Supplementary InformationIon (M+H)+ (M+Na)+ (M+K)+ 550.2192 Calc m/z 512.2643 534.2462 550.2202...
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1 Supplementary I nformation First total synthesis and solution structure of a polypeptin, PE2, a cyclic lipopeptide with broad spectrum antibiotic activity. Simon J. Mountford, a Biswaranjan Mohanty, a Kade D. Roberts, a Heidi Y. Yu, b Martin J. Scanlon, a Roger L. Nation, b Tony Velkov, b Jian Li, b Philp E. Thompson* a a Medicinal Chemistry and b Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Australia Contents Page Figure S1 HPLC and ESI-MS of crude and purified (inset) peptides, 7 and 7A 2 Figure S2 Chiral HPLC traces for RS-15 (a) and S-15 (b). 4 Figure S3 Overlay of 2D [ 15 N, 1 H N ]-SOFAST-HMQC spectra of 7A and 7 5 Figure S4 Overlay of 1D 1 H and 2D [ 15 N, 1 H N ]-SOFAST-HMQCs spectra of 7 (synthesized via 12) and 7 (synthesized via S-12) 6 Figure S5 1D 1 H-NMR spectra of amide regions of 7 and 7A in water and D2O. 7 Table S1 MIC values for 7A and polymyxin B 8 NMR spectra for Compounds 10-12, 15-16 9 HR-MS for compounds 12, S-12, 16 19 Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is © The Royal Society of Chemistry 2017
Transcript of Supplementary InformationIon (M+H)+ (M+Na)+ (M+K)+ 550.2192 Calc m/z 512.2643 534.2462 550.2202...
1
Supplementary Information
First total synthesis and solution structure of a polypeptin, PE2, a cyclic lipopeptide with
broad spectrum antibiotic activity.
Simon J. Mountford, a Biswaranjan Mohanty,a Kade D. Roberts,a Heidi Y. Yu,b Martin J.
Scanlon,a Roger L. Nation,b Tony Velkov,b Jian Li,b Philp E. Thompson*a
aMedicinal Chemistry and bDrug Delivery, Disposition and Dynamics, Monash Institute of
Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Australia
Contents
Page Figure S1 HPLC and ESI-MS of crude and purified (inset) peptides, 7 and 7A 2 Figure S2 Chiral HPLC traces for RS-15 (a) and S-15 (b). 4 Figure S3 Overlay of 2D [15N,1HN]-SOFAST-HMQC spectra of 7A and 7 5 Figure S4 Overlay of 1D 1H and 2D [15N,1HN]-SOFAST-HMQCs spectra of 7
(synthesized via 12) and 7 (synthesized via S-12) 6
Figure S5 1D 1H-NMR spectra of amide regions of 7 and 7A in water and D2O. 7 Table S1 MIC values for 7A and polymyxin B 8 NMR spectra for Compounds 10-12, 15-16 9 HR-MS for compounds 12, S-12, 16 19
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2017
2
A 7A 7
7
7A
3
Figure S1 HPLC and ESI-MS of crude and purified (inset) peptides 7 and 7A. (A) Synthesis from 12. (B) Synthesis from S-12.
B 7
7
4
Figure S2 Chiral HPLC traces for RS-15 (a) and S-15 (b).
5
Chemical shift assignments:
Sequence specific chemical shift assignments of 7 (FADDI-491B) and 7A (FADDI-491A)
peptides are provided in the supplementary information (see two excel files).
Figure S3 Overlay of 2D [15N,1HN]-SOFAST-HMQC spectra of 7A and 7 (3RS-12). Spectra
were acquired from each sample with peptide concentration of ~ 1.7 mM in 7% 2H2O and 93%
H2O at pH 4.35. All data were collected on a 600 MHz spectrometer equipped with CryoProbe
at 10 °C. The side-chain amide peaks for 7 are labelled in magenta. I, L, T, Z, V and F indicates
Ile, Leu, Thr, Dab, D-Val and D-Phe residues.
6
Figure S4 Overlay of 1D 1H and 2D [15N,1HN]-SOFAST-HMQCs spectra enabling comparison
of 7 (synthesized via 12) and 7 (synthesized via S-12). Spectra were acquired from each sample
with peptide concentration of ~ 1.7 mM in 7% 2H2O and 93% H2O at pH 4.35. All data were
collected on a 600 MHz spectrometer equipped with CryoProbe at 10 °C. I, L, T, Z, V and F
indicates Ile, Leu, Thr, Dab, D-Val and D-Phe residues.
7
Figure S5 1D 1H-NMR spectra of 7 and 7A in water and D2O. Only amide region is shown for
clarity. Spectra were acquired from each sample with peptide concentration of ~ 1.7 mM. The
dead time between the addition of D2O to the lyophilized peptides and the beginning of data
acquisition was ~ 8 minutes. The data acquisition time of each spectrum took ~ 7 minutes. All
data were collected on a 600 MHz spectrometer equipped with CryoProbe at 10 °C. I, O and
sc indicate unassigned peaks from sample impurities, overlapped peaks and side-chain amide
peaks, respectively. I, L, T, Z, V and F indicates Ile, Leu, Thr, Dab, D-Val and D-Phe residues.
8
Table S1 MIC values for 7A and polymyxin B
Bacterial species Strain 7A
MIC (µg/mL) Polymyxin B MIC (µg/mL)
Gram Negative
P. aeruginosa Pa ATCC 27853 >32 0.5 FADDI-PA021 >32 0.5 FADDI-PA025 >32 1 FADDI-PA070 >32 >32 FADDI-PA060 >32 4 FADDI-PA090 >32 2
Gram Negative
A. baumannii Ab ATCC 19606 >32 0.5 FADDI-AB34 >32 0.5 Ab ATCC 17978 >32 1 FADDI-AB065 >32 >32 FADDI-AB156 >32 8 FADDI-AB167 >32 8
Gram Negative
K. pneumonia Kp ATCC 13883 >32 1 FADDI-KP032 16 0.5 FADDI-KP027 >32 >32 FADDI-KP003 >32 >32 FADDI-KP012 >32 >32
Gram Negative
E. cloacae FADDI-EC006 32 0.5 FADDI-EC001 32 0.25 FADDI-EC003 32 0.25
Gram Positive VREa ATCC 700221 >32 >32 MRSA ATCC 43300 >32 >32 VISA ATCC 700698 >32 >32 VRSA ATCC 700699 >32 >32
Qualitative C
ompound R
eport
MS Spectrum
MS Zoom
ed Spectrum
z111
--- End Of Report ---
45588.23
Formula
C29H37N
O7
C29H37N
O7
C29H37N
O7
Ion(M
+H
)+(M
+N
a)+(M
+K)+
550.2192
Calc m/z
512.2643534.2462550.2202
Diff(ppm
)3.91.731.81
Mass
511.2561
MS Spectrum
Peak List
m/z
512.2623534.2453
Abund
55654.672025250.44
DB
Formula
C29 H37 N
O7
Com
pound Label
Cpd 1: C29 H37 N
O7
m/z
534.2453R
T0.127
Algorithm
Find By Formula
Tgt Mass
511.257
Diff
(ppm)
-1.76M
FG Form
ulaC29 H
37 N O
7M
ass511.2561
Abund2025250
Formula
C29 H37 N
O7
Com
pound Table
FALSE
Com
pound LabelCpd 1: C29 H
37 N O
7R
T0.127
Info.A
cquisition SW
Version
Column4
6200 series TOF/6500 series
Q-TO
F B.05.01 (B5125.1)
Sample G
roupForm
ula
Column4
C29H37N
O7
IRM
Calibration Status
SuccessD
A M
ethod
Monash_Accuracy.m
Com
ment
Column3
Column3
Instrument N
ame
Instrument 1
User N
ame
Dr Jason D
angA
cq Method
Monash_D
irect.mA
cquired Time
23-Feb-15 11:24:29 AM
Data File
SJM-402-50.d
Sample N
ame
SJM-402-50
Sample Type
Sample
Position
P1-A4
Page 1 of 1Printed at: 11:26 AM
on:23-02-15
Com
pound Table
MS Spectrum
MS Zoom
ed Spectrum
MS Spectrum
Peak List
--- End Of Report ---
Data File
SJM-402-82.d
Sample N
ame
SJM-402-82
Sample Type
Sample
Position
P1-A3
Instrument N
ame
Instrument 1
User N
ame
Dr Jason D
ang
Acq M
ethodM
onash_Direct.m
Acquired Tim
e10-N
ov-16 12:02:43 PM
IRM
Calibration Status
SuccessD
A M
ethodM
onash_Accuracy.m
Com
ment
Sample G
roupInfo.
Formula
C29H37N
O7
Stream N
ame
LC 1
Acquisition SW
Version
6200 series TOF/6500 series Q
-TOF B.06.01 (B6172 SP1)
Com
pound LabelR
TM
assA
bundForm
ulaTgt M
assD
iff (ppm)
Cpd 1: C29 H37 N
O7
0.122511.2567
48147C29 H
37 N O
7511.257
-0.55
Com
pound Labelm
/zR
TA
lgorithmM
assCpd 1: C29 H
37 N O
7512.2644
0.122Find By Form
ula511.2567
5x10
0 2 4 6 8
Counts vs. M
ass-to-Charge (m
/z)250
300350
400450
500550
600650
700750
800850
900950
Cpd 1: C
29 H37 N
O7: +E
SI S
can (rt: 0.089-0.205 min, 8 scans) F
rag=160.0V S
JM-402-82.d S
ub…
456.2023
5x10
0 2 4 6 8
Counts vs. M
ass-to-Charge (m
/z)600
700800
9001000
11001200
13001400
1500
Cpd 1: C
29 H37 N
O7: +E
SI S
can (rt: 0.089-0.205 min, 8 scans) F
rag=160.0V S
JM-402-82.d S
ub…
1045.5033(2[C
29H37N
O7]+N
a)+
m/z
Calc m
/zD
iff(ppm)
zA
bundForm
ulaIon
534.2471534.2462
-1.621
994797.22C29H
37NO
7(M
+N
a)+550.2196
550.22021.03
158800.23
C29H37N
O7
(M+
K)+1045.5033
1045.5032-0.07
1603627.94
C29H37N
O7
(2M+
Na)+
Qualitative C
ompound R
eport
Printed at 12:04 PM on 10-N
ov-20161
Page 1 of
Com
pound Table
MS Spectrum
MS Zoom
ed Spectrum
MS Spectrum
Peak List
--- End Of Report ---
Data File
SJM-402-80.d
Sample N
ame
SJM-402-80
Sample Type
Sample
Position
P2-B4
Instrument N
ame
Instrument 1
User N
ame
Dr Jason D
ang
Acq M
ethodM
onash_Direct.m
Acquired Tim
e29-N
ov-16 11:37:42 PM
IRM
Calibration Status
SuccessD
A M
ethodM
onash_Accuracy.m
Com
ment
Sample G
roupInfo.
Formula
C36H43N
O7
Stream N
ame
LC 1
Acquisition SW
Version
6200 series TOF/6500 series Q
-TOF B.06.01 (B6172 SP1)
Com
pound LabelR
TM
assA
bundForm
ulaTgt M
assD
iff (ppm)
Cpd 1: C36 H43 N
O7
0.124601.3052
30568C36 H
43 N O
7601.304
2.12
Com
pound Labelm
/zR
TA
lgorithmM
assCpd 1: C36 H
43 N O
7602.3113
0.124Find By Form
ula601.3052
5x10
0 1 2 3 4 5 6
Counts vs. M
ass-to-Charge (m
/z)250
300350
400450
500550
600650
700750
800850
900950
Cpd 1: C
36 H43 N
O7: +E
SI S
can (rt: 0.091-0.224 min, 9 scans) F
rag=160.0V S
JM-402-80.d S
ub…
624.2948([C
36H43N
O7]+N
a)+
5x10
0 1 2 3 4 5 6
Counts vs. M
ass-to-Charge (m
/z)650
700750
800850
900950
10001050
11001150
1200
Cpd 1: C
36 H43 N
O7: +E
SI S
can (rt: 0.091-0.224 min, 9 scans) F
rag=160.0V S
JM-402-80.d S
ub…
m/z
Calc m
/zD
iff(ppm)
zA
bundForm
ulaIon
624.2948624.2932
-2.651
632140.37C36H
43NO
7(M
+N
a)+640.2675
640.2671-0.59
137994.11
C36H43N
O7
(M+
K)+1225.5987
1225.5971-1.25
1228312.75
C36H43N
O7
(2M+
Na)+
Qualitative C
ompound R
eport
Printed at 11:39 PM on 29-N
ov-20161
Page 1 of
