Design,Synthesis And Biological Evaluation Of NanoLuc-type Bioluminescent Substrates

Bioluminescence is a common natural phenomenon that attracted the general public from ancient to present,and encouraged scientists to unravel the mystery of bioluminescence.Reasons for bioluminescence include interspecific communication,attracting prey,hunting,and self-defense.Although bioluminescence has been observed for thousands of years,the mechanism of bioluminescence was gradually deciphered with the progress of molecular biology.The essence of bioluminescence is a special type of chemiluminescence,which refers to the process that the luciferase in the organism catalyzes the enzymatic oxidation of the corresponding luciferin to convert almost all of the chemical energy into light energy.NanoLuc-furimazine bio luminescent system was introduced in 2012,which is a novel luminescent system.The system includes a novel substrate-furimazine,and a bright and stable enzyme-NanoLuc luciferase.NanoLuc was derived from a deep-sea shrimp Oplophorus gracilirostris.Later,Promega used directed evolution technology to genetically engineer it.And then optimized and screened a large number of substrates to obtain the substrate-furimazine.Finally,a bioluminescence system with excellent performance was formed.Compared with the traditional bioluminescent system,this bioluminescent system has the following advantages:luminescence does not aepeina on ATP,Mg2+and other auxiliary factors;NanoLuc has a small molecular weight at only 19 kDa;the stability of NanoLuc and substrate is enhanced;the most prominent is that the bioluminescent intensity of this system is 150 times more than other systems and can produce glowing blue light signals.However,the bioluminescence system also has the following shortcomings:First,the bioluminescence wavelength for in vivo imaging needs to be more than 600 nm,while the emission wavelength of the system is only 460 nm,which is easy to be absorbed and scattered by tissues;Second,the substrate is uniqueand there is no suitable substitute.Although scientists modified the C-2,C-6,and C-8 sites of furimazine in recent years,the reasonable substrates have not been found since no systematic evaluation criterion is available for the modified substrates.In this study,the NanoLuc-furimazine bioluminescence system was well considered.Based on furimazine,the bioluminescent system was optimized by designing and synthesizing the novel substrates to obtain a bioluminescent substrate with excellent luminescence properties and substitution.This research topic can be divided into two parts:The first part:based on furimazine,the substitution at the C-6 site was modified to provide a series of novel NanoLuc-type bioluminescent substrates.The A series substratesare characterized by the replacement of the phenyl ring with different substituted groups at the C-6 position of the molecular skeleton imidazo[1,2-a]pyrazine.The NanoLuc luciferase level,cell level and in vivo biological evaluation was carried out for such compounds.The results disclosed that most of the compounds had good bioluminescent activity:the emission wavelength of all new substrates at the enzyme level was redshifted and the half-life of most substrates with luciferase had increased,especially the half-life of A2 was more than 2 hours,and the luminescent intensity of compounds A6 and All was comparable to that of furimazine;at the cell level,the luminescent intensity of all the compounds was comparable to that of furimazine.However,the luminescence intensity of compounds A6 and A11 was stronger than that of other compounds.At animal level,the luminescence intensity of substrate A11 was stronger than that of furimazine,and the luminescence duration was more than 1 hour.Overall,compound A11 is expected to be a new alternative substrate for NanoLuc bioluminescence system.The second part:based on the substrate furimazine,a series of B bioluminescent substrates were designed and synthesized by modifying its C-8 position.Systemic activity evaluations were then performed at the enzyme level,cell level,and animal level in terms of luminescence intensity,duration,and emission wavelength.The results displayed that B series compounds reacted with NanoLuc luciferase:at the enzyme level,the bioluminescence intensities of B1 and B2 were higher than furimazine,and the biological emission wavelength indicated a certain red shift,while the luminescence duration decreased;at cell level,compared with furimazine,the bioluminescence intensity of B2 has been greatly improved;in animal imaging,compounds B1 and B2 exhibited excellent luminescence properties,and the luminescence intensity and duration were much larger than furimazine,especially the B2 luminescence intensity was 51 times more than furimazine.Therefore,B2 has the potential to be commercialized and can replace furimazine as the optimal NanoLuc-type of luminescent substrate for in vivo imaging.From the perspective of structure-activity relationship,we analyzed the effects of the structure of two series of A and B compounds on the activity of NanoLuc luciferase.The following conclusions can be drawn:the introduction of macromolecular conjugated groups at the C-6 substitution position,although the emission wavelength shows a great red shift,the intensity of luminescence decreases significantly,This is because the "pocket"of luciferase reacts with the substrate is small.If the molecular structure was too large,the compound is not conducive to entering the binding "pocket" and therefore cannot produce high intensity luminescence.The introduction of a fluorine atom at the C-6 phenyl group can significantly increase the luminescence time.The luminescence half-life of A2 is longer than 2 h.The introduction of electron-donating substituents at the C-6 substitution position can significantly reduce the luminescence intensity and should be avoided as much as possible.The introduction of oxygen and sulfur atoms at the C-8 substitution position can form a p-π conjugate with the heterocycle.The emission wavelength can be red-shifted,and the introduction of a sulfur atom can form a disulfide bond with the enzyme to make it more firmly bonded,and the luminescence intensity can be significantly enhanced.Compared with the modification at C-6 position,that is more suitable at the C-8 position to significantly improve the light emission characteristics of the substrate.If connected with a substituent group of a hydrogen bond donor or acceptor base,also allows a significant change in light emission characteristics.In summary,based on the NanoLuc-furimazine bioluminescence system,we designed and synthesized two series of new luminescent substrates based on the small molecular structure,and systematically compounded the compounds at the enzyme level,cell level and animal level.Activity evaluation,obtained some alternative substrates with stronger luminescence,longer duration and red shift of emission wavelength.These new substrates will help NanoLuc bioluminescence system to be well applied in biomedical fields such as disease detection and luminescence imaging.In addition,we can use the principle of prodrugs to link specific recognition groups to the 3-position carbonyl of furimazine,that not only to improve the stability of furimazine and its analogues,but also to enhance sensitivity and selectivity,so that broaden the application range of NanoLuc bioluminescent system.It is expected that our research results will help increase the range of NanoLuc bioluminescence system and provide useful information for subsequent optimization of bioluminescence systems.