A Chemo-Enzymatic Approach Towards Gamma-Thio-Nucleoside-5'-Triphosphates
Lead Research Organisation:
Durham University
Department Name: Chemistry
Abstract
Context of Proposed Research
Our methods will give a simpler route towards nucleoside-5'-triphosphates (NTPs), which are key molecules in a range of biotechnological and basic research applications. Currently there are two main approaches. The first is readily accessible to biologists, but offers only small amount of material at high cost. The other requires highly trained chemists to provide the materials, where the chemistry is challenging and often unreliable. Our approach represents a new biotechnology
Aims and Objectives
We aim to provide two related simpler approaches that should allows biologists to access the desired NTPs with relative easy in also allow them to access more useful amounts of these materials in a cost effective manner. Our objectives centre on using an enzyme system, nucleoside diphosphate kinase (NDPK), to facilitate the production of NTPs.
Potential Applications and Benefits
The specific class of NTP that we will address in this one-year proof-of-concept proposal has a range of applications in determining how enzymes work and also how signaling pathway operate within biological systems. Our approach will benefit research workers in these areas and the specialist chemical companies that prepare NTPs. With our concept proven, we hope to expand our strategy towards a broader range of nucleotide systems, and generate a generic, broad platform towards this class of molecule.
Our methods will give a simpler route towards nucleoside-5'-triphosphates (NTPs), which are key molecules in a range of biotechnological and basic research applications. Currently there are two main approaches. The first is readily accessible to biologists, but offers only small amount of material at high cost. The other requires highly trained chemists to provide the materials, where the chemistry is challenging and often unreliable. Our approach represents a new biotechnology
Aims and Objectives
We aim to provide two related simpler approaches that should allows biologists to access the desired NTPs with relative easy in also allow them to access more useful amounts of these materials in a cost effective manner. Our objectives centre on using an enzyme system, nucleoside diphosphate kinase (NDPK), to facilitate the production of NTPs.
Potential Applications and Benefits
The specific class of NTP that we will address in this one-year proof-of-concept proposal has a range of applications in determining how enzymes work and also how signaling pathway operate within biological systems. Our approach will benefit research workers in these areas and the specialist chemical companies that prepare NTPs. With our concept proven, we hope to expand our strategy towards a broader range of nucleotide systems, and generate a generic, broad platform towards this class of molecule.
Technical Summary
The five technical objectives will be addressed as follows:
OBJECTIVE 1: ACCESS ACTIVE NUCLEOSIDE DIPHOPHATE KINASES (NDPKs)
His-tagged WT NDPKs and His->Gly active site mutants will be expressed, and activities will be assessed using HPLC, where the rate of equilibration between UTP and ATP will be measured (imidazole will be added to rescue His->Gly mutants).
OBJECTIVE 2: EXPLORE IMIDAZOLE THIOPHOSPHORYLATION AND Im RESCUE OF -HIS MUTANT NDPKs
Literature shows that His->Gly NDPK competently transfers the gamma-thio-P group of gamma-thio-ATP to imidazole: we envisage the reverse process will proceed smoothly. Thiophosphorylated imidazole will be prepared, exposed to NDPK plus ADP, and gamma-thio-ATP generation will be confirmed by HPLC. Thereafter, imidazole will be phosphorylated in situ and the thiophosphorylation of ADP will be measured.
OBJECTIVE 3: EXPLORE DIRECT HIS THIOPHOSPHORYLATION OF WT NDPKs
Immobilsed NDPK will be exposed to thiophosphodichloridate ion or thiophosphoryl chloride to generate thiophosphorylated-NDPK. Phosphoryl transfer activity of this NDPK to ADP will be measured by HPLC, and protein thiophosphorylation will be confirmed by MS. These procedures will be repeated concurrently to demonstrate a continuous process.
OBJECTIVE 4: EXPLOIT THE MOST PROMISING RESULTS FROM OBJECTIVE 2 & 3 ACROSS A RANGE OF COMMERCIAL NDP SUBSTRATES.
HPLC will be used to screen a range of NDP substrates, and products will be compared to reference compounds and/or confirmed using MS and 31-P NMR methods
OBJECTIVE 5: CONFIRM UTILITY OF THIO-NTP SYSTEMS
NTP-based enzyme systems avilable in each of our labs will be assayed to gauge the 'quality' of representative gamma-thio-NTPs. These will include T7 RNA polymerase (DRWH lab), adenylyl cyclases (MJC lab), and Ire1 systems (MS lab). NDPK will also serve as its own test vehicle, where materials generated by NDPK will be re-assayed in the reverse direction via HPLC.
OBJECTIVE 1: ACCESS ACTIVE NUCLEOSIDE DIPHOPHATE KINASES (NDPKs)
His-tagged WT NDPKs and His->Gly active site mutants will be expressed, and activities will be assessed using HPLC, where the rate of equilibration between UTP and ATP will be measured (imidazole will be added to rescue His->Gly mutants).
OBJECTIVE 2: EXPLORE IMIDAZOLE THIOPHOSPHORYLATION AND Im RESCUE OF -HIS MUTANT NDPKs
Literature shows that His->Gly NDPK competently transfers the gamma-thio-P group of gamma-thio-ATP to imidazole: we envisage the reverse process will proceed smoothly. Thiophosphorylated imidazole will be prepared, exposed to NDPK plus ADP, and gamma-thio-ATP generation will be confirmed by HPLC. Thereafter, imidazole will be phosphorylated in situ and the thiophosphorylation of ADP will be measured.
OBJECTIVE 3: EXPLORE DIRECT HIS THIOPHOSPHORYLATION OF WT NDPKs
Immobilsed NDPK will be exposed to thiophosphodichloridate ion or thiophosphoryl chloride to generate thiophosphorylated-NDPK. Phosphoryl transfer activity of this NDPK to ADP will be measured by HPLC, and protein thiophosphorylation will be confirmed by MS. These procedures will be repeated concurrently to demonstrate a continuous process.
OBJECTIVE 4: EXPLOIT THE MOST PROMISING RESULTS FROM OBJECTIVE 2 & 3 ACROSS A RANGE OF COMMERCIAL NDP SUBSTRATES.
HPLC will be used to screen a range of NDP substrates, and products will be compared to reference compounds and/or confirmed using MS and 31-P NMR methods
OBJECTIVE 5: CONFIRM UTILITY OF THIO-NTP SYSTEMS
NTP-based enzyme systems avilable in each of our labs will be assayed to gauge the 'quality' of representative gamma-thio-NTPs. These will include T7 RNA polymerase (DRWH lab), adenylyl cyclases (MJC lab), and Ire1 systems (MS lab). NDPK will also serve as its own test vehicle, where materials generated by NDPK will be re-assayed in the reverse direction via HPLC.
Planned Impact
Academic and industrial researchers will benefit from this research. Our approach will make important research and biotechnology chemicals more readily available to a wider range of workers. We shall protect IP and engage with industrial users in order to allow reap economic benefits. Our research will be published to allow the general research community to benefit from our findings.
Description | We have proven the basic concept of using an enzyme-based approach and a cheap readily accessible chemical for the preparation of nucleoside triphosphate (NTPs). These data are now being expanded by a MSc student. More straightforward method towards one of the starting materials have been further optimised and developed. A patent was published: WO 2020/008209A1, Title: TRIPHOSPHORYLATION REACTION. Priority date: 6th July 2018. |
Exploitation Route | A MSc student took over the reins of this project, completing several aspects relevant to NTP (re-)generation. A Pathfinder award was sought and won. This supported a follow-on funding application which was also successful. A PDRA is now in post to continuing the translation of this work under Follow-on funding scheme. |
Sectors | Chemicals Healthcare Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | Patent filing 6 July 2018, published 9 January 2020 WO 2020/008209A1. Pathfinder Award won. Follow-on Funding awarded (September 2019). PDRA is in post since August 2020, but progress hampered by COVID situation. |
First Year Of Impact | 2020 |
Sector | Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
Description | Biotechnology applications of novel chemo-enzymatic approaches to synthesise nucleoside triphosphates (NTPs) |
Amount | £9,587 (GBP) |
Funding ID | BB/S009671/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2018 |
End | 11/2018 |
Description | Development of a Nucleoside Diphosphate Platform for the Chemo-Enzymatic Synthesis of Nucleoside Triphosphates |
Amount | £201,759 (GBP) |
Funding ID | BB/T004134/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 06/2020 |
End | 08/2022 |
Title | Chemo-enzymatic NTP synthesis using phosphoryl Imidazole |
Description | We have demonstrated that a His->Gly mutant NDPK can utilise phosphoryl imidazole and NDP substrate to make an NTP. |
Type Of Material | Technology assay or reagent |
Year Produced | 2018 |
Provided To Others? | No |
Impact | These preliminary results are being expanded by two PhD students. |
Description | Academic visitor from Turku, Finland (Satu Mikkola) |
Organisation | University of Turku |
Country | Finland |
Sector | Academic/University |
PI Contribution | The visitor received training in protein production |
Collaborator Contribution | The visitor has provided capillary electrophoresis expertise that will be used in an upcoming publication of the work |
Impact | No outputs |
Start Year | 2018 |
Title | TRIPHOSPHORYLATION REACTION |
Description | The disclosure provides a method of producing a triphosphate compound. The method comprises (a) dissolving imidazole in an aqueous alkaline solution to make an imidazole solution; (b) dissolving phosphoryl halide in an organic solvent to make an organic solution; (c) contacting the imidazole solution and the organic solution to produce a solution comprising phosphorylated imidazole (PIm); (d) removing the organic solvent from the solution comprising PIm; and (e) contacting the PIm with a mutant of nucleoside diphosphate kinase (NDPK) and a diphosphate compound, and thereby producing a triphosphate compound. |
IP Reference | WO2020008209 |
Protection | Patent application published |
Year Protection Granted | 2020 |
Licensed | No |
Impact | Interaction with potential industrial users to identify key additional works that must be performed to explore financial viability of the approach. These findings were gained through Pathfinder funding, which also supported a successful Follow-on Funding bid. This additional funding will seek to make the system more robust and translate to a solution for industrial biocatalysts. |
Description | Conference presentation at ICPC 2018, Budapest |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | I gave a 25 minute keynote lecture. This led to discussions with other audience members and the development of potential collaborations |
Year(s) Of Engagement Activity | 2018 |
URL | http://www.icpc22.mke.org.hu/keynote-lectures.html |
Description | Invited Departmental Seminar, Department of Chemistry, Helsinki |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | I delievered a research lecture containing material related to several projects. This may lead to the development of new collaborations. |
Year(s) Of Engagement Activity | 2018 |
Description | Invited Departmental Seminar, University of Turku, Finland |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | I delivered a lecture to colleagues in University of Turku Finland. The audience included a colleague involved in other BBSRC-funded research. Future collaborations are developing with other colleagues. |
Year(s) Of Engagement Activity | 2018 |
Description | Invited lecture, Department of Chemistry, Texas Christian University, USA |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | I delivered a lecture. Future collaboration may result. |
Year(s) Of Engagement Activity | 2019 |
Description | Level 4 undergraduate lecture |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Undergraduate students |
Results and Impact | I incorporated the NDPK story into an undergraduate lecture focused on bio organic chemistry of phosphorus. |
Year(s) Of Engagement Activity | 2019 |
Description | Presentation at EuCHEMS Liverpool |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | I delivered a lecture. Several questions arose and also reinforced developing collaboration in this area. |
Year(s) Of Engagement Activity | 2018 |
URL | https://www.euchems2018.org |