Two solvents (Dioxane, DMAc), Strong Base (NaOt-Bu) Step-by-Step Guide for Buchwald-Hartwig Amination Reaction Screening Kit

1 base (NaOt-Bu, 2 M in THF)
2 solvents (1,4 Dioxane and DMAc)
12 pre-weighed catalysts

Materials required - set up:

• 1 mylar bag from the KitAlysis™ Buchwald-Hartwig Amination Reaction Screening Kit and you will use the following components
  - 12 x 2 pre-weighed catalysts in glass vials loaded with stir bars and topped with cap mat
  - 2 empty 4 mL reaction vials
• 1 NEW KitAlysis™ 24-Well Reaction Block Replacement Film
• -1 (2 mL) ampule each of 1,4-Dioxane and DMAc (in ampule boxes)
• 2 NEW stir bars
• KitAlysis™ 24-Well Reaction Block (sold separately)
• KitAlysis™ Benchtop Inertion Box (sold separately) or glove box/glove bag
• KitAlysis™ Torque Screwdriver Set (sold separately)

Additional (not included) items needed:

• Pipette (0-100 µL) and tips
• 2 (1 mL) syringes with long needles
• Aryl halide and amine
• Nitrogen (or Argon): from hood line or tank
• 1 hot plate with stir capabilities
• 1 stir plate (or hot plate to be used without heat)
• HPLC vials, 96-well HPLC auto sampler block, or TLC plates.

Solutions & slurries to make: Go to the online user set-up page to enter molecular weights into the downloadable excel file.

Set Up Procedure:
(See KitAlysis™ video for useful visuals)

• Preheat a hot plate to 100 °C. Use an oil bath or second reaction block to hold temperature and avoid spiking.
• If base sensitive substrate, reduce temperature to between 60 °C and 80 °C.
• Place a NEW KitAlysis 24-Well Reaction Block Replacement Film on reaction block lid and verify all holes, including the temperature probe hole, line up with the corresponding holes on the film
• Check all screws to ensure they are not stripped. Replace any stripped screws with provided replacements.
• Place the KitAlysis™ Benchtop Inertion Box with tubing connected to inert gas onto the second, non-heated stir plate (see KitAlysis Benchtop Inertion Box set-up for details)
• Place KitAlysis™ 24-Well Reaction Block with lid into KitAlysis Benchtop Inertion Box. Start nitrogen flow and purge 5 minutes. Leave nitrogen flowing for remainder of set up.
• Make substrate mixtures directly into the empty 4 mL reaction vials according to recipe (provided in the downloadable excel file) omitting solvent and NaOt-Bu solution. Add one stir bar to each vial mixture. Label as “Dioxane Substrate Mixture A” and “DMAc Substrate Mixture B”). If amine is volatile, add it last (after solvent mixtures have been added) directly to requisite reaction vials via syringe from a separate vial that has been quickly purged and then capped before placing into Inertion Box.
• Partially open the lid on the KitAlysis™ Benchtop Inertion Box. Place the “Dioxane Substrate Mixture A” in the center hole on the left hand side of the Inertion Box diffuser tray. Place the “DMAc Substrate Mixture B” in the center hole on the right hand side of the plate. This vial placement allows for the best flow of inert gas (remove lids from the solid mixtures before placing them into the recommended holes, keeping the lids in the Inertion Box for later use if needed).
• Transfer the capped, 24-vial, preloaded catalysts into the reaction block making sure to load it according to the matching diagram on the packaging and the Reaction Block. Leave the mat on.
• Using an ampule cracker, open 1 ampule each of Dioxane, DMAc, and NaOt-Bu solution and quickly place into ampule holes located along the bottom of Inertion Box, below the Reaction Block.
• Once all components are in the KitAlysis™ Benchtop Inertion Box, close the lid and purge for an additional 5 minutes. Leave nitrogen flowing for remainder of set up.
• Purge needle and syringe in a nitrogen diffuser hole two times by pulling and then pushing plunger. Using purged needle and syringe, add required solvent amounts to open substrate mixture vials. DO NOT add NaOt-Bu solution to either substrate mixture yet-it is added separately last.
• Stir mixtures until in solution (1-2 min). For slurries, see “additional tips” below.
• While mixtures are stirring, carefully remove the cap mat from the 24-vial, preloaded catalysts in the reaction block.
• Dose 100 µL of Dioxane solution mixture to vials A1-A6 and vials B1-B6 according to scheme below. You may have a very small amount of excess solution remaining. Save it as a reaction standard for HPLC/TLC later.
• Dose 100 µL DMAc solution mixtures to vials C1-C6 and vials D1-D6 according to scheme below.

• After solvents mixtures are added, dose 15 µL of the NaOt-Bu solution to each of the 24 vials.
• After all substrate mixtures and base have been dosed according to recipe, take the KitAlysis 24-Well Reaction Block lid and line up the screws with the holes in the plate. Ensure the temperature probe holes line up on both the lid and the block.
• Screw on lid according to directions and pattern shown in the “additional tips” section below. Before removing KitAlysis™ 24-Well Reaction Block from the Inertion Box, ensure that the lid is evenly sealed onto the base. Do this visual check to avoid having to unscrew the lid and screw again.
• Once completely sealed, remove the KitAlysis™ 24-Well Reaction Block from the Inertion Box. Place on preheated hot plate with probe through the lid and inserted into the block. Heat at 100 °C overnight stirring at or near 300 rpm. If substrate is sensitive to base, lower the temperature to between 60 °C and 80 °C. Turn off nitrogen flow to box and dispose of any unused chemicals.
• Make the quench solution in a bottle with a replaceable lid following the recipe below for HPLC analysis. TLC is also possible.

Quench Solution Recipe

-49 mL CH₃CN

-1 mL AcOH

-15.4 mg Biphenyl (KitAlysis™ Internal Standard provided in the kit)

Note: This recipe makes 50 mL which is enough stock solution for all four screening set in the KitAlysis™ Buchwald-Hartwig Amination Reaction Screening Kit

• Follow the below Work-up Procedure

Work-up Procedure and Analysis

• Cool Reaction Block. Remove lid using small, non-torque KitAlysis screwdriver.
• Check each vial for solvent loss, and record.
• Aliquot 500 µL of prepared quench solution to each vial.
• Replace lid, tighten middle screw and stir on stir plate (NO HEAT) for 2-3 minutes. DO NOT INVERT BLOCK.
• After 5 minutes of stirring, let plate rest (without stirring) for 5 minutes to allow insoluble material to settle out of solution.
• While plate is resting, add 700 µL acetonitrile to each 24 individually labeled (A1, A2 etc,) HPLC vial or to each of 24 wells of a 96-well HPLC/UPLC auto sampler block.
• Remove lid on KitAlysis™ 24-Well Reaction Block carefully.
• Using a clean pipette each time, remove a 25 µL aliquot from each vial into corresponding HPLC vials or HPLC block . Be careful to pull material from the top of the vials to avoid any precipitate.
• Run on HPLC auto sampler. May need to optimize ratio of sample to dilution factor for your unique system.

Additional Tips:

Sealing the Plate-screwing down the cover

Sealing the plate properly is critical to success. The key is light even pressure to the lid of the block to keep the cover flat while sealing.

1. Line-up screws making sure that the base and lid temperature probe holes line-up in the KitAlysis™ 24-Well Reaction Block.
2. Initially flush: Using your thumb and forefinger, press the lid of the box until it becomes flush with the vials. Then, using the KitAlysis™ Torque Screwdriver, insert screws until flush, but not tight, with the top of the box, following the cross pattern provided below. Check to see that the lid is evenly sealed onto the base on all sides. Do this visual check to avoid having to unscrew the lid and screw again.
3. Tighten: Repeat the same pattern until the KitAlysis™ Torque Screwdriver “clicks” indicating complete tightness. Go around the block once more for a final check to ensure all screws are tight.

Probe for hot plate does not fit into reaction block hole

Use a small oil bath or other metal block (for best results) in the back of the hot plate and place the probe in there. Place reaction block as close to the center of the hot plate as possible for more even stirring.

Slurry Additions

Due to narrow opening of pipette tips, slurry additions will result in blockages. To aid in uniform dosing, snip off the end of the tip at the first marker (~10 mm). To ensure an evenly dispersed aliquot, it is critical that the mixture is stirring while you draw aliquots for dosing into the corresponding reaction vial.

Materials