From Sequence-Defined Macromolecules to Macromolecular Pin Codes.

Dynamic sequence-defined oligomers carrying a chemically written pin code are obtained through a strategy combining multicomponent reactions with the thermoreversible addition of 1,2,4-triazoline-3,5-diones (TADs) to indole substrates. The precision oligomers are specifically designed to be encrypted upon heating as a result of the random reshuffling of the TAD-indole covalent bonds within the backbone, thereby resulting in the scrambling of the encoded information. The encrypted pin code can eventually be decrypted following a second heating step that enables the macromolecular pin code to be deciphered using 1D electrospray ionization-mass spectrometry (ESI-MS). The herein introduced concept of encryption/decryption represents a key advancement compared with current strategies that typically use uncontrolled degradation to erase and tandem mass spectrometry (MS/MS) to analyze, decipher, and read-out chemically encrypted information. Additionally, the synthesized macromolecules are coated onto a high-value polymer material, which demonstrates their potential application as coded product tags for anti-counterfeiting purposes.


Matrix-Assisted Laser Desorption/Ionization Tandem Mass Spectrometry (MALDI-MS/MS). For the MALDI measurements a stock solution of the matrix, trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-
propenylidene]malonitrile (DCTB, 30 mg/ml) was prepared and the samples where solubilised in either tetrahydrofuran or acetonitrile (10 mg/ml). 45 µl of the matrix solution and 15 µl of the sample solution where mixed and subsequently spotted on the MALDI plate. The spots were dried at room temperature and loaded into an Applied Biosystems Sciex 4800+ MALDI-TOF/TOF analyser, controlled by 4000 Series Explorer software (Applied Biosystems, Germany). The instrument was operated in linear positive ion mode. Fragmentation (MS/MS) was performed in positive ion mode at 1 kV using air as collision gas with the 'metastable suppressor' and 'optimised precursor ion' options turned on.
High Resolution Mass Spectroscopy (HRMS). HRMS spectra were collected using an Agilent 6220 accurate-mass time-of-flight (TOF) analyzer equipped with a multimode ionization (MMI) source.

Infrared (IR).
Measurements were recorded on a Perkin Elmer FTIR SPECTRUM 1000 spectrometer with Attenuated Total Reflection (ATR) with a PIKE Miracle ATR unit in a frequency range from 4000 to 600 cm -1 .

(ii) Synthesis of 5-bromo-2-phenyl-1H-indole (l2c)
A mixture of 4-bromophenylhydrazine hydrochloride (12.6 g, 56.6 mmol, 1.1 equiv.) and acetophenone (6.00 mL, 51.4 mmol, 1.0 equiv.) in 200 mL of a 9:1 v% ethanol:acetic acid solution was placed under inert atmosphere and stirred under reflux (90 °C) overnight. The resulting mixture was cooled to room temperature and the solvent was removed in vacuo to give a yellow-brown residue. To this, S11 polyphosphoric acid (PPA) (ca. 100 g) was added and the resulting mixture was heated to 110 °C for 2 hours. The dark brown mixture was poured into ice-water (800 mL) whilst hot and stirred vigorously overnight at room temperature. 400 mL of ethyl acetate was added to the resulting heterogenous mixture and the organic phase was separated. The aqueous phase was extracted with another 2 x 400 mL ethyl acetate and the collected organic phases were washed with 2 x 200 mL brine, followed by drying over magnesium sulfate and solvent removal in vacuo. The resulting dark brown oil was purified by column chromatography (silica, hexane:ethyl acetate 4:1, RF = 0.35) to give 5-bromo-2-phenyl-1H-indole l2c as a pale brown powder. Yield = 47 % (6.52 g).

S20
The reaction mixture was stirred at room temperature for 48 hours and the solvent was removed in vacuo at < 35 °C. The crude product was purified by column chromatography (silica, hexane:ethyl acetate 2:1  0:1, RF (ethyl acetate = 0.43), yielding a viscous yellow oil that solidified upon further drying in vacuo. Yield = 83 % (902 mg). From SEC analysis, a small amount of the remaining trimer was observed to have co-eluted. Therefore, the product was re-purified by column chromatography    Mass (m/z)

Encryption model study (i) Aliphatic macromolecular pin code
A solution of PC1 (58.7 mg, 0.020 mmol, 1.0 equiv.) in DMSO-d6 (1 mL) was transferred into an NMR tube and placed in a pre-heated oil bath at 150 °C. After heating for 15 minutes, 0.20 mL of the mixture was taken out and diluted with 0.40 mL tetrahydrofuran prior to SEC analysis (see Figure 3c).

(ii) Aromatic macromolecular pin code
A solution of PC2 (59.2 mg, 0.020 mmol, 1.0 equiv.) in DMSO-d6 (1 mL) was transferred into an NMR tube and placed in a pre-heated oil bath at 120 °C. After heating for 15 minutes, 0.20 mL of the mixture was taken out and diluted with 0.40 mL tetrahydrofuran prior to SEC analysis (see Figure 3d).

Decryption model study (i) Aliphatic macromolecular pin code
A solution of PC1 (58.7 mg, 0.020 mmol, 1.0 equiv.) and sorbic alcohol (6.5 mg, 0.066 mmol, 3.3 equiv.) in 1 mL DMSO-d6 was transferred into an NMR tube and placed in a pre-heated oil bath at 120 °C, 135 °C or 150 °C. At distinct time intervals, the NMR tube was taken out of the oil bath and cooled immediately under running tap water before being submitted to NMR analysis (see Figure S3), from which the conversion of PC1 could be determined as a function of time (see Figure S4).

(ii) Aromatic macromolecular pin code
A solution of PC2 (59.2 mg, 0.020 mmol, 1.0 equiv.) and sorbic alcohol (6.5 mg, 0.066 mmol, 3.3 equiv.) in 1 mL DMSO-d6 was transferred into an NMR tube and placed in a pre-heated oil bath at 90 °C, 105 °C or 120 °C. At distinct time intervals, the NMR tube was taken out of the oil bath and cooled immediately under running tap water before being submitted to NMR analysis (see Figure S5), from which the conversion of PC2 could be determined as a function of time (see Figure S6). The kinetic reversibility profiles of the decryption model studies for both the aliphatic and aromatic pin codes (i.e. PC1 and PC2, respectively) depicted in Figure S4 and Figure S6, respectively, express the fraction of retro-TAD-indole reaction that has occurred upon heating the sequence-defined oligomers at a well-defined temperature for a distinct period of time.
The presence of sorbic alcohol (HDEO) as a kinetic trap for the in situ released TAD at elevated temperatures, and the therewith associated fast reaction kinetics with regard to the TAD-indole recombination reaction (i.e. kTAD-HDEO > 10 3 kTAD-indole), 3-4 allows for a simplified expression of the observed overall decryption reaction rate coefficient kobs.
The fraction of TAD-indole adducts remaining at specific time intervals, i.e. ln could be derived from the decryption conversion via integration of the respective 1 H NMR spectra. The observed rate coefficient was hence determined by a linear regression fit for each of the three temperatures investigated. Using the rearranged Arrhenius equation below, the observed overall activation energy (Ea, obs) for the decryption of the sequence-defined pin codes could eventually be derived from the Arrhenius plot, obtained by a best-fit linear interpolation of ln kobs as a function of T -1 (refer to   Table S1). With the gas constant R = 8.314 J K -1 mol -1 and a expressed in K, this gives Ea, obs in J mol -1 .   showing the absence of the expected monomer fragments of PC1, except that of A1, which can be identified because of the irreversible Diels-Alder adduct formed with the use of S1 at the beginning of the sequence. c) LC chromatogram following encryption of PC1. Apart from A1, no other isolatable or readable fragments can unambiguously be deciphered. The fragments observed relate to recombined fragments of the macromolecular pin code without the S1 trapping agent, which are unreadable. Retention time / min.

Decryption and read-out of PC1 and PC2
Besides kinetic 1 H NMR measurements of the decryption process, the thermal dissociation of the sequence-defined macromolecular pin codes was also monitored by means of LCMS analysis. For this, an anhydrous butyl acetate (1 mL) solution of PC1 (58.7 mg, 0.020 mmol, 1.0 equiv.) or PC2 (59.2 mg, 0.020 mmol, 1.0 equiv.) in the presence of conjugated diene S1 (24.1 mg, 0.066 mmol, 3.3 equiv.) was heated at 120 °C for 5 hours or 2 hours, respectively. Subsequently, the solvent was removed in vacuo and the resulting residue was re-dissolved in either acetonitrile or tetrahydrofuran and subjected to LC-ESI-MS analysis (see Figure S12 and Figure S13). The different monomer fragments could be readily identified by means of the identified mass fragments from the ESI-MS spectrum, whereby the initial order of the fragments within the sequence could be derived from the distinct isotopic pattern (as illustrated for PC1 in Figure S14).  Figure S12. a) Structures of the isolated monomers obtained after decryption of PC1 upon heating for 5 hours at 120 °C in the presence of S1. b) LC chromatogram (λ = 214 nm), indicating the successful separation of the four different monomer units. c) ESI-MS spectrum following the decryption process allows for the mass of the four monomer units to be identified. The monomers can be related to their respective structure based on the differences in mass, while using the isotopic pattern of the indole markers, their original order in the sequence can also be determined.  Figure S13. a) Structures of the isolated monomers obtained after decryption of PC2 upon heating for 5 hours at 120 °C in the presence of S1. b) LC chromatogram (λ = 214 nm), indicating the successful separation of the four different monomer units. c) ESI-MS spectrum following the decryption process allows for the mass of the four monomer units to be identified. The monomers can be related to their respective structure based on the differences in mass, while using the isotopic pattern of the indole markers, their original order in the sequence can also be determined.

Proof-of-concept demonstration on polymer banknotes
A 2016 series Bank of England £5 note and a 2016 series Reserve Bank of Australia $5 note were first individually suspended in stirring ethanol overnight to verify that the banknotes did not dissolve or discolor in the solvent (see Figure S15). Upon removal of the ethanol in vacuo, the resulting residue was submitted for ESI-MS analysis whereby a background spectrum was collected (see Figure S16). Figure S15. Macromolecular tagging of banknotes for anti-counterfeiting. a) Original $5 and £5 banknotes prior to coating with encrypted pin codes PC1 and PC2, respectively. b) Suspension of the banknotes in ethanol to test that they did not dissolve or discolor in the solvent (here shown for the £5). c) Banknotes after suspension in ethanol and prior to coating, showing that they remained unchanged by the solvent. Following the blank experiment, the sequence-defined macromolecular pin codes were next applied to the surface of the polymer banknotes. Thus, PC1 (7.64 mg, 2.6 µmol) and PC2 (9.67 mg, 3.3 µmol) were each dissolved in anhydrous butyl acetate (1 mL) and encrypted by heating for 2 hours at 120 °C.
The solvent was then removed in vacuo and the residues were re-dissolved in ethanol (2 mL) and then were deposited on the surface of either the £5 or $5 banknotes, respectively. S43 stirring ethanol for 5 hours and the solvent was removed in vacuo. A solution of conjugated diene S1 (3.13 mg, 8.6 µmol) in butyl acetate (1.5 mL) was added to the extracted PC1, while a solution of conjugated diene S1 (3.93 mg, 10.8 µmol) in butyl acetate (1.5 mL) was added to the extracted PC2.
The two solutions were finally heated for 4 hours at 120 °C, followed by quick cooling under running tap water and solvent removal in vacuo and the resulting decrypted macromolecular pin codes were submitted for ESI-MS analysis (Figure S17 and Figure S18). Figure S17. ESI-MS spectrum of the decrypted macromolecular pin code PC1 extracted from the £5 banknote.