Peptide-assisted design of peptoid sequences: One small ...€¦ · , with k = 1.38 x 10-23, T =...

Supporting Information

Peptide-assisted design of peptoid sequences: One small step in structure and distinct leaps in functions

Corresponding Author: Hans G. Börner*

Other Authors: Eva Maron, Zdravko Kochovski, Ronald N. Zuckermann

*to whom correspondence is to be addressed

Prof. Dr. Hans Börner,

Humboldt-Universität zu Berlin

Laboratory for Organic Synthesis of Functional Systems

Brook-Taylor-Str. 2, 12489 Berlin, Germany

E-Mail: [email protected]

Phone: +49 (0)30-2093 7348

Fax: +49 (0)30 2093-7500

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TABLE OF CONTENTS

1. MATERIALS .............................................................................................................. 3

2. INSTRUMENTATION AND SAMPLE PREPARATION ........................................ 3

3. PROCEDURE FOR THE SYNTHESIS OF PEPTOID-PEG- CONJUGATES ......... 5

4. ANALYSIS OF PEPTOID-PEG CONJUGATES ...................................................... 6

5. SOLUBILIZATION OF M-THPC ............................................................................ 22

6. DRUG RELEASE STUDIES .................................................................................... 25

7. REFERENCES .......................................................................................................... 25

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1. MATERIALS

All chemicals and solvents were purchased and used without purification unless specified.

m-THPC was kindly provided by Prof. Mathias O. Senge (School of Chemistry, SFI

Tetrapyrrole Laboratory, Trinity College, Dublin, Ireland). Peptides were synthesized and used

as published previously.1, 2

Peptoids: The amines including benzyl amine (99%), isobutyl amine (99%) isopropyl

amine (99.5%), methylamine (40% solution in water) and glycinamide hydrochloride (98%)

were purchased from Sigma Aldrich. The solvents N,N-dimethylformamid (DMF, 99.9 %) and

dichloromethane (DCM, 99.5 %) for the peptoid synthesis were purchased from Merck and

reagents described 2-bromoacetic acid (99%), thioanisole, trifluoroacetic acid (TFA, 99%)

were purchased from Sigma Aldrich. N,N'-Diisopropylcarbodiimid (DIC, 99.5%) was

purchased from Chem-Impex. Solid phase support Tentagel PAP resin (particle size: 90 µm,

capacity: 0.2 - 0.25 mmol/g, loaded PEG Mn = 3200, Đ = 1.04) was obtained from Rapp

Polymere GmbH (Tübingen, Germany). All peptoids and peptide-peptoid-hybrids were

synthesized using automated solid-phase submonomer synthesis on a Symphony X peptide

synthesizer at a scale of 200 mg Rink amide resin (0.64 mmol/g).

2. INSTRUMENTATION AND SAMPLE PREPARATION

Electrospray ionization mass spectrometry (ESI-MS) was performed using an LTQ

Orbitrap velos pro mass spectrometer (Thermo Fischer Scientific) equipped with an

atmospheric pressure ionization source operating in the nebulizer assisted electrospray mode.

The instrument was calibrated in the m/z range 220-2000 using a standard solution containing

caffeine, MRFA and Ultramark 1621. A constant spray voltage of 5 kV was used and nitrogen

at a dimensionless sheath gas flow-rate of 7 was applied. The capillary voltage, the tube lens

offset voltage and the capillary temperature were set to 25 V, 120 V and 275 °C, respectively.

For manual measurements a polymer solution with concentration of 10 µg/mL was injected. A

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mixture of THF and methanol (THF:MeOH = 3:2), all HPLC grade, was used as solvent.

Spectra were analyzed in Thermo Xcalibur Qual Browser software.

(MALDI-TOF MS) spectra were obtained on a Bruker ulfrafleXtreme MALDI-TOF

instrument. An accelerating voltage of 20 kV was applied, and spectra were obtained in

reflection mode (500 shots). The peptoid-PEGs were dissolved in a water/acetonitrile solution

(1:1) to yield a concentration of ~1 mg/mL. The matrix was α-cyano-4-hydroxycinnamic acid

in water as a 40 mg/mL solution. The two solutions (each 2 µL) were combined in a 1:1 solution

(polymer: α-cyano-4-hydroxycinnamic acid), mixed and drop-casted onto a MALDI plate and

allowed to dry before analysis.

UV-Vis spectroscopy was performed using a Shimadzu UV-2501 PC spectrometer

(Shimadzu Corp., 604-8511 Kyoto, Japan) using PS-cuvettes with 10 mm path.

Steady-state fluorescence emission spectra and kinetics were recorded on a Synergy

MXmicroplate reader (BioTek, Winooski, VT 05404, USA) in black polystyrene 96-well

plates.

Dynamic Light Scattering (DLS) data were collected on a Malvern Zetasizer nano-ZS

(Malvern Instruments Ltd., Worcestershire, UK) equipped with a He-Ne-Laser (λ = 632.8 nm)

on 400 µL aliquots of polymer-solutions. The sample concentration is provided in Tab. S5.

DLS experiments were repeated three times. Hydrodynamic radii RH were calculated using

Stokes-Einstein equation:

𝑅

, with k = 1.38 x 10-23, T = 293 K, η = Viscosity, μ = Diffusion coefficient.

Samples for cryo-electron microscopy (cryo-TEM) were prepared by applying a 4 μl

droplet of sample suspension to lacey carbon copper grids (200 mesh, Science Services) and

plunge frozen into liquid ethane using a Vitrobot Mark IV(FEI, Eindhoven, Netherlands) set at

4°C and 95 % humidity. The grids were mounted on a cryo transfer holder (Gatan 914, Gatan,

Munich, Germany) and transferred into a JEOL JEM-2100 (JEOL GmbH, Eching, Germany)

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transmission electron microscope for imaging. The microscope was operated at an acceleration

voltage of 200 kV and micrographs were recorded with a bottom-mounted 4*4k CMOS camera

(TemCam-F416, TVIPS, Gauting, Germany) at a magnification of 50 000x, corresponding to

a pixel size of 2.32 Å at the specimen level. Total electron dose for each micrograph was kept

below 20 e-/Å2.

3. PROCEDURE FOR THE SYNTHESIS OF PEPTOID-PEG- CONJUGATES

Resin (100 mg, 0.025 mmol) was incubated with dichloromethane (3 mL) for 30 minutes.

The resin is then drained and washed 6× (2.5 mL DMF, N2 mixing, 30 seconds each).

Bromoacetylation of the growing peptoid chains were performed using 0.8 M bromoacetic acid

and 0.8 M diisopropylcarbodiimide in DMF (500 μL each) under N2 mixing for 20 min. The

resin was drained and washed 6× (2.5 mL DMF, N2 mixing, 30 seconds each). Displacements

were performed using 1 M amines in DMF (1 mL) under N2 mixing for 1 hour. For

displacements by glycinamide hydrochloride, the salt was treated previously with an aqueous

KOH solution (17.8 M, 0.95 equival.), the precipitating salt was centrifuged and the obtained

solutions were diluted by DMF to get 1 M amine solutions. The resin was then drained and

washed 6× (2 mL DMF, N2 mixing, 30 seconds each). After the final displacement, the resin

is incubated with 20% 4-methylpiperidine in DMF (2 mL) under N2 mixing for 15 minutes.

The resin is then drained and washed 6× (2.5 mL DMF, N2 mixing, 30 seconds each). A

dichloromethane wash (3 mL × 10 minutes) is performed after 6 x (2.5 mL DMF, N2 Mixing,

30 seconds each) followed by resin drying under N2 stream for 10 minutes. For cleaving the

product from the resin, a premixed, freshly prepared cleavage cocktail was prepared

TFA/trimethylsilyl bromide/thioanisole (94:1:5) and was added to the resin (10 mL/g) in a

glass vial outfitted with a solvent resistant cap and shaken for 1 to 1.5 hours at room

temperature. The resin was removed via a filter syringe and the resin washed with

dichloromethane (3-4 mL) followed by evaporation. The raw product was taken in 1 mL of

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methanol and precipitated in cold diethyl ether, centrifuged and supernatant diethyl ether was

removed. Precipitates were dried in N2 stream, dissolved in deionized water with HCl (0,1 M).

Subsequent, peptide conjugates were dialyzed against deionized water (500-1000 Da MWCO,

regenerated cellulose), followed by lyophilization of conjugate solutions.

4. ANALYSIS OF PEPTOID-PEG CONJUGATES

Figure S1. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidAFFLFFQ-PEG.

Mpeak[M+H]+ = 3767.42 (m/z) Mcalc[M+H]+ = 3766.18 (m/z)

Mpeak[M+Na]+ = 3788.31 (m/z) Mcalc[M+Na]+ = 3788.16 (m/z)

Mpeak[M+K]+ = 3805.40 (m/z) Mcalc[M+K]+ = 3804.13 (m/z)

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Figure S2. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidQAFLFFQ-PEG.


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Figure S3. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidQFALFFQ-PEG.



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Figure S4. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidQFFAFFQ-PEG.

Mpeak[M+TFA-H2O+H]+ = 3728.19 (m/z) Mcalc[M+TFA-H2O+H]+ = 3731.04 (m/z)

Mpeak[M+TFA-H2O+Na]+ = 3753.67(m/z) Mcalc[M+TFA-H2O+Na]+ = 3754.03(m/z)

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Figure S5. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidQFFLAFQ-PEG.



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Figure S6. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidQFFLFAQ-PEG.


Mpeak[M+TFA-H2O+Na]+ = 4028.91 (m/z) Mcalc[M+TFA-H2O+Na]+ = 4028.23 (m/z)

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Figure S7. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidQFFLFFA-PEG.


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Figure S8. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidQFFLFFQ-PEG.



2500 3000 3500 4000 4500 5000

[M+K]+

38

74.8

7

39

34.6

2

38

89.8

1

37

41.7

3

36

97.5

7

39

19.0

4

384

6.9

5

380

2.7

8

371

5.7

5

m/z

[M+Na]+

37

87.2

0

38

30.0

6

375

9.2

7

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Figure S9. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidQFFVFFQ-PEG.



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Figure S10. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidFFLFF-PEG.

Mpeak[M+Na]+ = 3735. 98 (m/z) Mcalc[M+Na]+ = 3735.16 (m/z)


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Figure S11. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidFFVFF-PEG.



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Figure S12. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidFFAFF-PEG.



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Figure S13. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidQLFFFFQ-PEG.



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Figure S14. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidQFLFFFQ-PEG.




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Figure S15. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidQFFFLFQ-PEG.




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Figure S16. MALDI-TOF-MS analysis of Peptoid-PEG conjugate PeptoidQFFFFLQ-PEG.




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5. SOLUBILIZATION OF M-THPC

General procedure: The peptoid-PEG-conjugates (1.47 μmol) were dissolved in 1 mL

Millipore water. To these solutions 1 mL of m-THPC-solutions in ethanol (1.47 μmol/mL)

were added. These mixtures were slightly shaken for 1 h at room temperature. Subsequently

the ethanol/water solutions were frozen in lq. N2 and the solvent was removed by freeze-drying.

The residues were suspended in 1 mL of Millipore water. These suspensions were shaken

slightly over 1 h at room temperature and afterwards not soluble m-THPC was removed by

centrifugation (10 min, 13 000 rpm), to make sure no solid is left in the solution, centrifugation

was repeated 3 times. For concentration determination of solubilized drug, UV-Vis absorption

spectra of each supernatant diluted in ethanol (1 : 99 v/v) were recorded on a Shimadzu UV-

2501 PC spectrometer (Shimadzu Corp., 604-8511 Kyoto, Japan). Concentration of m-THPC

solubilized by each carrier was calculated through comparison of the absorption maximum at

650 nm to a calibration curve of the free drug in EtOH (0.0001, 0.0005, 0.001, 0.005, 0.01

mg/mL). Maximum payload capacity of each carrier is summarized in Table S3.

Table S1. DLS-analyzed hydrodynamic radii and dispersity of m-THPC loaded nanoparticles.

Sequence RH [nm] PDI

PeptoidAFFLFFQ 101 0.043

PeptoidQAFLFFQ 162 0.167

PeptoidQFALFFQ 203 0.361

PeptoidQFFAFFQ 90 0.034

PeptoidQFFLAFQ 99 0.053

PeptoidQFFLFAQ 103 0.062

PeptoidQFFLFFA 114 0.031

PeptoidQFFLFFQ 85 0.045

PeptoidQFFVFFQ 103 0.140

PeptoidQFFAFFQ 90 0.034

PeptoidFFLFF 80 0.009

PeptoidFFVFF 79 0.007

PeptoidFFAFF 94 0.002

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Figure S17. Cryo-TEM micrographs of unloaded PeptoidQFFLFFQ-PEG with average radius of

particles r = 16 nm.

Figure S18. Cryo-TEM micrographs of m-THPC loaded nanoparticles of PeptoidQFFLFFQ -

PEG with average radius of particles r = 76 nm

Figure S19. Cryo-TEM micrographs of m-THPC loaded nanoparticles of PeptoidQFFLFFA-

PEG with average radius of particles r = 74.

Table S2. Comparion loaded and not loaded Sequences by Cryo-TEM and DLS

Sequence RH, DLS [nm] PDIDLS Rcryo TEM [nm]

PeptoidQFFLFFA + m-THPC 114 0.031 74

PeptoidQFFLFFA - m-THPC 76 0.284 -

PeptoidQFFLFFQ + m-THPC 84 0.045 76

PeptoidQFFLFFQ - m-THPC -* - 33

*multimodal, sample can not be interpreted

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Table S3. Solubilization of m-THPC by using standard solubilization experiment with

indicated used polymer, molar mass, and resulting end concentrations of polymer solutions

and molar ratio of drug to polymer.

Precision segment

Sequence-PEG conc.a)

[mM]

Payload Initial release rate e) b) [mmol] c) [g] d)

PeptoidAFFLFFQ A F F L F F Q 1.47 1 : 3.3 0.302 0.22 1375

PeptoidQAFLFFQ Q A F L F F Q 1.47 1 : 53.0 0.019 0.01 -

PeptoidQFALFFQ Q F A L F F Q 1.47 1 : 20.1 0.050 0.04 -

PeptoidQFFAFFQ Q F F A F F Q 1.47 1 : 78.0 0.013 0.01 907

PeptoidQFFLAFQ Q F F L A F Q 1.47 1 : 13.7 0.073 0.05 -

PeptoidQFFLFAQ Q F F L F A Q 1.47 1 : 49.5 0.020 0.02 -

PeptoidQFFLFFA Q F F L F F A 1.47 1 : 1.9 0.535 0.39 353

PeptoidQFFLFFQ Q F F L F F Q 1.47 1 : 15.1 0.066 0.05 1586

PeptoidQFFVFFQ Q F F V F F Q 1.47 1 : 30.9 0.032 0.02 236

PeptoidQFFAFFQ Q F F A F F Q 1.47 1 : 63.1 0.016 0.01 907

PeptoidFFLFF - F F L F F - 1.47 1 : 4.1 0.245 0.22 346

PeptoidFFVFF - F F V F F - 1.47 1 : 5.6 0.178 0.163 749

PeptoidFFAFF - F F A F F - 1.47 1 : 47.7 0.021 0.020 250

PeptoidQLFFFFQ Q L F F F F Q 1.47 1 : 29.2 0.034 0.02 1071

PeptoidQFLFFFQ Q F L F F F Q 1.47 1 : 17.9 0.056 0.04 1245

PeptoidQFFLFFQ Q F F L F F Q 1.47 1 : 15.1 0.066 0.05 1586

PeptoidQFFFLFQ Q F F F L F Q 1.47 1 : 17.5 0.057 0.04 1444

PeptoidQFFFFLQ Q F F F F L Q 1.47 1 : 16.9 0.059 0.04 1448

a) polymer concentration used in solubilization experiment, b) mol ratio drug:carrier, c) molarity of solubilized drug per mmol polymer [mmol], d) mass of solubilized drug per g of functional segment [g], e) determined by 1st order derivative at t = 0 min.

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Drug release studies

For fluorescence-measurement, drug-loaded polymer solutions were dissolved in Millipore

water to obtain 280 µL of 0.1 µM m-THPC concentrations that were filled in a 96-well plate.

The release was initiated by adding 20 µL aqueous BSA-solution (100 µM). Additionally same

drug loaded solutions were treated with 20 µL aqueous Triton-X solution (0.1%) to get a top

fluorescence release-experiment. Top-experiments were normalized to fluorescence

measurement of 0.1 µM m-THPC solutions with 0.1% Triton-X solution. Samples were excited

at 417 nm and emission was recorded at 654 nm every 2 min for 18 h (gain 80, slit 20,

measurement).

6. REFERENCES

(1) Wieczorek, S.; Krause, E.; Hackbarth, S.; Röder, B.; Hirsch, A. K. H.; Börner, H. G., Exploiting specific interactions toward next-generation polymeric drug transporters. J. Am. Chem. Soc. 2013, 135 (5), 1711-4. (2) Wieczorek, S.; Remmler, D.; Masini, T.; Kochovski, Z.; Hirsch, A. K. H.; Börner, H. G., Fine-tuning Nanocarriers Specifically toward Cargo: A Competitive Study on Solubilizing Related Photosensitizers for Photodynamic Therapy. Bioconjugate Chem. 2017, 28 (3), 760-767.

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