Sequence-defined positioning of amine and amide residues to control catechol driven wet adhesion

Catechol and amine residues, both abundantly present in mussel adhesion proteins, are known to act cooperatively by displacing hydration barriers before binding to mineral surfaces. In spite of synthetic efforts toward mussel-inspired adhesives, the effect of positioning of the involved functional groups along a polymer chain is not well understood. By using sequence-defined oligomers grafted to soft hydrogel particles as adhesion probes, we study the effect of catechol–amine spacing, as well as positioning relative to the oligomer terminus. We demonstrate that the catechol–amine spacing has a significant effect on adhesion, while shifting their position has a small effect. Notably, combinations of non-charged amides and catechols can achieve similar cooperative effects on adhesion when compared to amine and catechol residues. Thus, these findings provide a blueprint for the design of next generation mussel-inspired adhesives.


Oligomer synthesis
All oligomers were synthesized using the building blocks EDS, TrDS and CDS as previously described. [1] The oligomers were assembled via iterative deprotection and amide coupling on a Tentagel® S RAM resin. For deprotection, the resin was treated with 20% piperidine in DMF (2x 15 min) and washed with DMF (10x). For the coupling step, the building block (5 eq.) and PyBOP (5 eq.) were dissolved in DMF and DIPEA (10 eq.) was added. The resin was treated with the coupling solution for 1 hr with subsequent DMF washing (10x). After assembly of the full sequence, the trityl groups were cleaved by treating the resin with 0.1 M HCl in trifluorethanol (2x1.5 h). Then the resin was washed with DMF (5x) and the free amines were deprotonated with 20% DIPEA in DMF for 10 minutes. For introducing the side chains, the resin was treated for 1 hr with a solution of solution of either succinamic acid or 4-(dimethylamino)butyric acid (5 eq.), PyBOP (5 eq.) and DIPEA (10 eq.) in DMF and washing in DMF (10x) afterward. The structures were cleaved from solid support with a solution of TFA/TIPS (95/5), precipitated in diethyl ether and the precipitate was lyophilized. All oligomers with a protected catechol moiety were deprotected by treatment with 16 eq. trifluoromethanesulfonic acid and 8 eq. thioanisole per methyl ether in TFA for 16 h. Afterward the

Supporting Information
3 reaction solution was precipitated in diethyl ether and the deprotected oligomers were lyophilized. The chemical analysis of the building block and oligomers are shown in the supporting information S1-S4)

Freeze Dryer
Lyophilization of the final structures was conducted on an Alpha 1-4 LD plus instrument from Martin Christ Freeze Dryers GmbH. The lyophilization was done at a pressure of 0.1 mbar.

S3 Building Block Synthesis and Chemical Analysis
The building block EDS was synthesized according to literature. [4] Synthesis Route for Functional Building Blocks Figure S1. Overview of building block synthesis route: a) 0.25 eq. trityl chloride in DCM; b) 1 eq.
Functional building blocks were synthesized with the new synthesis route shown in Figure S1. b) The crude product of a) was dissolved in THF and triethylamine (3 eq.) and a solution of Fmoc-OSu (1 eq.) in THF was added over 2 h at -78°C. Afterwards the activated acid (1 eq.) in THF was added and the reaction was stirred for 16 h at room temperature. The reaction mixture was extracted with a saturated NaCl solution (3x) and the organic phase was dried with MgSO 4 and the solvent was evaporated under reduced pressure to give the crude product as a brown foam.
c) The crude product of b) was dissolved in DCM and triethylsilane (10 eq.) and 10 vol-% TFA were added. The reaction was stirred at room temperature for 1 h. Afterwards the solvent was evaporated under reduced pressure and the product was precipitated in diethyl ether. The precipitate was dissolved in DCM and triethylamine (3 eq.) and succinic anhydride (1 eq.) were added. The reaction was stirred for 2 h at room temperature and afterwards extracted with a citric acid solution (3x). The organic phase was dried with MgSO 4 and the solvent was evaporated under reduced pressure to give the crude product as a brown foam. 6

14-oic acid (CDS) (2)
CDS (2) was synthesized following the synthesis route in Figure S1. The crude product was recrystallized in acetone and DCM (1:1) to give a white powder with a yield of 17 g (60%).

S4 Oligomer Synthesis and Chemical Analysis
All oligomers were synthesized on solid support according to literature [1] using the building blocks EDS, TrDS and CDS.

On Resin Deprotection Of Trityl
The resin was treated with 0.1 M HCl in trifluorethanol (2x1.5 h). Afterwards the resin was washed with DMF (5x) and the free amines were deprotonated with 20% DIPEA in DMF for 10 minutes.

Side Chain Coupling
After trityl deprotection the resin was treated for 1 h with a solution of 5 eq. acid, 5 eq. PyBOP and 10 eq. DIPEA in DMF. Afterwards the resin was washed with DMF (10x).

Deprotection Of Catechols
All oligomers with a protected catechol moiety were deprotected in solution. For this they were treated with 16 eq. trifluoromethanesulfonic acid and 8 eq. thioanisole per methyl ether in TFA for 16 h. Afterwards the reaction solution was precipitated in diethyl ether and the deprotected oligomers were freeze dried.

Oligomer Chemical Analysis
(3) protected Compound 3 was obtained with a yield of 32% after deprotection, purification by preparative RP-HPLC and lyophilization.
Compound 6 was obtained with a yield of 26% after deprotection, purification by preparative RP-HPLC and lyophilization.

Supporting Information
(9) protected Compound 9 protected was obtained with a yield of 67% after cleavage from solid support and lyophilization.    (9) Compound 9 was obtained with a yield of 27% after deprotection, purification by preparative RP-HPLC and lyophilization.     (10) protected Compound 10 protected was obtained with a yield of 61% after cleavage from solid support and lyophilization.
Compound 10 was obtained with a yield of 19% after deprotection, purification by preparative RP-HPLC and lyophilization.  RP-HPLC (gradient from 0% to 50% eluent B over 30 min at 25°C): t r =9.8 min, purity 89%.   (11) protected Compound 11 protected was obtained with a yield of 72% after cleavage from solid support and Compound 11 was obtained with a yield of 28% after deprotection, purification by preparative RP-HPLC and lyophilization.      Additionally, 500 µL of Oligomer in MES buffer is added. The amount of Oligomer was equal to a 10 fold excess in comparison to carboxylic acid groups on the particles (see Table S1). To start the reaction 100 µL of a solution of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) with concentration 100 mg mL -1 in ultrapure water is added. The reaction solution is shaken for 2 h before the reaction solution was removed via centrifugation (13500 rpm, 5 min) and replaced with a fresh reaction solution. After an additional reaction time of 2 h the supernatant is removed and the particle are washed with ultrapure water via centrifugation (13500 rpm, 5 min). The functionalization degree was determined via microscope based TBO titration. Table S1: Overview over molecular weight and amount of the oligomers that were used per reaction step for PEG-CA-SCP functionalization and the functionalization degree of the particles determined via microscope based TBO titration.

Crotonic Acid Titration via UV-VIS Spectroscopy
The determination of carboxylic acid groups on the PEG-CA particles was done in triplicates.

Determination of oligomer functionalization degree via microscope based TBO titration
For the determination of functionalization degree of oligomer functionalized SCPs 100 µL of SCP solution was washed via centrifugation (13500 rpm, 5 min) with sodium hydroxide solution pH 10-11.
After removing the supernatant 125 µL of TBO solution with 0.3125 mmol L -1 were added, wrapped in aluminum foil and shaken overnight. Next, the TBO solution was removed and the particles were washed three times with 1 mL of sodium hydroxide solution with pH 10-11 and afterwards dissolved in 125 µL. The same procedure was done for PEG-CA particles and non-functionalized PEG particles.
Next, for all particle solutions the grey value was determined for 20 particles per batch to calculate the functionalization degree as following: Where D OGF is the oligomer functionalization degree, ΔG B is the difference of grey values between non-functionalized and carboxylic acid functionalized SCPs (ΔG B > 0), G N is the average grey value of non-functionalized SCPs and G SCP is the average grey value of oligomer functionalized SCPs.

S6 Determination of the SCPs elastic modulus
Force-indentation measurement with a NanoWizard 2 AFM provided the elastic modulus of the SCPs.
A silica bead with a raduis of 2.3 µm was glued with an epoxy glue onto a tipless, non-coated cantilever (spring constant 0.32 N/m; NanoAndMore GmbH). Several force curves were recorded from different particles and analyzed with the novel contact model developed by Glaubitz et al. [5] . The model considers deformation of the object at two sites: the indentation site of the AFM probe and at the contact with the solid support. The respective deformation (δ) -force (F) dependence reads: For all SCPs except for the diamine oligomer (4) carrying SCPs the elastic moduli were similar, around 71.9 ± 10.5 kPa. The elastic modulus for the diamine oligomer (4) functionalized SCPs was 103 ± 14.4 kPa. The increase in elastic modulus for the diamine carrying SCP is probably the to an extended conformation of the of the oligomer stiffening the PEG network. But overall, the rather low variations of the elastic moduli for the different SCPs are expected due to the low density of oligomers in the SCP.
About 13.5-14.2 wt% of the SCPs material are oligomers. Due to the high SCP swelling degree the oligomer concentration within the SCP network is 11 mmol l -1 . Figure S69. Typical AFM indentation-force curves for the analysis with the contact model developed by Glaubitz et al. [5] The solid lines are fits to the data.

S7 Reflection Interference Contrast Microscopy (RICM) measurements Setup
RICM on an inverted microscope (Olympus IX73) was used to obtain the contact area between the microparticles and a hard glass surface. For illumination a monochromatic (530 nm) collimated LED (Thorlabs, Germany, M530L2-C1) was used. An UPlanFL N 60x/0.90 dry objective (Olympus Corporation, Japan), additional polarizers and a quarter waveplate (Thorlabs, germany) to avoid internal reflections and a monochrome CMOS camera (DMK 33UX174, The Imaging Source Europe GmbH, Germany) were used to image the RICM patterns.

Determination of the Contact Radius
RICM was used to measure the contact radius formed by the SCPs resting on the polymer surface ( Figure S2). Polarized light waves reflected from the upper glass surface (I 1 ) and the surface of the bead (I 2 ) interact to create an interference image. The intensity at a given position in the image depends on the separation h(x) between the two surfaces: I(x) = I 1 + I 2 + 2•sqrt(I 1 • I 2 ) cos[2k•h(x) + π], where k = 2πn/λ, and n and λ are the index of refraction of water and the wavelength of the monochromatic light, respectively. In order to detect the interference pattern, stray light was reduced by an 'antiflex' technique. This is accomplished by crossed polarizer and analyzer filter with a λ/4-plate placed between the objective lens and the analyzer. [6] Figure S70.Schematic drawing of the RICM principle.

Correction Factors
For analysis of the RICM patterns correction factors must be determined for finite aperture and geometry effects. To obtain the correction factors, we imaged hard, non-deformable glass beads on a glass surface in RICM mode with a known size and curvature. We recorded 5 glass beads with a diameter in the range of 20-40 µm (polysciences) and extracted the intensity profile. Using the profiles, we reconstructed the shape of the beads and compared it to the known spherical shapes of the glass beads (glass bead radius R measured by light microscope), and determined the correction factors, see Pussak et al. [7] Contact radius determination To determine the contact radius a of the SCP on the polymer surface we reconstructed the height profile of the particles from the RICM images (see Figure S3). This was done by determining the lateral x(i) positions of the i-th minima and maxima by a self-written IgorPro procedure (Wavemetrics, USA).
Next, the vertical position y(i) of the maxima and minima were determined by i c n where n is the refractive index and  the wavelength. The height profile was then reconstructed by plotting y(i) vs x(i) and fitting the data by a circle equation representing the assumed shape of the SCP: where R is the independently measured SCP radius and y 0 the vertical shift of the SCP center due to flattening of the SCP upon adhesion. The fit with y 0 as the only free fit parameter intersects with the xaxis and gives the contact radius a.

S8 Stability of the catechol group
Compound 12 was used as a model for the investigation of the catechol stability. For this 1 mg was dissolved in 500 µl water and the mixture was measured via RP-HPLC directly after dissolving and after 12 days. Figure S72. RP-HPLC of compound 12 directly after dissolving and after 12 days. Peak 1 shows compound 12.

S9 Non-normalized and oligomer concentration normalized adhesion energy values
Supporting Information