Step-by-Step Guide for KitAlysis™ High-Throughput Base Reaction Screening Kit

Step-by-Step Guide for KitAlysis High-Throughput Base Reaction Screening Kit

7 pre-weighed bases
4 base solutions (DBU (neat), Et3N (neat), LiHMDS (1.0 M in THF), and NaOtBu (2.0 M in THF))
4 solvents (MeOH, Toluene, DMF, and THF)

Kit 7 Reaction Scheme

Materials Required - Set up:

  • 1 mylar bag from the KitAlysis™ High-Throughput Base Screening Kit and you will use the following components
    - 7 x 2 pre-weighed catalysts in glass vials loaded with stir bars and topped with cap mat
    - 4 empty 4 mL reaction vials
  • 1 NEW KitAlysis™24-Well Reaction Block Replacement Film
  • 1 (2 mL) ampule each of 2 of the following: MeOH, Toluene, DMF, THF (in ampule boxes)
  • 1 (2 mL) ampule each of DBU, Et3N, LiHMDS (1.0 M in THF), and NaOtBu (2.0 M in THF) (in ampule boxes and can (Et3N))
  • 4 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) & tips
  • 4 (1 mL) syringes with long needles
  • Chemist’s Choice base
  • Your substrates, ligands, catalysts
  • 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 desired temperature if heating required (use oil bath or second reaction block to hold temperature and avoid spiking).
  • Place a NEW KitAlysis™ 24-Well Reaction Block Replacement Film on reaction block lid (make sure 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.
  • Weigh desired substrates, ligands, catalysts directly into two empty 4 mL reaction vials according to recipe (provided in the downloadable excel file) omitting solvent. Add one stir bar to each vial mixture. Label as “Solvent 1 Substrate Mixture A” and “Solvent 2 Substrate Mixture B”).
  • Weigh Chemist’s Choice base (30 µmol) into remaining two empty 4 mL reaction vials.
  • Partially open the lid on the KitAlysis™ Benchtop Inertion Box. Place the “Solvent 1 Substrate Mixture A” in the center hole on the left hand side of the Inertion Box diffuser tray. Place the “Solvent 2 Substrate Mixture B” in the center hole on the right hand side of the plate. Place Chemist’s Choice base vials in open holes next to Reaction Block. 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. Note: B6 and D6 should be empty and can be used to check orientation.
  • Using an ampule cracker, open 1 ampule each of the 2 selected solvents and 4 base solutions and quickly place into the holes located along the bottom and side of the Inertion Box, below and next to 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 2x by pulling and then pushing plunger. Using purged needle and syringe, add required solvent amounts to open substrate mixture vials and Chemist’s Choice base vials.
  • 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 bases in the reaction block.
  • Using a pipette, dose 4 base solutions and Chemist’s Choice solution into appropriate vials according to scheme below.
  • Dose stock solutions
    a) Dose 100 µL of “Solvent 1 Substrate Mixture A” to vials A1-A6 and B1-B6 according to scheme below.
    b) Dose 100 µL of “Solvent 2 Substrate Mixture B” into vials C1-C6 and D1-D6 according to scheme below.

You may have a very small amount of excess solution remaining for each mixture.
Save it as a reaction standard for HPLC/TLC later.

KitAlysis™ 24-Well Reaction Block

Base Vials
K3PO4 A1,C1
K2CO3 A2,C2
KOAc A3,C3
KHCO3 A4,C4
KOH A5,C5
NaOH A6,C6
Cs2CO3 B1,D1
LiHMDS B2,D2
NaOtBu B3,D3
DBU B4,D4
Et3N B5,D5
Chemist's
Choice
B6,D6
  • 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 as you go along 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 (if necessary) with probe through the lid and inserted into the block. If necessary, heat at 60 °C for desired reaction time stirring at or near 300 rpm.
  • Make the quench solution in a bottle with a replaceable lid following the recipe below for HPLC analysis. TLC is also possible.
  • At reaction completion, follow the below Work-Up Procedure

Quench Solution Recipe

  • 24.5 mL CH3CN
  • 0.5 mL AcOH

Note: This recipe makes 25 mL which is enough stock solution for both screening sets in the KitAlysis™ High-Throughput Base Screening Kit.

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 2-3 minutes of stirring, let plate rest (without stirring) for 5 minutes to allow insoluble material to settle out of solution to the bottom of the vials.
  • While plate is resting, add 700 µL of acetonitrile to each 24 individually labeled (A1, A2 etc,) HPLC vials or to each of 24 wells of a 96-well HPLC/UPLC auto sampler block (see “additional recommended materials” below for suggestions on the auto sampler block and cap mat.
  • 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. You may need to adjust the amount of acetonitrile from the suggested 700 µL to accommodate your unique HPLC 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.

Sealing the Plate-screwing down the cover

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, simply 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 well while you draw aliquots for dosing into the corresponding reaction vial.

snip at ~ 10mm

Materials