Synthesis and Mechanochemical Activity of Peptide-Based Cu(I) Bis(N-heterocyclic carbene) Complexes.

With the class of shock-absorbing proteins, nature created some of the most robust materials combining both mechanical strength and elasticity. Their excellent ability to dissipate energy to prevent surrounding cells from damage is an interesting property that regularly is exploited for applications in biomimetic materials. Similar to biomaterials, where mechanical stimuli are transmitted into a (bio)chemical response, mechanophoric catalysts transform mechanical energy into a chemical reaction. Force transmission is realized commonly by polymeric handles directing the applied force to the mechanophoric bond, which in turn leads to stress-induced activation of the catalyst. Therefore, shock-absorbing proteins able to take up and store mechanical energy elastically for subsequent force transduction to the labile bond seem to be perfect candidates to fulfill this task. Here, we report on the synthesis of two different latent mechanophoric copper(I) bis(N-heterocyclic carbene) complexes bearing either two carboxyl groups or two amino groups which allow conjugation reactions with either the N- or the C-terminus of amino acids or peptides. The chosen catalysts can be activated, for instance, by applying external mechanical force via ultrasound, removing one N-heterocyclic carbene (NHC) ligand. Post-modification of the mechanophoric catalysts via peptide coupling (Gly, Val) and first reactions showed that the mechanoresponsive behavior was still present after the coupling. Subsequent polycondensation of both catalysts lead to a polyamide including the Cu(I) moiety. Mechanochemical activation by ultrasound showed conversions in the copper(I)-catalyzed alkyne-azide "click" reaction (CuAAC) up to 9.9% proving the potential application for the time and spatial controlled CuAAC.


Introduction
Even though the rupture of macromolecules using strong shear forces has been already described by Staudinger in 1930 [1] and Eyring in 1940 [2], mechanochemistry is a comparably new field of research investigating the impact of mechanical forces to defined chemical changes in macromolecules [3][4][5]. Introducing selectively cleavable bonds into polymers enables chain scission in a chemically productive use via mechanochemistry even allowing reaction pathways to be altered [6]. Mechanophores are used for a widespread range of mechanochemical transformations such as color changes [7], changes in fluorescence [8][9][10], activation of latent catalysts [9,11], biased reactivity [6], release of small molecules [12], generation of protons [13], stabilization of radicals [14], remodeling of polymers [15,16], and also for molecular synthesis [17]. Exploiting such transformations, mostly in the fields of self-healing, stress sensing and catalysis can lead to a redirection of mechanochemistry from being "destructive to (being) productive" [18].
Gel permeation chromatography (GPC) measurements were performed on a Viscotek GPCmax VE 2001 (Malvern Panalytical Ltd., Crowthorne, UK) using a HHR-H Guard-17369 (Malvern Panalytical Ltd.) and a GMHHR-N-18055 column (Malvern Panalytical Ltd.) with DMF containing 10 mM LiTf2N as eluent at 60 °C and via detection of the refractive index with a Viscotek VE 3580 RI

Mechanochemical Activation
Ultrasonication experiments were conducted by placing the latent mechanocatalyst (7.50 µmol) into a 10 mL reaction vessel with two additional side necks attached to a VCX 500 ultrasonic processor Control experiments without ultrasound were carried out in two-necked flasks at room temperature as well as at 60 • C to prove the activation of the catalyst by ultrasound.

Synthesis of the Mechanophoric Catalysts
We started with the synthesis of Cu(I) bis(NHC) complexes, bearing appropriate functional groups to allow a subsequent extension by amino acids via peptide-coupling. Therefore two different Cu(I) bis(NHC) complexes were designed: one bearing two carboxylic acid moieties (4), the second bearing two terminal amino groups (8), both allowing peptide/protein attachment via the N-or C-terminus of amino acids/peptides. The carboxyl-functionalized complex ([Cu(C 10 COOH-NHC) 2 ]Br) (4) was synthesized in a four-step synthesis (Scheme 1a). In a first step, 11-bromoundecanoic acid was protected via a methyl ester to prevent a complexation with the copper(I) in the following steps which would lower the yield of the desired complex significantly (5-10%, see Supplementary Materials). The obtained product 1 was subsequently quaternized using N-methylimidazole forming thus the NHC precursor 2 in quantitative yields which in turn is able to act as ligand for the desired Cu(I) complexes. For deprotonation and complexation, Cu 2 O was chosen as internal base and copper(I) source at once [59] due to the lability of the methyl ester group under basic conditions realizing thus the synthesis of the [Cu(C 10 COOMe-NHC) 2 ]Br complex (3) instead of the commonly used method which involves strong bases such as potassium bis(trimethylsilyl)amide (KHMDS) or sodium tert-butoxide [9]. Finally, LiOH was used for deprotection yielding the carboxyl-functionalized catalyst [Cu(C 10 COOH-NHC) 2 ]Br (4) quantitatively. copper(I) source at once [59] due to the lability of the methyl ester group under basic conditions realizing thus the synthesis of the [Cu(C10COOMe-NHC)2]Br complex (3) instead of the commonly used method which involves strong bases such as potassium bis(trimethylsilyl)amide (KHMDS) or sodium tert-butoxide [9]. Finally, LiOH was used for deprotection yielding the carboxylfunctionalized catalyst [Cu(C10COOH-NHC)2]Br (4) quantitatively.  For the synthesis of the amine-functionalized ([Cu(C3NH2-NHC)2]Br) complex (8) (Scheme 1b), 3-bromopropan-1-amine hydrobromide was protected in a first step with a Boc group to avoid side reactions with the primary amines during the subsequent complex formation and enabling an efficient purification process. The Boc-protected imidazolium precursor 6 was obtained after a For the synthesis of the amine-functionalized ([Cu(C 3 NH 2 -NHC) 2 ]Br) complex (8) (Scheme 1b), 3-bromopropan-1-amine hydrobromide was protected in a first step with a Boc group to avoid side reactions with the primary amines during the subsequent complex formation and enabling an efficient purification process. The Boc-protected imidazolium precursor 6 was obtained after a quaternization with N-methylimidazole in a yield of 80% and was subsequently transformed to the [Cu(C 3 NHBoc-NHC) 2 ]Br complex (7) using the same Cu 2 O method described above. The deprotection was achieved with trifluoroacetic acid yielding the amino-functionalized complex [Cu(C 3 NH 2 -NHC) 2 ]Br (8)

Peptide Coupling Reactions
Peptide coupling reactions were performed to probe the attachment of amino acids onto the Cu(I) bis(NHC)-complexes 4 and 8. Firstly, it was crucial to understand if a proper attachment via conventional peptide coupling strategies could be effected. Secondly, it was important to investigate the effect of attached amino acids toward the catalytic activity of the mechanoresponsive copper(I) catalysts in CuAAC "click" reactions. Therefore, glycine methyl ester hydrochloride (9) and L-valine methyl ester hydrochloride (10) were coupled to catalyst 4 using common coupling agents [61,62] such as DCC or EDC•HCl and pentafluorophenol as shown in Scheme 2a,b.

Peptide Coupling Reactions
Peptide coupling reactions were performed to probe the attachment of amino acids onto the Cu(I) bis(NHC)-complexes 4 and 8. Firstly, it was crucial to understand if a proper attachment via conventional peptide coupling strategies could be effected. Secondly, it was important to investigate the effect of attached amino acids toward the catalytic activity of the mechanoresponsive copper(I) catalysts in CuAAC "click" reactions. Therefore, glycine methyl ester hydrochloride (9) and L-valine methyl ester hydrochloride (10) were coupled to catalyst 4 using common coupling agents [61,62] such as DCC or EDC·HCl and pentafluorophenol as shown in Scheme 2a,b. Both, the glycine-(11) and the L-valine-modified mechanophores (12) could be obtained in yields of around 50%. The 1 H NMR spectrum of catalyst 11 is shown in Figure 4a. The NH of the formed peptide bond was detected at 6.06 ppm, while protons H11 (-NHCH2-) and H13 (-OCH3) from the coupled glycine methyl ester were present at 4.03 and 3.75 ppm, respectively. The signals of protons H3 and H4 (-NCH=CHN-) of the Cu(I) NHC ring at 6.15 and 6.06 ppm verify that the complex was not destroyed during the coupling. Analogous the 1 H NMR spectrum of the L-valine-modified complex (12) (Figure 4b) showed the successful formation of the peptide bond at 5.96 ppm (NH) as well as all signals belonging to the introduced L-valine.
Extending the concept of peptide-linking to the Cu(I) bis(NHC) complexes and elongating the force transmitting polymeric scaffold with nondistortive peptide bonds, a coupling of dicarboxylic telechelic catalyst 4 with catalyst 8, bearing two amino groups were performed (Scheme 2c). Due to the bifunctional character of both catalysts, an equimolar polycondensation under the formation of amide bonds took place, embedding the Cu(I) bis(NHC) complex within a polyamide chain to ensure remaining mechanoresponsive behavior [60]. The polycondensation was monitored via GPC ( Figure  5) observing a dimeric structure formation (1100 g/mol) at the early stage of the reaction after 1 h, while higher condensates (5400 g/mol up to 13,200 g/mol) can be observed after ongoing reaction time (up to 75 h) due to the step-growth character of the polycondensation leading also to broader molecular weight distributions. Figure 4c shows the purified 1 H NMR spectrum of the coupled catalyst 13. The signal of the NHgroup at 7.02 ppm clearly showed the successful peptide coupling and, likewise, for the other catalysts the resonances at 6.20 and 6.16 ppm coming from the -NCH=CHN-protons of the catalysts (H3, H4, H14, and H15) proved the presence of the undestroyed Cu(I) bis(NHC) structure. Both, the glycine-(11) and the L-valine-modified mechanophores (12) could be obtained in yields of around 50%. The 1 H NMR spectrum of catalyst 11 is shown in Figure 4a. The NH of the formed peptide bond was detected at 6.06 ppm, while protons H 11 (-NHCH 2 -) and H 13 (-OCH 3 ) from the coupled glycine methyl ester were present at 4.03 and 3.75 ppm, respectively. The signals of protons H 3 and H 4 (-NCH=CHN-) of the Cu(I) NHC ring at 6.15 and 6.06 ppm verify that the complex was not destroyed during the coupling. Analogous the 1 H NMR spectrum of the L-valine-modified complex (12) (Figure 4b) showed the successful formation of the peptide bond at 5.96 ppm (NH) as well as all signals belonging to the introduced L-valine.
Extending the concept of peptide-linking to the Cu(I) bis(NHC) complexes and elongating the force transmitting polymeric scaffold with nondistortive peptide bonds, a coupling of dicarboxylic telechelic catalyst 4 with catalyst 8, bearing two amino groups were performed (Scheme 2c). Due to the bifunctional character of both catalysts, an equimolar polycondensation under the formation of amide bonds took place, embedding the Cu(I) bis(NHC) complex within a polyamide chain to ensure remaining mechanoresponsive behavior [60]. The polycondensation was monitored via GPC ( Figure 5) observing a dimeric structure formation (1100 g/mol) at the early stage of the reaction after 1 h, while higher condensates (5400 g/mol up to 13,200 g/mol) can be observed after ongoing reaction time (up to 75 h) due to the step-growth character of the polycondensation leading also to broader molecular weight distributions.

Investigation of Catalytic Activity of the Cu(I) bis(NHC) Complexes
Subsequently, the catalytic activity of the synthesized catalysts were investigated using a model CuAAC "click" reaction of benzyl azide (14) and phenylacetylene (15). Cu(I) bis(NHC) complexes are known for their mechanoresponsive behavior since they can be switched from their latent, inactive state to their active state after exposure to mechanical stress, for example in the form of ultrasound (in solution) or by compression (in bulk) [9,60,63]. In its latent state the Cu(I) center is surrounded by the two shielding NHC ligands which prevent the alkyne from coordination to the copper(I) center. When ultrasound is applied, one of the NHC ligands is cleaved from the Cu by rupturing the copper-carbene bond, in turn enabling the replacement by an alkyne under formation of the copper acetylide which is known to be the crucial step in the "click" reaction [64]. The principle of the catalyst activation is shown in Figure 6.
The activation of the latent catalyst to its active monocarbene form was observed by monitoring the subsequent CuAAC "click" reaction (see Figure 6) of 14 and 15 via 1 H NMR spectroscopy. Observing the shifts from the origin methylene group of 14 from 4.35 to 5.59 ppm for the "click" product 16 as well as an increasing triazole resonance at 8.11 ppm enable the calculation of the "click" conversion in dependence of the applied sonication time.
Biomimetics 2019, 4, x FOR PEER REVIEW 13 of 20 Figure 5. The peptide coupling between the two bifunctional catalysts 4 and 8 can be followed via GPC. It is expected that the reaction follows a step-growth mechanism due to its polycondensation character. This is proven by GPC where the formation of the dimer is observed early during the reaction. The formation of higher molecular weight species is only observed, if most of the monomer is consumed (75 h).

Investigation of Catalytic Activity of the Cu(I) bis(NHC) Complexes
Subsequently, the catalytic activity of the synthesized catalysts were investigated using a model CuAAC "click" reaction of benzyl azide (14) and phenylacetylene (15). Cu(I) bis(NHC) complexes are known for their mechanoresponsive behavior since they can be switched from their latent, inactive state to their active state after exposure to mechanical stress, for example in the form of ultrasound (in solution) or by compression (in bulk) [9,60,63]. In its latent state the Cu(I) center is surrounded by the two shielding NHC ligands which prevent the alkyne from coordination to the copper(I) center. When ultrasound is applied, one of the NHC ligands is cleaved from the Cu by rupturing the copper-carbene bond, in turn enabling the replacement by an alkyne under formation of the copper acetylide which is known to be the crucial step in the "click" reaction [64]. The principle of the catalyst activation is shown in Figure 6.
The activation of the latent catalyst to its active monocarbene form was observed by monitoring the subsequent CuAAC "click" reaction (see Figure 6) of 14 and 15 via 1 H NMR spectroscopy. Observing the shifts from the origin methylene group of 14 from 4.35 to 5.59 ppm for the "click" product 16 as well as an increasing triazole resonance at 8.11 ppm enable the calculation of the "click" conversion in dependence of the applied sonication time. Figure 5. The peptide coupling between the two bifunctional catalysts 4 and 8 can be followed via GPC. It is expected that the reaction follows a step-growth mechanism due to its polycondensation character. This is proven by GPC where the formation of the dimer is observed early during the reaction. The formation of higher molecular weight species is only observed, if most of the monomer is consumed (75 h).
In compliance with the literature [9], for all Cu(I) bis(NHC) catalysts an increase in the "click" conversion with increasing sonication time was observed (Table 1 and Figure 7). In case of the [Cu(C 10 COOMe-NHC) 2 ]Br (3) and its deprotected pendant [Cu(C 10 COOH-NHC) 2 ]Br (4) a similar catalytic activity could be determined after activation of the catalysts by ultrasound. Both catalysts showed a latency period up to the 5th sonication cycles. Thus, both catalysts revealed only a minimal conversion of 0.5% after the 5th cycle which increased afterward linearly up to a conversion of 3.5% after the 17th cycle.
Biomimetics 2019, 4, x FOR PEER REVIEW 14 of 20 Figure 6. Activation of the latent catalyst is achieved by ultrasonication which cleaves one of the two shielding NHC ligands. This allows the alkyne to coordinate to the Cu(I) and the "click" reaction can take place. As a model reaction, the "click" reaction between benzyl azide (14) and phenylacetylene (15) was chosen.
In compliance with the literature [9], for all Cu(I) bis(NHC) catalysts an increase in the "click" conversion with increasing sonication time was observed (Table 1 and Figure 7). In case of the [Cu(C10COOMe-NHC)2]Br (3) and its deprotected pendant [Cu(C10COOH-NHC)2]Br (4) a similar catalytic activity could be determined after activation of the catalysts by ultrasound. Both catalysts showed a latency period up to the 5th sonication cycles. Thus, both catalysts revealed only a minimal conversion of 0.5% after the 5th cycle which increased afterward linearly up to a conversion of 3.5% after the 17th cycle.
The Boc-protected amine-based catalyst [Cu(C3NHBoc-NHC)2]Br (7) showed a significantly higher activity than its deprotected pendant [Cu(C3NH2-NHC)2]Br (8). A conversion of 4.8% could Figure 6. Activation of the latent catalyst is achieved by ultrasonication which cleaves one of the two shielding NHC ligands. This allows the alkyne to coordinate to the Cu(I) and the "click" reaction can take place. As a model reaction, the "click" reaction between benzyl azide (14) and phenylacetylene (15) was chosen.
The Boc-protected amine-based catalyst [Cu(C 3 NHBoc-NHC) 2 ]Br (7) showed a significantly higher activity than its deprotected pendant [Cu(C 3 NH 2 -NHC) 2 ]Br (8). A conversion of 4.8% could already be detected after the 3rd cycle and increased linearly during the next cycles up to 9.9% after the 17th cycle. The deprotection of the catalyst to [Cu(C 3 NH 2 -NHC) 2 ]Br (8) had a bigger influence on the mechanoresponsivity in comparison to [Cu(C 10 COOH-NHC) 2 ]Br (4): an activation could only be detected after the 10th cycle (2.9% conversion) subsequently increasing linearly to a conversion of 4.8% after the 17th cycle which is only the half of the Boc-protected catalyst 7. It can be hypothesized that the sterically demanding Boc groups caused a pre-stretching of the copper-carbene bond and facilitated its cleavage [60,65]. The higher conversion using the [Cu(C 3 NH 2 -NHC) 2 ]Br catalyst (8) instead of the carboxyl-based catalyst (4) can be explained by the ionic end group structure (-NH 3 + TFA − ; see Figure 2) of catalyst 8 which is able to facilitate the protonation of the cleaved free NHC and accelerates thus the copper(I) acetylide formation. The peptide coupling between [Cu(C 10 COOH-NHC) 2 ]Br (4) and methyl ester-protected L-valine (10) leading to [Cu(C 10 COOH-Val-NHC) 2 ]Br (12) does not affect the catalytic activity, which is still present in "click" conversions of 3.4% after the 17th cycle. Thus, a successful peptide coupling which involves several coupling agents as well as a base, could be performed, causing no deactivation of the Cu(I) bis(NHC) catalyst. Likewise, the methyl ester-protected glycine modified [Cu(C 10 COOH-Gly-NHC) 2 ]Br catalyst (11) revealed catalytic activity which could be detected after the 5th cycle with a conversion of 2.9% increasing linearly to 6.9% after the 17th cycle.  To exclude undesired site reactions and proving the activation of the catalyst only by ultrasonication, test reactions were performed at room temperature using, apart from ultrasound, the same conditions for the "click" reaction. Therefore, mixtures containing the catalysts 3, 4, 7, 8 and 12 were stirred at room temperature for 42.5 h observing in all cases no conversion. Additionally, reactions were conducted at 60 °C to exclude thermal influences on the catalyst activation revealing  (15) and benzyl azide (14) to the "clicked" product 16 as calculated from 1 H NMR spectroscopy with a standard deviation of ±1%. Samples were taken after the cycles 0, 3, 5, 10, 14, and 17.
To exclude undesired site reactions and proving the activation of the catalyst only by ultrasonication, test reactions were performed at room temperature using, apart from ultrasound, the same conditions for the "click" reaction. Therefore, mixtures containing the catalysts 3, 4, 7, 8 and 12 were stirred at room temperature for 42.5 h observing in all cases no conversion. Additionally, reactions were conducted at 60 • C to exclude thermal influences on the catalyst activation revealing only very small conversions (1-2%) compared to the ultrasound-initiated "click" reactions in the range of uncatalyzed Huisgen 1,3-dipolar reactions [9,66].
The coupling of both bifunctional copper(I) complexes 4 and 8 yielded a polymeric catalyst 13 that still possessed mechanoresponsive behavior and only showed activation after application of ultrasound. Thus, the potential of peptide-based Cu(I) bis(NHC) complexes for a time and spatial controlled activation via mechanical force could be proven which opens the opportunity for a new class of mechanoresponsive molecules.

Conclusions
We successfully synthesized Cu(I) bis(NHC) mechanophoric catalysts bearing either two carboxyl groups (4) or two amino groups (8) and linked them via peptide bonds to the amino acids glycine (11) and L-valine (12). The chosen groups (-COOH and -NH 2 ) are perfect candidates for subsequent modification reactions with peptides since they allow the coupling to both the C-and N-terminus. A direct peptide coupling between the carboxyl-(4) and amino-functionalized (8) Cu(I) bis(NHC) catalysts generated peptide-based mechanophores.
The mechanophoric behavior of the low molecular weight complexes was proven via ultrasonication both for the protected ([Cu(C 10 COOMe-NHC) 2 ]Br (3), [Cu(C 3 NHBoc-NHC) 2 ]Br (7)) and the unprotected catalysts ([Cu(C 10 COOH-NHC) 2 ]Br (4), [Cu(C 3 NH 2 -NHC) 2 ]Br (8)). While the deprotection of the [Cu(C 10 COOMe-NHC) 2 ]Br (3) catalyst did not show significant impact on the mechanoresponsive behavior with a conversion around 3.5%, in both cases the deprotection of [Cu(C 3 NHBoc-NHC) 2 ]Br (7) reduced conversion from 9.9% to 4.8% for [Cu(C 3 NH 2 -NHC) 2 ]Br (8). The mechanoresponsive behavior was still present after coupling of single methyl ester protected amino acids to the [Cu(C 10 COOH-NHC) 2 ]Br (4) complex. However, while the coupling of glycine led to a slightly higher conversion of 6.9% the coupling of L-valine did not increase the conversion which still was at 3.4%. The polymeric catalyst (13) still showed mechanoresponsive behavior after application of ultrasound opening thus the opportunity to couple not only single amino acids but also whole peptide chains to our low molecular weight catalysts. The coupling of peptides such as elastin and titin that are known for their elasticity and their "molecular spring" behavior will be exploited for an efficient force transmission along the backbone to facilitate catalyst activation.

Conflicts of Interest:
The authors declare no conflict of interest.