

contribution are displayed in red and marked with solid. Cross peaks in the loading plot are

numbered as follows: 1, lupulone; 2, humulone; 3, colupulone; 4, cohumulone; 5, humulone

series; 6, lupulone series; 7, total humulones and lupulones.

6 4a5 3a 2

14

3

125

5

5

5

5

7

7

7

7

7

55

5

5

6

6

3

4

7

7

25

6

7

3.5 3.0 2.5 2.0 1.5 1.0 0.5

200

150

100

500

F2 (1H) ppm22

018

014

010

080

6040

20

F1 (1

3 C) p

pm

Supplementary Figure 7

Loading plot for PC2 components displaying the region from 1H -0.8 - 4.9 ppm and

13C -10 - 240 ppm showing cross peaks contributing to PC2 variance. Signals with positive

contribution to PC2 are displayed as green dots and with solid arrows, while those with

negative contribution are shown as red dots and with dashed arrows. Some correlation peaks

are labelled and assigned to specific hop constituents as follows,

(a) cross peaks for 2-methylbut-3-en-2-ol; (b) linalool; (c) fatty acids and (d) total bitter acids.

Supplementary Figure 8

PCA scoring plots of PC1 and PC2 of hop resin samples using 2D-1H- HMBC versus 1D-1H- NMR as datasets. Note the comparable positioning of hop samples placed in circles across PC1, especially for most distant samples with negative score values i.e. HHE & HSE; with positive score values i.e. HTU and HHS.

-100 -50 0 50

-40

-20

020

40

PC1 (67.5%)

CZAG EHM

ENB HHE

HHM ∆

HHS ▪HHT

HPE ○

HSE

HTU

TPE

PC

2 (1

8.1%

)

2D-1H - HMBC

HHS ▪HTU

HHT

HSE

HHE

ENB

EHM

ATHM

ATPE

ATPEATHM

HHM ∆

CZAG

HPE ○TPE

PC1 (61.3%)

PC

2 (2

6.0%

)

1D-1H-NMR

-20 0 20 40

-10

1020

0

Supplementary Figure 9Loading plot showing cross peaks contributing to PC1 variance and quantification of H3-16/21 crosspeak assigned for total lupulones (L) across samples.

a) Loading plot for PC1 components of the region from 1H 0.4-2.6 ppm and 13C 0-230 ppm showing cross peaks contributing to PC1 variance. Signals with positive contribution to PC1 are displayed in green, while those with a negative contribution are displayed in red.

b) Bar plot of total lupulones (L) crosspeak intensities assigned for H3-16/21 appearing at 1H (1.56 ppm) and 13C (25.5 ppm). The HHE cultivar showed the highest level as denoted in red colour, versus the HTU cultivar exhibiting the lowest amounts. This is in full agreement with absolute quantifications of total lupulones using 1H-NMR, (Farag et al., 2012).

2.5 2.0 1.5 1.0F2 Chemical Shift (ppm)

20

40

60

80

100

120

140

160

180

200

220

F1 C

hem

ical

Shi

ft (p

pm)

2.5 2.0 1.5 1.0

200

150

100

500

most importend loadings PC1

F2 (1H)

F1 (1

3C)

220

180

140

100

8060

4020

high in HHEhigh in HTU

overlay spektrum and loadings

H1.56/C25.5

ATH

M

ATP

E

CZA

G_A

CZA

G_B

CZA

G_C

EH

M

EN

B

HH

E

HH

M

HH

S

HH

T

HP

E

HS

E_B

HS

E_C

HS

E_D HTU TP

E

H1.58/C25.5

050

100

150

H1.56/C25.5

H1.56/C25.5

a bMost important loadings PC1

c)