DOI: 10.1002/open.201600032 Read the full text of the article at 10.1002/open.201500200
Comparison of Reductive Ligation-Based Detection Strategies for Nitroxyl (HNO) and S-Nitrosothiols
The group of Professor S. Bruce King
Invited for this month’s cover picture is the group of Prof. S. Bruce King at the Department of Chemistry of Wake Forest University. The cover picture shows a prefluorescent phosphine-based probe reacting with nitroxyl (HNO) and S-nitrosothiol (RSNO), nitrogen oxide-derived biological signals. Both species react with the prefluorescent probe, but only the product from the HNO reaction can complete a further chemical ligation pathway that results in fluorescence, indicating the presence of HNO. The product of the probe with RSNO does not complete this ligation and does not generate a fluorescent species. These phosphine-based probes thus demonstrate a selectivity for HNO over RSNO based on their chemical reactivity and can be used in biological systems to differentiate these species. For more details, see the Communication on p. 110 ff. were not selective? Zhengrui Miao noticed that no one had ever really directly compared the response of these probes to HNO and RSNO. He did this comparison and found fluorescence generation from HNO treatment was greatly enhanced compared with RSNO, and these findings initiated the study. The overall lesson is to keep an open mind and don’t plan the outcome of your experiments!
What are the exciting developments in your field? Redox biological signaling has significantly developed over the last couple of decades with small molecules such as NO, CO, H2S, and H2O2 being identified as competent signaling agents. Nitroxyl (HNO), the one-electron reduced/protonated form of NO, has been discussed as a signaling agent, but the lack of specific & selective detection methods hampers a better understanding of its biology. Significant advances in HNO detection have occurred with the development of new copper- and phosphine-based fluorescent probes and new electrochemical & mass spectrometric methods. With these mechanistically different ways to detect HNO, questions regarding HNO’s biology and endogenous formation can be approached.
Acknowledgement
This work was supported by Wake Forest University and the Center for Molecular Communication and Signaling of Wake Forest University. Flow cytometry services were supported by the Comprehensive Cancer Center of Wake Forest University (NCI CCSG P30CA012197).
What is the most significant result of this study?
The most significant result is that the aza-ylide intermediate generated from the reaction of these probes and HNO rapidly and reliably undergo a Staudinger ligation resulting in fluorescence and a stable amide by-product. Reaction of these probes with RSNO gives a similar aza-ylide, but in this case, the Staudinger ligation does not occur or proceeds inefficiently. This difference allows these types of probes to detect HNO without interference from RSNO. Flow cytometry experiments show that these reactions also occur in cells and that these probes only detects HNO in cells.
What was the biggest challenge/surprise on the way to the results presented in this paper?
One of the challenges starting this project was to not let our previous ideas about the chemistry dictate what experiments to do. We knew that RSNO reacts with phosphines to yield aza-ylides that should undergo ligations and yield fluorescence. Should we continue if we were going to see that our probes ChemistryOpen 2016, 5, 89
89
Ó 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Comparison of Reductive Ligation-Based Detection Strategies for Nitroxyl (HNO) and S-Nitrosothiols
The group of Professor S. Bruce King
Invited for this month’s cover picture is the group of Prof. S. Bruce King at the Department of Chemistry of Wake Forest University. The cover picture shows a prefluorescent phosphine-based probe reacting with nitroxyl (HNO) and S-nitrosothiol (RSNO), nitrogen oxide-derived biological signals. Both species react with the prefluorescent probe, but only the product from the HNO reaction can complete a further chemical ligation pathway that results in fluorescence, indicating the presence of HNO. The product of the probe with RSNO does not complete this ligation and does not generate a fluorescent species. These phosphine-based probes thus demonstrate a selectivity for HNO over RSNO based on their chemical reactivity and can be used in biological systems to differentiate these species. For more details, see the Communication on p. 110 ff. were not selective? Zhengrui Miao noticed that no one had ever really directly compared the response of these probes to HNO and RSNO. He did this comparison and found fluorescence generation from HNO treatment was greatly enhanced compared with RSNO, and these findings initiated the study. The overall lesson is to keep an open mind and don’t plan the outcome of your experiments!
What are the exciting developments in your field? Redox biological signaling has significantly developed over the last couple of decades with small molecules such as NO, CO, H2S, and H2O2 being identified as competent signaling agents. Nitroxyl (HNO), the one-electron reduced/protonated form of NO, has been discussed as a signaling agent, but the lack of specific & selective detection methods hampers a better understanding of its biology. Significant advances in HNO detection have occurred with the development of new copper- and phosphine-based fluorescent probes and new electrochemical & mass spectrometric methods. With these mechanistically different ways to detect HNO, questions regarding HNO’s biology and endogenous formation can be approached.
Acknowledgement
This work was supported by Wake Forest University and the Center for Molecular Communication and Signaling of Wake Forest University. Flow cytometry services were supported by the Comprehensive Cancer Center of Wake Forest University (NCI CCSG P30CA012197).
What is the most significant result of this study?
The most significant result is that the aza-ylide intermediate generated from the reaction of these probes and HNO rapidly and reliably undergo a Staudinger ligation resulting in fluorescence and a stable amide by-product. Reaction of these probes with RSNO gives a similar aza-ylide, but in this case, the Staudinger ligation does not occur or proceeds inefficiently. This difference allows these types of probes to detect HNO without interference from RSNO. Flow cytometry experiments show that these reactions also occur in cells and that these probes only detects HNO in cells.
What was the biggest challenge/surprise on the way to the results presented in this paper?
One of the challenges starting this project was to not let our previous ideas about the chemistry dictate what experiments to do. We knew that RSNO reacts with phosphines to yield aza-ylides that should undergo ligations and yield fluorescence. Should we continue if we were going to see that our probes ChemistryOpen 2016, 5, 89
89
Ó 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
