Computational planning of the synthesis of complex natural products.

Training algorithms to computationally plan multistep organic syntheses has been a challenge for more than 50 years1-7. However, the field has progressed greatly since the development of early programs such as LHASA1,7, for which reaction choices at each step were made by human operators. Multiple software platforms6,8-14 are now capable of completely autonomous planning. But these programs 'think' only one step at a time and have so far been limited to relatively simple targets, the syntheses of which could arguably be designed by human chemists within minutes, without the help of a computer. Furthermore, no algorithm has yet been able to design plausible routes to complex natural products, for which much more far-sighted, multistep planning is necessary15,16 and closely related literature precedents cannot be relied on. Here we demonstrate that such computational synthesis planning is possible, provided that the program's knowledge of organic chemistry and data-based artificial intelligence routines are augmented with causal relationships17,18, allowing it to 'strategize' over multiple synthetic steps. Using a Turing-like test administered to synthesis experts, we show that the routes designed by such a program are largely indistinguishable from those designed by humans. We also successfully validated three computer-designed syntheses of natural products in the laboratory. Taken together, these results indicate that expert-level automated synthetic planning is feasible, pending continued improvements to the reaction knowledge base and further code optimization.

Strengthening Security for Gene Synthesis: Recommendations for Governance.

Since the inception of gene synthesis technologies, there have been concerns about possible misuse. Using gene synthesis, pathogens-particularly small viruses-may be assembled "from scratch" in the laboratory, evading the regulatory regimes many nations have in place to control unauthorized access to dangerous pathogens. Progress has been made to reduce these risks. In 2010, the US Department of Health and Human Services (HHS) published guidance for commercial gene synthesis providers that included sequence screening of the orders and customer screening. The industry-led International Gene Synthesis Consortium (IGSC) was formed in 2009 to share sequence and customer screening methods, and it now includes the major international gene synthesis providers among its members. Since the 2010 HHS Guidance was released, however, there have been changes in gene synthesis technologies and market conditions that have reduced the efficacy of these biosecurity protections, leading to questions about whether the 2010 HHS Guidance should be updated, what changes could make it more effective, and what other international governance efforts could be undertaken to reduce the risks of misuse of gene synthesis products. This article describes these conditions and recommends actions that governments should take to reduce these risks and engage other nations involved in gene synthesis research.

Automated production of [18 F]FTHA according to GMP.

14-(R,S)-[18 F]fluoro-6-thia-heptadecanoic acid is a tracer for fatty acid imaging by positron emission tomography. High demand for this tracer required us to replace semiautomatic synthesis with a fully automated procedure. An automated synthesis device was constructed in-house for multistep nucleophilic 18 F-fluorination and a control system was developed. The synthesis device was combined with a sterile filtration unit and both were qualified. 14-(R,S)-[18 F]fluoro-6-thia-heptadecanoic acid was produced according to good manufacturing practice guidelines set by the European Union. The synthesis includes an initial nucleophilic labelling reaction, deprotection, preparative HPLC separation, purification of the final product, and formulation for injection. The duration and temperature of the reaction and hydrolysis were optimized, and the radiochemical stability of the formulated product was determined. The rotary evaporator used to evaporate the solvent after HPLC purification was replaced with solid phase extraction purification. We also replaced the human serum albumin used in the earlier procedure with a phosphate buffer-ascorbic acid mixture in the final formulation solution. From 2011 to 2016, we performed 219 synthesis procedures, 94% of which were successful. The radiochemical yield of 14-(R,S)-[18 F]fluoro-6-thia-heptadecanoic acid, decay-corrected to the end of bombardment, was 13% ± 6.3%. The total amount of formulated end product was 1.7 ± 0.8 GBq at end of synthesis.

Highly efficient solid phase supported radiosynthesis of [11 C]PiB using tC18 cartridge as a "3-in-1" production entity.

Pittsburgh compound B ([11 C]PiB) is the gold standard positron emission tomography (PET) tracer for the in vivo imaging of amyloid plaques. Currently, it is synthesized by either solution chemistry or using a "dry loop" approach followed by HPLC purification within 30 minutes starting from [11 C]CO2 . Here, we report a novel, highly efficient solid phase supported carbon-11 radiolabeling procedure using commercially available disposable tC18 cartridge as a "3-in-1" entity: reactor, purifier, and solvent replacement system. [11 C]PiB is synthesized by passing gaseous [11 C]CH3 OTf through a tC18 cartridge preloaded with a solution of precursor. Successive elution with aqueous ethanol solutions allows for nearly quantitative separation of the reaction mixture to provide chemically and radiochemically pure PET tracer. [11 C]PiB suitable for human injection is produced within 10 minutes starting from [11 C]CH3 OTf (20 min from [11 C]CO2 ) in 22% isolated yield not corrected for decay and molar activity of 190 GBq/µmol using 0.2 mg of precursor. This technique reduces the amount of precursor and other supplies, avoids use of preparative HPLC and toxic solvents, and decreases the time between consecutive production batches. Solid phase supported technique can facilitate [11 C]PiB production compliant with Good Manufacturing Practice (GMP) and improve synthesis reliability.

A systematic comparison of error correction enzymes by next-generation sequencing.

Gene synthesis, the process of assembling gene-length fragments from shorter groups of oligonucleotides (oligos), is becoming an increasingly important tool in molecular and synthetic biology. The length, quality and cost of gene synthesis are limited by errors produced during oligo synthesis and subsequent assembly. Enzymatic error correction methods are cost-effective means to ameliorate errors in gene synthesis. Previous analyses of these methods relied on cloning and Sanger sequencing to evaluate their efficiencies, limiting quantitative assessment. Here, we develop a method to quantify errors in synthetic DNA by next-generation sequencing. We analyzed errors in model gene assemblies and systematically compared six different error correction enzymes across 11 conditions. We find that ErrASE and T7 Endonuclease I are the most effective at decreasing average error rates (up to 5.8-fold relative to the input), whereas MutS is the best for increasing the number of perfect assemblies (up to 25.2-fold). We are able to quantify differential specificities such as ErrASE preferentially corrects C/G transversions whereas T7 Endonuclease I preferentially corrects A/T transversions. More generally, this experimental and computational pipeline is a fast, scalable and extensible way to analyze errors in gene assemblies, to profile error correction methods, and to benchmark DNA synthesis methods.

Rapid and accurate synthesis of TALE genes from synthetic oligonucleotides.

Custom synthesis of transcription activator-like effector (TALE) genes has relied upon plasmid libraries of pre-fabricated TALE-repeat monomers or oligomers. Here we describe a novel synthesis method that directly incorporates annealed synthetic oligonucleotides into the TALE-repeat units. Our approach utilizes iterative sets of oligonucleotides and a translational frame check strategy to ensure the high efficiency and accuracy of TALE-gene synthesis. TALE arrays of more than 20 repeats can be constructed, and the majority of the synthesized constructs have perfect sequences. In addition, this novel oligonucleotide-based method can readily accommodate design changes to the TALE repeats. We demonstrated an increased gene targeting efficiency against a genomic site containing a potentially methylated cytosine by incorporating non-conventional repeat variable di-residue (RVD) sequences.

Assembly-line synthesis of organic molecules with tailored shapes.

Molecular 'assembly lines', in which organic molecules undergo iterative processes such as chain elongation and functional group manipulation, are found in many natural systems, including polyketide biosynthesis. Here we report the creation of such an assembly line using the iterative, reagent-controlled homologation of a boronic ester. This process relies on the reactivity of α-lithioethyl tri-isopropylbenzoate, which inserts into carbon-boron bonds with exceptionally high fidelity and stereocontrol; each chain-extension step generates a new boronic ester, which is immediately ready for further homologation. We used this method to generate organic molecules that contain ten contiguous, stereochemically defined methyl groups. Several stereoisomers were synthesized and shown to adopt different shapes-helical or linear-depending on the stereochemistry of the methyl groups. This work should facilitate the rational design of molecules with predictable shapes, which could have an impact in areas of molecular sciences in which bespoke molecules are required.

Long-term operation of biomass-to-liquid systems coupled to gasification and Fischer-Tropsch processes for biofuel production.

Long-term operation of the biomass-to-liquid (BTL) process was conducted with a focus on the production of bio-syngas that satisfies the purity standards for the Fischer-Tropsch (FT) process. The integrated BTL system consisted of a bubbling fluidized bed (BFB) gasifier (20 kW(th)), gas cleaning unit, syngas compression unit, acid gas removing unit, and an FT reactor. Since the raw syngas from the gasifier contains different types of contaminants, such as particulates, condensable tars, and acid gases, which can cause various mechanical problems or deactivate the FT catalyst, the syngas was purified by passing through cyclones, a gravitational dust collector, a two-stage wet scrubber (packing-type), and a methanol absorption tower. The integrated system was operated for 500 h over several runs, and stable operating conditions for each component were achieved. The cleaned syngas contained no sulfur compounds (under 1 ppmV) and satisfied the requirements for the FT process.

The central role of chemistry in 'quality by design' approaches to drug development.

The quality of medicines reaching the consumer is strictly controlled and maintained by the regulatory agencies of the world. Pharmaceutical companies have to meet and maintain these regulatory quality standards. For this purpose, an increasing number of processes are incorporating quality by design (QbD) principles. Implementation of QbD involves chemistry in several ways, such as in the development of new synthetic and analytical methods, avoiding formation of genotoxic impurities and designing drug-like compounds to improve the quality of biological profile of medicines. A combined effort from regulatory authorities, pharmaceutical industries and academic research groups could also facilitate QbD implementation.

Effective use of a new quality and safety checklist for the steady and safe supply of fluorine-18 fluorodeoxyglucose for positron emission tomography/computed tomography.

Fluorine-18-fluorodeoxyglucose ((18)F-FDG ) positron emission tomography/computed tomography (PET/CT) with the in-hospital synthesis of (18)F-FDG was initiated in our hospital on April 1, 2010. We aim to perform stable supply of (18)F-FDG for patients and to avoid unnecessary radiation exposure due to mis-preparation of (18)F-FDG. Pharmacists perform quality control tests to determine whether (18)F-FDG meets official regulations. After the quality control test, we give (18)F-FDG that conforms to these standards to patients to conduct (18)F-FDG PET/CT. After a quality control test is initiated, various problems can occur including leakage and staff radiation exposure. We recorded daily radiation exposure in the hot lab and calculated the average daily radiation exposure on a monthly basis for a period of one year. We developed a checklist to safely and quickly synthesize(18)F-FDG for patients. The total radiation exposure of the three pharmacists was 394, 180, and 214µSv/y and overall lower than the occupational maximum values (≤50mSv/year and ≤100mSv/5years for males). In conclusion, using the new checklist, pharmacists and the operator of the Sumitomo Heavy Industries Accelerator service Co., Ltd. were able to practice their daily work effectively during the synthesis and quality control testing of (18)F-FDG. Notably the usual radiation exposure reported in the present study was quite lower than the allowable maximum.

Description of high purity and high specific activity of [11C]Choline synthesis using TRACERlab FXc module, and detailed report of quality controls.

We proposed a method of synthesis to produce [11C]Choline using TRACERlab FXc module that utilized gas phase iodination. The product had radiochemical purity of 99.79 ± 0.14 % and specific activity of 45.7 ± 7.59 GBq/µmol. [11C]Choline did not have at the moment a specific monograph in European Pharmacopeia therefore we used, when possible, as quality controls reference the monograph of [18F]FDG and we proposed suitable methods to verify radiochemical purity and to quantify residual DMAE and choline amounts.

Automated GMP synthesis of [(18)F]ICMT-11 for in vivo imaging of caspase-3 activity.

INTRODUCTION: Isatin-5-sulfonamide ([(18)F]ICMT-11) is a sub-nanomolar inhibitor of caspase-3 previously evaluated as an apoptosis imaging agent. Herein, an alternative radiosynthesis of [(18)F]ICMT-11 with increased purity and specific activity is presented. Finally, a GMP-applicable automated radiosynthesis of [(18)F]ICMT-11 is described. METHODS: The preparation of [(18)F]ICMT-11 was evaluated under a variety of reaction conditions, including reaction solvent, by employing alternative phase transfer catalysts and under different deprotection conditions. Following initial investigations, the process was transferred onto a fully automated GE FASTlab synthesis platform for further development and optimisation. RESULTS: The synthesis of [(18)F]ICMT-11 was successfully validated under GMP conditions, resulting in a yield of 4.6 ± 0.4 GBq with a radiochemical purity of >98% at EOS and a specific activity of 685 ± 237 GBq/µmol within 90 min. Quality control was carried out in accordance with the European Pharmacopoeia and demonstrated that [(18)F]ICMT-11 can be consistently manufactured on the FASTlab to meet specifications. CONCLUSIONS: A simplified methodology for the synthesis of the apoptosis imaging agent, [(18)F]ICMT-11, has been achieved by the S(N)2 displacement of a tosylate leaving group with [(18)F]fluoride ion. This results in an increased purity and specific activity over the original copper catalysed "Click" synthetic stratagem reaction involving 2-[(18)F]fluoroethylazide with an alkyne precursor and is now suitable for routine clinical application.

Synthesis of ApoSense compound [18F]2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid ([18F]NST732) by nucleophilic ring opening of an aziridine precursor.

INTRODUCTION: The small molecule 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid (NST732) is a member of the ApoSense family of compounds, capable of selective targeting, binding and accumulation within cells undergoing apoptotic cell death. It has application in molecular imaging and blood clotting particularly for monitoring antiapoptotic drug treatments. We are investigating a fluorine-18-radiolabeled analog of this compound for positron emission tomography studies. METHODS: We prepared the tosylate precursor methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(tosyloxymethyl)butanoate (4) to synthesize fluorine-18-labeled NST732. Fluorination reaction of the tosylate precursor in 1:1 acetonitrile:dimethylsulfoxide with tetrabutyl ammonium fluoride proceeds through an aziridine intermediate (4A) to afford two regioisomers: 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-fluorobutanoate (5) and methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoate (6). Acid hydrolysis of the fluoromethylbutanoate (6) isomer produced NST732. As the fluorination reaction of the tosylate precursor proceeds through an aziridine intermediate (4A) and the fluorination conceivably could be done directly on the aziridine, we have separately prepared an aziridine precursor (4A). Fluorine-18 labeling of the aziridine precursor (4A) was performed with [(18)F]tetrabutyl ammonium fluoride to afford the same two regioisomers (5 and 6). The [18F]2-((5-dimethylamino)naphthalene-1-sulfonamido)methyl)-2-fluorobutanoic acid (NST732) was then obtained by the hydrolysis of corresponding [18F]-labeled ester (6) with 6 N hydrochloric acid. RESULTS: Two regioisomers obtained from the fluorination reaction of aziridine were easily separated by high-performance liquid chromatography. The total radiochemical yield was 15%±3% (uncorrected, n=18) from the aziridine precursor in a 70-min synthesis time with a radiochemical purity>99%. CONCLUSION: Fluorine-18-labeled ApoSense compound [18F]NST732 is prepared in moderate yield by direct fluorination of an aziridine precursor.

[Towards the estimation of survival curves parameters and geroprotectors classification].

The following parameters are proposed to estimate in survival curves analysis: Median Life Span, Hill slope, Expected Maximal Life Span. Those estimates enable to classify geroprotectors as weak, moderate and strong.