www.sciencemag.org/cgi/content/full/science.aar6236/DC1

Supplementary Materials for

Mapping the dark space of chemical reactions with extended nanomole

synthesis and MALDI-TOF MS

Shishi Lin, Sergei Dikler, William D. Blincoe, Ronald D. Ferguson, Robert P. Sheridan,

Zhengwei Peng, Donald V. Conway, Kerstin Zawatzky, Heather Wang, Tim Cernak, Ian

W. Davies, Daniel A. DiRocco, Huaming Sheng,* Christopher J. Welch,* Spencer D.

Dreher*

*Corresponding author. Email: huaming.sheng@merck.com (H.S.); christopher@welchinnovation.com

(C.J.W.); spencer_dreher@merck.com (S.D.D.)

Published 24 May 2018 on Science First Release

DOI: 10.1126/science.aar6236

This PDF file includes:

Materials and Methods

Figures S1 to S55

Tables S1 to S8

References

Other Supplementary Material for this manuscript includes the following:

(available at www.sciencemag.org/cgi/content/full/science.aar6236/DC1)

Data S1 to S5

1.0 General information.

1.1 General materials and methods.

1.2 UPLC-MS analysis.

2.0 Description of extended nanomole-scale chemistry platform.

2.1 Reaction and source plates.

2.2 Robotics dosing.

2.2.1 Mosquito robotics dosing.

2.2.2. Thermo matrix Platemate robotics dosing.

2.3 Heterogeneous solid addition.

2.4 Aluminum plate design and compatibility.

2.4.1 COC plastic plate chemistry.

2.4.2 Glass plate chemistry.

2.4.3 Photoredox plastic plate chemistry.

2.5 Nanophotoredox plate heating and irradiation.

2.6 Standard reaction heating and agitation.

3.0 MALDI reaction assessment development.  

3.1 Instrumentation.

3.1.1 Testing sample preparation conditions using a Bruker Autoflex MALDI-TOF.  

3.1.2 Ultrahigh-throughput screening of 1536 format targets using a Bruker Rapiflex MALDI-TOF/TOF.  

3.2 Sample preparation.

3.2.1 Sandwich spotting method.  

3.2.2 Sample spot drying method. 

3.2.3 Automated target preparation for ultrahigh-throughput screening by MALDI-TOF.  

3.3 Results and discussion.

3.3.1 Sample spot drying selection.  

3.3.2 Matrix screening and selection. 4.0 Chemistry experimentation.

4.1 Nanomole-scale photoredox platform build.  

4.1.1 Chemistry set-up.  

4.1.2 Analytical.  

4.2 Glass plates platform build.  

4.2.1 Chemistry set-up.  

4.2.2 Analytical.  

4.3 Fragment poisons analysis.  

4.3.1 Chemistry fragments source plate preparation.

4.3.1.1 384-well DMSO source plate preparation.  

4.3.1.2 384-well dioxane source plate preparation.  

4.3.2 Ir/Ni catalyzed reactions (100 nmol scale, 1 uL volume).  

4.3.3 Ru/Ni catalyzed reactions (100 nmol scale, 1 uL volume).  

4.3.4 Cu/oxamate ligand catalyzed reactions (250 nmol scale, 2.5 uL volume).  

4.3.5 Pd/ RuPHOS catalyzed reactions (250 nmol scale, 2.5 uL volume).  

4.3.6 Analytical.

4.3.6.1 UPLC-MS analysis.  

4.3.6.2 MALDI-TOF MS analysis.  

4.3.7 Results/Discussion.

4.3.7.1 Value of internal standard normalization in MALDI-TOF MS crude reaction analysis.  

4.3.7.2 Binary MALDI-TOF MS thresholds in fragments analysis.  

4.3.7.3 Single functional fragment poisons analysis.  

4.3.7.4 Polyfunctional fragment poisons analysis.  

4.4 Whole molecules, simplest-partner evaluation.  

4.4.1 Whole molecules source plate preparation.  

4.4.1.1 384-well DMSO bromide/amine source plate preparation.  

4.4.1.2 384-well dioxane bromide/amine source plate preparation.  

4.4.2 Ir/Ni catalyzed reactions (100 nmol scale, 1 uL volume).  

4.4.3 Ru/Ni catalyzed reactions (100 nmol scale, 1 uL volume). 

4.4.4 Cu/oxamate ligand catalyzed reactions (250 nmol scale, 2.5 uL volume).  

4.4.5 Pd/RuPHOS catalyzed reactions (250 nmol scale, 2.5 uL volume).  

4.4.6 Analytical.

4.4.6.1 UPLC-MS nnalysis.  

4.4.6.2 MALDI-TOF MS analysis.  

4.4.7 Results/Discussion.

4.4.7.1 MALDI-TOF MS correlation with UPLC-MS analytical methods.  

4.4.7.2 MALDI-TOF MS whole-molecule reaction thresholding with no product standard using the simplest partner approach.  

4.4.7.3 Binary success/failure binning.  

4.4.7.4 Comparison of binary MALDI thresholding reactivity trends versus standard UPLC-MS metrics.  

4.4.7.5 Binary MALDI failure rates for whole molecules parameters that lead to failure for each synthetic method.  

4.4.7.6 Binary MALDI cumulative average failure rates for whole molecules parameters for all synthetic method.

4.5 Selected Scale-up Experiments Validating Whole Molecule Trend Analysis. 4.5.1 Chemistry Set-Up.

4.5.2 Scale-up reactions source-plate prep. 4.5.2.1 96-well DMSO amine source plate preparation. 4.5.2.2 96-well dioxane amine source plate preparation.

4.5.3 Cu/oxamate ligand catalyzed reactions (10 umol scale, 100 uL volume).

4.5.4 Pd/RuPHOS catalyzed reactions (10 umol scale, 100 uL volume).

4.5.5 UPLC-MS analysis.

4.5.6 Results/discussion. 4.6 Whole molecules randomized cross-over experiment.

4.6.1 Chemistry set-up. 4.6.2 384-well DMSO bromide/amine source plate preparation. 4.6.3 Dual Ru/Ni catalyzed reactions (100 nmol scale, 1 uL volume). 4.6.4 Cu/oxamate ligand catalyzed reactions (250 nmol scale, 2.5 uL volume). 4.6.5 UPLC-MS analysis. 4.6.6 Results/Discussion.

5.0 All fragment additive and whole molecule structures. Index of data in attached Excel document.

Data Tab S1 Fragment additive full data set. Data Tab S2 Whole molecule simplest partner full data set. Data Tab S3 Thresholded binary MALDI reactivity data. Data Tab S4 Full-factorial chemistry validation study data.

Data Tab S5 Scale-up examples .

1.0 General information. 1.1 General reagents and methods. All reagents were used as purchased from commercial suppliers. Solvents were purchased from Sigma Aldrich, anhydrous, sure-seal quality, and used with no further purification. All reactions were set up and sealed inside an MBraun glovebox operating with a constant N2-purge (oxygen typically <5 ppm). The experimental design was accomplished using Free-Slate Library Studio. Analytical chemicals and reagents: Dimethyl sulfoxide (99.9%, Fisher certified ACS), acetonitrile (Fisher Optima LC/MS Grade), water (Fisher Optima LC/MS Grade), and hydrochloric acid (Fisher certified ACS PLUS) were obtained from Fisher Scientific (Waltham, MA, USA). Trifluoroacetic acid (≥99.0%), 7,7,8,8-tetracyanoquinodimethane (98%), dithranol (≥98.0%), 3-aminoquinoline (98%), 4-hydroxy-3-methoxycinnamic acid (≥98.5%) 2,5-dihydroxybenzoic acid (≥99.5%), 2,4,6-trihydroxyacetophenone (≥99.5%), 3-hydroxypicolinic acid (≥99.0%), sinapic acid and α-cyano-4-hydroxycinnamic acid (≥99.0%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Informer compounds were synthesized as described in literature (32).

 

1.2 UPLC-MS analysis. Reactions were monitored using a Waters Acquity UPLC I-Class system (Waters Corp., Milford, MA, USA) equipped with a binary pump, a FTN sampler, column manager, a photodiode array detector, SQD detector 2 with electrospray ionization (ESI) source in the positive mode and MassLynx® software. Separations were performed on a Waters CORTECS UPLC C18+ column (30 × 2.1mm, 1.6µm). Conditions; mobile phase = 1:1 A:B where A = 90% ACN in H2O (2 mM HCOONH4) and B = H2O (2 mM HCOONH4). Gradient: 0-1.8 min 10-100% A, 1.8-2.0 min 100%A. Flow rate 0.7 mL/min; T = 40 ºC. Acetonitrile (HPLC grade), water (HPLC grade) were purchased from Fisher Scientific. Formic acid (HCOOH) and ammonium formate (HCOONH4) were purchased from Sigma–Aldrich. Solutions containing 2 mM ammonium formate (HCOONH4) in water (pH 3.5) and 2 mM ammonium formate in acetonitrile (pH 3.5): 12.6 g ammonium formate (HCOONH4) and 7.9 mL formic acid (HCOOH) were dissolved in 1 L of HPLC grade water. A 100-fold dilution of this stock solution was performed in either pure water or a 90:10 acetonitrile–water mixture to prepare the 2 mM solutions. High throughput data analysis to produce Excel spreadsheets was done with Virscidian Analytical StudioTM software. Conversion to product was analyzed by UPLC-UV/MS. An internal standard was used and product/IS ratio at 210 nm calculated. For samples where overlaps in the UV trace were detected, EIC traces were used instead. 2.0 Description of extended nanomole-scale chemistry platform. Nanomole-scale chemistry reactions, to this point, have been run using plastic plates with low-volatility solvents and homogeneous components at room temperature. These plates are compatible with polar aprotic solvents such as DMSO and NMP. In this section, the engineering advancements made to enable extension of nanomole-scale chemistry to a much wider assortment of chemistry methods are described.

2.1 Reaction and source plates. AdvantageTM 384-well plates (Analytical-Sales, Cat. No. 38120, polypropylene, 120 µL-wells, flat bottom, clear) were used as source plates for stock solutions and for analytical plates on UPLC-MS. Corning® 1536-well plates (Corning EchoTM qualified, Cat. No. 3730, Cyclic Olefin-Copolymer COC, 12.5 µL-wells, flat bottom, clear) and Invenios 1536-well glass plates were used as reaction plates. For product information on 1536-well glass plates, please contact Invenios (320 North Nopal Street, Santa Barbara, CA 93103-3225, USA, 1.805.962.3333). 2.2 Robotics dosing. The reactions in this work were dosed with either the TTP Labtech Mosquito® robot or the Thermo Matrix® 2x2 Platemate robot. 2.2.1 Mosquito robotics dosing. Dosing of reaction components into the analytical 384- and reaction 1536-well plates in reactions with low-volatility solvents was accomplished in the glovebox using a Mosquito® HTS (“Mosquito”) liquid handling robot (Figure S1, TTP Labtech, 4.5 mm pitch tip spool) with no special modifications, and using the TTP Labtech native software.

Figure S1. Left to right. (A) TTP Labtech Mosquito HTS liquid handling robot. (B) 16-tip dosing mechanism. 2.2.2. Thermo matrix Platemate robotics dosing. For all other experiments which involve volatile reagents or solvents, dosing of reaction components into the 384- and 1536-well plates was accomplished in the glovebox using a Matrix 2x2 Platemate (“Matrix”) liquid handling robot from Thermo Scientific with no special modifications, and using the ControlMate native software. This robot enables faster dosing with simultaneous 384-tip additions, and greater flexibility with multiple dosing modes (see Fig. S2). For all experiments in this work, 30 uL tips in disposable magazines from Thermo Scientific (Catalog # 5316) were used.

Figure S2. Versatile dosing modes of the Thermo Matrix Platemate liquid handling robot with 384-tip dosing. 2.3 Heterogeneous solid addition. The heterogeneous inorganic bases Cs2CO3 and K3PO4 used in this study were made up as 0.5 M slurries in tert-amyl alcohol. When this mixture is irradiated on a Resodyn acoustic mixing LabRam (PharmaRAM II Mixer) at room temperature for 12 h at 18G in the presence of an approximately equal weight of Norstone YTZ Grinding Media (0.5 mm), the resulting slurries do not significantly settle within 15 minutes on standing. These slurries can be dosed evenly using the Mosquito or Thermo Matrix liquid handling robots and subsequently used for reactions upon evaporation of the solvent (Fig. S3).

Figure S3. Left to right. (A) Settled inorganic base with grinding beads before agitation. (B) Inorganic base as a slurry after agitation (grinding beads are settled at the bottom). (C) Dried Cs2CO3 dosed onto a flat calibration block to demonstrate uniformity of spots. (D) Inorganic base slurry dosed into 384 wells of a 1536-well plate.

16-tip protocol 24-tip protocol

384-tip protocol

2.4 Aluminum plate design and compatibility. Heating plastic and glass 1536-well plates required designing a new high-quality sealing mechanism. The aluminum blocks described below were custom-made for this task and are now commercially available from Analytical Sales and Services. Contact Analytical Sales for additional aluminum block product information for all blocks described below (Analytical Sales and Services, Inc., 179 US Rt 206, Flanders, NJ 07836 USA. 1.973.616.0700). 2.4.1 COC plastic plate chemistry. In this work, the Cu catalysis chemistry experiments (see experiments 4.3.4 and 4.4.4 below) were run heated at 100 °C with the plastic COC plate, using the aluminum sealing block shown in Figure S4, below. These blocks provide a high quality seal that prevents solvent loss.

Figure S4. Left to right, top then bottom. (A) Aluminum top for nanomole-scale chemistry, (B) Aluminum top underside with silicon rubber mat and PFA sheet, (C) COC 1536-well plate (D) Side view of sealed nanomole-scale chemistry plate set-up, (E) Standard aluminum bottom, also used for glass plates. 2.4.2 Glass plate chemistry. We developed commercially-available Invenios glass 1536-well plates for use in nanomole-scale chemistry that can tolerate a much broader range of organic reagents and solvents. This approach was demonstrated in the Pd catalysis conditions used in this work (dioxane solvent at 80 °C, see experiments 4.3.5 and 4.4.5, below). These plates, in conjunction with rapid 384-tip Matrix liquid handling dosing and the effective sealing provided by the aluminum blocks enables use of volatile reaction components (solvents and reagents). These glass plates were used with the standard nanomole-scale chemistry set-up as shown above. The silicon rubber mat and PFA sheet provide a good sealing mechanism when the set-up is tightened as shown in Figure S5. The quality of the seal is illustrated by the indentations seen on the PFA sheet when the aluminum top is removed after the stipulated reaction time.

Figure S5. Left to right, top then bottom. (A) Aluminum top with silicon rubber mat and PFA sheet. (B) Invenios 1536-well plate glass plate. (C) Side view of sealed glass plate set-up. (D) Indentations on the PFA sheet after reaction, demonstrating effective sealing mechanism. 2.4.3 Photoredox plastic plate chemistry. In order to extend the scope and compatibility to encompass a wider range of organic transformations, we developed a platform for nanomole-scale photoredox chemistry. The nanomole-scale photoredox chemistry reaction plate, used in the Ir/Ni and Ru/Ni photoredox reactions (see experiments 4.3.2, 4.3.3, 4.4.2 and 4.4.3 below) is shown in Figure S6. The acrylic bottom (9 mm thick) enables light penetration, while providing a solid bottom for the COC 1536-well plate to rest. The set-up provides a good seal to minimize solvent loss. 2.5 Nanophotoredox plate heating and irradiation. The sealed nanophotoredox plate is then placed inside a vacuum oven (Fisher Scientific, Isotemp vacuum oven, Model 281A) lined with reflective aluminum foil on the interior for maximal light exposure. The plate sits on a borosilicate crystalizing dish to suspend it from the bottom of the oven. This approach eliminates edge effects since light is reflected all around instead of focusing directly on the bottom of the plate. The set-up is run under N2 purge with a slight vacuum. A H150W tuna blue Kessil lamp (P/N: H150-blue, S/N: L4C3DG0006, 24 VDC, 1.5A, 34W) placed outside the vacuum oven was used to illuminate the reaction and the temperature of the reactions in this work was maintained at 55 ºC, as shown in Figure S7.

Figure S6. Left to right, top then bottom. (A) Aluminum top for nanomole-scale chemistry, (B) Aluminum plate bottom with acrylic plate for photoredox chemistry, (C) Bottom view of photoredox plate with 1536-well plate, (D) Side view of sealed nanomole-scale chemistry plate set-up.

Figure S7. Left to right. (A) Interior set-up of vacuum oven. (B) Irradiation with Kessil lamp. 2.6 Standard reaction heating and agitation. The set-up for heating and mixing heterogeneous reactions is as shown below. This system consists of two custom-made heating plates: the top heating plate is set 10 °C above the desired temperature while the bottom heating plate is set 10 °C below. The resulting temperature of the reaction is the average of the two temperatures. Because the top is kept warmer than the bottom, volatile reaction components are contained at the bottom of the plate during the reaction process. For reactions run outside the glovebox, the 1536-well nanoplate is wrapped in 2 oxygen impermeable Mylar bags in the glovebox to maintain an inert atmosphere. Even though the aluminum plate sealing mechanism is effective in

keeping solvents and reagents within the plate, we found that air exchange through the Invenios glass plates is still possible across the set-up. The use of Mylar bags effectively overcomes this issue of inertion. Alternatively, the LabRam can be located within the glovebox and no Mylar bags are necessary. The spacer enables even heat transfer while the silicon rubber mats provide a cushioning effect throughout the agitation process. The reactions are typically run at 5G on the LabRam with the heating mechanism secured with the LabRam vice clamp as shown in Figure S8. Upon reaction completion, the bottom plate is cooled rapidly to room temperature by sitting on a bed of dry ice, while the top remains hot. This is done to push any potential condensation of solvent back down to the bottom of the plate before opening to quench.

Figure S8. LabRam mixing set-up with custom heating elements.  

3.0 MALDI reaction assessment development. This section describes the development effort towards an effective MALDI-TOF method for routine chemistry reaction profiling. The following sample preparation parameters were analyzed to investigate their impact on the MALDI signal-to-noise (S/N) ratio of the peak of interest: analyte and matrix plate spotting techniques, matrix selection, and sample spot drying process.

Figure S9. Development of an automated uHT MALDI-TOF MS workflow for the profiling of nanomole-scale chemistry reactions. 3.1 Instrumentation. 3.1.1 Testing sample preparation conditions using a Bruker Autoflex MALDI-TOF. A Bruker Autoflex III MALDI-TOF mass spectrometer equipped with a Smartbeam laser operating at 200 Hz was used for initial testing of sample preparation conditions. The laser was set to acquire 1000 laser shots per spot. Positive ion mode was used for all the experiments. Peak intensity was recorded for all the peaks of interest. Each S/N ratio value below was the average of three repeats. The sample preparation conditions were tested using MTP 384 ground steel BC target obtained from Bruker. 3.1.2 Ultrahigh-throughput screening of 1536 format targets using a Bruker Rapiflex MALDI-TOF/TOF. Ultrahigh-throughput screening of two 1536 format MALDI targets (fragment additives and whole molecules) was performed using a Bruker Rapiflex MALDI PharmaPulse (MPP) MALDI-TOF/TOF system equipped with a scanning Smartbeam 3D laser operating at 10 kHz. The instrument was operated in the reflector positive ion mode in the mass range 100-1500 Da. The 10-bit digitizer was set to a sampling rate of 1.25 GS/s. The fastest readout speed of 8 min 22s (averaged from 3 measurements) for a 1536 format MALDI target was achieved acquiring 200 laser shots per spectrum. Most of the measurements were done with 1000 laser shots per spectrum requiring 10-11 min to measure a target in 1536 format. All automated runs were set up, triggered and processed using MALDI PharmaPulse 2.0 software (Bruker Daltonics; Billerica, MA, USA). In the whole molecule analysis experiment a custom flexAnalysis processing script was used to simultaneously extract nearly 400 masses, peak intensities and peak areas of reaction products and the internal standard into a single spreadsheet in addition to standard MPP 2.0 processing. S/N 5 was used as a threshold for labeling and extracting signal data. See experimental sections 4.3 and 4.4 and the respective Excel Data Tab S1 and Tab S2 for more details on plate preparation and analysis of results.

3.2 Sample preparation. 3.2.1 Sandwich spotting method. A 1 µL aliquot of a 1 mg/mL solution of analyte in DMSO was spotted on the MALDI plate and solvent was evaporated. Next a 1 µL aliquot of a 4 mg/mL solution of the matrix in 0.1% TFA in 50/50 (v/v) ACN/H2O was spotted on top of the evaporated analyte solution. The matrix solvent was evaporated then sample was analyzed on the Autoflex MALDI.

3.2.2 Sample spot drying method. Due to the high boiling point of DMSO, a few drying strategies were used to allow for rapid evaporation of solvent in preparation for MALDI analysis. A 1 µL aliquot of DMSO was spotted onto the MALDI plate then dried in one of three ways:

1. Air dried at ambient temperature and pressure. 2. Placing the plate in a vacuum chamber and lowering pressure with an in-house vacuum to

0.2 bar. 3. Placing the plate in a glove box vacuum chamber and lowering pressure with a vacuum

pump to 0.01 bar.

For the glove box experimentation, a MBraun Glove Box Workstation equipped with a Labconco 195 LPM rotary vane pump was used. For both vacuum methods, the pressure was ramped down to the target pressure over the course of 1 minute. This was to avoid solvent movement on the plate due to a sudden pressure change. 3.2.3 Automated target preparation for ultrahigh-throughput screening by MALDI-TOF. All reaction mixtures were spotted using a Mosquito HTS liquid handling robot on Bruker HTS MALDI targets with barcodes (1.0 mm thickness) in 1536 format [HTS MALDI plate 1.0 mm, BC, Part# 1833280]. The targets were mounted on Bruker HTS MALDI adapters (Part#1847571). 175 nL of reaction mixture was deposited and allowed to dry followed by depositing 150 nL of a 4 mg/mL solution of α-cyano-4-hydroxycinnamic acid in 0.1% TFA, 50% acetonitrile/H2O.

Figure S10. HTS MALDI target showing 1536 spots from nanomole-scale chemistry reactions. 3.3 Results and discussion

3.3.1 Sample spot drying selection. Three methods of drying the 1 µL of analyte in DMSO were used as described above. Air drying at ambient temperature gave a drying time of 8 h. The

in-house vacuum decreased drying time to 30 minutes and the glove box decreased drying time further to 2 minutes. See Table S1 for a summary of the information. Table S1: 1 µL DMSO drying methods and drying times

Drying Method Drying Time Air dry at ambient temperature and pressure 8 h

Place under in-house vacuum at 0.2 bar 30 min Place in glove box vacuum at 0.01 bar 2 min

The in-house vacuum method was chosen for all further experimentation since 30 minutes drying time was adequate for the method development performed in this present study. The rapid drying time achieved under the glove box vacuum demonstrated the possibility of even faster sample preparation if desired. 3.3.2 Matrix screening and selection. Nine different matrices were screened for compatibility with six informer compounds (X2, X3, X6, X8, X13 and X15) (32) with structures shown in Figure S11. Informer compounds were prepared in 1 mg/mL solutions in DMSO. These compounds were prepared for MALDI analysis as described above using the “sandwich” method. The results were recorded as shown in Table S2. Boxes marked in green corresponds to S/N ratio of the protonated X compounds greater than 200, boxes in yellow are 30-200 S/N and boxes in red showed S/N <30. S1 α-cyano-4-hydroxycinnamic acid provided the best S/N ratio of protonated product ion for each informer compound tested compared to the other matrices. Moreover, S1 was the matrix that provided most reproducible crystallization pattern during spot drying process. Thus it was determined to use S1 α-cyano-4-hydroxycinnamic acid as the matrix for the ultra-high throughput reaction monitoring. Table S2: Matrix screening results. Boxes in green are S/N of protonated X compounds greater than 200, boxes in yellow are S/N between 30-200 and boxes in red are S/N smaller than 30.

Compound S1 S2 S3 S4 S5 S6 S7 S8 S9

X2 X3 X6 X8 X13 X15

CN

CN

NC

CN

S4: 7,7,8,8-tetracyanoquinodimethaneN

NH2

S5: 3-aminoquinoline

OH

HO OH

O

S6: 2,4,6-trihydroxyacetophenone

OH OHO

S7: Dithranol

O

O

HO

S8: 4-hydroxy-3-methoxycinnamic acid

NOH

O

OH

S9: 3-hydroxypicolinic acid

OH

O

OH

O

HO

O

S3: Sinapic Acid

O

OH

OH

HO

S2: 2,5-dihydroxybenzoic acid

HO

NC

O

OH

S1 -cyano-4-hydroxycinnamic acid

MALDI Matrices

Informer Compounds

N

N

N

O

O

Br

O

X2

SO

O

O

O

N

Br

ON

ClBr

N

O O

N

Br

OO

N O

OOO

N O

O

HN N

O

SBr

N

NI

S

NH

OO

O

[M+H]+ = 364.029X3

[M+H]+ = 438.001X6

[M+H]+ = 461.063

X8[M+H]+ = 503.081

X13[M+H]+ = 402.048

X15[M+H]+ = 616.113

Figure S11. Structures of matrix and informer compounds used in matrix evaluation. Informer compounds shown with standard numbers from original publication (32).  

4.0 Chemistry experimentation.

4.1 Nanomole-scale photoredox platform build. Validation of the 1536 nanomole-scale photoredox screening tool is described below. The goal was to demonstrate reasonable reactivity and minimal effects of light intensity on plate positions.

4.1.1 Chemistry set-up. A stock solution containing all of the reaction components was made as follows: 3-bromo-5-phenylpyridine S10 (1 equiv), 4-phenylpiperidine S11 (1.5 equiv), DABCO (1.8 equiv), NiCl2 glyme (0.05 equiv) and Ir(dF-CF3ppy2)(dtbbpy)(PF6) (0.0002 equiv) in 0.1 M DMSO. This stock solution was dispensed to a 384-well source plate, which was placed onto the Mosquito deck. The Mosquito robot was used to dose all combined reaction components in 1 uL into 384 wells of a 1536-well plate. Once the dosing was completed, the 1536-well plate was covered with a PFA film, sealed in the aluminum plate with acrylic bottom to allow light penetration and placed in a vacuum oven at 55 °C for 16 hours under N2 with a slight vacuum. The interior of the vacuum oven was fully lined with aluminum foil to provide a reflective surface. A 34W blue Kessil lamp placed outside the vacuum oven was used to illuminate the reaction. After reaction, the plate was quenched with 3 uL of a DMSO stock solution of acetic acid (1 vol%) and trimethoxybenzene (0.003 M) using the Mosquito. The Mosquito then sampled 1 uL from the quenched reaction plate into a 384-well plate containing 75 uL of DMSO per well. The 384-well plate was then heat-sealed and shaken. 4.1.2 Analytical. The quenched analysis plate was then subjected to chromatographic analysis by UPLC-MS. The ratio of the UV LC area counts of product over internal standard was used to determine quantitative assay yields of these reactions, showing 45% average yield of S12 with 3.59 relative standard deviation across the plate.  

4.2 Glass plates platform build. To validate the glass plates platform, one reaction was run 384 times using a RuPHOS/ Pd catalyzed Cs2CO3 N-arylation validation experiment (250 nmol, 2.5 uL volume).

4.2.1 Chemistry set-up. In a N2-purged glovebox, Cs2CO3 (637 mg, 1.96 mmol), grinding beads (~500 mg) and 5.4 mL of t-amyl alcohol were added into an 8 mL capped vial. This mixture was irradiated on a Resodyn LabRam (18 G) at room temperature for 12 h. The resulting slurry did

not significantly settle within 15 minutes of standing. This slurry was distributed to 12 x 1 mL vials in a row with a 1 mL single-tip pipettor and then 40 uL was added to each well of a 384-well source plate with a 12-channel pipettor. In the glovebox, a Thermo-Matrix liquid handling robot with 384 dosing tips was used to dose 1.5 uL of Cs2CO3 slurry to 384 positions of an Invenios glass 1536 well-plate. This plate then sat for ~30 minutes to evaporate the solvent, thus plating 0.75 umol Cs2CO3, 3 equiv) into each well. Then 3-bromo-5-phenylpyridine S10 (585 mg, 2.5 mmol, 1 eq) and RuPHOS Pd G2 precatalyst (194 mg, 0.25 mmol, 10 mol%) were added to a 40 mL vial, followed by dioxane (25 mL) and finally piperidine S13 (319 mg, 370 uL, 3.75 mmol, 1.5 equivalents). This stock solution was similarly distributed to 12 x 1 mL vials, then dosed into a 384 well plate (40 uL per well) with a 12-channel pipettor. Using the Matrix Robot with 384-tips, 1.25 uL of dioxane was aspirated from a 384-well plate, then 1.25 uL of the above-mentioned stock solution was aspirated into the same tip, and then this was dosed into the 1536 well-plate containing Cs2CO3. The plate was sealed in an aluminum sealing plate within 30 seconds of completion of dosing, wrapped successively in 2 Mylar bags in the glovebox, and then irradiated on the LabRam outside the glovebox at 5G for 16 h. The entire process for source plate prep, Matrix dosing and plate sealing can be completed in <5 minutes, minimizing loss of volatile components. The bottom plate was heated to 70 °C, while the top plate was heated to 90 °C, giving an internal temperature of 80 °C for the reaction plate. After reaction, the plate was removed from the LabRam and the bottom plate cooled with dry ice to room temperature while the top plate was still hot. The plate was then taken into the glovebox, and the Matrix robot was used to quench the reaction and prepare analytical plates. Into four 384-well analytical/quench plate was added 75 uL of DMSO solution per well which contained 1,3,5-trimethoxy benzene (0.264 mM) and acetic acid (1 vol%). The Matrix robot added 5 uL of this quench to the reaction plate, then removed 8 uL from the reaction plate and added this back to the analytical plate. Then 5 uL from the analytical plate was added back to the reaction plate and this was mixed 3 times, then added back to the analytical plate. 4.2.2 Analytical. The analytical plate was then run on UPLC-MS using a 2-minute method to analyze reaction consistency across the plate. All reactions went to 100% conversion with an average yield of 73% of S14 across all 384 samples. 4.3 Fragment poisons analysis. The fragments poisons analysis used 383 Fragments (+1 control) x 4 chemistry conditions (described below) in the N-arylation coupling. 142 simple, single-functional fragments and 241 poly-functional fragments were utilized (for identities of fragments, see figures S40 to S47 in SI section 5.0, and Excel Data Tab S1).

Note: Parts of this workflow involve the use of volatile reagents/solvents in extremely small volumes. The extended nanomole-scale chemistry approach also requires robotics training on the 1536-glass plates in order to align the 384-tips to the desired well positions. As such, speed and accuracy are crucial in the reaction set-up process. Taking this into account, two chemists worked together to facilitate the entire process. 4.3.1 Chemistry fragments source plate preparation. Into two sets of 383 x 8-mL vials was weighed 0.12 mmol of each fragment. 4.3.1.1 384-well DMSO source plate preparation. Using a TECAN liquid handling robot, 0.6 mL of DMSO was added to each vial with a septa-piercing needle. These vials were then shaken vigorously for 2 hours. Samples that were not immediately soluble were heated to 60 °C with shaking for 1h. Nafion grinding beads were added to the remaining insoluble samples and put on the LabRam at 10 G for several hours. Samples that resulted in persistent slurries were used as is. The remaining insoluble samples were further diluted to 2.4 mL (0.05 M), put on the LabRam for another 2 hours, were dosed into the 1536-well plates and the solvent was evaporated. The TECAN robot was then used to dose 40 uL of solution from each 8 mL vial into a dry 384-well plate in a custom-built inertion chamber containing the 384-well plate. DMSO was used in place of the samples for the compounds which were pre-plated. The plate was used directly as described in the experiments below. 4.3.1.2 384-well dioxane source plate preparation. Inside the glovebox, using a Rainin 6-channel 1 mL spreadable multi-addition pipettor, 0.6 mL of dioxane was added to each fragment vial. These vials were then sealed and shaken vigorously for 2 hours. Samples that were not immediately soluble were heated to 60 °C with shaking for 1h. Nafion grinding beads were added to the remaining insoluble samples and put on the LabRam at 10 G for several hours. Samples that resulted in persistent slurries were used as is. The remaining insoluble samples were further diluted to 2.4 mL (0.05 M), put on the LabRam for another 2 hours, were dosed into

Four Catalytic Methods: Cu: DiF oxamate ligand (50%), CuI (25%), K

3PO

4, DMSO, 100 °C, 20h

Pd: RuPhos G2 Precatalyst (10%), Dioxane, Cs2CO

3, 80 °C, 20h

Ir/Ni (photoredox): Ir(dFCF3-ppy

2)(dtbbpy)(PF

6) (0.05%), NiCl

2 glyme (5%), DABCO,

DMSO, 55 °C, blue light, 20h Ru/Ni (photoredox): Ru(bpy)

3(PF

6)

2 (0.1%), NiCl

2 glyme (5%), DABCO, DMSO, blue light,

55 °C, 20h

the 1536-well plates and the solvent was evaporated. In the glovebox, the 383 vials were transferred to 4 x glass 96-well plates with a spreadable 6-channel pipettor. The 4 plates were then transferred to a single 384-well plate using a Rainin Liquidator 96-channel pipettor. The plate was used directly as described in the experiments below. 4.3.2 Ir/Ni catalyzed reactions (100 nmol scale, 1 uL volume). In the glovebox, a stock solution containing each of the reaction components was made as follows: Ir(dFCF3-ppy2(dtbbpy)(PF6) (0.0001 M in DMSO, 0.0005 equiv), NiCl2 glyme (0.01 M in DMSO, 0.05 equiv), DABCO (0.6 M in DMSO, 3 equiv) and 3-bromo-5-phenylpyridine S10 (0.2 M in DMSO, 1 equiv). Piperidine S13 (1 equiv) was added last into the above stock solution as a neat liquid. This solution was distributed to 12 x 1 mL vials, then dosed into a 384 well plate (40 uL per well) with a 12-channel pipettor. The DMSO fragment source plate described above and this reagents source plate were placed on the Matrix robot deck. Using a 384-tip dosing head the Matrix robot aspirated 0.5 uL of fragments followed by 0.5 uL of reagent mixture and this was dosed into a plastic-COC 1536-well plate. This was then sealed in the aluminum reactor block with acrylic bottom, put into the oven reactor at 55 °C, then irradiated with a blue Kessil Lamp for 16h. The reaction plate was then cooled to ambient temperature, and quenched with the general protocol. In a 384-well analytical quench plate was added 75 uL of DMSO solution/ well which contained 1,3,5-trimethoxy benzene (0.264 mM) and acetic acid (1 vol%). The Matrix robot added 5 uL of this quench to the reaction plate, then removed 8 uL from the reaction plate and added this back to the analytical plate. Then 5 uL from the analytical plate was added back to the reaction plate and this was mixed 3 times, then added back to the analytical plate.

4.3.3 Ru/Ni catalyzed reactions (100 nmol scale, 1 uL volume). In the glovebox, a stock solution containing each of the reaction components was made as follows: Ru(bpy)3(PF6)2 (0.001 M in DMSO, 0.005 equiv), NiCl2 glyme (0.01 M in DMSO, 0.05 equiv), DABCO (0.6 M in DMSO, 3 equiv) and 3-bromo-5-phenylpyridine S10 (0.2 M in DMSO, 1 equiv). Piperidine S13 (1 equiv) was added last into the above stock solution as a neat liquid. This solution was distributed to 12 x 1 mL vials, then dosed into a 384 well plate (40 uL per well) with a 12-channel pipettor. The DMSO fragment source plate described above and this reagents source plate were placed on the Matrix robot deck. Using a 384-tip dosing head the Matrix robot aspirated 0.5 uL of fragments followed by 0.5 uL of reagent mixture and this was dosed into a plastic-COC 1536-well plate. This was then sealed in the aluminum reactor block with acrylic bottom, put into the oven reactor at 55 °C, then irradiated with a blue Kessil Lamp for 16h. The reaction plate was then cooled to ambient temperature, and quenched with the general protocol. In a 384-well analytical quench plate was added 75 uL of DMSO solution/ well which contained 1,3,5-trimethoxy benzene (0.264 mM) and acetic acid (1 vol%). The Matrix robot added 5 uL of this quench to the reaction plate, then removed 8 uL from the reaction plate and added this back to the analytical plate. Then 5 uL from the analytical plate was added back to the reaction plate and this was mixed 3 times, then added back to the analytical plate.

4.3.4 Cu/oxamate ligand catalyzed reactions (250 nmol scale, 2.5 uL volume). In a glovebox, 0.5 M K3PO4 slurry solution in t-amyl alcohol was prepared as previously described and plated in the first quadrant of a 1536-well COC plate (see Section 2.3) with the Matrix robot. A stock solution containing each of the reaction components was made as follows: CuI (0.05 M in DMSO, 0.25 equiv) and 3-bromo-5-phenylpyridine S10 (0.2 M in DMSO, 1 equiv). Piperidine

S13 (1 equiv) was added into the above stock solution as a neat liquid, followed by oxamate ligand S15 (0.5 equiv) as a solid. This solution was distributed to 12 x 1 mL vials, then dosed into a 384 well plate (40 uL per well) with a 12-channel pipettor. The DMSO fragment source plate described above and this reagents source plate were placed on the Matrix robot deck. Using a 384-tip dosing head the Matrix robot aspirated 1.25 uL of fragments followed by 1.25 uL of reagent mixture and this was dosed into the 1536-well plate containing K3PO4. The plate was sealed in an aluminum sealing plate within 30 seconds of completion of dosing, wrapped successively in 2 Mylar bags in the glovebox, and then irradiated on the LabRam outside the glovebox at 5G for 16 h. The bottom plate was heated to 90 °C, while the top plate was heated to 110 °C, giving an internal temperature of 100 °C for the reaction plate. After reaction, the plate was removed from the LabRam and the bottom plate was cooled with dry ice to room temperature while the top plate was still hot. This was then taken in the glovebox, and the Matrix robot was used to quench the reaction and to prepare an analytical plate. In a 384-well quench plate, was added 75 uL of DMSO solution/well which contained 1,3,5-trimethoxy benzene (0.663 mM) and acetic acid (2.5 vol%). The Matrix robot added 5 uL of this quench to the reaction plate, then removed 8 uL from the reaction plate and added this back to the quench plate. Then 5 uL from the quench plate was added back to the reaction plate and this was mixed 3 times, then added back to the quench plate. From this plate, the matrix robot aspirated 30 uL of this reaction mixture and added this to 45 uL DMSO in a 384-well analytical plate.

4.3.5 Pd/ RuPHOS catalyzed reactions (250 nmol scale, 2.5 uL volume). In a glovebox, 0.5 M Cs2CO3 slurry solution in t-amyl alcohol was prepared and plated in the first quadrant of a 1536-well Invenios glass plate (see Section 2.3) with the Matrix robot. Stock solutions of each of the reaction components were made as follows: RuPhos Pd G2 (0.02 M in dioxane, 0.1 equiv) and 3-bromo-5-phenylpyridine S10 (0.2 M in dioxane, 1 equiv). Piperidine S13 (1 equiv) was added last into the above stock solution as a neat liquid. This was distributed to 12 x 1 mL vials, then to a 384 well reagents source plate (20 uL/ well) with a 12-channel pipettor. The dioxane fragment source plate described above and this reagents source plate were placed on the Matrix robot deck. Using a 384-tip dosing head the Matrix robot aspirated 1.25 uL of fragments followed by 1.25 uL of reagent mixture and this was dosed into the above glass plate containing the plated Cs2CO3. The plate was sealed in an aluminum sealing plate within 30 seconds of completion of dosing, and wrapped successively in 2 Mylar bags in the glovebox. The entire process for source plate prep, Matrix dosing and plate sealing can be completed in <5 minutes, minimizing loss of volatile components. The wrapped plates were then irradiated on the LabRam outside the glovebox at 5G for 16 h. The bottom plate was heated to 70 °C, while the top plate was heated to 90 °C, giving an internal temperature of 80 °C for the reaction plate. After reaction, the plate was then removed from the LabRam and the bottom plate was cooled with dry ice to room temperature (while the top plate was still hot). This was then taken in the glovebox, and the Matrix robot was used to quench the reaction and to prepare an analytical plate. In a 384-well quench plate, was added 75 uL of DMSO solution/well which contained 1,3,5-trimethoxy

benzene (0.663 mM) and acetic acid (2.5 vol%). The Matrix robot added 5 uL of this quench to the reaction plate, then removed 8 uL from the reaction plate and added this back to the quench plate. Then 5 uL from the quench plate was added back to the reaction plate and this was mixed 3 times, then added back to the quench plate. From this plate, the matrix robot aspirated 30 uL of this reaction mixture and added this to 45 uL DMSO in a 384-well analytical plate. 4.3.6 Analytical. 4.3.6.1 UPLC-MS analysis. The 1536 reactions (383 additives and one control run with 4 sets of conditions) were analyzed using a 2 minute UPLC-MS analysis and the samples were manually curated carefully using Virscidian Analytical StudioTM (cutting overlapping peak shoulders, re-assigning misassigned peaks, etc.). Product area counts at UV wavelength 210 nm and product extracted ion counts (EIC) for the desired product peak were extracted from Analytical Studio to Excel. 4.3.6.2 MALDI-TOF MS analysis. The four 384-well analytical plates prepared in Experiment 4.3.2, 4.3.3, 4.3.4 and 4.3.5 above were placed onto the Mosquito deck, together with the MALDI target plate. 0.05 umol of the deuterated product standard S16 (structure below) was added to each well. The Mosquito dosed 175 nL of the reaction mixture, combining four 384 plates into one 1536 MALDI plate. This was allowed to dry for 1 hr. Following that, 150 nL of a 4 mg/mL solution of α-cyano-4-hydroxycinnamic acid S1 in 0.1% TFA, 50% acetonitrile was spotted using the Mosquito over the dried reaction spots. The plate was dried in the glove box and analyzed. The MALDI data is reported as normalized MALDI product response (signal intensity of S14/signal intensity of internal standard S16.

The combined fragment analysis data containing fragments structures, control reactivity, UPLC-MS data and MALDI data is shown in the attached Excel Data Tab S1. 4.3.7 Results/Discussion. 4.3.7.1 Value of internal standard normalization in MALDI-TOF MS crude reaction analysis. In Figure S12A/C, both the UPLC-MS EIC (extracted ion count) and UV210 nm product, both show poor correlation with the raw MALDI product signal (R2=0.24 and 0.16, respectively). This is likely due to spotting variability and inhibition of MALDI-MS signal by reaction components and fragment additives. The value of normalizing the MALDI signal by dividing the product MALDI by the internal standard MALDI response across all four synthetic methods can be seen very clearly in Figure S12 B/D. The correlation for normalized MALDI data with EIC and UV210 are now R2=0.85 and R2=0.73 respectively. When closely inspecting the UPLC-MS data, in some cases the UV210 nm product peaks overlapped with fragment

additives, or other reaction reagents or side products in the chromatogram. Manual integration of these peaks resulted in UV peaks that were sometimes larger and sometimes smaller than the likely accurate value. For this reaction set, the EIC-product data was found to be much more reliable as a quantitative measure of reaction performance.

Figure S12. (A,C) Comparative scatter plot with UPLC-MS EIC-product and UPLC-UV210 product responses versus MALDI-MS product responses for 1536 fragment-additive reactions shows low correlation (R2=0.24 and R2=0.16, respectively). (B,D) The same UPLC-MS EIC-product and UPLC-UV210 product responses compared against internal standard normalized MALDI data show much better correlation (R2=0.85 and 0.73, respectively). In Figure S13, the normalized MALDI data for each method is ranked from highest to lowest. The corresponding EIC responses are shown below, for comparison, demonstrating how the effects of different chemistry conditions on MALDI-MS can be overcome with a normalizing internal standard. This study demonstrates that ultrahigh-throughput MALDI-TOF MS analysis

can provide routine reaction profiling that gives similar results to LC-MS and LC-UV-based analysis but 400 times faster.

Figure S13. The ranked, normalized MALDI responses for each of the 4 synthetic methods, and the corresponding UPLC-MS EIC-product responses, demonstrating that each method and the diverse fragment additives are effectively normalized with the internal standard. 4.3.7.2 Binary MALDI-TOF MS thresholds in fragments analysis. In order to create useful definition of reaction poisoning, we established the control reaction (no additives) performance in terms of UV210, EIC and normalized MALDI product response for each of the four synthetic protocols (Table S3). These values were then divided by 2 to represent 50% knockdown of the reaction and then the 383 fragment additive reactions were binned as higher or lower than these values. Using the control reaction to set the maximum value normalizes chemistry performance of reaction conditions and allows for focusing on the fragments’ impact instead of the inherent reactivity of the system (which was relatively similar for all 4 conditions). Binary binning of the normalized MALDI signal correlates strongly with the EIC signal (95%) and UV 210 nm signal

(88%) across all 4 reaction sets. The quality of binning for each of the 4 methods is also shown in Figures S14 to S17. Table S3. Use of binary MALDI threshold to bin fragment poisons.

Figure S14. Cu EIC vs MALDI. Horizontal and vertical lines are 50% knockdown of control reactivity for product EIC counts and MALDI, respectively. Control reaction is circled.

MALDI 

control

EIC‐area 

counts 

control

1/2 MALDI 

signal

1/2 EIC 

signal

True 

Positive

True 

Negative

False 

Positive

False 

Negative

% 

correlation

Cu 1.17 2910887 0.59 1455444 333 43 3 4 98

Ir 1.49 3107043 0.75 1553522 139 219 8 17 93

Pd 1.47 3084328 0.74 1542164 163 197 6 17 94

Ru 1.55 3435580 0.78 1717790 168 188 1 26 93

803 647 18 64 95

MALDI 

control

UV210‐area 

counts 

control

1/2 MALDI 

signal

1/2 UV210 

signal

True 

Positive

True 

Negative

False 

Positive

False 

Negative

% 

correlation

Cu 1.17 62680 0.59 31340 315 46 22 0 94

Ir 1.49 90077 0.75 45039 85 231 63 4 83

Pd 1.47 73611 0.74 36806 135 207 39 2 89

Ru 1.55 107217 0.78 53609 123 205 46 9 86

658 689 170 15 88

Figure S15. Ir EIC vs MALDI. Horizontal and vertical lines are 50% knockdown of control reactivity for product EIC counts and MALDI, respectively. Control reaction is circled.

Figure S16. Pd EIC vs MALDI. Horizontal and vertical lines are 50% knockdown of control reactivity for product EIC counts and MALDI, respectively. Control reaction is circled.

Figure S17. Ru EIC vs MALDI. Horizontal and vertical lines are 50% knockdown of control reactivity for product EIC counts and MALDI, respectively. Control reaction is circled. A summary of the binary pass/fail analysis in terms of the overall failure rates, failure for single functional fragments and for polyfunctional fragments for all four synthetic methods is shown in Figure S18.

Figure S18. The effects of 383 fragment additives on C-N coupling reaction performance using MALDI-TOF MS analysis are depicted for the 4 examined synthetic methods. In (A) the failure rate of each method across all 383 additives is shown. In (B), the failure rate for the 142 single functional fragments is shown. In (C) the failure rate for 241 polyfunctional fragments is shown.

4.3.7.3 Single functional fragment poisons analysis. Diving into this data, the 142 single-functionality fragment additives were binned according to similar functional groups. Figures S19 and S20 show the percentage of failure for each of the binned functionality by synthetic method. Those highlighted in red are functional groups where >50% of the binned fragments show >50% MALDI signal knockdown. Repeated failure across structural variants helps to substantiate the reactivity trends. Once identified, these problematic functional groups can be subjected to more detailed study using uHT experimentation coupled with atomic and quantum molecular physical descriptors to enable structure-based predictive machine-learning.

Figure S19. Single functional group fragments for Cu and Ir catalytic methods. Highlighted in red are poisons, where >50% of examples resulted in >50% signal knockdown. MR= membered-ring, pri=primary, sec=secondary, open=open-chained, het=heterocycle, 1 N= 1 nitrogen.

Figure S20. Single functional group fragments for Pd and Ru catalytic methods. Highlighted in red are poisons, where >50% of examples resulted in >50% signal knockdown. MR= membered-ring, pri=primary, sec=secondary, open=open-chained, het=heterocycle, 1 N= 1 nitrogen. 4.3.7.4 Polyfunctional fragment poisons analysis. Next, we used SpotfireTM to systematically highlight which of the 241 poly-functional fragment additives contain or do not contain the poisons identified above. Table S4 shows that across the 4 synthetic conditions, for the 251 examples where poisons are present in a complex fragment [i.e. a Pd reaction with a fragment additive with both a carboxylic acid (a poison) and an alcohol (a non-poison)], poisoning results nearly 90% of the time. More importantly, of the 707 poly-functional fragments that do not contain individual poisons, almost half (~46%) still results in reaction poisoning, indicating cooperative poisoning in the more complex fragments. These polyfunctional fragment trends are depicted in Figure S21.

Table S4. Overall polyfunctional fragment additives effects. Displayed are polyfunctional fragments which do contain or do not contain single functional poisons and whether these act as poisons.

Figure S21. Polyfunctional fragment additives effects for each synthetic method. A) % Knockdown with polyfunctional fragments that do contain individual poisons. B) % Knockdown with polyfunctional fragments that do not contain individual poisons. Spots below the threshold line represent >50% knock-down from the control reaction and are considered poisons.

Polyfunctional 

fragments with  

poisons

Hits Misses % fail

Polyfunctional 

fragments with  no 

poisons

Hits Misses % fail

Cu 6 0 6 100.0 238 208 30 12.6

Ir 101 10 91 90.1 137 49 88 64.2

Pd 80 9 71 88.8 158 71 87 55.1

Ru 64 12 52 81.3 174 56 118 67.8

Total 251 31 220 87.6 707 384 323 45.7

Some representative structures of polyfunctional fragment additives showing poisonous and non-poisonous interactions for Pd are presented in Figures S22 and S23. Subtle differences in structures or functional groups can lead to poisoning effects. Full polyfunctional reactivity for all 4 catalytic methods is found in Excel Data Tab S1.

 Figure S22. Pd polyfunctional fragment poisons composed from non-poisons.

NHN

O

O

NNS

OH

N

HO

N

O

N

O

O O

N

HNN

NHN

NHN

NN

HO

HN

O

O

OH

N

OHOH

NH

O

O

HN

O

O N

OO

NH

O O

HN

ON+O

O-

N

OHN

O

HN

NH

N

O

O

NHOH

O

OHN

O

ONH

O

O

O

ON

O

O

N

OH

O

NO

N OH

HN

N

S

OH

NHO

N

HN

O

O

NH

HN

HN

OS

NNN

ON

HN

O

N

N

ON

NN

O

O

N

ON

NO

O

HN

N

O

O

O

S

N

N

N

OH

N

O

N

OO

HO

ON

N

OH

NS

OHNH

O

O

O

NH

NHO

F

N

OHN

O

O

Heterocycles + sec-anilines

Other Heterocycles

Pd non-poisonouspolyfunctional fragments

Heterocycles with esters/ ketones/ amides/ alkenes

Heterocycles with alcohols/ amines

Combinations of alcohols, amines, alkenes, amides, ketones, esters, nitriles, sulfonamides, nitro

 Figure S23. Pd polyfunctional fragment non-poisons composed from non-poisonous single functionality.

4.4 Whole molecules, simplest-partner evaluation. 192 N-heterocycle-containing aryl bromides and 192 aliphatic, cyclic secondary amines were evaluated with simple coupling partners (S10 and S11) against the four previously described synthetic conditions.

The heteroaryl-containing bromides and aliphatic, cyclic secondary amines were chosen over a range of molecular weights and were selected to cover diverse bulk properties and functional groups. In general, the following classes of compounds were omitted in the selection process: known more reactive functionalities that could lead to isomeric products (such as primary amines or primary anilines), highly hindered compounds, compounds in salt forms, known strong poisons (for example thiols) or highly reactive electrophiles (such as acyl halides or epoxides), and more reactive iodides and poly-bromides. Note: Parts of this workflow involve the use of volatile reagents/solvents in extremely small volumes. The extended nanomole-scale chemistry approach also requires robotics training on the 1536-glass plates in order to align the 384-tips to the desired well positions. As such, speed and accuracy are crucial in the reaction set-up process. Taking this into account, two chemists worked together to facilitate the entire process. 4.4.1 Whole molecules source plate preparation. Into two sets of 384 x 8-mL vials was weighed 0.12 mmol of each of 192 aryl bromides and 192 secondary amines. Note: About 10% of amine and bromide structures have IP-considerations, and for these, the entire structures cannot be revealed. We have noted these in blue in Figures S48 to S55 and also Excel Data Tab S2 and have drawn the local environment around the amine or bromide and have added R-groups to hide the exact peripheral structures. The structural parameters used for calculations of trends are tabulated in Excel Data Tab S2. 4.4.1.1 384-well DMSO bromide/amine source plate preparation. Using a TECAN liquid handling robot, 0.6 mL of DMSO was added to each vial with a septa-piercing needle. These vials were then shaken vigorously for 2 hours. Samples that were not immediately soluble were heated to 60 °C with shaking for 1h. Grinding beads were added to the remaining insoluble samples and put on the LabRam at 10 G for several hours. Samples that resulted in persistent slurries were used as is. The remaining insoluble samples were further diluted to 2.4 mL (0.05 M), put on the LabRam for another 2 hours, were dosed into the 1536-well plates and the solvent was evaporated. The TECAN robot was then used to dose 40 uL of solution from each 8 mL vial into a dry 384-well plate in a custom-built inertion chamber containing the 384-well plate. The plate was used directly as described in the experiments below. 4.4.1.2 384-well dioxane bromide/amine source plate preparation. Inside the glovebox, using a Rainin 6-channel spreadable multi-addition pipettor, 0.6 mL of dioxane was added to each bromide/amine vial. These vials were then shaken vigorously for 2 hours. Samples that were not immediately soluble were heated to 60 °C with shaking for 1h. Nafion grinding beads were

Four Catalytic Methods: Cu: DiF oxamate ligand (50%), CuI (25%), K

3PO

4, DMSO, 100 °C, 20h

Pd: RuPhos G2 Precatalyst (10%), Dioxane, Cs2CO

3, 80 °C, 20h

Ir/Ni (photoredox): Ir(dFCF3-ppy

2)(dtbbpy)(PF

6) (0.05%), NiCl

2 glyme (5%), DABCO,

DMSO, 55 °C, blue light, 20h Ru/Ni (photoredox): Ru(bpy)

3(PF

6)

2 (0.1%), NiCl

2 glyme (5%), DABCO, DMSO, 55 °C,

blue light, 20h

added to the remaining insoluble samples and put on the LabRam at 10 G for several hours. Samples that resulted in persistent slurries were used as is. The remaining insoluble samples were further diluted to 2.4 mL (0.05 M), put on the LabRam for another 2 hours, were dosed into the 1536-well plates and the solvent was evaporated. Then, in the glovebox, the 384 vials were transferred to 4 x glass 96-well plates with a spreadable 6-channel pipettor. The 4 plates were then transferred to a single 384-well plate using a Rainin Liquidator 96-channel pipettor. The plate was used directly as described in the experiments below. 4.4.2 Ir/Ni catalyzed reactions (100 nmol scale, 1 uL volume). In the glovebox, stock solutions containing of each of the reaction components was made as follows: Ir(dFCF3-ppy2)(dtbbpy)(PF6) (0.0001 M in DMSO, 0.0005 equiv), NiCl2 glyme (0.01 M in DMSO, 0.05 equiv), DABCO (0.6 M in DMSO, 3 equiv) and either 3-bromo-5-phenylpyridine S10 (0.2 M in DMSO, 1 equiv), or 4-phenylpiperidine S11 (0.2 M in DMSO, 1 equiv) (depending on whether the reaction was crossed with an amine or a bromide in the experiment). This was distributed to 12 x 1 mL vials, then to a 384 well reagents source plate (20 uL/ well) with a 12-channel pipettor. The DMSO aryl bromide/amines source plate described above and this reagents source plate were placed on the Matrix robot deck. Using a 384-tip dosing head the Matrix robot aspirated 0.5 uL of bromide/amine followed by 0.5 uL of reagent mixture and this was dosed into a plastic-COC 1536-well plate. This was then sealed in the aluminum reactor block with acrylic bottom, put into the oven reactor at 55 °C, then irradiated with a blue Kessil Lamp for 16h. The reaction plate was then cooled to ambient temperature, and quenched with the general protocol. In a 384-well analytical quench plate was added 75 uL of DMSO solution/ well which contained 1,3,5-trimethoxy benzene (0.264 mM) and acetic acid (1 vol%). The Matrix robot added 5 uL of this quench to the reaction plate, then removed 8 uL from the reaction plate and added this back to the analytical plate. Then 5 uL from the analytical plate was added back to the reaction plate and this was mixed 3 times, then added back to the analytical plate. 4.4.3 Ru/Ni catalyzed reactions (100 nmol scale, 1 uL volume). In the glovebox, stock solutions containing of each of the reaction components was made as follows: Ru(bpy)3(PF6)2 (0.001 M in DMSO, 0.005 equiv), NiCl2 glyme (0.01 M in DMSO, 0.05 equiv), DABCO (0.6 M in DMSO, 3 equiv) and either 3-bromo-5-phenylpyridine S10 (0.2 M in DMSO, 1 equiv), or 4-phenylpiperidine S11 (0.2 M in DMSO, 1 equiv) (depending on whether the reaction was crossed with an amine or a bromide in the experiment). This was distributed to 12 x 1 mL vials, then to a 384 well reagents source plate (20 uL/ well) with a 12-channel pipettor. The DMSO fragment source plate described above and this reagents source plate were placed on the Matrix robot deck. Using a 384-tip dosing head the Matrix robot aspirated 0.5 uL of bromide/amine followed by 0.5 uL of reagent mixture and this was dosed into a plastic-COC 1536-well plate. This was then sealed in the aluminum reactor block with acrylic bottom, put into the oven reactor at 55 °C, then irradiated with a blue Kessil Lamp for 16h. The reaction plate was then cooled to ambient temperature, and quenched with the general protocol. In a 384-well analytical quench plate was added 75 uL of DMSO solution/ well which contained 1,3,5-trimethoxy benzene (0.264 mM) and acetic acid (1 vol%). The Matrix robot added 5 uL of this quench to the reaction plate, then removed 8 uL from the reaction plate and added this back to the analytical plate. Then 5 uL from the analytical plate was added back to the reaction plate and this was mixed 3 times, then added back to the analytical plate.

4.4.4 Cu/oxamate ligand catalyzed reactions (250 nmol scale, 2.5 uL volume). In a glovebox, 0.5 M K3PO4 slurry solution in t-amyl alcohol was prepared and plated into the plastic 1536-well COC reaction plate (see Section 2.3). Stock solutions containing each of the reaction components were made as follows: CuI (0.05 M in DMSO, 0.25 equiv), oxamate ligand S15 (0.1 M in DMSO, 0.5 equiv) and either 3-bromo-5-phenylpyridine S10 (0.2 M in DMSO, 1 equiv), or 4-phenylpiperidine S11 (0.2 M in DMSO, 1 equiv) (depending on whether the reaction was crossed with an amine or a bromide in the experiment). This was distributed to 12 x 1 mL vials, then to a 384 well reagents source plate (20 uL/ well) with a 12-channel pipettor. The DMSO fragment source plate described above and this reagents source plate were placed on the Matrix robot deck. Using a 384-tip dosing head the Matrix robot aspirated 1.25 uL of bromide/amine followed by 1.25 uL of reagent mixture and this was dosed into the above 1536-well plate containing the plated K3PO4. The plate was sealed in an aluminum sealing plate within 30 seconds of completion of dosing, wrapped successively in 2 Mylar bags in the glovebox, and then irradiated on the LabRam outside the glovebox at 5G for 16 h. The bottom plate was heated to 90 °C, while the top plate was heated to 110 °C, giving an internal temperature of 100 °C for the nano plate. After reaction, the plate was then removed from the LabRam and the bottom plate was cooled with dry ice to room temperature while the top plate was still hot. This was then taken in the glovebox, and the Matrix robot was used to quench the reaction and to prepare an analytical plate. In a 384-well quench plate, was added 75 uL of DMSO solution/ well which contained 1,3,5-trimethoxy benzene (0.663 mM) and acetic acid (2.5 vol%). The Matrix robot added 5 uL of this quench to the reaction plate, then removed 8 uL from the reaction plate and added this back to the quench plate. Then 5 uL from the quench plate was added back to the reaction plate and this was mixed 3 times, then added back to the quench plate. From this plate, the matrix robot aspirated 30 uL of this reaction mixture and added this to 45 uL DMSO in a 384-well analytical plate.

4.4.5 Pd/RuPHOS catalyzed reactions (250 nmol scale, 2.5 uL volume). In a glovebox, 0.5 M Cs2CO3 slurry solution in t-amyl alcohol (see Section 2.3) was prepared and plated into the 1536-well Invenios glass plate. Stock solutions containing each of the reaction components were made as follows: RuPhos Pd G2 (0.02 M in dioxane, 0.1 equiv) and either 3-bromo-5-phenylpyridine S10 (0.2 M in dioxane, 1 equiv), or 4-phenylpiperidine S11 (0.2 M in dioxane, 1 equiv) (depending on whether the reaction was crossed with an amine or a bromide in the experiment). This was distributed to 12 x 1 mL vials, then to a 384 well reagents source plate (20 uL/ well) with a 12-channel pipettor. The dioxane fragment source plate described above and this reagents source plate were placed on the Matrix robot deck. Using a 384-tip dosing head the Matrix robot aspirated 1.25 uL of bromide/amine followed by 1.25 uL of reagent mixture and this was dosed into the above glass plate containing the plated Cs2CO3. The plate was sealed in an aluminum sealing plate within 30 seconds of completion of dosing, and wrapped successively in 2 Mylar bags in the glovebox. The entire process for source plate prep, Matrix dosing and plate sealing can be completed in <5 minutes, minimizing loss of volatile components. The wrapped plates were then irradiated on the LabRam outside the glovebox at 5G for 16 h. The bottom plate was heated to 70 °C, while the top plate was heated to 90 °C, giving an internal temperature of 80 °C for the reaction plate. After reaction, the plate was then removed from the LabRam and the bottom plate was cooled with dry ice to room temperature while the top plate was still hot. This was then taken in the glovebox, and the Matrix robot was used to quench the reaction and to prepare an analytical plate. In a 384-well quench plate, was added 75 uL of DMSO solution/ well

which contained 1,3,5-trimethoxy benzene (0.663 mM) and acetic acid (2.5 vol%). The Matrix robot added 5 uL of this quench to the reaction plate, then removed 8 uL from the reaction plate and added this back to the quench plate. Then 5 uL from the quench plate was added back to the reaction plate and this was mixed 3 times, then added back to the quench plate. From this plate, the matrix robot aspirated 30 uL of this reaction mixture and added this to 45 uL DMSO in a 384-well analytical plate. 4.4.6 Analytical. 4.4.6.1 UPLC-MS analysis. The 1536 reactions (192 amines x 1 simple bromide partner, 192 bromides x 1 simple amine partner, 4 methods) were analyzed using a 2 minute UPLC-MS analysis and the samples were carefully manually curated using Virscidian Analytical StudioTM (cutting overlapping peak shoulders, re-assigning misassigned peaks, etc.) to provide high quality UV and MS data. UV wavelength 210 nm area counts, total wavelength counts (TWC) and extracted ion counts (EIC) for the desired product peak were extracted from Analytical Studio to Excel. 4.4.6.2 MALDI-TOF MS Analysis. The four 384-well analytical plates prepared in the whole molecules experiments above were placed onto the Mosquito deck, together with the MALDI target plate. 0.05 umol internal standard S16 used in the fragments plate above was added to each well. The Mosquito dosed 175 nL of the reaction mixture, combining four 384 plates into one 1536 MALDI plate. This was allowed to dry for 1 hr. Following that, 150 nL of a 4 mg/mL solution of α-cyano-4-hydroxycinnamic acid S1 in 0.1% TFA, 50% acetonitrile was spotted using the Mosquito over the dried reaction spots. The plate was dried in the glove box and analyzed. The MALDI data is reported as the normalized MALDI product response (Product signal intensity/ IS signal intensity). The combined whole molecule analysis data containing compound structures, UPLC-MS data and MALDI data is shown in the attached Excel Data Tab S2. 4.4.7 Results/Discussion. 4.4.7.1 MALDI-TOF MS correlation with UPLC-MS analytical methods. The complete UPLC-MS and MALDI analytical data for all whole molecule reactions are compiled along with calculated molecular parameters in Excel Data Tab S2. In these reactions a single internal standard (S16) was used for normalization, as using tailored, reaction-specific internal standards for all 384 products in the set is unfeasible. We hoped that the use of a single internal standard, while clearly not controlling for structure-based variations in MALDI signal intensity, might still be useful for normalizing the effects of MALDI spotting variability. Figure S24 shows that the UPLC-MS EIC and UPLC-MS total wavelength chromatogram (TWC) do not correlate well with the normalized MALDI product response (R2=0.32 and R2=0.29, respectively). Interestingly, these UPLC-MS analytics actually correlated slightly better with the raw MALDI product signal than the normalized signal (EIC R2=0.48, TWC R2=0.34), however, in the binary trends analysis described in 4.4.7.4 below, the pass/fail rate for raw MALDI versus normalized MALDI are very similar. Because it is common practice to report MS data as ratios with internal standards, we chose to use the normalized MALDI data for our subsequent trends analysis.

Figure S24. Comparative scatter-plots showing how raw MALDI-product responses correlate with (A) EIC-product and (B) TWC-product and how internal-standard normalized MALDI responses correlate with (C) EIC-product and (D) TWC-product. 4.4.7.2 MALDI-TOF MS whole-molecule reaction thresholding with no product standard using the simplest partner approach. MALDI has been previously reported for the accurate quantification of small molecules in biochemical assays but has not been reported to date for analysis of small-molecules in crude synthetic chemistry reactions without covalent labels or immobilization/semi-purification (such as the self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) approach). This is important for reaction profiling, as reactions need to be run with relevant molecules as close to their reported, optimal operating conditions as possible. Our use of MALDI in the fragment additive evaluation exemplifies how a good correlation to standard analytical approaches can be achieved when using an optimized MALDI matrix, a normalizing internal standard and a very high number of laser shots/sample. The more provocative aspect of the work is the use of MALDI to assign

reaction success/failure without having a product standard in hand to calculate response factors and without using closely related internal standards for optimal MALDI normalization for each reaction. The problem of lack of product standard is the same as that existing for UV-based and MS-based analysis. MALDI presents the added complication of more variability in the data caused by ionization inhibition due to the presence of complicated reagents/catalysts and spot-to-spot variability. In order to begin to use uHT analysis to look for broad reactivity trends in large swaths of complex substrate space, we considered that the simplest partner approach, wherein a single MS-active coupling partner is used in every reaction may provide some normalization of the MS-response for all products. This normalization, coupled with large reactivity data sets with potentially reinforcing data, could then enable us to see structural reactivity trends even with the limitations described for MALDI above. Thus, in the discussion below, we define an arbitrary normalized MALDI-product threshold above which reactions are deemed successful and below which they are counted as failures. This data is then compared to similar threshold evaluations with UPLC-MS data to confirm that MALDI-MS is providing the same value as these well-established analytical methods. 4.4.7.3 Binary success/failure binning. In order to create a binary measure of success/ failure, we chose an arbitrary threshold of 20% of the average analytical response for all of the methods compared below. These are shown in Table S5. Table S5. Average responses for 4 analytical metrics described in experiments below. Experiments with higher than the 20% Binary thresholds were deemed successful, those with lower than the threshold were counted as failures.

4.4.7.4 Comparison of binary MALDI thresholding reactivity trends versus standard UPLC-MS metrics. The 384 substrates (192 amines and 192 bromides) that were used in 1536 reactions run with 4 synthetic methods were first clustered according to similar structural parameters (functional group counts and bulk whole molecule properties such as H-bond donor/ acceptor counts, molecular weight and total polar surface area). Those parameters with at least 10 examples are listed in Tables S6 and S7.

Analytical metric Average across set 20% Binary threshold

Raw MALDI‐product signal 170026 34005.2

Normalized MALDI‐product signal 0.427 0.0854

EIC‐product 1669726 333945.2

TWC‐product 228041 45608.2

Table S6. Bulk whole molecule properties with at least 10 examples in the whole molecule simplest partner test for amines and bromides.

Table S7. Functional group counts with at least 10 examples in the whole molecule simplest partner test for amines and bromides.

Amine Bulk Physical Parameters # Bromide Bulk Physical Parameters #

H‐Bond Acceptors (1 to 3) 137 H‐Bond Acceptors (1 to 3) 127

H‐Bond Donors (1) 135 H‐Bond Donors (0) 106

Nitrogens, Mid (2 to 3) 100 Nitrogens, low (<=1) 85

MW, low <200 93 TPSA low (<28) 75

clogP high (>3.07) 73 H‐Bond Donors (1) 69

TPSA high (>48) 66 MW, high >250 67

TPSA low (<28) 63 MW, mid 67

TPSA mid (>28, <48) 63 Nitrogens, Mid (2 to 3) 66

clogP mid (>1.87, <3.07) 62 clogP high (>3.07) 65

MW, high >250 60 clogP low (>1.87) 64

clogP low (>1.87) 57 TPSA high (>48) 64

H‐Bond Donors (>=2) 57 clogP mid (>1.87, <3.07) 63

H‐Bond Acceptors (>=4) 55 MW, low <200 58

Nitrogens, High (>=4) 44 TPSA mid (>28, <48) 53

MW, mid 39 Hinderance (all) 51

Nitrogens, low (<=1) 37 H‐Bond Acceptors (>=4) 50

Hinderance (all) 28 Hinderance, binding 30

Hinderance, binding 18 Nitrogens, High (>=4) 23

Hinderance, non binding 19

H‐Bond Donors (>=2) 17

H‐Bond Acceptors (0) 15

Amine Functional Group Count # Bromide Functional Group Count #

Piperidines 102 5 MR Heterocycles (all) 101

Piperazines 85 6 MR Heterocycles (all) 97

Amides (all) 51 5MR Heterocycles no NH 54

5 MR Heterocycles (all) 45 3/4‐pyridyl‐type 6 MR heterocycles 49

Tertiary amines 45 5 MR NH Heterocycles 33

Piperazines N‐Carbonyl 37 Amides (all) 33

5MR Heterocycles no NH 36 Esters 27

Piperazines N‐Alkyl 28 Remote 6 MR Heterocycles 27

6 MR Heterocycles (all) 24 2‐pyridyl‐type 6 MR heterocycles 26

Amides‐ NHR 21 5 MR NH Heterocycles >1N 22

Carbamates 18 Amides‐ NHR 22

Esters 16 N‐O, N‐S Bond 20

Anilines (all) 15 Anilines (all) 19

Sulfonamides 15 5MR NX Heterocycles 17

3‐substituted‐N‐carbonyl piperazines 12 Anilines‐ NHR 14

Ureas 11 Alcohols 13

Piperazines N‐Het 10 5 MR NH Heterocycles 1N 11

Carbamates 11

Phenols 10

Next the percent failure rates for these parameters were examined using the binary success/ failure thresholds described above. Finally, the similarity of failure rates for the different compared synthetic methods was evaluated. In Figure S25, the failure rates of clustered structural parameters are compared for normalized MALDI-product response versus EIC and TWC metrics. In Table S8, the correlation for normalized MALDI data is shown compared with responses that are not normalized with an internal standard.

Figure S25. Scatter plots show correlation for normalized MALDI-product response trends from binary thresholds failure analysis compared with standard UPLC-MS analysis (EIC and TWC).

Table S8. Correlation data for normalized and non-normalized MALDI-product trends from binary thresholds failure analysis compared with standard UPLC-MS analysis (EIC and TWC).

4.4.7.5 Binary MALDI failure rates for whole molecules parameters that lead to failure for each synthetic method. The effects of diverse functional groups and bulk whole molecule properties (with at least 10 examples) in the amine and bromide substrate sets on binary MALDI failure rate for each synthetic method are presented in Figures S26 to S33 below (see Excel Data Tab S3). This analysis will help in determining the likelihood of failure in synthesis for different conditions based on specific substrate structure. 

Synthetic 

Method

R2 for % Failure  

normalized MALDI 

vs. EIC

R2 for % Failure 

normalized MALDI 

vs. TWC

R2 for % Failure  

raw MALDI 

response vs. EIC

R2 for % Failure 

raw MALDI 

response vs. TWC

Cu 0.86 0.84 0.80 0.68

Ir 0.81 0.88 0.81 0.85

Pd 0.90 0.90 0.87 0.88

Ru 0.76 0.82 0.75 0.69

Figure S26. Failure rates for bulk whole molecule properties using MALDI binary binning for 384 Cu reactions.

Figure S27. Failure rates for functional groups using MALDI binary binning for 384 Cu reactions.

Figure S28. Failure rates for bulk whole molecule properties using MALDI binary binning for 384 Ir/Ni reactions.

Figure S29. Failure rates for functional groups using MALDI binary binning for 384 Ir/Ni reactions.

Figure S30. Failure rates for bulk whole molecule properties using MALDI binary binning for 384 Pd reactions.

Figure S31. Failure rates for functional groups using MALDI binary binning for 384 Pd reactions.

Figure S32. Failure rates for bulk whole molecule properties using MALDI binary binning for 384 Ru/Ni reactions.

Figure S33. Failure rates for functional groups using MALDI binary binning for 384 Ru/Ni reactions. 4.4.7.6 Binary MALDI cumulative average failure rates for whole molecules parameters for all synthetic method. The average failure rates for diverse functional groups and bulk whole molecule properties (with at least 10 examples) in the amine and bromide substrate sets for all 4 synthetic methods can be depicted in a single scatter plot, as shown in Figure S34. This figure also shows how these reactivity trends correlate with failure rates determined by EIC-product and TWC-product. The color of each dot represents which synthetic method performs the best

for the given structural parameter. These parameters are listed in descending order of failure rate in bromides in Figure S35 and amines in Figure S36. This cumulative trends analysis will be helpful in choosing between conditions for synthesis based on substrate structure and in highlighting structural elements that tend to fail across synthetic methods.

Figure S34. Cumulative average failure rates for different structural parameters for all four synthetic methods by MALDI binary thresholding, compared against (A) UPLC-MS EIC-product and (B) TWC product responses. The color of each dot represents which synthetic method has the lowest failure rate for each structural parameter.

Figure S35. Cumulative binary MALDI failure rates for bromides across 4 methods for (A) bulk whole molecule properties and (B) functional groups. Blue colored bars represent reactions where Cu has the lowest failure rate, red bars where Pd has the lowest failure rate.

Figure S36. Cumulative binary MALDI failure rates for amines across 4 methods for (A) bulk whole molecule properties and (B) functional groups. Blue colored bars represent reactions where Cu has the lowest failure rate, red bars where Pd has the lowest failure rate and green bars where Ir/Ni has the lowest failure rate. 4.5 Selected scale-up experiments validating whole molecule trend analysis. Analysis of the fragment and whole molecule reactivity data suggests that Cu provides a strong synthetic advantage over the other methods tested in handling potential single- and polyfunctional polar structural poisons and also in a wide variety of aryl bromide parameters. At the same time, Figures S34 and S36 suggest that there are trends in amines where Pd outperforms Cu. Figure S36 shows that certain substituted piperazine analogues (3-substituted-N-carbonyl piperazines and N-heterocycle appended piperazines) appear to be problematic for Cu. A deep dive on the individual reaction data points from nanomole-scale profiling in a comparison of Cu and Pd

methods (Figure S37) shows that normalized MALDI product response, EIC and TWC all substantiate the greater reactivity of Pd in these systems. In order to demonstrate how interesting trends can be confirmed, we selected several examples of the substrates from the nanomole-scale profiling and ran these along with an additional collection of piperazines that fit the trends on 10 umol scale (40x). Figure S39 shows how the relative TWC and EIC for these reactions all support that Pd is the superior method to Cu.

Figure S37. (A) Scatter plot with comparative Cu and Pd normalized MALDI data from nanomole-scale profiling for 3-substituted-N-carbonyl piperazines and N-heterocycle appended piperazines show a preference for Pd for nearly all substrates. These MALDI trends are similar to UPLC-MS data (B) EIC-product and (C) TWC-product from the nanomole-scale experiments. Compounds with blue squares were repeated in micro-scale experiments (see Figure S39). Compound identification can be found in Figure S38.

4.5.1 Chemistry set-up. 27 substrates were selected to demonstrate the scalability of the nanomole-scale chemistry platform and to demonstrate how identified trends can be validated. These scale-up reactions were run in 1 mL glass vials at 10 umol scale (40x scale-up), in a 96-well HTE set up as described in the experiments below. Manual liquid handling of solutions or slurries was done using single and multi-channel Eppendorf pipettors. Figure S38. 27 compounds run on 10 umol scale as confirmation of Pd versus Cu reactivity trends discussed in Figures S37 and S39. Compounds in boxes were reproduced from nanomole-scale screening, while those without boxes are additional new molecules that fit the structural type.

4.5.2 Scale-up reactions source-plate prep. 50 umol of each of the 27 amines was weighed into 4 mL vials. 4.5.2.1 96-well DMSO amine source plate preparation. Inside the glovebox, DMSO was added to each 50 umol amine vial to make 0.3 M solutions. These vials were then shaken vigorously for 2 hours. Samples that resulted in persistent slurries were used as is. These solutions were transferred to a glass 96-well plate with a spreadable 6-channel pipettor. The plate was used directly as described in the experiments below. 4.5.2.2 96-well dioxane amine source plate preparation. Inside the glovebox, dioxane was added to each 50 umol amine vial to make 0.3 M solutions. These vials were then shaken vigorously for 2 hours. Samples that resulted in persistent slurries were used as is. These solutions were transferred to a glass 96-well plate with a spreadable 6-channel pipettor. The plate was used directly as described in the experiments below. 4.5.3 Cu/oxamate ligand catalyzed reactions (10 umol scale, 100 uL volume). In a glovebox, a 0.5 M K3PO4 slurry in t-amyl alcohol was prepared and 60 uL (30 umol) was plated to each 1 mL vial of the 96-well plate. In separate vials, stock solutions containing each of the reaction components were made as follows: CuI (0.15 M in DMSO, 0.25 equiv), oxamate ligand (0.3 M in DMSO, 0.5 equiv) and 3-bromo-5-phenyl pyridine (0.3 M in DMSO, 1 equiv). Each of the solutions was distributed to 12 x 1 mL vials. 33.3 uL of amine, 33.3 uL of bromide, 16.6 uL of CuI and 16.6 uL of oxamate ligand S15 were added to the 96-well plate with a 12-channel pipettor in that order. The plate was sealed and heated to 100 °C for 20 hr. on a tumble stirrer in the glove box. After reaction, the plate was cooled to room temperature and quenched with 100 uL of DMSO containing 1,3,5-trimethoxybenzene (0.02 M) and 10% acetic acid. From this mixture, 20 uL of the solution was diluted into 730 uL DMSO in a 96-well analytical plate. This analytical plate was then run on UPLC-MS with a 2 minute method to analyze reaction consistency across the plate. 4.5.4 Pd/RuPHOS catalyzed reactions (10 umol scale, 100 uL volume). In a glovebox, a 0.5 M Cs2CO3 slurry in t-amyl alcohol was prepared and 60 uL was plated to each 1 mL vial of the 96-well plate. In separate vials, stock solutions of each of the reaction components were made as follows: 3-bromo-5-phenylpyridine (0.3 M in dioxane, 1 equiv), and RuPhos Pd G2 (0.03 M in dioxane, 0.1 equiv). 33.3 uL of the amine, 33.3 uL of the bromide and 33.3 uL of RuPhos Pd G2 were added to each vial in that order with a 12-channel pipettor. The plate was sealed and heated to 80 °C for 20 hr. on a tumble stirrer in the glove box. After reaction, the plate was cooled to room temperature and quenched with 100 uL of DMSO containing 1,3,5-trimethoxybenzene (0.02 M) and 10% acetic acid. From this mixture, 20 uL solution was diluted into 730 uL DMSO in a 96-well analytical plate. This analytical plate was then run on UPLC-MS with a 2 minute method to analyze reaction consistency across the plate. 4.5.5 UPLC-MS analysis. The above reactions were analyzed using a 2 minute UPLC-MS analysis and the samples were carefully manually curated using Virscidian Analytical StudioTM (cutting overlapping peak shoulders, re-assigning misassigned peaks, etc.) to provide high quality UV and MS data. UV wavelength 210 area counts, total wavelength chromatogram

(TWC) counts and extracted ion counts (EIC) for the desired product peak were extracted from Analytical Studio to Excel. The confirmatory UPLC-MS reactivity data for these reactions is in Excel Data Tab S4.

Figure S39. (A) Comparative Cu and Pd TWC-product data for 3-substituted-N-carbonyl piperazines and N-heterocycle appended piperazines from 10 umol scale-up confirmation experiments. Compounds with blue squares were repeats from nanomole-scale experiments. Compound identification can be found in Figure S38. In (B) the same reactions are shown to generally favor Pd using EIC-product analysis as well.

4.5.6 Results/Discussion. The scale-up experiments using both randomly repeated substrates from the nanomole-scale profiling as well as additional substrates with similar structural features confirmed the reproducibility of the trends identified from the nanomole-scale chemistry reactions. Structure reactivity trends identified in such manner can be promoted for higher-order machine learning studies to begin to build predictive models. 4.6 Whole molecules randomized cross-over experiment. In these experiments, we hoped to assign hit/miss to the full factorial space imagined in the simplest partner test (Experiment 4.4), using the simple logic that if either building block in a complex pair fails (low normalized MALDI product response) when reacted with a simple partner, then the complex pair will fail, and if both building blocks have high normalized MALDI product responses the reaction will be successful. To test this idea, we randomly paired 144 amines with 144 bromides and ran 2 of the synthetic methods featured in this work. 4.6.1 Chemistry set-up. 144 Aryl bromides and 144 amines were randomly selected using an Excel randomization process. 0.12 mmol of these bromides and amines were weighed into a set of 8-mL vials was weighed 144 aryl bromides and 144 secondary amines separately. 4.6.2 384-well DMSO bromide/amine source plate preparation. Inside the glovebox, using a Rainin 6-channel spreadable multi-addition pipettor, 0.3 mL of DMSO was added to each bromide/amine vial. These vials were then shaken vigorously for 2 hours. Samples that were not immediately soluble were heated to 60 °C with shaking for 1h. Grinding beads were added to the remaining insoluble samples and put on the LabRam at 10 G for several hours. Samples that resulted in persistent slurries were used as is. The remaining insoluble samples were further diluted to 2.4 mL (0.05 M), put on the LabRam for another 2 hours and plated directly onto the 1536-well plates. Then, in the glovebox, the 288 vials were transferred to 4 x glass 96-well plates with a spreadable 6-channel pipettor. The 4 plates were then transferred into a single 384-well plate using a Rainin Liquidator 96-channel pipettor. The plate was used directly as described in the experiments below. 4.6.3 Dual Ru/Ni catalyzed reactions (100 nmol scale, 1 uL volume). In the glovebox, a stock solution containing of each of the reaction components was made as follows: Ru(bpy)3(PF6)2 (0.001 M in DMSO, 0.005 equiv), NiCl2 glyme (0.01 M in DMSO, 0.05 equiv) and DABCO (0.6 M in DMSO, 3 equiv). This was distributed to 12 x 1 mL vials, then to a 384 well reagents source plate (20 uL/ well) with a 12-channel pipettor. The DMSO bromide and amine source plate described above and this reagents source plate were placed on the Matrix robot deck. Using a 384-tip dosing head the Matrix robot aspirated 0.5 uL of the bromides and amines mixture followed by 0.5 uL of reagent mixture and this was dosed into a COC 1536-well plate. This was then sealed in the aluminum reactor block with acrylic bottom, put into the oven reactor at 55 °C, then irradiated with a blue Kessil Lamp for 16h. The reaction plate was then cooled to ambient temperature, and quenched with the general protocol. In a 384-well analytical/quench plate, was added 75 uL of DMSO solution/ well which contained 1,3,5-trimethoxy benzene (0.264 mM) and acetic acid (1 vol%). The Matrix robot added 5 uL of this quench to the reaction plate, then removed 8 uL from the reaction plate and added this back to the analytical plate. Then 5 uL from

the analytical plate was added back to the reaction plate and this was mixed 3 times, then added back to the analytical plate. 4.6.4 Cu/oxamate ligand catalyzed reactions (250 nmol scale, 2.5 uL volume). In a glovebox, 0.5 M K3PO4 slurry solution in t-amyl alcohol was prepared and plated to every other well of a 1536-well COC plate (see Section 2.3). Separate stock solutions of each of the reaction components were made as follows: CuI (0.0833 M in DMSO, 0.25 equiv) and oxamate ligand S15 (0.25 M in DMSO, 0.5 equiv). This was distributed to a 384 well reagents plate (20 uL/ well) with a 12-channel pipettor. The DMSO bromide and amine source plate described above and these two reagents source plate were placed on the Matrix robot deck. Using a 384-tip dosing head the Matrix robot aspirated 1.25 uL of the bromides and amines mixture followed by 0.75 uL of CuI and 0.5 uL of oxamate ligand stock solutions and this was dosed into above 1536-well plate containing the plated K3PO4. The plate was sealed in an aluminum sealing plate within 30 seconds of completion of dosing, wrapped successively in 2 Mylar bags in the glovebox, and then irradiated on the LabRam outside the glovebox at 5G for 16 h. The bottom plate was heated to 90 °C, while the top plate was heated to 110 °C, giving an internal temperature of 100 °C for the nano plate. After reaction, the plate was then removed from the LabRam and the bottom plate was cooled with dry ice to room temperature while the top plate was still hot. This was then taken in the glovebox, and the Matrix robot was used to quench the reaction and to prepare an analytical plate. In a 384-well quench plate, was added 75 uL of DMSO solution/ well which contained 1,3,5-trimethoxy benzene (0.663 mM) and acetic acid (2.5 vol%). The Matrix robot added 5 uL of this quench to the reaction plate, then removed 8 uL from the reaction plate and added this back to the quench plate. Then 5 uL from the quench plate was added back to the reaction plate and this was mixed 3 times, then added back to the quench plate. From this plate, the matrix robot aspirated 30 uL of this reaction mixture and added this to 45 uL DMSO in a 384-well analytical plate. 4.6.5 UPLC-MS analysis. The 288 reactions were analyzed using a 2 minute UPLC-MS analysis and the samples were carefully manually curated using Virscidian Analytical StudioTM (cutting overlapping peak shoulders, re-assigning misassigned peaks, etc.) to provide high quality UV and MS data. 4.6.6 Results/Discussion. We examined a number of ways to compare the previously obtained MALDI data for each amine and bromide from the simplest-partner test with the new UPLC-MS data for the 288 cross-over reactions run in this experiment. We settled on a comparison of the UPLC-MS metric [TIC A% (total ion count product area percent) x TWC A% (total wavelength count product area percent)], with the product of the amine normalized MALDI response and the bromide normalized MALDI response. (TIC A% x TWC A% versus MALDI amine x MALDI bromide). TIC A% x TWC A% is a useful measure of reaction performance that requires identification of both sizeable UV and MS peaks for reaction success. Hence, we set binary failure thresholds for each the MALDI and TIC A% and TWC A%, and then looked to see if we could reasonably assign success/failure to the 288 cross-over reactions. The MALDI binary failure threshold was set at 0.0073 (the square of the previously defined failure threshold of 0.0854 from experiment 4.4.7.3, above). Using this value, we found the threshold for TIC A% x TWC A% that provides the best MALDI predictivity to the cross-over reactions to be 80. In practical terms this corresponds to a reaction where the product is almost 10 area percent of all

UV peaks and also of all MS peaks. Note: we removed catalyst/ligand peaks of well-known UV or MS retention time and solvents from these analyses to minimize their impact on area percent measurements. With these thresholds, we found that 92% of reactions predicted by the MALDI amine x MALDI bromide metric to fail in the cross-over experiments did indeed fail by UPLC-MS analysis (128 of 139). In terms of predicting success in cross-over space, we found that a MALDI x MALDI threshold of 0.167 allowed for predicting TIC A% x TWC A% success 84% of the time (31 of 37). Overall, using these low and high thresholds from the simplest partner test, we could assign success/failure to >50 % of the cross-over space with about 90% accuracy. The remaining experiments with middling MALDI x MALDI product values (between 0.0073 and 0.167), will require experimentation to determine reaction outcome. Refer to Excel Data Tab S5 for complete results.

5.0 All fragment additive and whole molecule structures

 

Figure S40. 384 fragment additives, source plate location A1 to H6.  

 

1 2 3 4 5 6

A Chemistry 0 Chemistry 16 Chemistry 32 Chemistry 48 Chemistry 64 Chemistry 80

B Chemistry 1 Chemistry 17 Chemistry 33 Chemistry 49 Chemistry 65 Chemistry 81

C Chemistry 2 Chemistry 18 Chemistry 34 Chemistry 50 Chemistry 66 Chemistry 82

D Chemistry 3 Chemistry 19 Chemistry 35 Chemistry 51 Chemistry 67 Chemistry 83

E Chemistry 4 Chemistry 20 Chemistry 36 Chemistry 52 Chemistry 68 Chemistry 84

F Chemistry 5 Chemistry 21 Chemistry 37 Chemistry 53 Chemistry 69 Chemistry 85

G Chemistry 6 Chemistry 22 Chemistry 38 Chemistry 54 Chemistry 70 Chemistry 86

H Chemistry 7 Chemistry 23 Chemistry 39 Chemistry 55 Chemistry 71 Chemistry 87

N2H

O OH

NH

O

FF

FNH2

NH

NH

N2H

O

 

Figure S41. 384 fragment additives, source plate location A7 to H12.  

7 8 9 10 11 12

A Chemistry 96 Chemistry 112 Chemistry 128 Chemistry 144 Chemistry 160 Chemistry 176

B Chemistry 97 Chemistry 113 Chemistry 129 Chemistry 145 Chemistry 161 Chemistry 177

C Chemistry 98 Chemistry 114 Chemistry 130 Chemistry 146 Chemistry 162 Chemistry 178

D Chemistry 99 Chemistry 115 Chemistry 131 Chemistry 147 Chemistry 163 Chemistry 179

E Chemistry 100 Chemistry 116 Chemistry 132 Chemistry 148 Chemistry 164 Chemistry 180

F Chemistry 101 Chemistry 117 Chemistry 133 Chemistry 149 Chemistry 165 Chemistry 181

G Chemistry 102 Chemistry 118 Chemistry 134 Chemistry 150 Chemistry 166 Chemistry 182

H Chemistry 103 Chemistry 119 Chemistry 135 Chemistry 151 Chemistry 167 Chemistry 183

NHN

FF

S

HN

NH

OO

Chiral

NNH

N

N

HO

 

Figure S42. 384 fragment additives, source plate location A13 to H18.  

13 14 15 16 17 18

A Chemistry 192 Chemistry 208 Chemistry 224 Chemistry 239 Chemistry 255 Chemistry 271

B Chemistry 193 Chemistry 209 Chemistry 225 Chemistry 240 Chemistry 256 Chemistry 272

C Chemistry 194 Chemistry 210 Chemistry 226 Chemistry 241 Chemistry 257 Chemistry 273

D Chemistry 195 Chemistry 211 Chemistry 227 Chemistry 242

Chemistry 258

Chemistry 274

E Chemistry 196 Chemistry 212 Chemistry 228 Chemistry 243 Chemistry 259 Blank control

F Chemistry 197 Chemistry 213 Chemistry 229 Chemistry 244 Chemistry 260 Chemistry 275

G Chemistry 198 Chemistry 214 Chemistry 230 Chemistry 245 Chemistry 261 Chemistry 50

H Chemistry 199 Chemistry 215 Chemistry 231 Chemistry 246 Chemistry 262 Chemistry 276

O

O

NH

N

N

N

NHO

O

 

Figure S43. 384 fragment additives, source plate location A19 to H24.  

19 20 21 22 23 24

A Chemistry 281 Chemistry 70 Chemistry 303 Chemistry 315 Chemistry 327 Chemistry 338

B Chemistry 282 Chemistry 4 Chemistry 304 Chemistry 316 Chemistry 328 Chemistry 11

C Chemistry 9 Chemistry 290 Chemistry 305 Chemistry 14 Chemistry 329 Chemistry 339

D Chemistry 35 Chemistry 291 Chemistry 306 Chemistry 317 Chemistry 330 Chemistry 340

E Chemistry 283 Chemistry 292 Chemistry 92 Chemistry 95 Chemistry 331 Chemistry 341

F Chemistry 284 Chemistry 293 Chemistry 307 Chemistry 318 Chemistry 332 Chemistry 61

G Chemistry 285 Chemistry 294 Chemistry 308 Chemistry 319 Chemistry 15 Chemistry 91

H Chemistry 5 Chemistry 295 Chemistry 309 Chemistry 101 Chemistry 333 Chemistry 8

N

S NH

N2H

HN

N

S

HS

 

Figure S44. 384 fragment additives, source plate location I1 to P6.  

1 2 3 4 5 6

I Chemistry 8 Chemistry 24 Chemistry 40 Chemistry 56 Chemistry 72 Chemistry 88

J Chemistry 9 Chemistry 25 Chemistry 41 Chemistry 57 Chemistry 73 Chemistry 89

K Chemistry 10 Chemistry 26 Chemistry 42 Chemistry 58 Chemistry 74 Chemistry 90

L Chemistry 11 Chemistry 27 Chemistry 43 Chemistry 59 Chemistry 75 Chemistry 91

M Chemistry 12 Chemistry 28 Chemistry 44 Chemistry 60 Chemistry 76 Chemistry 92

N Chemistry 13 Chemistry 29 Chemistry 45 Chemistry 61 Chemistry 77 Chemistry 93

O Chemistry 14 Chemistry 30 Chemistry 46 Chemistry 62 Chemistry 78 Chemistry 94

P Chemistry 15 Chemistry 31 Chemistry 47 Chemistry 63 Chemistry 79 Chemistry 95

O

S

O

O

NHO

NHO

N

N2H

O

NH2

NH

N

NNH

 

Figure S45. 384 fragment additives, source plate location I7 to P12.  

7 8 9 10 11 12

I Chemistry 104 Chemistry 120 Chemistry 136 Chemistry 152 Chemistry 168 Chemistry 184

J Chemistry 105 Chemistry 121 Chemistry 137 Chemistry 153 Chemistry 169 Chemistry 185

K Chemistry 106 Chemistry 122 Chemistry 138 Chemistry 154 Chemistry 170 Chemistry 186

L Chemistry 107 Chemistry 123 Chemistry 139 Chemistry 155 Chemistry 171 Chemistry 187

M Chemistry 108 Chemistry 124 Chemistry 140 Chemistry 156 Chemistry 172 Chemistry 188

N Chemistry 109 Chemistry 125 Chemistry 141 Chemistry 157 Chemistry 173 Chemistry 189

O Chemistry 110 Chemistry 126 Chemistry 142 Chemistry 158 Chemistry 174 Chemistry 190

P Chemistry 111 Chemistry 127 Chemistry 143 Chemistry 159 Chemistry 175 Chemistry 191

O

HN

S

Chiral

NN

O

N O

OHO

OH

O

NN2H

O

NNH

O

N

ON2HN

N

NO

O

 

Figure S46. 384 fragment additives, source plate location I13 to P18.  

13 14 15 16 17 18

I Chemistry 200 Chemistry 216 Chemistry 232 Chemistry 247 Chemistry 263 Chemistry 277

J Chemistry 201 Chemistry 217 Chemistry 332 Chemistry 248 Chemistry 264 Chemistry 3

K Chemistry 202 Chemistry 218 Chemistry 233 Chemistry 249 Chemistry 265 Chemistry 12

L Chemistry 203 Chemistry 219 Chemistry 234 Chemistry 250 Chemistry 266 Chemistry 18

M Chemistry 204 Chemistry 220 Chemistry 235 Chemistry 251 Chemistry 267 Chemistry 278

N Chemistry 205 Chemistry 221 Chemistry 236 Chemistry 252 Chemistry 268 Chemistry 279

O Chemistry 206 Chemistry 222 Chemistry 237 Chemistry 253 Chemistry 269 Chemistry 280

P Chemistry 207 Chemistry 223 Chemistry 238 Chemistry 254 Chemistry 270 Chemistry 37

NHO

O

NH

OH

NH

O

O

NH2

O

N

 

Figure S47. 384 fragment additives, source plate location I19 to P24.  

19 20 21 22 23 24

I Chemistry 0 Chemistry 296 Chemistry 48 Chemistry 320 Chemistry 23 Chemistry 63

J Chemistry 286 Chemistry 297 Chemistry 28 Chemistry 321 Chemistry 334 Chemistry 75

K Chemistry 287 Chemistry 298 Chemistry 310 Chemistry 322 Chemistry 335 Chemistry 72

L Chemistry 20 Chemistry 299 Chemistry 311 Chemistry 323 Chemistry 39 Chemistry 66

M Chemistry 68 Chemistry 300 Chemistry 73 Chemistry 324 Chemistry 57 Chemistry 16

N Chemistry 87 Chemistry 301 Chemistry 312 Chemistry 325 Chemistry 336 Chemistry 29

O Chemistry 288 Chemistry 77 Chemistry 313 Chemistry 326 Chemistry 337 Chemistry 6

P Chemistry 289 Chemistry 302 Chemistry 314 Chemistry 26 Chemistry 78 Chemistry 32

NH

O

HO

S

NH

O

NH

O

NNH

OO O

Chiral

 

Figure S48. 192 aryl bromides, source plate location A1 to H6. Compounds in light blue are IP-protected and the structures are partially concealed.  

1 2 3 4 5 6

A Chemistry 335

Chemistry 210 Chemistry 320 Chemistry 211 Chemistry 221 Chemistry 195

B Chemistry 379

Chemistry 380 Chemistry 348 Chemistry 365 Chemistry 193 Chemistry 342

C Chemistry 312

Chemistry 269 Chemistry 323 Chemistry 243 Chemistry 203 Chemistry 284

D Chemistry 272

Chemistry 327 Chemistry 299 Chemistry 343 Chemistry 340 Chemistry 364

E Chemistry 324

Chemistry 225 Chemistry 267 Chemistry 349 Chemistry 224 Chemistry 316

F Chemistry 214

Chemistry 298 Chemistry 332 Chemistry 367 Chemistry 372 Chemistry 265

G Chemistry 326

Chemistry 260 Chemistry 258 Chemistry 357 Chemistry 292 Chemistry 301

H Chemistry 213

Chemistry 295 Chemistry 339 Chemistry 318 Chemistry 253 Chemistry 361

 

Figure S49. 192 aryl bromides, source plate location A7 to H12. Compounds in light blue are IP-protected and the structures are partially concealed.  

7 8 9 10 11 12

A

Chemistry 239 Chemistry 262 Chemistry 208 Chemistry 302 Chemistry 222 Chemistry 291

B

Chemistry 268 Chemistry 275 Chemistry 264 Chemistry 223 Chemistry 289 Chemistry 366

C

Chemistry 297 Chemistry 218 Chemistry 196 Chemistry 345 Chemistry 257 Chemistry 314

D

Chemistry 313 Chemistry 273 Chemistry 278 Chemistry 375 Chemistry 300 Chemistry 230

E

Chemistry 194 Chemistry 236 Chemistry 351 Chemistry 358 Chemistry 248 Chemistry 250

F

Chemistry 259 Chemistry 355 Chemistry 325 Chemistry 277 Chemistry 293 Chemistry 207

G

Chemistry 229 Chemistry 359 Chemistry 215 Chemistry 246 Chemistry 322 Chemistry 261

H

Chemistry 252 Chemistry 370 Chemistry 204 Chemistry 274 Chemistry 197 Chemistry 294

N

O

N

Br NH

Chiral

N

R

N

N

Br

 

Figure S50. 192 aryl bromides, source plate location I1 to P6. Compounds in light blue are IP-protected and the structures are partially concealed.  

1 2 3 4 5 6

I Chemistry 288

Chemistry 202 Chemistry 254 Chemistry 344 Chemistry 331 Chemistry 330

J Chemistry 205

Chemistry 336 Chemistry 374 Chemistry 242 Chemistry 383 Chemistry 311

K Chemistry 356

Chemistry 283 Chemistry 199 Chemistry 353 Chemistry 382 Chemistry 233

L Chemistry 376

Chemistry 232 Chemistry 266 Chemistry 319 Chemistry 305 Chemistry 337

M Chemistry 227

Chemistry 281 Chemistry 263 Chemistry 378 Chemistry 217 Chemistry 255

N Chemistry 206

Chemistry 377 Chemistry 237 Chemistry 317 Chemistry 216 Chemistry 309

O Chemistry 279

Chemistry 234 Chemistry 235 Chemistry 369 Chemistry 346 Chemistry 338

P Chemistry 241

Chemistry 381 Chemistry 228 Chemistry 373 Chemistry 270 Chemistry 368

 

Figure S51. 192 aryl bromides, source plate location I7 to P12. Compounds in light blue are IP-protected and the structures are partially concealed.  

7 8 9 10 11 12

I

Chemistry 231 Chemistry 244 Chemistry 363 Chemistry 209 Chemistry 238 Chemistry 251

J

Chemistry 249 Chemistry 350 Chemistry 362 Chemistry 333 Chemistry 360 Chemistry 184

K

Chemistry 303 Chemistry 247 Chemistry 307 Chemistry 352 Chemistry 371 Chemistry 341

L

Chemistry 198 Chemistry 271 Chemistry 354 Chemistry 328 Chemistry 245 Chemistry 286

M

Chemistry 321 Chemistry 280 Chemistry 304 Chemistry 296 Chemistry 219 Chemistry 192

N

Chemistry 306 Chemistry 287 Chemistry 285 Chemistry 201 Chemistry 226 Chemistry 212

O

Chemistry 240 Chemistry 308 Chemistry 256 Chemistry 347 Chemistry 276 Chemistry 282

P

Chemistry 220 Chemistry 315 Chemistry 200 Chemistry 334 Chemistry 290 Chemistry 329

Br

O

NH

ONH

 

Figure S52. 192 secondary amines, source plate location A13 to H18. Compounds in light blue are IP-protected and the structures are partially concealed.  

13 14 15 16 17 18

A

Chemistry 108 Chemistry 13 Chemistry 60 Chemistry 147 Chemistry 45 Chemistry 91

B

Chemistry 126 Chemistry 124 Chemistry 43 Chemistry 174 Chemistry 45 Chemistry 160

C

Chemistry 120 Chemistry 56 Chemistry 80 Chemistry 64 Chemistry 94 Chemistry 90

D

Chemistry 49 Chemistry 183 Chemistry 17 Chemistry 36 Chemistry 94 Chemistry 106

E

Chemistry 164 Chemistry 69 Chemistry 39 Chemistry 14 Chemistry 138 Chemistry 176

F

Chemistry 166 Chemistry 77 Chemistry 142 Chemistry 22 Chemistry 178 Chemistry 135

G

Chemistry 150 Chemistry 84 Chemistry 74 Chemistry 9 Chemistry 163 Chemistry 158

H

Chemistry 83 Chemistry 75 Chemistry 11 Chemistry 9 Chemistry 181 Chemistry 72

NH

O

N

S

NH

RNH

NN

NR

NH

O

N

HN

O

O

HN

O

O

 

Figure S53. 192 secondary amines, source plate location A19 to H24. Compounds in light blue are IP-protected and the structures are partially concealed.  

19 20 21 22 23 24

A

Chemistry 55 Chemistry 169 Chemistry 185 Chemistry 134 Chemistry 62 Chemistry 1

B

Chemistry 143 Chemistry 51 Chemistry 161 Chemistry 157 Chemistry 76 Chemistry 25

C

Chemistry 37 Chemistry 144 Chemistry 78 Chemistry 104 Chemistry 88 Chemistry 173

D

Chemistry 27 Chemistry 92 Chemistry 53 Chemistry 167 Chemistry 146 Chemistry 54

E

Chemistry 109 Chemistry 171 Chemistry 85 Chemistry 188 Chemistry 47 Chemistry 40

F

Chemistry 28 Chemistry 116 Chemistry 71 Chemistry 42 Chemistry 179 Chemistry 159

G

Chemistry 46 Chemistry 133 Chemistry 152 Chemistry 100 Chemistry 191 Chemistry 59

H

Chemistry 93 Chemistry 107 Chemistry 155 Chemistry 156 Chemistry 29 Chemistry 113

HNNH

N

NH

S

OO

SN

HN

O

N

NN NH

HN

N

 

Figure S54. 192 secondary amines, source plate location I13 to P18. Compounds in light blue are IP-protected and the structures are partially concealed.  

13 14 15 16 17 18

I

Chemistry 24 Chemistry 182 Chemistry 20 Chemistry 9 Chemistry 67 Chemistry 175

J

Chemistry 145 Chemistry 2 Chemistry 11 Chemistry 9 Chemistry 102 Chemistry 118

K

Chemistry 154 Chemistry 110 Chemistry 172 Chemistry 52 Chemistry 7 Chemistry 38

L

Chemistry 127 Chemistry 117 Chemistry 139 Chemistry 114 Chemistry 168 Chemistry 123

M

Chemistry 121 Chemistry 35 Chemistry 18 Chemistry 50 Chemistry 115 Chemistry 73

N

Chemistry 48 Chemistry 82 Chemistry 86 Chemistry 12 Chemistry 151 Chemistry 3

O

Chemistry 23 Chemistry 149 Chemistry 111 Chemistry 98 Chemistry 137 Chemistry 187

P

Chemistry 70 Chemistry 129 Chemistry 68 Chemistry 19 Chemistry 141 Chemistry 8

RNH

NN

NR

NH

O

RNH

NN

NR

NH

O

N

R O

HN

NH

O

O

N

HN

N

NH

Chiral

 

Figure S55. 192 secondary amines, source plate location I19 to P24. Compounds in light blue are IP-protected and the structures are partially concealed.  

19 20 21 22 23 24

I

Chemistry 136 Chemistry 87 Chemistry 170 Chemistry 79 Chemistry 21 Chemistry 26

J

Chemistry 103 Chemistry 31 Chemistry 65 Chemistry 57 Chemistry 0 Chemistry 165

K

Chemistry 189 Chemistry 130 Chemistry 125 Chemistry 184 Chemistry 5 Chemistry 112

L

Chemistry 119 Chemistry 58 Chemistry 30 Chemistry 63 Chemistry 148 Chemistry 190

M

Chemistry 153 Chemistry 32 Chemistry 6 Chemistry 15 Chemistry 33 Chemistry 131

N

Chemistry 122 Chemistry 97 Chemistry 61 Chemistry 16 Chemistry 41 Chemistry 89

O

Chemistry 99 Chemistry 105 Chemistry 132 Chemistry 4 Chemistry 44 Chemistry 101

P

Chemistry 95 Chemistry 81 Chemistry 186 Chemistry 96 Chemistry 140 Chemistry 10

N

HN

N HN

HO

OHN

HN

H

F

F

Chiral

R

NH

O N

O

N

HN

N

O S

HN

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