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)