Supporting Information

Improved Synthesis of a Cyclic Glutamine Analog Used in

Antiviral Agents Targeting 3C and 3CL Proteases Including
SARS-CoV-2 Mpro

Wayne Vuong and John C. Vederas*

*To whom correspondence should be addressed.

Email: john.vederas@ualberta.ca

S1
Contents
LCMS Method for Tracking Reaction Progress of Compound 6: ................................................................ S3

1
H and 13C NMR Spectra of Compounds 2—7: ............................................................................................ S4

S2
LCMS Method for Tracking Reaction Progress of Compound 6:

LCMS was performed using an Agilent 1200 SL HPLC System equipped with a Phenomenex Kinetex

(2.1x50 mm, 1.7 µm particle size, 100 Å) (Phenomenex, Torrance, CA, USA) column thermostated at 50

°C followed by mass spectrometric detection. An aliquot of 5 µL was loaded onto the column at a flow

rate of 0.50 mL/min and an initial buffer composition of 98% mobile phase A (0.1% formic acid in H2O)

and 2% mobile phase B (0.1% formic acid in CH3CN). The following method was then used:

Table S1: LCMS gradient for monitoring of reaction progress in the formation of compound 6.

Time (min) A (%) B (%)

0.00 98 2
5.00 5 95
8.00 5 95

Mass spectra were acquired in fast polarity switching using an Agilent 6220 Accurate-Mass TOF

HPLC/MS system (Santa Clara, CA, USA) equipped with a dual sprayer electrospray ionization source.

Mass spectrometric conditions were as follows: drying gas 13 L/min at 325 °C, nebulizer 1553 Torr, mass

range 100 – 1100 Da, acquisition rate of ~1.03 spectra/sec, fragmentor 30 V, capillary 4000 V (+ve) /

3500 V (-ve). Data acquisition was performed using the OpenLab CDS C.01.07[27]. Analysis of the LCMS

data was performed using MassHunter Analytical Studio Reviewer (ver. B.02.01). The starting material 5

was found to elute at 5.32 min, and the desired product 6 was found to elute at 4.51 min. A

representative spectra is shown below:

Figure S1: Example LCMS trace for the formation of product 6.

S3
1H and 13C NMR Spectra of Compounds 2—7:

S4
 Department of Chemistry, University of Alberta

Recorded on: v700, Apr 16 2021 Sweep Width(Hz): 8389.26 Acquisiton Time(s): 5 Relaxation Delay(s): 0.1
Pulse Sequence: PRESAT Digital Res.(Hz/pt): 0.13 Hz per mm(Hz/mm): 29.23 Completed Scans 8

Wayne, WV-5-148
699.762 MHz H1 1D in cdcl3 (ref. to CDCl3 @ 7.26 ppm)
temp 27.5 C -> actual temp = 27.0 C, coldid probe

7.260

5.719
5.714
5.709
5.704
5.694
5.690
5.684
5.680
5.670
5.086
5.084
5.061
5.059
5.057
5.042
4.957
4.946
4.358
4.347
3.773
3.724
3.665
2.582
2.572
2.562
2.553
2.373
2.363
2.353
2.342
2.320
2.311
2.040
2.008
1.998
1.988
1.581
1.497
1.435
9 8 7 6 5 4 3 2 1 ppm
1.00

2.04
0.83

0.85

3.01
3.00

1.01

2.03

1.94

9.08
File: /home/vnmr1/vnmrsys/data/Wayne/2021.04/2021.04.16.v7_WV-5-148_loc70_11.18_H1_1D
 Department of Chemistry, University of Alberta

Recorded on: v700, Apr 16 2021 Sweep Width(Hz): 46296.3 Acquisiton Time(s): 1 Relaxation Delay(s): 1
Pulse Sequence: s2pul Digital Res.(Hz/pt): 0.35 Hz per mm(Hz/mm): 147.61 Completed Scans 128

Wayne, WV-5-148
175.976 MHz C13{H1} 1D in cdcl3 (ref. to CDCl3 @ 77.06 ppm)
temp 27.5 C -> actual temp = 27.0 C, coldid probe

80.089
77.241
77.060
76.877

52.433
52.174
51.789

41.890

36.464
33.770

28.342
175.505
172.866

155.391

134.416

117.706

180 160 140 120 100 80 60 40 20 ppm

File: /home/vnmr1/vnmrsys/data/Wayne/2021.04/2021.04.16.v7_WV-5-148_loc70_11.18_C13_1D
 Department of Chemistry, University of Alberta

Recorded on: v700, Feb 23 2021 Sweep Width(Hz): 8389.26 Acquisiton Time(s): 5 Relaxation Delay(s): 0.1
Pulse Sequence: PRESAT Digital Res.(Hz/pt): 0.13 Hz per mm(Hz/mm): 29.25 Completed Scans 8

Wayne, WV-5-092
699.762 MHz H1 1D in cdcl3 (ref. to CDCl3 @ 7.26 ppm)
temp 27.5 C -> actual temp = 27.0 C, coldid probe

7.260

5.746
5.732
5.722
5.707
5.085
5.083
5.061
5.059
5.039
5.038
5.036
5.024
5.023
5.022
4.973
4.965
4.960
4.952
3.705
3.650
2.587
2.361
2.351
2.321
2.313
2.305
2.303
2.300
2.292
2.284
2.279
2.266
2.254
2.246
1.565
1.526
1.499
1.485
1.452
9 8 7 6 5 4 3 2 1 ppm
1.01

1.03
1.02
1.01

3.02
3.01

1.01
1.06
3.08

18.00
File: /home/vnmr1/vnmrsys/data/Wayne/2021.02/2021.02.23.v7_WV-5-092_loc2_11.13_H1_1D
 Department of Chemistry, University of Alberta

Recorded on: v700, Feb 23 2021 Sweep Width(Hz): 36764.7 Acquisiton Time(s): 1 Relaxation Delay(s): 1
Pulse Sequence: s2pul Digital Res.(Hz/pt): 0.28 Hz per mm(Hz/mm): 146.43 Completed Scans 128

Wayne, WV-5-092
175.971 MHz C13{H1} 1D in cdcl3 (ref. to CDCl3 @ 77.06 ppm)
temp 27.5 C -> actual temp = 27.0 C, coldid probe

83.338
77.242
77.060
76.878

56.627
52.281
51.739

42.823

36.660

31.991
28.012
175.369
171.067

151.780

134.855

117.361

180 160 140 120 100 80 60 40 20 ppm

File: /home/vnmr1/vnmrsys/data/Wayne/2021.02/2021.02.23.v7_WV-5-092_loc2_11.14_C13_1D
 Department of Chemistry, University of Alberta

Recorded on: v700, Mar 3 2021 Sweep Width(Hz): 8389.26 Acquisiton Time(s): 5 Relaxation Delay(s): 0.1
Pulse Sequence: PRESAT Digital Res.(Hz/pt): 0.13 Hz per mm(Hz/mm): 29.23 Completed Scans 8

Wayne, WV-5-098
699.762 MHz H1 1D in cdcl3 (ref. to CDCl3 @ 7.26 ppm)
temp 27.5 C -> actual temp = 27.0 C, coldid probe
9.740

7.260

5.005
4.997
4.991
4.984
3.719
3.714
3.709
3.695
3.685
2.966
2.897
2.896
2.884
2.883
2.872
2.870
2.859
2.857
2.727
2.726
2.720
2.720
2.701
2.695
2.335
2.326
2.322
2.312
2.283
2.275
2.272
2.264
2.262
2.038
1.498
1.492
1.253
9 8 7 6 5 4 3 2 1 ppm
0.87

0.96

3.00
2.85

0.93
0.96
0.94

1.05
0.93

18.10
File: /home/vnmr1/vnmrsys/data/Wayne/2021.03/2021.03.03.v7_WV-5-098_loc8_00.28_H1_1D
 Department of Chemistry, University of Alberta

Recorded on: v700, Mar 3 2021 Sweep Width(Hz): 36764.7 Acquisiton Time(s): 1 Relaxation Delay(s): 1
Pulse Sequence: s2pul Digital Res.(Hz/pt): 0.28 Hz per mm(Hz/mm): 146.36 Completed Scans 1316

Wayne, WV-5-098
175.971 MHz C13{H1} 1D in cdcl3 (ref. to CDCl3 @ 77.06 ppm)
temp 27.5 C -> actual temp = 27.0 C, coldid probe

83.636
77.242
77.060
76.878

55.911
52.420
52.240

45.106

36.440
32.028
27.988
174.681
170.815

151.941

180 160 140 120 100 80 60 40 20 ppm

File: /home/vnmr1/vnmrsys/data/Wayne/2021.03/2021.03.03.v7_WV-5-098_loc8_00.29_C13_1D
 Department of Chemistry, University of Alberta

Recorded on: u500, Jul 18 2021 Sweep Width(Hz): 6009.62 Acquisiton Time(s): 5 Relaxation Delay(s): 0.1
Pulse Sequence: PRESAT Digital Res.(Hz/pt): 0.09 Hz per mm(Hz/mm): 20.9 Completed Scans 8

Wayne, WV-6-012
499.787 MHz H1 1D in cdcl3 (ref. to CDCl3 @ 7.26 ppm)
temp 27.7 C -> actual temp = 27.0 C, colddual probe

7.346
7.334
7.322
7.260
6.684
6.673
6.663
5.005
4.999
4.993
4.988
4.986
4.976
4.971
3.855
3.788
3.711
3.680
3.676
2.739
2.735
2.727
2.723
2.720
2.637
2.626
2.594
2.583
2.508
2.497
2.492
2.480
2.458
2.447
2.435
2.417
2.321
2.308
2.296
2.295
2.290
2.281
2.274
1.572
1.498
9 8 7 6 5 4 3 2 1 ppm
0.55

0.42

1.00

1.30
1.69
3.02
1.32
1.68

1.00
2.09

2.04

18.10
File: /home/vnmr1/vnmrsys/data/Wayne/2021.07/2021.07.18.u5_WV-6-012_loc12_17.27_H1_1D
 Department of Chemistry, University of Alberta

Recorded on: u500, Jul 18 2021 Sweep Width(Hz): 33783.8 Acquisiton Time(s): 1 Relaxation Delay(s): 1
Pulse Sequence: s2pul Digital Res.(Hz/pt): 0.26 Hz per mm(Hz/mm): 105.09 Completed Scans 128

Wayne, WV-6-012
125.685 MHz C13{H1} 1D in cdcl3 (ref. to CDCl3 @ 77.06 ppm)
temp 27.7 C -> actual temp = 27.0 C, colddual probe

83.486
77.314
77.060
76.806

61.764
61.450
56.374
56.322
52.348
52.097
52.019
40.583
40.179
32.285
32.193
31.852
28.064
28.000
174.718
174.683
170.928
170.897

151.737
147.985
147.446

180 160 140 120 100 80 60 40 20 ppm

File: /home/vnmr1/vnmrsys/data/Wayne/2021.07/2021.07.18.u5_WV-6-012_loc12_17.28_C13_1D
 Department of Chemistry, University of Alberta

Recorded on: v700, Jul 21 2021 Sweep Width(Hz): 8389.26 Acquisiton Time(s): 5 Relaxation Delay(s): 0.1
Pulse Sequence: PRESAT Digital Res.(Hz/pt): 0.13 Hz per mm(Hz/mm): 29.24 Completed Scans 8

Wayne, WV-6-018
699.762 MHz H1 1D in cdcl3 (ref. to CDCl3 @ 7.26 ppm)
temp 27.5 C -> actual temp = 27.0 C, coldid probe

7.260

5.542
5.048
5.041
5.033
5.026
3.722
3.341
3.333
3.331
3.328
3.327
3.306
3.296
3.293
2.515
2.500
2.495
2.485
2.480
2.372
2.367
2.362
2.352
2.119
2.104
2.042
1.828
1.823
1.594
1.583
1.505
1.500
1.257
1.252
9 8 7 6 5 4 3 2 1 ppm
0.99

0.98

3.01

2.01

1.03
1.97

0.99

1.05

18.00
File: /home/vnmr1/vnmrsys/data/Wayne/2021.07/2021.07.21.v7_WV-6-018_loc18_12.57_H1_1D_
 Department of Chemistry, University of Alberta

Recorded on: v700, Jul 21 2021 Sweep Width(Hz): 46296.3 Acquisiton Time(s): 1 Relaxation Delay(s): 1
Pulse Sequence: s2pul Digital Res.(Hz/pt): 0.35 Hz per mm(Hz/mm): 147.11 Completed Scans 128

Wayne, WV-6-018
175.976 MHz C13{H1} 1D in cdcl3 (ref. to CDCl3 @ 77.06 ppm)
temp 27.5 C -> actual temp = 27.0 C, coldid probe

83.460
77.243
77.060
76.879

56.179
52.317

40.047
37.914
31.751
28.089
28.023
179.207

171.183

152.113

180 160 140 120 100 80 60 40 20 ppm

File: /home/vnmr1/vnmrsys/data/Wayne/2021.07/2021.07.21.v7_WV-6-018_loc18_12.58_C13_1D
 Department of Chemistry, University of Alberta

Recorded on: v700, Jul 22 2021 Sweep Width(Hz): 8389.26 Acquisiton Time(s): 5 Relaxation Delay(s): 0.1
Pulse Sequence: PRESAT Digital Res.(Hz/pt): 0.13 Hz per mm(Hz/mm): 29.23 Completed Scans 8

Wayne, WV-6-022
699.762 MHz H1 1D in cdcl3 (ref. to CDCl3 @ 7.26 ppm)
temp 27.5 C -> actual temp = 27.0 C, coldid probe

7.882
7.873

7.333
7.306
7.300
7.260

6.307

5.447
5.435
5.088
4.467
4.462
4.356
4.348
3.716
3.311
3.297
3.280
3.270
2.799
2.367
2.358
2.040
1.886
1.826
1.810
1.796
1.744
1.734
1.725
1.683
1.672
1.664
1.523
1.516
1.264
1.252
0.959
0.955
0.877
9 8 7 6 5 4 3 2 1 ppm
0.98

5.13

0.99

0.82

1.91

0.99
0.87

3.10

2.04

2.02
0.95

1.07
1.63
1.48
1.19
1.10

6.00
File: /home/vnmr1/vnmrsys/data/Wayne/2021.07/2021.07.22.v7_WV-6-022-repurified_loc22_17.39_H1_1D
 Department of Chemistry, University of Alberta

Recorded on: v700, Jul 22 2021 Sweep Width(Hz): 46296.3 Acquisiton Time(s): 1 Relaxation Delay(s): 1
Pulse Sequence: s2pul Digital Res.(Hz/pt): 0.35 Hz per mm(Hz/mm): 147.22 Completed Scans 128

Wayne, WV-6-022
175.976 MHz C13{H1} 1D in cdcl3 (ref. to CDCl3 @ 77.06 ppm)
temp 27.5 C -> actual temp = 27.0 C, coldid probe

77.243
77.060
76.879

66.917

53.383
52.431
51.586
42.434
40.527
38.443
32.949
29.737
28.410
24.669
22.934
22.105
179.845

172.940
172.149

156.126

136.411

128.536
128.141
128.010

180 160 140 120 100 80 60 40 20 ppm

File: /home/vnmr1/vnmrsys/data/Wayne/2021.07/2021.07.22.v7_WV-6-022-repurified_loc22_17.40_C13_1D