Common use of Xxxxxx Xxxx X Clause in Contracts

Xxxxxx Xxxx X. Xxxxxxxx, Xxxxxx X. Xxxxxxxxx, Xxx Xxx, and Xxxxxxx X. X. Xxxx* Abstract: The creation of adaptive matter is heavily inspired by biological systems. However, it remains challenging to design complex material responses that are governed by reaction networks, which lie at the heart of cellular complexity. The main reason for this slow progress is the lack ofa general strategy to integrate reaction networks with materials. Herein we use a systematic approach to preprogram the response of a hydrogel to a trigger, in this case the enzyme trypsin, which activates a reaction network embedded within the hydrogel. A full characterization of all the kinetic rate constants in the system enabled the construction of a computational model, which predicted different hydrogel responses depending on the input concentration of the trigger. The results of the simulation are in good agreement with experimental findings. Our methodology can be used to design new, adaptive materials of which the properties are governed by reaction networks of arbitrary complexity. Living systems are adaptive and use enzymatic reaction networks to detect changes in their environment, process input information, and determine an appropriate response.[1] Materials science has recently taken a keen interest in the adaptivity of living systems,[2] and has created new materials with life-like properties such as self-healing,[3] camouflaging,[4] and control over surface characteristics.[5] Impressive exam- ples include the incorporation of the oscillating Belousov– Zhabotinsky reaction into a self-walking gel,[6] and the work of Xxxxxxxxx and co-workers,[7] who used chemo–mechanico– chemical feedback loops to produce a homeostatic material. Others have pioneered control over hydrogel lifetimes with preprogrammed feedback loops using organic[8] or enzy- matic[9] reactions. However, progress towards “life-like” materials has been slow as we lack a general framework for constructing materials with autonomous behavior and preprogrammed responses to external stimuli. Designing materials with complex responses requires the incorporation of chemical reaction networks, where the kinetics within the system are suitably balanced.[10] Here, we present a systematic approach to program the complex response of hydrogels, inspired by our previous work on enzymatic reaction networks.[11,12] First, we developed a polyacrylamide (PAAm)-based hydrogel that [*] S. G. J. Xxxxxx, I. N. Vialshin, X. X. Xxxxxxxxx, X. Xxx, Prof. W. T. S. Huck Radboud University, Institute for Molecules and Xxxxxxxxx Xxxxxxxxxxxxxx 000, 0000 XX Xxxxxxxx (Xxx Xxxxxxxxxxx) E-mail: x.xxxx@xxxxxxx.xx.xx Supporting information for this article can be found under: xxxx://xx.xxx.xxx/10.1002/anie.201610875. contains two orthogonal types of crosslinks that can be degraded or formed, respectively, through enzymatic activity (Figure 1 A). When a trigger, the endopeptidase trypsin (Tr), is applied to the gel, degradation of the initial crosslinks 1 (C1) proceeds rapidly. Simultaneously,a crosslink precursor (a copolymerized thioester) is slowly cleaved, creating thiols that very quickly react with an added linker to form new crosslinks 2 (C2). Thus,a gel–liquid–gel transition takes place and a new gel is formed with potentially different properties such as shape and stiffness as compared to the initial gel. Next, we introduced an enzymatic reaction network into the hydrogel of which the components all have their own specific function. When all components are present in the right concentrations, the network is able to sense the input concentration of Tr, and determine the corresponding hydro- gel response. Importantly, the kinetics in the network are fully charac- terized, and the programmed response of the network is predicted by a computational model that is in good agreement with our experimental results. In this way, we provide a systematic approach for integrating reaction networks within adaptive materials. We synthesized PAAm gels with two orthogonal cross- linkers (Figure 1 A): C1 is susceptible to cleavage by Tr, which will be used as a trigger. Tr also triggers the cleavage of thioester 3, revealing the cryptic thiol groups, which can react with the poly(ethylene glycol)-bis-maleimide crosslinker 4 (MW= 2000 g mol@1) that is present in the gel, forming C2. Tr rapidly cleaves amide bonds at the C-terminal end of positively charged amino acids, and therefore we synthesized C1 with an arginine–serine moiety in the middle of the molecule (Figure 1A; see section S2 of the Supporting Information (SI) for complete details on molecular structures and synthesis of all molecules used). We measured a value for kcat/KM (a measure of catalytic efficiency) of @ 24600 mm@1 h@1 for Tr cleaving C1, confirming that Tr will rapidly degrade gels containing C1 (details of all kinetic studies are in section S3 of the SI). For the cryptic crosslink precursor, we studied a number of amino acids that could serve as Tr-cleavable protecting groups of thiol side groups. Interestingly, due to the relatively high reactivity of thioesters, we found that the reactivity of Tr towards lysine thioesters was as high as the arginine amide bonds in C1 (kcat/KM > 29400 mm@1 h@1). Therefore, the less reactive leucine thioester 3 was prepared (Tr hydrolysis kcat/ KM = 575 mm@1 h@1). Importantly, thioester 3 is also quickly cleaved by another enzyme, chymotrypsin (Cr; kcat/KM = 34400 mm@1 h@1), enabling more complex responses when using both enzymes in an enzymatic reaction network as we will show below. 1794 T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2017, 5G, 1794 –1798

Xxxxxx Xxxx X. XxxxxxxxVialshin, Xxxxxx X. Xxxxxxxxx, Xxx Xxx, and Xxxxxxx X. X. Xxxx* Abstract: The creation of adaptive matter is heavily inspired by biological systems. However, it remains challenging to design complex material responses that are governed by reaction networks, which lie at the heart of cellular complexity. The main reason for this slow progress is the lack ofa general strategy to integrate reaction networks with materials. Herein we use a systematic approach to preprogram the response of a hydrogel to a trigger, in this case the enzyme trypsin, which activates a reaction network embedded within the hydrogel. A full characterization of all the kinetic rate constants in the system enabled the construction of a computational model, which predicted different hydrogel responses depending on the input concentration of the trigger. The results of the simulation are in good agreement with experimental findings. Our methodology can be used to design new, adaptive materials of which the properties are governed by reaction networks of arbitrary complexity. Living systems are adaptive and use enzymatic reaction networks to detect changes in their environment, process input information, and determine an appropriate response.[1] Materials science has recently taken a keen interest in the adaptivity of living systems,[2] and has created new materials with life-like properties such as self-healing,[3] camouflaging,[4] and control over surface characteristics.[5] Impressive exam- ples include the incorporation of the oscillating Belousov– Zhabotinsky Xxxxxxxxxxx reaction into a self-walking gel,[6] and the work of Xxxxxxxxx and co-workers,[7] who used chemo–mechanico– chemical feedback loops to produce a homeostatic material. Others have pioneered control over hydrogel lifetimes with preprogrammed feedback loops using organic[8] or enzy- matic[9] reactions. However, progress towards “life-like” materials has been slow as we lack a general framework for constructing materials with autonomous behavior and preprogrammed responses to external stimuli. Designing materials with complex responses requires the incorporation of chemical reaction networks, where the kinetics within the system are suitably balanced.[10] Here, we present a systematic approach to program the complex response of hydrogels, inspired by our previous work on enzymatic reaction networks.[11,12] First, we developed a polyacrylamide (PAAm)-based hydrogel that [*] S. G. J. Xxxxxx, I. N. VialshinXxxxxxxx, X. X. Xxxxxxxxx, X. Xxx, Prof. W. T. S. Huck Radboud University, Institute for Molecules and Xxxxxxxxx Xxxxxxxxxxxxxx 000, 0000 XX Xxxxxxxx (Xxx Xxxxxxxxxxx) E-mail: x.xxxx@xxxxxxx.xx.xx Supporting information for this article can be found under: xxxx://xx.xxx.xxx/10.1002/anie.201610875. contains two orthogonal types of crosslinks that can be degraded or formed, respectively, through enzymatic activity (Figure 1 A). When a trigger, the endopeptidase trypsin (Tr), is applied to the gel, degradation of the initial crosslinks 1 (C1) proceeds rapidly. Simultaneously,a crosslink precursor (a copolymerized thioester) is slowly cleaved, creating thiols that very quickly react with an added linker to form new crosslinks 2 (C2). Thus,a gel–liquid–gel transition takes place and a new gel is formed with potentially different properties such as shape and stiffness as compared to the initial gel. Next, we introduced an enzymatic reaction network into the hydrogel of which the components all have their own specific function. When all components are present in the right concentrations, the network is able to sense the input concentration of Tr, and determine the corresponding hydro- gel response. Importantly, the kinetics in the network are fully charac- terized, and the programmed response of the network is predicted by a computational model that is in good agreement with our experimental results. In this way, we provide a systematic approach for integrating reaction networks within adaptive materials. We synthesized PAAm gels with two orthogonal cross- linkers (Figure 1 A): C1 is susceptible to cleavage by Tr, which will be used as a trigger. Tr also triggers the cleavage of thioester 3, revealing the cryptic thiol groups, which can react with the poly(ethylene glycol)-bis-maleimide crosslinker 4 (MW= 2000 g mol@1) that is present in the gel, forming C2. Tr rapidly cleaves amide bonds at the C-terminal end of positively charged amino acids, and therefore we synthesized C1 with an arginine–serine moiety in the middle of the molecule (Figure 1A; see section S2 of the Supporting Information (SI) for complete details on molecular structures and synthesis of all molecules used). We measured a value for kcat/KM (a measure of catalytic efficiency) of @ 24600 mm@1 h@1 for Tr cleaving C1, confirming that Tr will rapidly degrade gels containing C1 (details of all kinetic studies are in section S3 of the SI). For the cryptic crosslink precursor, we studied a number of amino acids that could serve as Tr-cleavable protecting groups of thiol side groups. Interestingly, due to the relatively high reactivity of thioesters, we found that the reactivity of Tr towards lysine thioesters was as high as the arginine amide bonds in C1 (kcat/KM > 29400 mm@1 h@1). Therefore, the less reactive leucine thioester 3 was prepared (Tr hydrolysis kcat/ KM = 575 mm@1 h@1). Importantly, thioester 3 is also quickly cleaved by another enzyme, chymotrypsin (Cr; kcat/KM = 34400 mm@1 h@1), enabling more complex responses when using both enzymes in an enzymatic reaction network as we will show below. 1794 T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2017, 5G, 1794 –1798