SUPPORTING INFORMATION
Chiral Effects in Perylene Diimide Nanofibers
Galen L. Eakins,†,‡ Jonathan P. Wojciechowski,§ Adam D. Martin,§ James E. A. Webb,§
Pall Thordarson,§ Justin M. Hodgkiss*,†,‡
†
MacDiarmid Institute for Advanced Materials and Nanotechnology
P.O. Box 600, Wellington, New Zealand
‡
School of Chemical and Physical Sciences, Victoria University of Wellington,
P.O. Box 600, Wellington, New Zealand
§
School of Chemistry, the Australian Centre for Nanomedicine and the ARC Centre of
Excellence for Convergent Bio-Nano Science and Technology
The University of New South Wales, Sydney, NSW 2052, Australia
TABLE OF CONTENTS
SECTION A: Synthesis and Purification Details............................................................ S2
SECTION B: NMR Spectra of the Synthesized Compounds ........................................... S5
SECTION C: MALDI Mass Spectra of Synthesized PDIs .............................................S10
SECTION D: IR Spectra of Synthesized PDIs ..............................................................S14
S1
Section A: Synthesis and Purification Details
Synthesis and purification was performed as previously described (1). For reference, the
procedures used are briefly restated here.
General Solid-Phase Peptide Synthesis
Synthesis employed standard Fmoc-based solid-phase peptide synthesis (SPPS)
protocols (2). A sample of Fmoc-Glu(OtBu)-Wang resin or Fmoc-D-Glu(OtBu)-Wang
resin (0.50 g, loading 0.52-0.56 mmol/g) was prepared by swelling the resin in
dichloromethane (DCM) (7 mL) for 30 minutes and then washing the resin with
dimethylformamide (DMF) (7 mL). Sequentially alternating Fmoc-deprotection and
amino acid coupling reactions with the desired Fmoc-protected amino acids afford the
desired peptide sequence. Fmoc-deprotection reactions were performed using 30% (v/v)
piperidine/DMF (7 mL) with gentle shaking for 30 minutes followed by thorough
washes of the resin with DMF. Amino acid coupling reactions were performed using a
mixture of the desired amino acid (4 molar equivalents), O-benzotriazole-N,N,N’,N’tetramethyluronium hexafluorophosphate (HBTU) (3.95 molar equivalents), and N,Ndiisopropylethylamine (DIPEA) (6 molar equivalents) in molar excess relative to the
loading of the Wang resin. The coupling reaction mixtures were gently shaken for 30
minutes followed by a thorough wash of the resin. A Kaiser test was conducted after
each coupling reaction to confirm completion. Upon removal of the terminal Fmoc
protecting group from the completed peptide sequence, the resin was washed
thoroughly with DMF followed by several washes with DCM. Cleavage of the
completed peptide sequence from the Wang resin was achieved by gently shaking the
resin for 3 hours in a mixture of trifluoroacetic acid (TFA) and DCM (7 mL, 3:4
TFA/DCM). The cleavage solution was collected, the resin was washed with DCM (5
mL), and the wash was added with the collected cleavage solution. Cold diethyl ether
(40 mL, 0-5 °C) was added to the cleavage solution and chilled for 30 minutes to
precipitate the peptide product. The precipitate was collected by filtration, washed with
cold diethyl ether, and dried under vacuum to yield the peptide product.
S2
General Perylene Diimide Synthesis
Synthesis of the peptide-substituted perylene diimides outlined in Scheme S1 was based
on standard amine condensation methods for the synthesis of perylene diimide
derivatives as reported in literature (3). For the N,N’-dipeptide perylene diimides, a
mixture of the desired peptide and perylene 3,4,9,10-tetracarboxylic acid dianhydride
(PTCDA) were combined in a 2:1 molar ratio (with a slight peptide excess) in imidazole
(0.80-1.0 g) and stirred at 124-135 °C for 24 hours under nitrogen. Synthesis of Nhexyl-N’-peptide perylene diimides was performed by analogous procedures using a
mixture of the desired peptide and perylene-3,4-monoanhydride-9,10-hexylimide (4)
(hexyl-PTCAI) in a 1:1 molar ratio with a slight peptide excess. Following the coupling
reaction, the mixture was allowed to cool to room temperature with continued stirring
and the addition of 3 mL of water to the mixture upon reaching 90 °C. Upon cooling,
the solution was loaded onto an HP20ss column with the addition of small amounts of
water. Once fully loaded onto the resin, the mixture was eluted and recycled through the
column using dilute aqueous acetic acid with an increasing acid gradient until all dark
red/purple components were retained within the resin and the eluted solvent became
yellow to colourless. The dark solids retained in the resin were then thoroughly washed
with 7% (v/v) acetic acid (700-1200 mL) until the eluted wash changed from yellow to
nearly colourless. A subsequent wash with acetone (150-300 mL) was made to remove
traces of any reddish impurity. Removal of the desired product from the resin following
the washes was accomplished using 20% (v/v) pyridine in water. Removal of the
solvent under reduced pressure afforded the dark coloured peptide-substituted perylene
diimides.
Scheme S1. Synthesis of peptide-substituted perylene diimides
O
O
X
O
peptide—NH2
imidazole
O
O
X = O or N—hexyl
124-135 °C
24 hours
O
O
R1 N
N
O
O
R2
R1 = hexyl or R2
R2 = peptide
S3
Purification
Our purification protocol capitalized on the low solubility and pH-triggered aggregation
of the desired final products to accomplish separation. The reverse-phase HP20ss resin
allows charged species to pass through the column while retaining uncharged species.
The use of the acetic acid washes easily protonates and removes species such as the
imidazole and free peptide while simultaneously triggering aggregation and retention of
the desired PDI products within the resin. The product is then easily removed by
flushing with basic washes to ionize the glutamic acid units. During the dipeptide
compound syntheses, the formation of monopeptide-chromophores was expected to be
minimal since the reactions are conducted with a slight excess of the peptides; however,
any monopeptide-chromophores formed would have greater solubility than the desired
products. As such, the monopeptide-chromophores would release from the HP20ss resin
more readily than the desired dipeptide products during the successive acetic
acid/acetone flushes during the purification protocol. Similarly, undesired perylene
species formed from aberrant peptide sequence impurities present in the peptide
samples used can be easily screened due to differences in the solubility and resin
affinity for the perylene species formed. While the strongly aggregating products are not
amenable to quantification by HPLC, we estimate purity via 1H NMR spectra to be
>95% in all cases.
Synthesis and Characterization References
(1) Eakins, G. L.; Gallaher, J. K.; Keyzers, R. A.; Falber, A.; Webb, J. E. A.; Laos, A.;
Tidhar, Y.; Weissman, H.; Rybtchinski, B.; Thordarson, P.; Hodgkiss, J. M. J. Phys.
Chem. B 2014, 118, 8642–8651.
(2) Coin, I.; Beyermann, M.; Bienert, M. Nat. Protoc. 2007, 2, 3247-3256.
(3) Wicklein, A.; Lang, A.; Muth, M.; Thelakkat, M. J. Am. Chem. Soc. 2009, 131,
14442-14453.
(4) Wang, R.; Shi, Z.; Zhang, C.; Zhang, A.; Chen, J.; Guo, W.; Sun, Z., Dyes and
Pigments 2013, 98, 450-458.
S4
Normalized Intensity
8
180
160
51.865
51.697
51.395
48.306
48.219
40.228
30.152
29.996
27.560
27.407
26.254
18.492
18.240
18.042
9
171.216
170.933
165.600
174.129
173.862
173.182
172.293
171.968
171.686
Normalized Intensity
4.410
4.395
4.381
4.281
4.265
4.249
4.236
4.172
3.575
3.571
3.563
3.555
2.282
2.264
2.246
2.230
1.953
1.904
1.771
1.756
1.224
1.211
1.205
1.199
1.191
8.542
8.528
8.232
8.217
8.208
7.971
7.960
7.919
7.903
Section B: NMR Spectra of Synthesized Compounds
0.065
0.060
0.055
0.050
0.045
0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0
7
6
140
5
4
Chemical Shift (ppm)
120
100
80
Chemical Shift (ppm)
3
60
2
40
1
20
0
Figure S1. 1H NMR spectrum of P2E, NH2-Gly-(D-Ala)3-(D-Glu)3, in DMSO-d6.
0.10
0.05
0
-20
Figure S2. 13C NMR spectrum of P2E, NH2-Gly-(D-Ala)3-(D-Glu)3, in DMSO-d6.
S5
(A)
4.410
4.395
4.381
4.281
4.265
4.249
4.236
4.172
3.575
3.571
3.563
3.555
2.282
2.264
2.246
2.230
1.953
1.904
1.771
1.756
1.224
1.211
1.205
1.199
1.191
8.542
8.528
8.232
8.217
8.208
7.971
7.960
7.919
7.903
Section B: NMR Spectra of Synthesized Compounds
0.065
0.060
0.055
Normalized Intensity
0.050
0.045
0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0
6
5
4
Chemical Shift (ppm)
3
2
1
0
2.634
2.619
2.262
2.228
1.622
1.576
1.570
1.541
1.531
1.521
1.510
1.435
1.427
7
5.253
5.236
5.210
4.735
4.724
4.697
4.681
4.667
4.656
4.644
4.634
(B)
8
7.855
7.845
7.785
7.735
7.647
7.607
7.494
7.448
7.270
8.852
8.841
8.816
8.803
9
1.0
0.9
Normalized Intensity
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
9
8
7
6
5
4
Chemical Shift (ppm)
3
2
1
0
Figure S3. 1H NMR spectral comparison of: (A) NH2-Gly-(D-Ala)3-(D-Glu)3 peptide
(P2E) in DMSO-d6 and (B) N,N'-di[-Gly-(D-Ala)3-(D-Glu)3]perylene-3,4,9,10tetracarboxylic acid diimide (2E) in 2:1 CDCl3/TFA.
S6
(A)
4.410
4.395
4.381
4.281
4.265
4.249
4.236
4.172
3.575
3.571
3.563
3.555
2.282
2.264
2.246
2.230
1.953
1.904
1.771
1.756
1.224
1.211
1.205
1.199
1.191
8.542
8.528
8.232
8.217
8.208
7.971
7.960
7.919
7.903
Section B: NMR Spectra of Synthesized Compounds
0.065
0.060
0.055
Normalized Intensity
0.050
0.045
0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
8
5
4
Chemical Shift (ppm)
3
2
1
0
1.847
1.831
1.561
1.553
1.538
1.522
1.508
1.406
1.399
1.393
0.947
0.933
0.920
9
6
2.666
7
5.240
5.117
4.820
4.757
4.690
4.625
4.336
4.321
4.305
8
7.895
7.755
7.467
7.417
7.402
7.332
(B)
9
8.891
8.875
8.852
8.832
8.816
0
0.010
0.009
Normalized Intensity
0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
7
6
5
4
Chemical Shift (ppm)
3
2
1
0
Figure S4. 1H NMR spectral comparison of: (A) NH2-Gly-(D-Ala)3-(D-Glu)3
peptide (P2E) in DMSO-d6 and (B) N-hexyl-N'-[-Gly-(D-Ala)3-(D-Glu)3]perylene3,4,9,10- tetracarboxylic acid diimide (2HE) in 2:1 CDCl3/TFA.
S7
3.549
3.541
2.270
2.266
2.262
2.245
2.231
2.224
2.217
2.212
1.873
1.204
1.193
1.182
1.174
(A)
4.397
4.385
4.373
4.266
4.254
4.245
4.234
4.223
4.152
8.534
8.521
8.231
8.219
8.205
8.193
7.980
7.967
7.956
Section B: NMR Spectra of Synthesized Compounds
1.0
0.9
Normalized Intensity
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
6.5
6.0
5.5
5.0
4.5
4.0
Chemical Shift (ppm)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
2.167
1.641
1.627
1.600
1.544
1.530
1.518
1.464
1.316
1.302
7.0
2.659
2.647
2.365
7.5
5.287
5.261
5.232
4.826
4.813
4.799
4.783
4.751
4.738
4.695
4.682
(B)
8.0
7.888
7.811
7.757
7.653
7.542
7.461
7.406
7.312
8.891
8.877
8.5
0.0035
Normalized Intensity
0.0030
0.0025
0.0020
0.0015
0.0010
0.0005
9
8
7
6
5
4
Chemical Shift (ppm)
3
2
1
0
Figure S5. 1H NMR spectral comparison of: (A) NH2-Gly-(Ala)3-(Glu)3 peptide
(P2) in DMSO-d6 and (B) N-[-Gly-(L-Ala)3-(L-Glu)3]-N'-[-Gly-(D-Ala)3-(D-Glu)3]perylene-3,4,9,10- tetracarboxylic acid diimide (2M) in 2:1 CDCl3/TFA.
S8
3.549
3.541
2.270
2.266
2.262
2.245
2.231
2.224
2.217
2.212
1.873
1.204
1.193
1.182
1.174
4.397
4.385
4.373
4.266
4.254
4.245
4.234
4.223
4.152
(A)
8.534
8.521
8.231
8.219
8.205
8.193
7.980
7.967
7.956
Section B: NMR Spectra of Synthesized Compounds
1.0
0.9
Normalized Intensity
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
8.5
(B)
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
Chemical Shift (ppm)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Figure S6. 1H NMR spectral comparison of: (A) peptide (P2) and (B) N-[-Gly(Ala)3-(Glu)3]perylene-3,4-imide-9,10-bis(n-butyl)ester (7) in DMSO-d6.
S9
Section C: MALDI Mass Spectra
(A)
(B)
Figure S7. MALDI mass spectra of PDI 2E: (A) Primary ion spectrum; (B)
[M+Na]+ ion fragmentation spectrum.
S10
Section C: MALDI Mass Spectra
(A)
(B)
Figure S8. MALDI mass spectra of PDI 2HE: (A) Primary ion spectrum; (B)
[M+Na]+ ion fragmentation spectrum with assigned ion fragments.
S11
Section C: MALDI Mass Spectra
(A)
(B)
Figure S9. MALDI mass spectra of PDI 2M: (A) Primary ion spectrum; (B)
[M+H]+ ion fragmentation spectrum.
S12
Section C: MALDI Mass Spectra
(A)
(B)
Figure S10. MALDI mass spectra of perylene imide bisester 7: (A) Primary ion
spectrum; (B) [M+Na]+ ion fragmentation spectrum with assigned ion fragments.
S13
Section D: IR Spectra of Synthesized Perylene Imides
(A)
(B)
Figure S11. IR spectra of compound 2E: (A) Spectral range 4000-500 cm−1; (B)
Fingerprint region spectral comparison showing anhydride to imide conversion.
S14
Section D: IR Spectra of Synthesized Perylene Imides
(A)
(B)
Figure S12. IR spectra of compound 2HE: (A) Spectral range 4000-500 cm−1; (B)
Fingerprint region spectral comparison showing anhydride to imide conversion.
S15
Section D: IR Spectra of Synthesized Perylene Imides
(A)
(B)
Figure S13. IR spectra of compound 2M: (A) Spectral range 4000-500 cm−1; (B)
Fingerprint region spectral comparison showing anhydride to imide conversion.
S16