Supplementary Table and Figures
Supplementary Table 1
Expected HMBC correlation patterns for hop bitter acid acyl side chain signals
Compound
Me group
2
(1H signal multiplicity)
J & 3J HMBC to C
Humolone,
4’ (d)
2’ (CH2);
3’ (CH);
5’ (CH3)
Lupolone
5’ (d)
2’ (CH2);
3’ (CH);
4’ (CH3)
Cohumulone,
3’ (d)
1’ (C=O); 2’ (CH2); 4’ (CH3)
Colupolone
4’ (d)
1’ (C=O); 2’ (CH2); 3’ (CH3)
Adhumulone,
4’ (t)
2’ (CH);
3’(CH2)
Adlupolone
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 2
HMBC 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 
13
C 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
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 customwritten procedures (Supplementary Word File, or Download from ipb-halle.de) to produce
scores and loading plots for classification of samples.
1
2
TPE 
HPE ○
40
A
HTU 
0

EHM
HHT
ENB 
-20
HHE 
-40
PC2 (18.1%)
20
HSE 
CZAG 
HHS ▪
Pixel position
Intensity
H0.74/C208.3 0,741 H0.74/C208.3
208,3085
0,155 0,741
0,145
ATPE 
ATHM 
HHM ∆
-100
-50
PC1 (67.5%)
4
0
50
3
208,3085
0,1
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
4a 3
a
6
5
4
1
6
50
3
2
5
1
5
7
5
7
6
5
5
150
7
6
5
5
7
5
140
7
100
80
100ppm
F1 (13C)
2
2
40
5
60
7
20
0
series; 6, lupulone series; 7, total humulones and lupulones.
5
180
7
7
3.5
3.0
220
200
3
4
2.5
F2 (1H)
2.0ppm
1.5
1.0
0.5
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-1HNMR 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.
TPE 
HPE ○
20
HSE 
HTU 
0

HHT
EHM
ENB 
CZAG 
-20
HHE 
HHS ▪
 ATPE
 ATHM
HHM ∆
-40
PC2 (18.1%)
40
2D-1H - HMBC
-100
-50
PC1 (67.5%)
0
50
10
HHT 
CZAG 
 EHM
HSE 
 ENB
HTU 
HHS ▪
TPE 
HPE ○
ATPE 
0
-10
PC2 (26.0%)
20
1D-1H-NMR
HHM ∆
ATHM 
HHE 
-20
0
PC1 (61.3%)
20
40
Supplementary Figure 9
Loading 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).
a
b
high in HHE
high in HTU
0
Most
important loadings PC1
most importend loadings PC1
20
H1.58/C25.5
H1.56/C25.5
40
20
60
100
100
F1 Chemical Shift (ppm)
150
140
140
100
120
180
50
180
160
200
c)
overlay spektrum and loadings
TPE
HTU
HSE_D
HSE_C
HPE
HSE_B
HHT
HHS
HHM
ENB
HHE
CZAG_B
1.0
CZAG_C
1.5
F2 (1H)
ATPE
2.0
1.0
CZAG_A
2.5
1.5
F2 Chemical Shif t (ppm)
ATHM
2.0
EHM
220
2.5
0
200
220
F1 (13C)
80
100
80
H1.56/C25.5
H1.56/C25.5
150
60
50
40