Dissecting the Reaction of Phase II Metabolites of ... · Protein mass spectrometry. The protein...

Monrad et al. page S1

SUPPLEMENTARY INFORMATION

Dissecting the Reaction of Phase II Metabolites of Ibuprofen

and Other NSAIDS with Human Plasma Protein

Rune Nygaard Monrad,a James C. Errey,a Conor S. Barry,a Mazhar Iqbal,b Xiaoli Meng,b Lisa

Iddon,b Jennifer A. Perrie,b John R. Harding,c Ian D. Wilson,c Andrew V. Stachulski,b

and Benjamin G. Davis*a

Table of Contents

Page Additional experimental methods. ....................................................................................................... 2 Raw and processed protein mass spectra ............................................................................................. 5 Mass spectra of tryptic peptides ........................................................................................................ 18 Reactive Accessibility of HSA .......................................................................................................... 29 Control Reaction of HSA with D-glucuronic acid ............................................................................ 31 General Synthetic Methods for AGs .................................................................................................. 33 (4-Isobutyl)phenylacetyl 1β-acyl glucuronide (ibufenac acyl glucuronide) 1a ................................ 34 [(2R)-2-Methyl-(4-isobutyl)]phenyl]acetyl 1β-acyl [(R)-ibuprofen acyl glucuronide) 1b ............... 34 [(2S)-2-Methyl-(4-isobutyl)]phenyl]acetyl 1β-acyl [(S)-ibuprofen acyl glucuronide) 1c ................ 35 [2,2-Dimethyl-(4-isobutyl)]phenyl]acetic acid .................................................................................. 35 [2,2-Dimethyl-(4-isobutyl)]phenyl]acetyl 1β-acyl glucuronide 1d ................................................... 36 4-Bromobenzoyl 1β-acyl glucuronide 2 ............................................................................................ 36 [3-(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-1-yl]acetyl 1β-acyl glucuronide ...................... 36 (Ponolrestat 1β-acyl glucuronide) 3 .................................................................................................. 36 References .......................................................................................................................................... 37

Electronic Supplementary Material (ESI) for Chemical Science.This journal is © The Royal Society of Chemistry 2014


Additional experimental methods.

Size-exclusion chromatography. Desalting and removal of excess reagents were carried out by

PD10 size-exclusion chromatography (10 kDa molecular weight cutoff). The PD10 column was

washed with buffer or water (25 mL), the protein sample (2.5 mL) was loaded and eluted with

buffer or water (3.5 mL).

Water. Water (H2O) was purified using a Milli-Q purification system.

Determination of protein concentration. Protein concentrations were measured by UV

absorbance. Either based on the extinction coefficient at 280 nm (ε280nm = 28730 M-1 cm-1 for

unmodified HSA) calculated from the amino acid sequence using the following protein

concentration calculater: http://www.mrc-lmb.cam.ac.uk/ms/methods/proteincalculator.html. The

concentration of protein was also determined using Bradford assay with BSA as a standard, which

agreed favourably with the concentration obtained by A280.

Protein mass spectrometry. The protein solutions were analysed under denaturing conditions by

liquid chromatography (Waters, Milford, MA) coupled to positive electrospray ionization time of

flight mass spectrometry (ESI+ TOF MS, Micromass, UK) using a Phenomenex Jupiter 5u C4

300Å 250 x 4.6 mm column. The protein solution was injected and eluted at 1 mL/min using a 35

min linear gradient method from solvent A (water/5% acetonitrile/0.1% formic acid) and solvent B

(acetonitrile/0.1% formic acid), see Table 1 below. All solvents were degassed by sonication for 15

min prior to use. The output of the liquid chromatography was split 1:4 (mass spectrometer:waste)

and injected into the mass spectrometer with a scan range of 300–1800 m/z, capillary voltage


2500 V, cone voltage of 30 V, source temperature of 80°C, and desolvation temperature of 200°C.

Albumin elutes at 13.5 min and is visualized as a charge envelope from 950 to 1750 m/z

representing +69 to +41 charges. The spectrum was then deconvolved to the uncharged parent mass

using MaxEnt 1 (Micromass). Myoglobin (horse heart) was used as a calibration standard and to

test the sensitivity of the system. The following section shows the raw protein spectra followed by

their deconvoluted spectra. Both the full spectrum and a zoom of the most interesting peaks are

included.

Table 1. Chromatographic conditions for protein mass spectrometry.

Time (min)

%A %B Flowrate mL/min

Curve

0.0 95 5 1 1 5.0 95 5 1 1 20.0 5 95 1 6 25.0 5 95 1 1 30.0 95 5 1 6 35.0 95 5 1 1

Tryptic digestion and MS/MS analysis. 50 µL of a solution of modified HSA (approx. 4 g/L) in

NH4HCO3 (50 mM, pH 8.0) was added trypsin (10 µg, 1 g/L in 50 mM NH4HCO3) and incubated at

37 ºC for 16 h. The resultant solution was added 1 % aq. formic acid (5 µL) and analysed by liquid

chromatography (Agilent) coupled to ESI+ TOF MS (Q-Tof microTM, Micromass, UK) using a

Phenomenex Jupiter 5u C18 300Å 150 x 0.5 mm column. The tryptic peptides were injected and

eluted at 15 µL/min using a 90 min linear gradient method from solvent A (water/0.1% formic acid)

and solvent B (acetonitrile/0.1% formic acid), see Table 2 below. The output of the liquid

chromatography was injected into the mass spectrometer with a scan range of 100–2800 m/z,


capillary voltage 3000 V, cone voltage of 35 V, source temperature of 80°C, and desolvation

temperature of 200°C.

Table 2. Chromatographic conditions for analysis of tryptic peptides.

Time (min)

%A %B Flowrate µL/min

0.0 95 5 15 5.0 95 5 15 30.0 60 40 15 60.0 5 95 15 70.0 95 5 15 90.0 95 5 15


Raw and processed protein mass spectra

The two most significant impurities (66487 and 66598 Da) in the purified HSA correspond to small

amounts of post-translationally modified protein. Both acetylation (∆MW = 42 Da) and

glycosylation with glucose or another hexose (∆MW = 162 Da, ~9% here) are found in HSA isolated

from humans.1-4

Human serum albumin

64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 3.07e366437

66487

66598

HSA, found 66437 HSA, expected 66438

Human serum albumin

950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650m/z 0

100

%

TOF MS ES+ 27211471127

11081090

10721055

10391023

1008

993978

978 950

1187 1209

12321255

13041279

1255

1256

1280

1330

1304

1305

1305

1305

1331

1358

1331

1333

14161386

1359

1362

1387

1387

1414

14161447

1417

1420

14801479

1451

1514

14801550

1587 1626

Human serum albumin

65600 65800 66000 66200 66400 66600 66800 67000 67200 67400 67600mass 0

100

%

TOF MS ES+ 3.07e366437

66487

66598


p-Bromobenzoic acid AG 5.0 mM

64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 1.80e366801

66441

HSA, found 66441HSA, expected 66438Glycation, found 66801Glycation, expected 66797


65750 66000 66250 66500 66750 67000 67250 67500 67750 68000 68250 68500mass 0

100

%

TOF MS ES+ 1.80e366801

66441

6661066744

66850

6697167171


950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650m/z 0

100

%

TOF MS ES+ 27211731153

1133

10961078

10611045

1045

1013

998983

1216

1238

1262

1262

1286

1262

1262

1312 13661338

1338

1339

13661395 1425

1395 142514561425

14891456

1523

1559



950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650m/z 0

100

%

TOF MS ES+ 370121611731114

10961078

1061

1045

1029

1013998 983

969

1153

123812621286

12621312

13111338

1366

1394

14241456

1489

1481 15231559

1524


64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 3.13e366798

66439

HSA, found 66439HSA, expected 66438Glycation, found 66798Glycation, expected 66797


66200 66400 66600 66800 67000 67200 67400 67600 67800mass 0

100

%

TOF MS ES+ 3.13e366798

66439

6648666603

66539 66742

66848

66898 66960


Statil AG 5.0 mM

950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700m/z 0

100

%

TOF MS ES+ 1541167

11271090

10731055

1023

1008993

978964

1187 12091232

130413301358

1331

1331

1358

1386

1359

1416

1387

1416

1447

1417

1480

14791514

Statil AG 5.0 mM

64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 1.18e366440

66812

66984

HSA, found 66440HSA, expected 66438Transacylation (TA), found 66812TA, expected 66811Glycation (Glyc.), found 66984Glyc., expected 66987Both TA and Glyc., found 67366Both TA and Glyc., expected 67360

67366

Statil AG 5.0 mM

65600 65800 66000 66200 66400 66600 66800 67000 67200 67400 67600mass 0

100

%

TOF MS ES+ 71266440

66491

66812

66602

6676066984

66864 67366


Statil AG 0.5 mM

950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650m/z 0

100

%

TOF MS ES+ 18111471127

11081090

1073

1056

10391023

1008

992978

964 950

12551187 1209

1232

1279

1255

1304

1280

13581330

1305

1305

13051332

1386

1358

1360

1416

1387

1447

1417

14801447 1514

15131550

1515 1587 1627

Statil AG 0.5 mM

64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 1.42e366441

6699566820

HSA, found 66641HSA, expected 66438Transacylation (TA), found 66820TA, expected 66811Glycation (Glyc.), found 66995Glyc., expected 66987

Statil AG 0.5 mM

65800 66000 66200 66400 66600 66800 67000 67200 67400mass 0

100

%

TOF MS ES+ 54866441

66488

6660366540

6699566820


Ibufenac AG 5.0 mM

950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700m/z 0

100

%

TOF MS ES+ 2131231

1127

1108

1090

10731056

10551023

1023

1008

11871146

1209 1255 1279

13041330

1357

14151385

1389 14451478

1449 15111486 1546

Ibufenac AG 5.0 mM

64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 2.52e366440

66610

66787

66957

67133

Ibufenac AG 5.0 mM

65600 65800 66000 66200 66400 66600 66800 67000 67200 67400 67600 67800 68000mass 0

100

%

TOF MS ES+ 1.65e366440

66610

66484

66787

6665366957

67133

HSA, found 66440HSA, expected 66438Transacylation (TA), found 66610TA, expected 66612Glycation (Glyc.), or double TA, found 66787Glyc., expected 66788Double TA, expected 66786Both Glyc. and TA, or triple TA, found 66957Both Glyc. and TA, expected 66962Triple TA, expected 66960Both Glyc. and double TA, or doubleGlyc., or quadruple TA, found 67133Both Glyc. and double TA, expected 67136Double Glyc., expected 67138Quadruple TA, expected 67134


Ibufenac AG 0.5 mM

950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750m/z 0

100

%

TOF MS ES+ 15311671147

1090

10731056

105610391023

1008993

125512311209

1279 1304

13301385

13571415

1386

1386

1446

1415 1478

1446 154615111479 1546

16221584 1622

Ibufenac AG 0.5 mM

64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 1.91e366444

66610

66792

HSA, found 66444HSA, expected 66438Transacylation (TA), found 66610TA, expected 66612Glycation (Glyc.), or double TA, found 66792Glyc., expected 66788Double TA, expected 66786

Ibufenac AG 0.5 mM

65000 65250 65500 65750 66000 66250 66500 66750 67000 67250 67500 67750 68000mass 0

100

%

TOF MS ES+ 1.12e366444

6648666610 66792


(R)-Ibuprofen AG 5.0 mM

900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750m/z 0

100

%

TOF MS ES+ 1221146.6

1127.21090.2

1072.7

1039.1

1023.3

1007.7

978.1964.0

1187.51166.6 1254.71209.0

1278.81303.8 1329.9

1357.0 1385.2

1414.7 1445.5

1477.61511.2 1546.3

1583.1 1621.81662.4


64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 2.44e366442

6662766802

HSA, found 66442HSA, expected 66438Transacylation (TA), found 66627TA, expected 66626Glycation (Glyc.), found 66802Glyc., expected 66802Both TA and Glyc., found 66990Both TA and Glyc., expected 66990

66990


65800 66000 66200 66400 66600 66800 67000 67200 67400mass 0

100

%

TOF MS ES+ 1.32e366442

66627

66484

66540

66802

66990



950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750m/z 0

100

%

TOF MS ES+ 2161187.41166.6

1127.1

1108.3

1090.21072.6

1055.6

1039.2

1023.2

1007.7978.1

1209.01254.6

1303.8

1329.8 1356.9

1385.2

1414.61445.4

1477.5 1511.01546.3

1583.11621.7

1662.3 1705.0


64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 5.39e366438

66797


66637


66000 66100 66200 66300 66400 66500 66600 66700 66800 66900 67000mass 0

100

%

TOF MS ES+ 1.93e366438

66482

66537

66601 66797

66637


(S)-Ibuprofen AG 5.0 mM

950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750m/z 0

100

%

TOF MS ES+ 14812311167

1127

11081090

1090107210561039

10231008

993

1209

1254 12791304

13571330 1357

1385

1357 147714151386 1446

1419 14491478 1511

1511 15111547 1583


64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 2.40e366439

6662566801



66000 66200 66400 66600 66800 67000 67200 67400 67600 67800mass 0

100

%

TOF MS ES+ 93866439

66625

66482

66536

66801

6667266972 67147



1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700m/z 0

100

%

TOF MS ES+ 2281146.4

1127.2

1108.3

1090.21072.5

1055.6

1007.8

992.7978.2

1187.51254.51209.0

1278.7

1303.8 1329.81356.9

1385.4 1414.5

1445.4 1477.71511.2

1546.21583.2


64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 3.99e366442

66631

66802



66100 66200 66300 66400 66500 66600 66700 66800 66900 67000mass 0

100

%

TOF MS ES+ 1.50e366442

66481

6680266540 6663166607


Dimethyl Ibu-analogue AG 5.0 mM

65800 66000 66200 66400 66600 66800 67000 67200mass 0

100

%

TOF MS ES+ 80566439

66485

66540 6659766813

66635


950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700m/z 0

100

%

TOF MS ES+ 1031146.6

1127.11108.41090.2

1072.6

1055.7

1023.2

992.7978.1

950.2

1166.6 1209.01254.6

1278.7 1303.7

1329.8 1356.91385.21414.7 1445.4

1477.51511.0

1546.21583.01621.8

1662.3


64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 2.10e366439

66813


66635



1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750m/z 0

100

%

TOF MS ES+ 1931147

1127

11081090

1073

10561056

10391008993

978

11671255

1187 12311279 1304

1330

1304

1357

1330

1331

1385

1358

1415

1386

1386

1387

1445

1415

1418

1478

1446

1446

15461511

15111512

1512

1546

1583

15471583

1622


64000 65000 66000 67000 68000 69000 70000 71000mass 0

100

%

TOF MS ES+ 2.58e366442

HSA, found 66442HSA, expected 66438


65900 66000 66100 66200 66300 66400 66500 66600 66700 66800 66900 67000mass 0

100

%

TOF MS ES+ 64666442

6633066262 66384

66485

66602

66544


Mass spectra of tryptic peptides

K-137, K(+)YLYEIAR, Glycosylation, retention time 33.49 min

Y-ions

Y1 Y2 Y4 Y5 Y6 Y7

m/z predicted 175.1 246.2 488.3 651.3 764.4 927.5

m/z found 175.2 246.3 488.6 651.8 764.9 928.1

Internal ions

IA EI YE

m/z predicted 185.1 243.1 293.1

m/z found 185.2 243.3 293.3

Mass spectra K-137, K(+)YLYEIAR, Glycosylation, retention time 33.49 min 52Bw4-5-2

100 200 300 400 500 600 700 800 900 1000 1100m/z0

100

%

RNM_52Bw4-5 688 (28.365) Cm (666:1030) TOF MSMS 704.10ES+ 1.27e3380.4

120.2

261.3129.2

235.3233.3

226.3147.2

211.3

201.2

340.4

318.4

341.4

408.5

453.5

409.5

662.7454.5527.6

538.6582.6

712.9

814.9713.8

52Bw4-5-2

158 160 162 164 166 168 170 172 174 176 178 180 182 184 186 188 190 192 194 196 198 200 202 204m/z0

100

%

RNM_52Bw4-5 688 (28.365) Cm (666:1030) TOF MSMS 704.10ES+ 266201.2

183.3

175.2

158.2169.2

159.2 169.2162.2

159.2

160.2

167.2

166.2

165.2

165.2

165.2

169.2

169.2

173.2171.2

170.2

171.3

172.2174.3

181.2

181.2

176.2

179.2177.2

178.2

182.2

183.3187.2

185.2

184.3

186.2

197.3

187.2

195.2191.2

190.2

189.2

188.3

195.2

192.2 193.2

195.3

196.2

199.2

197.3

198.2

200.3203.2

202.2

204.3

52Bw4-5-2

483 484 485 486 487 488 489 490 491 492 493m/z0

100

%


487.5

484.5

484.5483.6

483.5

483.5

482.7

483.4483.4

483.6

484.4

483.6

483.7

483.7

484.3484.0

484.6486.5

485.6

484.6

485.4484.7

485.4

485.3

485.6

486.4

486.4486.3

485.9

486.6 487.4

486.6

487.4

487.1

487.6

488.6

488.6487.7

488.5

487.7

487.8487.8

488.3

491.5

489.5

488.7

489.4

488.9

490.5489.6

489.6

490.4

490.4

489.8

491.5

491.4490.6

490.7

491.4

491.4

491.4

491.2

491.6

492.6491.6

492.4

491.7492.4

491.9

492.6

492.7

492.7

52Bw4-5-2

646 647 648 649 650 651 652 653 654 655 656 657 658 659m/z0

100

%

0

100

%

RNM_52Bw4-5 688 (28.365) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (666:1030) TOF MSMS 704.10ES+ 28.8651.8

650.8649.7648.7647.7

646.7

645.9

652.8

658.7

657.7653.7 655.7

654.7 656.7


651.7650.8

648.8

647.8646.8

646.6

645.9

647.7

646.8

646.9

647.5

648.7

647.9

648.6

649.8648.8

649.6

649.5

649.3

650.7

649.9

650.6

650.5

650.8

650.9

651.0

651.8

652.7

651.8

652.7

652.6

652.6

652.3

652.7

657.7652.8

655.7

653.8

653.6

653.5

655.7654.6653.8

653.9

653.9

654.8

655.6

655.8 657.7

656.8

656.7

656.6

656.9

657.6

659.7658.7

657.8

657.9

658.5

658.8

658.8

659.5

52Bw4-5-2

756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778m/z0

100

%

0

100

%


763.9762.9756.8

756.0

761.9758.9757.9 759.9 760.9

765.9

775.9766.9

774.9

767.9 772.9771.9769.9768.9 770.9

773.9776.9

777.9


764.8

762.8756.9756.7 760.9759.0

757.9 760.7759.9

761.9

761.0 762.0

763.9

763.8

766.0

765.9 775.9766.9

766.9 775.8

775.8775.0772.9771.9767.9 769.9

769.8 771.0772.8 773.0 774.0

776.9778.0

52Bw4-5-2

920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939m/z0

100

%

0

100

%


921.0920.1

923.1

928.1

924.1

925.1927.1

926.1

929.1

930.1 932.0931.1 937.1936.1933.1 934.0 935.1 939.1938.1


922.0

921.8921.1920.0

923.1

922.2

922.3

924.1923.1

923.9

928.1

928.0

927.9925.1

925.0926.1 927.2

936.2929.1

931.9930.1931.1

936.0935.0933.9933.1

932.4 935.9

939.0937.1

939.0938.0 939.2

52Bw4-5-2

284 286 288 290 292 294 296 298 300 302 304 306 308 310m/z0

100

%

0

100

%

RNM_52Bw4-5 688 (28.365) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (666:1030) TOF MSMS 704.10ES+ 210284.3

283.3

293.3

290.3285.3

286.3

288.3287.3 289.3

291.3292.3

294.3

301.3295.3 299.4

298.3

297.3296.3

300.3 304.4

302.3

303.3

306.3305.3 309.3308.3307.3


283.3

293.3

290.3

285.3

286.3288.3

287.3 288.4

290.4

291.3292.3

294.3

301.3299.4295.3

299.3298.3

297.3296.3

301.3 304.4

302.3

303.3306.3

305.3 309.3308.3307.3

52Bw4-5-2

243 244 245 246 247 248 249 250 251 252m/z0

100

%


243.3

243.3

242.4

244.3

244.3

244.3

243.3 244.2

243.4

243.4

245.2

244.3

245.1

249.3245.3

249.2

246.3

246.2

247.3

246.4

247.3

248.3247.3

248.2 249.2

248.5

249.3

249.4251.3

250.3

250.4

250.4

252.3

251.3

252.2

251.4

252.3


K-195, ASSAK(+)QR, Transacylation, retention time 24.36 min

B- and Y-ions

B2 B6 (includes

modification)

Y1 Y2

m/z predicted 159.1 747.4 175.1 303.2

m/z found 159.3 748.2 175.3 303.5

Internal ions

SA SS K(+)Q AK(+)Q

m/z predicted 159.1 175.1 431.3 502.3

m/z found 159.3 175.3 431.7 502.8

Mass spectra K-195, ASSAK(+)QR, Transacylation, retention time 24.36 min 52Bw4-2

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z0

100

%


136.2

147.3

226.4

226.4

226.3

927.5

922.5

922.4

325.6

290.4

921.5373.6

373.6 921.4

905.4496.8444.7810.4506.8 776.3

826.3

928.6

928.6

928.7

928.7

928.8

1612.6928.91119.71004.6

1584.31249.0

52Bw4-2

156 157 158 159 160 161 162 163 164 165m/z0

100

%


157.3

156.3

156.2

155.3

156.2155.3

155.4

156.2155.8

157.2

156.3

157.2156.9

159.3

159.2158.3

157.3

158.2

157.3

158.2

158.0157.7

158.3

158.3

158.3

158.4

159.2158.8

164.3

159.3

163.3162.3161.3159.3

160.2

160.0159.8 161.2160.4

162.3

162.2161.8

163.2

163.1162.4

164.2163.3

163.7

165.3

164.3

165.3164.3

165.2164.9

165.3

52Bw4-2

743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758m/z0

100

%

0

100

%


747.2743.2

745.2744.1746.1

757.2

753.2749.1

752.2750.2

751.1 754.1 756.0755.3

758.1


748.1

748.0

747.1745.3744.3744.1

743.2

744.1

745.1

745.0

746.2

746.1

746.0

747.1 747.9

748.4

753.1

749.1

748.5

750.2749.2

750.1

750.0

752.2752.0751.3

750.9 753.0

757.2

757.2753.3

757.1

756.0

755.3755.1

754.2754.0

754.9

756.9758.3758.1

52Bw4-2

168 169 170 171 172 173 174 175 176 177 178 179 180 181 182m/z0

100

%

0

100

%


169.3

168.3

173.3171.3

170.2 172.3

181.3

176.3177.3

179.3


175.3

173.3

171.3169.3

169.3168.3

169.2 170.3 171.2

173.3

172.3171.3

172.4

175.3173.3

174.2174.1175.2

175.3

175.4181.3

181.2179.3177.3176.3

176.2 176.4 178.2177.4 179.2180.3

179.4 180.8

181.4182.3

182.2 182.3

52Bw4-2

301 302 303 304 305 306 307 308 309m/z0

100

%

0

100

%


301.4

306.4

303.5

304.4

305.5

307.5 308.5

309.5


302.4

301.4

301.4

301.4

301.3

301.5

302.1

303.5302.5

303.4

303.1

308.5306.5306.4303.5

304.5

304.4

303.6304.4

306.4305.5

305.4304.5

305.4

306.4305.5

306.3

307.5

307.4

307.1

308.4307.5

308.3307.6

308.0

309.5309.4308.6309.3

308.8

309.6

309.7

52Bw4-2

410 412 414 416 418 420 422 424 426 428 430 432 434 436 438 440 442m/z0

100

%

0

100

%

RNM_52Bw4-2 539 (25.476) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (464:540) TOF MSMS 921.80ES+ 11.3422.6412.6

411.6

409.6

413.6

421.6417.6416.6

415.6

419.6

418.6

427.6424.7

423.6425.7

441.7433.7430.7429.7

428.6

431.7

432.7

434.7

439.6435.6 438.7

437.7

436.7

440.7


412.6

412.6

411.6410.6

412.7422.6

413.7

421.6416.6

415.6 421.6417.6 419.6

418.7420.6

422.6

427.6422.6

424.7

424.6427.6

427.6

441.6439.7430.7429.7

428.6

433.8431.7

432.8

439.6438.7435.7 441.6

52Bw4-2

458 460 462 464 466 468 470 472 474 476 478 480 482 484 486 488 490 492 494 496 498 500 502 504m/z0

100

%

0

100

%


495.8

477.7476.7

471.7465.7461.7460.8458.7

459.7

464.7

463.7 468.7466.7

469.8

472.7 475.7

474.7

478.7

482.8

479.7 480.7489.7483.8

487.7485.8

491.7

490.7494.8

502.8497.8500.8

499.7

501.8 504.7

503.8


495.8

495.8478.7477.7

462.7460.8457.7

460.7

459.8

471.7465.7464.8

463.6 470.7468.7467.7

470.7

472.8

475.7

474.7

480.8

479.7

482.8

480.8

481.7

489.7483.8

487.8484.8

495.8

495.7491.8

493.8

502.7497.8 501.7

499.7

504.7

503.8 505.8


K-199, LK(+)CASLQK, Glycosylation, retention time 27.80 min

B- and Y-ions

B1 Y1 Y2 Y3 Y5

m/z predicted 114.1 147.1 275.2 388.3 546.3

m/z found 114.2 147.3 275.5 388.6 546.8

Internal ions

CA SL ASL K(+)CASL K(+)CASLQ

m/z predicted 175.1 201.1 272.2 427.7 (doubly charged) 491.8 (doubly charged)

m/z found 175.3 201.3 272.4 427.6, 428.2 491.7, 492.3

K-199, LK(+)CASLQK, Glycosylation, retention time 27.80 min 52Bw4-3

100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625m/z0

100

%


110.2

129.3

130.2

147.3 221.3

175.3

158.3

211.4

175.3

231.4

269.4258.4

258.4

285.4 324.5

286.4

306.4

340.5

460.8340.5

460.7373.6 389.6460.9

597.9

52Bw4-3

114 115 116 117 118m/z0

100

%


115.2

115.2

114.2

114.2

114.2

115.2114.2

115.0114.3

115.2

116.2

116.2115.3

116.2115.3

115.8

116.2

117.2116.2

117.2 117.2

52Bw4-3

385 386 387 388 389 390 391 392m/z0

100

%

RNM_52Bw4-3 621 (27.076) Cm (617:691) TOF MSMS 621.60ES+ 13389.6386.5

386.5384.5

384.5

385.6

385.6

384.7

385.5

385.3384.7

385.3

386.5

385.7

386.4386.1

389.5388.6

387.6386.6

386.6

387.5

386.7

387.5386.8

387.5

387.4

387.6

388.5387.7

388.4388.1

388.0

388.6

388.7

389.5

389.3389.1

389.1

389.6

391.6

390.7

390.5

390.5

390.5

390.4

390.2

391.5

391.5390.7

391.4

391.4

391.8

392.4391.8

392.3

52Bw4-3

541 542 543 544 545 546 547 548 549 550 551 552 553m/z0

100

%

0

100

%


542.8540.8

541.9

545.8

544.8

543.8

547.8

550.8548.8

549.9

552.9

552.0


540.8

542.7541.9

540.8

541.7

541.3542.7

544.8

543.8542.8

543.7

542.9

543.7

544.8

544.7

544.7

547.8546.9545.8

544.9

545.0

545.7

546.8545.9

545.9

546.7

547.0

547.8

552.9550.8550.7548.8547.9

548.8

548.6

548.4

550.6549.9549.8

549.7 550.6

552.8552.0

550.9

551.9551.2

552.1

552.7

552.9

553.1

553.7

52Bw4-3

192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208m/z0

100

%

0

100

%


199.3

195.3

194.3193.3192.3

197.3

196.3

198.3200.3

203.3

202.3

207.3

204.3205.3

206.3

208.3


201.3199.3

197.3

195.3

195.3

195.3

193.3192.3

193.3

193.0

194.4193.4 195.2

195.4

197.3

197.3196.3

195.4

196.3

196.2 197.1

199.3198.3

197.3

198.2

197.4

198.3

199.2

199.2

199.4

200.3

199.4

200.2

201.3

200.4

201.2

201.3

201.4204.4203.3

202.3

201.4

202.2

203.3

203.3

202.3

204.3

203.4

203.4

204.2

207.3205.3

205.3

204.9

207.3206.3

205.3

206.2

207.3

206.4

208.3

208.3

207.4

208.3

52Bw4-3

271 272 273 274 275 276 277 278 279 280 281 282 283m/z0

100

%

0

100

%


270.4

276.4

272.4 273.4

275.4274.4

282.4

277.4

280.4

279.4278.4

281.4

283.4


271.4

270.4271.4

270.5

270.5

271.3

276.4

273.3272.4271.5

271.5

272.3

271.5

272.3

272.1

272.5

272.5

276.4273.4

273.4

276.4275.5

274.4273.5

274.3

274.2

275.4274.5

275.3275.5

276.2

277.4

276.4

277.4

276.5

277.3

282.4

280.4279.4

279.4278.4

277.5

278.4

277.5

278.2

278.5

279.3

279.2

279.5

279.9

281.4

281.4280.4

281.4280.5

281.3

280.9

282.4

281.5

281.8

282.4

283.4282.5

282.5

283.3

283.5

52Bw4-3

490 491 492 493 494 495 496m/z0

100

%

0

100

%

RNM_52Bw4-3 621 (27.076) Sb (2,40.00 ); Cm (617:691) TOF MSMS 621.60ES+ 6494.8

493.7492.7491.7489.8

491.6490.7

490.7489.9

491.6

491.6491.0

491.8

492.7491.9

492.5492.3

493.7

492.8

493.6

494.6

494.2

493.8 494.6

495.7

495.0

495.6495.4

495.9

496.2


493.7492.7491.7489.8

491.6490.7

490.7489.9

490.5490.2

491.6

491.6491.0

491.5491.3

491.8

492.7491.9

492.5492.3

492.2

493.7

492.8

493.6

493.6493.3493.1

494.6

494.2

493.8

494.1

494.6

494.6

495.7

495.0

495.6495.4

495.3

495.9

496.2

496.3


K-205, LKCASLQK(+), Glycosylation, retention time 27.80 min

B- and Y-ions

B1 B4 B5 B6 Y1 (includes

modification)

Y3 (includes

modification)

m/z predicted 114.1 416.2 503.3 616.3 497.2 738.4

m/z found 114.2 416.7 503.7 617.1 497.7 738.2

Internal ions

CA SL CAS ASL KCA CASLQ KCASL

m/z predicted 175.1 201.1 262.1 272.2 303.1 503.2 503.3

m/z found 175.3 201.3 262.4 272.4 303.5 503.7 503.7

Mass spectra K-205, LKCASLQK(+), Glycosylation, retention time 27.80 min 52Bw4-3

100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625m/z0

100

%


110.2

129.3

130.2

147.3 221.3

175.3

158.3

211.4

175.3

231.4

269.4258.4

258.4

285.4 324.5

286.4

306.4

340.5

460.8340.5

460.7373.6 389.6460.9

597.9

52Bw4-3

114 115 116 117 118m/z0

100

%


115.2

115.2

114.2

114.2

114.2

115.2114.2

115.0114.3

115.2

116.2

116.2115.3

116.2115.3

115.8

116.2

117.2116.2

117.2 117.2

52Bw4-3

414 415 416 417 418 419 420 421 422 423 424m/z0

100

%

0

100

%

RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Sb (2,40.00 ); Cm (617:691) TOF MSMS 621.60ES+ 1.99423.6417.6

416.7

415.6413.6

414.6421.6

418.6419.6

420.6

422.6


413.6

413.6

413.6

413.5

416.6415.6

413.7

415.6414.7414.6

413.7

413.8

414.4

415.5414.7

415.5

415.1

415.6

415.7

416.1416.5

423.7

421.6418.5417.6417.6

416.7

417.5

417.4

417.7

417.7

417.7

417.7

418.5

419.6418.6

419.6418.6

418.8

419.4

421.6420.6

419.7

420.6419.7

420.5

420.5

421.6

421.5

421.2

423.6

423.6421.7

423.6422.7

422.5421.8

422.5

422.4

422.8

423.5

423.5

423.7

423.8

424.4

52Bw4-3

502 503 504 505 506 507m/z0

100

%

0

100

%

RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Sb (2,40.00 ); Cm (617:691) TOF MSMS 621.60ES+ 1

506.8

503.8

502.7

501.7504.7 505.7


503.7

502.8502.7

501.7

502.6501.8

502.1

502.6502.4

503.6

503.1

503.6503.4

506.7504.7

503.8

504.7503.8

503.9

504.6504.0

505.9505.8

505.7504.8

504.9

505.6505.4

506.6

506.4506.2

506.8

507.6

507.2

507.1 507.6

52Bw4-3

614 615 616 617 618 619 620 621 622 623m/z0

100

%

0

100

%

RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Sb (2,40.00 ); Cm (617:691) TOF MSMS 621.60ES+ 1

623.0

618.0617.1

615.0613.9 616.0

621.9

619.0

620.0 621.0


617.0615.0

613.9

614.2

614.9

616.9616.0615.9

615.7615.1

615.6 616.8616.4

623.0618.0

617.9

617.9617.1

617.6

623.0

622.9622.0621.8621.0620.0619.1

618.9618.0

618.8 619.9

619.7

620.8620.5

620.4

621.2

621.8

622.8622.2

622.7

623.1

623.6

52Bw4-3

494 495 496 497 498 499 500 501 502m/z0

100

%

0

100

%

RNM_52Bw4-3 621 (27.076) Sm (Mn, 2x5.00); Sb (2,40.00 ); Sb (2,40.00 ); Cm (617:691) TOF MSMS 621.60ES+ 1.56494.8

493.7

500.7

496.7

495.8

497.7498.7

499.8501.7

RNM_52Bw4-3 621 (27.076) Cm (617:691) TOF MSMS 621.60ES+ 7500.7494.8

493.7

493.7

493.6

493.6

494.6

494.2

493.8 494.6

494.6

499.7497.7496.7

495.7

495.0

495.6495.4

495.9

496.6

496.8

497.6496.9

497.3

498.8498.7497.7

498.6497.8

498.6497.9

498.4498.3

499.6

499.4498.8

499.2

500.7499.8

500.6

500.6499.9

500.4

500.8

501.7501.6

500.9

501.6500.9

501.6501.3

501.8

502.0

52Bw4-3

724 726 728 730 732 734 736 738 740 742 744 746 748 750 752 754m/z0

100

%

0

100

%


731.1

724.0 729.1727.4

730.3

735.2

732.2734.0 736.2

748.2

739.1 740.1744.3743.1

746.1746.8

751.3

750.9753.3

754.2


735.2731.1

730.3729.2728.0

727.0725.1

724.2

731.2

734.0732.3

734.0

738.1737.2

751.3748.2738.3

744.3743.1742.2

740.1

741.3748.1746.8

745.7

745.6 748.0

748.3

750.9749.2

750.8

753.3

752.1 754.2

52Bw4-3

155 160 165 170 175 180 185 190 195 200 205 210m/z0

100

%

0

100

%


156.3

155.3

153.2

158.3

166.3

159.3165.3

173.3

167.3171.3

211.4

183.3

181.3176.3

177.3

209.3201.3187.3

185.3

199.3

195.3191.3

189.3

203.3

207.3


158.3156.3

156.2

155.3

153.2

166.2

158.3165.2

159.3 164.3162.3

173.3167.2169.3

211.4

209.3175.3183.3

183.3175.3

183.3181.3176.3

181.3

181.2

201.3187.3

187.3201.3

197.3

195.3191.3

189.3195.3

195.3

201.3

204.4207.3

52Bw4-3

260 261 262 263 264 265 266 267 268 269 270 271 272 273 274m/z0

100

%

0

100

%


268.4

263.4260.4 262.4

261.4

267.4

265.4264.4 266.4

271.4

270.4 272.4 273.4


269.4

269.4

263.4

262.4

260.4

260.4

260.3

261.4260.5

261.2

262.3

262.1

262.4

263.3

262.5

269.3268.4265.4263.4

265.3264.4263.4

263.5264.4

264.5

267.4266.4265.5

266.3

266.5

267.3

268.3

267.5

268.0

268.5

268.5

268.8

269.4

271.4269.5271.4270.4

269.5

269.5

270.2

270.4

271.4

270.5

273.3272.4271.5

272.3

272.3

272.5

272.5

273.4

273.5

273.6

52Bw4-3

300 302 304 306 308 310 312 314 316 318 320 322 324 326 328 330 332 334 336m/z0

100

%

0

100

%


306.4

303.5

300.5302.4

301.4

305.5

304.5

314.5310.5

308.5

312.5

323.5

315.5 318.5316.5

321.5320.5

332.5

328.5

326.5

325.5327.5

329.5

330.5

331.5

333.5

334.5

336.5


324.4

306.4303.5

303.5302.5

300.5

302.4305.5

314.5312.4310.5

306.5

310.4

324.4

315.5323.5321.5

317.5316.5

320.5

320.4

321.5

324.5

333.5332.5

329.4328.5

326.5325.5

326.6

332.5332.4

331.5

333.5333.5334.5

337.5336.5

335.5


The obtained LC–MS/MS data (Table 2 in the manuscript and the K-199 and K-205 MS/MS spectra

above) with m/z 621.6 and a retention time of 27.80 min could in theory be ascribed glycosylation

at both lysines 199 and 205. Due to the typical inability of trypsin to tolerate a modification at

lysines at the P1 position we believe that the ion with m/z 621.6 and a retention time of 27.80 min

corresponds to a modification at K-199, but it could in principle be the K-205 modification - or both

simultaneously.


K-436, VGSK(+)CCK, Transacylation, retention time 24.16 min

B- and Y-ions

B1 B2 B3 B4 (includes

modification)

Y1 Y2

m/z predicted 100.1 157.1 244.1 546.3 147.1 250.1

m/z found 100.2 157.3 244.4 546.8 147.3 250.4

Internal ions

GS CC SK(+) K(+)C

m/z predicted 145.1 207.0 390.2 406.2

m/z found 145.3 207.3 390.5 407.6

Mass spectra K-436, VGSK(+)CCK, Transacylation, retention time 24.16 min 52Bw4-1-2

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580m/z0

100

%

0

100

%


120.2198.3

181.3

173.3

157.3

136.2

237.4

201.3

209.4

235.3

252.4

459.7320.4312.5269.4

270.4340.5

348.5417.6

546.8

476.7563.9

RNM_52Bw4-1 497 (24.660) Cm (454:535) TOF MSMS 451.20ES+ 89112.2 120.2

198.3120.2

181.3

181.2

173.3129.2

130.2

181.3

198.3237.4

201.3

201.3

209.4

252.4

546.8459.7252.4 312.4

269.4 280.5 320.4 340.5

357.5 417.7

546.8

476.8504.7

546.9

547.0563.9

52Bw4-1-2

100 101 102 103 104 105m/z0

100

%


100.2

100.2

100.2

101.2

100.3

102.1101.2

101.3

101.3

102.2

103.2

102.3104.2

103.2 105.2

52Bw4-1-2

148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165m/z0

100

%


153.3

153.2

153.2

152.3151.3

150.3149.2148.2

149.2150.2

151.2

152.3

151.3

151.3153.2

155.3

155.2

154.3153.3

154.3

154.2

155.2

155.2

155.3

156.2

155.3

156.2

156.2

156.3

157.2

157.3

157.3

157.3

158.3

158.2

158.2

158.2

159.3

159.2

158.3

159.2

159.1

159.3

165.3162.3160.3

160.2162.2

161.2160.3

161.3

164.3163.3

164.2 164.3

165.3

52Bw4-1-2

204 205 206 207 208 209 210 211m/z0

100

%

0

100

%


207.3206.3

205.3

204.3

208.4

211.4

210.3


209.3

207.3

206.3

205.3204.3

205.3204.4206.2

207.3

206.3207.3

206.4

209.3207.4

209.3208.4207.4

209.2208.4

209.4

209.4

211.4

210.3

210.3209.5

211.3

210.4211.2

211.4

211.4

52Bw4-1-2

235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255m/z0

100

%


235.3

235.3

234.4

234.4

237.3235.4

235.4

236.4

236.3 237.2

237.4

252.4

237.5

245.4

238.4

237.5

239.4

239.3

239.3

241.4

239.4240.4

239.4

241.4244.4

242.4

242.4 242.4 243.4 245.3

251.4

251.3250.4249.4245.4

246.4

246.3248.4

246.4 247.4248.4

250.3

250.4

250.4

251.4

251.4

251.5

252.4

254.4252.4

253.4

252.5

253.3

253.4

254.3

254.5

254.5 255.4

254.5

52Bw4-1-2

530 532 534 536 538 540 542 544 546 548 550 552 554 556 558 560 562 564 566 568 570 572 574m/z0

100

%


546.8

546.7

545.8545.8

539.8531.9530.8

535.9533.9 541.9

546.9

547.0

563.9547.8

563.9547.9

563.8

556.9555.8

548.8549.8

561.8558.0

573.8564.0

564.9

573.8568.0565.9

574.8

52Bw4-1-2

144 145 146 147 148 149 150 151 152m/z0

100

%


147.2

144.3

144.2

144.2143.9

147.2146.3

145.3

145.2144.3

145.2144.9

146.2

146.2145.9

146.3

147.2146.9

147.3

151.3

151.3150.3149.2148.2

147.3

148.2

148.2

148.2147.9

148.3

149.2148.3

149.2148.3

149.2

150.2

149.3

150.2149.3

150.2

151.2

151.2

151.1150.7

152.2

151.3

151.3

152.2152.0


K-525, K(+)QTALVELVK, Glycosylation, retention time 18.86 min

B- and Y-ions

B1 (includes

modification)

Y1 Y3 Y4 Y8

m/z predicted 479.2 147.1 359.3 488.3 872.5

m/z found 479.7 147.2 359.5 488.6 873.2

Internal ions

TA AL LV ALV TAL ELV VEL LVE TALV LVEL QTALV TALVE

m/z

predicted

173.1 185.1 213.2 284.2 286.2 342.2 342.2 342.2 385.2 455.3 513.3 514.3

m/z

found

173.3 185.3 213.3 284.4 286.4 342.5 342.5 342.5 385.5 455.6 513.7 514.7

Mass spectra K-525, K(+)QTALVELVK, Glycation, retention time 18.86 min 52Bw4-6-2

100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800m/z0

100

%


110.2

129.2

136.2

136.2

147.2

226.3

226.3

218.4147.3

186.3

175.3

187.3

249.4

321.4

249.4

318.4284.4

315.4

314.4

321.5

325.5

744.2

423.5330.5

394.5742.0

424.5744.3

744.3

52Bw4-6-2

145 146 147 148 149 150 151 152 153 154 155 156m/z0

100

%


147.2

147.2

147.2146.2145.3

144.2

147.3

155.3153.2152.3

148.3

148.2152.2

152.3153.2

153.3

154.3

156.2155.3

155.3

156.2

52Bw4-6-2

358 359 360 361 362 363m/z0

100

%

0

100

%


358.5

357.6

362.5361.5

360.5

363.5


359.5358.5

358.4

358.4

358.3357.6

358.2

357.7

358.1358.0

359.4

358.6

359.4

359.3

359.3359.0358.7

359.0

362.5

362.4360.5

359.6

360.4

360.4359.7

360.2359.7

360.2360.1

361.5361.4

360.5

361.4360.5

360.6

360.7

361.3361.2

362.3

361.6

362.3362.0

361.6

361.9 362.3

363.6362.5

363.5

363.4362.5

363.3362.6

363.3363.1

363.1362.9

363.6

363.7

363.9

52Bw4-6-2

485 486 487 488 489 490 491 492 493m/z0

100

%

0

100

%


486.6

485.7

487.7

490.6

489.6

492.6

491.7

493.2


485.7

485.6485.2

486.6

486.5485.8

485.9

486.5486.2

490.6488.6

488.5487.7

486.7

487.6486.7

487.5487.0

487.3

487.8

488.5487.8

488.3488.1

489.7488.7

489.7489.5

488.8

489.2

489.1

490.4

489.8

490.3490.1

493.6

490.6

493.5492.5491.8

490.7

491.7

491.6490.9

491.3

491.9

492.5492.2

492.7

493.2492.8

493.1 493.5


52Bw4-6-2

871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888m/z0

100

%

0

100

%

RNM_52Bw4-6 198 (18.852) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (176:231) TOF MSMS 740.70ES+ 1

880.3

879.2

873.2

871.3

872.3

874.2

873.4 878.1875.1

877.2

883.3882.5

885.2884.2

886.3

RNM_52Bw4-6 198 (18.852) Cm (176:231) TOF MSMS 740.70ES+ 4880.3879.3873.2

873.1

872.3871.6871.3

872.2 873.0

879.2

879.1878.3878.1877.2875.2874.3

874.0

875.0 877.2876.7 877.8 879.0

880.2

885.2884.2883.4883.3882.5

880.6

882.3881.6

884.1

884.4

885.1

886.4885.3

886.2888.6888.4

887.6

52Bw4-6-2

466 468 470 472 474 476 478 480 482 484 486 488 490 492 494 496 498m/z0

100

%

0

100

%


495.7

477.7

470.6

467.6

466.6465.6

469.7

468.7

476.6

475.6471.6

472.6

474.6

478.6

479.6

488.6

481.6480.6 486.6

482.6

485.7483.6484.7

487.7

494.6490.6

489.6

493.6

492.6

491.7

497.7


495.7477.7

477.6469.7

469.6467.6

467.6465.6

465.7

466.8

468.6

470.6

477.6476.6471.6

471.7

475.7

475.6474.6

477.6

477.5

486.6479.7478.7

482.6479.7

481.6480.7

482.6

485.7

483.7

483.7484.7

490.6488.6

488.5

487.6

489.7495.6

493.6

490.6

493.5492.5

495.6

494.7

496.6

497.7

497.6

497.7

498.7

52Bw4-6-2

162 164 166 168 170 172 174 176 178 180 182 184 186 188 190 192m/z0

100

%

0

100

%


186.3

175.3

173.3166.2

162.2

169.3167.3

168.3

171.3

170.3 174.3

185.3183.3

176.3 181.3

177.2 180.3 182.3

184.3191.3188.3

189.3


175.3173.3

166.2

166.2

165.3162.3

162.2 164.3

166.3

171.2169.3167.3

167.3

167.2168.3

168.2 169.2

170.3

171.2

171.3173.2

171.3

172.7

175.2

174.3

185.3183.3175.3

181.3176.3

177.2 181.2179.3178.3

183.2181.4

185.2183.3

183.4184.3

184.3

185.3

186.2

187.3

186.3

187.3

187.3188.3

188.3 191.3

191.3189.2

190.3192.2

52Bw4-6-2

208 209 210 211 212 213 214 215 216m/z0

100

%

0

100

%


207.3 209.3

208.3210.3

213.3

212.3

216.3

215.3

214.3


209.3207.3

209.2208.3

207.3

207.4

208.2207.4

208.3

208.3

209.2208.9

209.3

209.3

211.3210.3

209.4

210.3209.4

209.4

210.4211.2

211.2210.4

211.4

213.3

212.3

211.4

212.3

211.5

212.3

212.2

212.4

213.3

213.2212.4

213.2

213.2212.8

216.3215.3213.4

214.3

213.4

214.3213.4

214.3

214.2

214.2

215.3214.4

214.4

215.2

215.1

216.3215.3

215.4

216.3

216.2215.4

216.0215.8

216.4

216.4

216.4

216.4

216.8

52Bw4-6-2

278 279 280 281 282 283 284 285 286 287 288 289 290 291m/z0

100

%

0

100

%


282.4

278.4279.3

281.4280.4

283.4

290.4

288.4

286.4

285.4

287.4

289.4


284.4

284.4

281.4

278.4

280.4279.3

279.3

278.5

279.2

279.1

279.1

280.3279.4

279.5

280.3

280.3

280.2

281.3280.5

281.3280.5

281.3

281.2

281.2

284.3282.4

282.4

281.4

282.3

281.5

281.8

282.3

283.4282.4

283.3

283.3

282.5

283.2

283.2

284.3283.5

283.9

284.2

290.4284.4

290.4288.3

286.4

285.4284.5

285.3

284.5

285.3

285.3

285.1

285.4

286.4

285.5

286.3

285.5

286.2

288.3

287.4

287.3286.5

287.3286.5

287.3286.9

287.4

288.3

288.2

288.4

289.4288.4

289.4

288.5

288.5

289.3

289.1

290.3

289.5

289.5

290.3

289.6

290.0

290.4

290.5

290.5

290.7

291.0

52Bw4-6-2

379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394m/z0

100

%

0

100

%


381.5

379.5

380.5

385.5

383.5

382.5

384.5

388.6

387.5

386.6

389.6

393.5

391.4390.5

392.5


381.5

379.5

379.4

379.4

380.5379.6

380.5

380.4

379.7

380.4

380.3

381.4

381.4

380.6

380.6

380.7

381.3

381.3

383.5381.5

382.5

382.5

381.6

381.7

382.4

383.5

383.4

382.6

383.3

385.6

385.5

383.6

385.4384.6383.6

383.6

384.4

384.4

385.4

385.4

385.0

389.6

388.6

388.5385.6

387.5386.5

386.5

385.7

387.5

387.4

387.4

387.6

388.5

388.4

387.6

388.4

389.5

388.6

389.5

388.7

389.3

393.5389.6

392.5391.4

390.5389.6

390.4

390.3

391.4

391.4

391.3

392.5

391.6

391.7

392.4

393.4

393.4

393.3

393.3

394.4

393.6

394.4

393.6

394.3

394.6

52Bw4-6-2

448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464m/z0

100

%

0

100

%


452.6

448.6

451.6449.6

450.6

462.6461.7

460.6455.6

454.6 458.6456.6

457.6

459.6

463.6


452.6

448.6

448.6

448.5

448.4

452.5451.7

450.7449.6

449.6

449.5

449.5

449.6

450.6449.7

450.5

450.5

451.6

451.6

451.5

451.4

452.5

452.4

453.5

453.5

462.7

461.7

461.5

460.6458.6456.6455.6454.7453.7

454.6

454.6 455.6

455.5

455.5

455.6

456.5

456.5

456.5

458.6

457.6457.5

456.7

457.4

457.4

458.5

458.1

460.6459.5

458.7

458.7

458.8

459.4

460.5

460.5

460.5

461.5

461.4

462.5

461.8

461.9

462.5

462.4

463.6462.7

463.5

462.8

463.5

463.6

463.7

463.8

463.9

464.2

52Bw4-6-2

506 508 510 512 514 516 518 520 522 524 526 528 530 532 534 536m/z0

100

%

0

100

%


513.7

512.7

505.6

507.7506.7 509.7508.7 510.7

511.7

514.7

515.7

516.7518.7

517.7 520.7

519.7

522.7533.7524.7

523.7

530.8

525.7

527.8526.8 529.6

532.8531.7

534.7 535.7


514.7

513.7

513.7512.7

510.7

507.8505.6507.5506.6

508.7

509.7510.7

512.6511.7

513.7 521.8515.7

515.8

521.7518.6

517.8516.8

517.7520.7

518.8

519.7521.6

521.8

522.8

533.7531.8530.8524.7523.7524.6

529.6524.7

528.8525.8525.8

527.8525.9

527.7

530.7

531.7532.8

533.7

533.8

535.7534.7535.7


K-525, K(+)QTALVELVK, Transacylation, retention time 22.07 min

B- and Y-ions

B1 (includes

modification)

Y2 Y3 Y4

m/z predicted 303.2 246.2 359.3 488.3

m/z found 303.5 246.4 359.6 488.8

Internal ions

AL VE QT EL ELV VEL LVE ALVE QTAL VELV ALVEL

m/z predicted 185.1 229.1 230.1 243.1 342.2 342.2 342.2 413.2 414.2 441.3 526.3

m/z found 185.3 229.4 230.4 243.4 342.6 342.6 342.6 413.7 414.6 441.7 526.8

K-525, K(+)QTALVELVK, Transacylation, retention time 22.07 min 52Bw4-8

100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650m/z0

100

%


265.4175.3

136.2

112.2158.3

229.4

185.3 246.4 266.4

652.0

359.6

294.4342.6

406.6360.6

653.0

52Bw4-8

301 302 303 304 305 306 307 308m/z0

100

%


304.4303.5

300.5

301.5300.6

301.4300.6

301.3

303.4301.5

302.5302.4301.7 302.6

303.3302.8

303.5

304.4303.5

304.3

303.6304.3

307.5

307.4306.5

304.5

306.5

306.4

306.4

305.5

305.4304.5

305.4304.6

304.7

306.4305.5

306.3305.9

306.5

306.6

307.4306.6

306.6307.3

307.5

308.5

308.5

307.6

308.4307.6

308.1

308.6

308.6

52Bw4-8

470 472 474 476 478 480 482 484 486 488 490 492 494 496 498 500 502 504 506m/z0

100

%

RNM_52Bw4-8 364 (22.032) Cm (329:400) TOF MSMS 651.40ES+ 610

488.8471.8

470.7

477.7

472.8

485.8

478.7 486.8504.7

489.8505.8

52Bw4-8

225 226 227 228 229 230 231 232 233 234m/z0

100

%

0

100

%

RNM_52Bw4-8 364 (22.032) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (329:400) TOF MSMS 651.40ES+ 1.34e3229.4

230.4


230.4

52Bw4-8

242 243 244 245 246m/z0

100

%

0

100

%

RNM_52Bw4-8 364 (22.032) Sm (Mn, 2x5.00); Sb (2,40.00 ); Cm (329:400) TOF MSMS 651.40ES+ 127243.4

244.4

245.4


244.4

245.4244.5 245.9246.0

52Bw4-8

413 414 415 416 417 418m/z0

100

%

0

100

%


412.6

414.6

415.6416.6


413.6

413.5412.7

412.6

412.5412.1

412.5

413.4412.8

413.1

413.0

413.7

414.7

414.6

414.6

413.8

414.5

414.5

413.8

414.2414.0

414.4

414.7

415.7

415.5

414.7

414.7

415.5415.3

414.8

415.3415.1

416.7416.6

416.6

416.5416.1

415.7

416.0 416.4

418.7416.7

418.7417.7

417.6416.8

417.5417.2417.1

417.5

418.6418.5

418.4

52Bw4-8

440 441 442 443 444 445 446 447m/z0

100

%

0

100

%


443.7

442.7

446.7

444.7


441.5

439.7

441.5440.7440.6

439.7

440.5440.1439.7

440.0 440.4

441.2440.8

441.1 441.5

441.6

443.7441.7

442.6

441.8

442.6

441.8

442.5441.9

442.5442.2

443.6442.7

443.6442.7

443.6

443.6442.8

442.9

443.5

442.9

446.7

446.6

443.8

446.6

445.7444.8

444.7444.5

443.9

443.9

445.7

445.6445.5

445.4

446.0

446.5446.2

446.7

446.8

446.8

446.8

446.9

447.3447.3

52Bw4-8

525 526 527 528 529 530 531m/z0

100

%

0

100

%


524.8

527.8

528.8

530.8

529.9


526.8

526.7

524.8

524.7

524.7

524.3

526.7

525.9525.8524.8

525.7525.6525.2525.1

526.6526.3526.1

526.9

528.8527.8

527.8

527.8

527.7527.0

527.6527.3

528.8527.9

527.9

528.5528.4

530.8

530.7528.9

530.7529.9529.9

529.6 530.6530.4530.2

530.9

531.1

531.4


K-534, KQTALVELVK(+), Transacylation, retention time 22.07 min

B- and Y-ions

B1 B2 Y1 (includes

modification)

m/z predicted 129.1 257.2 321.1

m/z found 129.2 257.4 321.5

Internal ions

AL VE QT EL ELV VEL LVE ALVE QTAL VELV ALVEL

m/z predicted 185.1 229.1 230.1 243.1 342.2 342.2 342.2 413.2 414.2 441.3 526.3

m/z found 185.3 229.4 230.4 243.4 342.6 342.6 342.6 413.7 414.7 441.7 526.8

Mass spectra K-534, KQTALVELVK(+), Transacylation, retention time 22.07 min 52Bw4-8

100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725m/z0

100

%


265.4175.3

136.2

112.2158.3

229.4

185.3246.4 266.4

652.0

359.6

294.4342.6

406.6360.6

653.0

52Bw4-8

119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139m/z0

100

%


130.2

129.2133.2 137.2

52Bw4-8

254 255 256 257 258 259 260 261 262 263m/z0

100

%

RNM_52Bw4-8 364 (22.032) Cm (337:388) TOF MSMS 651.40ES+ 351

258.4

257.4256.4

52Bw4-8

320 321 322 323 324 325 326m/z0

100

%


321.4

320.5

320.4 320.5

324.5

324.5321.5

321.6 324.4

321.6

323.5322.4

321.7

323.5 323.6

323.6 324.2

325.5

324.7

325.6

326.5

326.4325.7326.6

52Bw4-8

185 190 195 200 205 210 215 220 225 230 235 240 245m/z0

100

%


185.3

186.3

215.3202.3187.3 204.3211.3

230.4

243.4

52Bw4-8

337 338 339 340 341 342 343 344 345 346 347 348m/z0

100

%


343.5

52Bw4-8

411 412 413 414 415 416 417 418 419 420 421m/z0

100

%


413.7411.6410.7

410.7

411.6

410.8411.6

413.6

411.7

411.7

413.5412.7

411.7

412.6411.8 413.4

413.7

414.7

414.6

413.8414.5

413.8

415.5

414.7

415.5

415.7416.6

416.5

416.5415.7

420.7419.6418.7417.7

416.8418.6 419.5 420.6

52Bw4-8

441 442 443 444m/z0

100

%

RNM_52Bw4-8 364 (22.032) Cm (337:388) TOF MSMS 651.40ES+ 13443.7441.7441.6

441.5

441.5440.7

441.2440.8

441.1441.0441.5

441.4

442.6

441.8

442.6

441.8

442.5441.9

442.5442.3442.2

442.6

443.6442.7

443.6

443.6442.7

443.6

442.8

442.9

443.5

442.9

443.5

443.7

443.8

443.9

444.5444.3444.1444.1

52Bw4-8

511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532m/z0

100

%

0

100

%


515.8

514.7510.8

518.8516.8

517.8520.7

519.8

526.8

522.8

523.8 524.8

527.8528.8 530.8


515.7

514.7512.8

511.8510.8 514.7

514.5

515.2

521.7515.8

521.7515.9

518.8516.8

516.8518.7

517.8

521.6520.7

519.7 520.7

526.8521.9522.7

521.9526.8

523.8526.7524.8

525.9

526.9

528.8527.8

531.8530.9528.9


The obtained LC–MS/MS data (Table 2 in the manuscript and the K-525 and K-534 MS/MS spectra

above) with m/z 651.4 and a retention time of 22.07 min could in theory be ascribed transacylation

at both lysines 525 and 534. Due to the typical inability of trypsin to tolerate a modification at

lysines at the P1 position we believe that the ion with m/z 651.4 and a retention time of 22.07 min

corresponds to a modification at K-525, but it could in principle be the K-534 modification - or both

simultaneously.


Reactive Accessibility of HSA The solvent accessibilities at probe sizes 1.0, 1.4 and 2.8 Å calculated using the NACCESS program5 as the relative % accessibility of each residue as the compared to the accessibility of that residue in an Ala-xxx-Ala tripeptide6are shown below. The calculations were performed on the A chain of dimeric HSA; the B strain gave nearly identical values. There is no clear correlation between reactivity and relative side-chain accessibility. For example, Lys199 has a very low relative side-chain accessibility; its reactivity might be attributed to a very low pKa of 7.9, which has been rationalised previously by close interactions with His242.7, 8 Lys525 and Lys534 also have quite low relative side-chain accessibility.


Control Reaction of HSA with D-glucuronic acid As a control experiment, D-glucuronic acid (0.5 or 5.0 mM) was incubated with HSA (33 g/L) according to the general procedure for incubation of AGs with HSA. Workup by size-exclusion chromatography and subsequent analysis by mass spectrometry (LC/MS) revealed a completely unmodified spectrum of HSA. At 0.5 mM


At 5 mM


General Synthetic Methods for AGs Allyl, benzyl and 4-methoxybenzyl α,β-D-glucuronates, as well as compound 1c ([(2S)-ibuprofen

acyl glucuronide] were prepared as described previously: Tetrahedron, 2007, 63, 7506-7805, ref.

15a in the main MS, and Org. Biomol. Chem., 2009, 7, 2525-2533. Below we summarise the

selective acylation method used for all the 1β-acyl glucuronides and appropriate deprotection

conditions for allyl, benzyl and 4-methoxybenzyl (PMB) esters. Characterisation data are finally

given for all compounds: 1a, 1b, 1c (for ease of comparison with 1b), 1d, 2 and 3.

Selective acylation:

The appropriate carboxylic acid (typically 0.5 mmol) was dissolved with allyl, benzyl or 4-

methoxybenzyl α,β-D-glucuronate (0.5 mmol) and HATU (0.5 mmol) in anhydrous acetonitrile (5

mL) and stirred with N-methylmorpholine (1.0 mmol) under N2 at 20°C. When the reaction was

judged complete by TLC analysis (10% EtOH-CH2Cl2), neutralization was effected by addition of

Amberlyst A-15 (H+, 2 eq.). The mixture was filtered, then the filtrate was evaporated and the

residue chromatographed on silica, eluting with 5-10% EtOH-CH2Cl2. Appropriate fractions were

combined and evaporated to give the 1β-acyl glucuronide as an allyl, benzyl or PMB ester, typically

as a foam, in 60-80% yield.

Deprotection of allyl esters:

PS–PPh3–Pd(0), purchased from Argonaut Technologies, was stirred in THF–DMF, 1:1 (1 mL per

0.1 g of resin) for 0.5 h, filtered, washed with further THF and dried before use. This material (0.2

eq.), the appropriate allyl ester (1 eq.) and morpholine (1 eq.) were stirred in THF : DMF (4:1, 4 mL

per mmol allyl ester) at 20 °C under N2 for 3 h. Amberlite IR-120 (H+) resin (1.5 eq.) was added

followed by stirring for 0.5 h, then the resin was filtered off and washed with the same solvent (10

mL) followed by evaporation of filtrate and washings to dryness. Chromatography of the crude

product, eluting with 15% and then 50% EtOH–CH2Cl2, afforded the acyl glucuronide as a foam on

evaporation of appropriate fractions.

Deprotection of benzyl esters:

A solution of the appropriate benzyl ester (typically 0.3 mmol) in THF: PriOH, 1:1 (10 mL) was

stirred at 60°C with cyclohexa-1,4-diene (1 mL) and 10% Pd-C (10 mg) for 1.5h. When reaction

was complete by TLC the mixture was cooled and filtered, then the combined filtrate and washings


were evaporated to dryness and the residue was triturated with ether. Removal of ether followed by

rigorous drying afforded the desired acyl glucuronide in high purity.

Deprotection of 4-methoxybenzyl (PMB) esters:

The PMB ester was treated with a solution of 10% CF3CO2H in CH2Cl2 at 0°C and stirred until

deprotection was complete by TLC. Solvent was removed in vacuo and the residue triturated with

CH2Cl2 to deliver the desired acyl glucuronide as a solid, after rigorous drying to remove acid

traces.

Carboxylic acids:

Ibufenac (precursor of 1a) and ponolrestat (precursor of 3) were supplied by Astra Zeneca;

commercial (R, S)-ibuprofen was used to acylate benzyl glucuronate (v. s.) and the diastereomers

were separated at the benzyl ester stage. As noted above, (S)- 1c has been described previously but

its characterisation is given below for clarity along with (R)-1b. [2,2-Dimethyl-(4-

isobutyl)]phenylacetic acid, the precursor of 1d, was obtained from ibuprofen as described below.

(4-Isobutyl)phenylacetyl 1β-acyl glucuronide (ibufenac acyl glucuronide) 1a (Via the benzyl ester) 1H NMR (400 MHz, (CD3)2CO): δ 0.88 (6H, d, J = 6.6 Hz, Me2CH), 1.81-

1.86 (1H, m, Me2CH), 2.45 [2H, d, J=7.2 Hz, ArCH2CH(CH3)2], 3.46-3.64 (3H, m, 2´-H, 3´-H and

4´-H), 3.72 (2H, s, ArCH2CO), 3.95 (1H, d, J = 9.5Hz, 5´-H), 5.57 (1H, d, J = 8.1Hz,1´-H), 7.12

(2H, d, J = 8 Hz, ArH) and 7.23 (2H, d, J = 8 Hz, ArH); 13C NMR (100 MHz, (CD3)2CO): δ 21.7,

30.0, 39.8, 44.7, 71.5, 72.4, 75.8, 76.1, 94.5, 128.9, 129.0, 129.3, 131.2, 140.2, 169.5 and 170.3;

m/z (ES +ve mode) 391 (MNa+). Found: m/z, 391.1364. C18H24O8Na requires m/z, 391.1369.

[(2R)-2-Methyl-(4-isobutyl)]phenyl]acetyl 1β-acyl [(R)-ibuprofen acyl glucuronide) 1b (Via the benzyl ester) 1H NMR (400 MHz, (CD3)2CO): δ 0.88 (6H, d, J = 6.5 Hz, Me2CH), 1.47 (3

H, d, J = 6.9 Hz, CH3CH), 1.84 (1H, m, Me2CH), 2.45 [2H, d, J=7.2 Hz, ArCH2CH(CH3)2], 3.41,

3.55 and 3.65 (3H, 3m, 2´-H, 3´-H and 4´-H), 3.81 (1 H, q, J = 6.9 Hz, CH3CH), 3.99 (1 H, d, J =

9.6 Hz, 5´-H), 5.57 (1H, d, J = 8 Hz,1´-H), 7.12 and 7.26 (4 H, dd, ArH); 13C NMR (100 MHz,

(CD3)2CO): δ 18.3, 21.7, 30.1, 44.7, 71.7, 72.5, 75.9, 76.3, 94.6, 127.2, 127.3, 129.1, 137.6, 140.3,


169.1 and 172.9; m/z (ES +ve mode) 405 (MNa+). Found: m/z, 405.1519. C19H26O8Na requires

m/z, 405.1525.

[(2S)-2-Methyl-(4-isobutyl)]phenyl]acetyl 1β-acyl [(S)-ibuprofen acyl glucuronide) 1c (Via the benzyl ester) 1H NMR (400 MHz, (CD3)2CO): δ 0.88 (6H, d, J = 6.8 Hz, Me2CH), 1.45 (3

H, d, J = 7.2 Hz, CH3CH), 1.84 (1H, m, Me2CH), 2.44 [2H, d, J=7.2 Hz, ArCH2CH(CH3)2], 3.43,

3.57 and 3.63 (3H, 3m, 2´-H, 3´-H and 4´-H), 3.81 (1 H, q, J = 7.2 Hz, CH3CH), 3.96 (1 H, d, J =

9.4 Hz, 5´-H), 5.59 (1H, d, J = 8 Hz,1´-H), 7.12 and 7.25 (4 H, dd, ArH); 13C NMR (100 MHz,

(CD3)2CO): δ 18.6, 21.7, 30.0, 44.6, 71.7, 72.5, 75.9, 76.3, 94.6, 127.3, 129.2, 137.7, 140.3, 169.0

and 172.8; m/z (ES +ve mode) 405 (MNa+). Found: m/z, 405.1519. C19H26O8Na requires m/z,

405.1525.

[2,2-Dimethyl-(4-isobutyl)]phenyl]acetic acid Ibuprofen methyl ester (1.42 g, 6.88 mmol) was added in portions under N2 to a stirred suspension

of NaH (1.28 g, 53.6 mmol) in dry THF (50 mL). After stirring at 20°C for 30 min, iodomethane

(2.0 cm3, 32.2 mmol) was added dropwise over 10 min. The mixture was stirred at 20°C for 2 days,

then filtered through Celite, and the filtrate was evaporated. The crude product was dissolved in a

minimum amount of EtOAc and purified by chromatography on SiO2, eluting with a gradient of

3% to 20% of diethylether in hexane. The first eluted product (640 mg, 40%) was the Me ester of

the title compound; it was hydrolysed by dissolving in dioxane: water, 9:1 followed by the addition

of 2M NaOH (13.6 cm3, 27.3 mmol) and heating at 50°C for 16h. After cooling, the bulk of solvent

was removed in vacuo and 2M HCl was added to give a pH of 2, followed by extraction with

diethyl ether (3 x 40 cm3). The combined organic extracts were washed with brine, dried over

MgSO4 and evaporated to dryness. Chromatography of the residue using diethyl ether-hexane

mixtures afforded the title acid (500 mg, 83% yield), Rf 0.15 (90:10, Hex:Et2O); 1H NMR [400

MHz, (CD3)2CO] δ 0.90 (6H, d, J = 6.5 Hz, 2CH3), 1.58 (6H, s, 2CH3), 1.85 (1H, m, CH2CHMe2),

2.44 (2H, d, J = 7.1 Hz, CH2), 7.11 (2H, ddd, J = 2.0, 2.2, 8.3 Hz, ArH), and 7.33 (2H, ddd, J = 2.0,

2.2, 8.3 Hz, ArH); 13C NMR (100 MHz, (CD3)2CO] δ 22.4, 26.2, 30.1, 44.9, 45.9, 125.5, 129.1,

140.3, 141.0 and 183.2; m/z ESI 238 [M + NH4]+. Found: m/z, 238.18036, C14H24O2N requires

238.18070; found, C 76.22, H 9.19; C14H20O2 requires C, 76.33, H 9.15%). This carboxylic acid

was coupled to benzyl glucuronate, followed by deprotection, as described above.


[2,2-Dimethyl-(4-isobutyl)]phenyl]acetyl 1β-acyl glucuronide 1d (Via the benzyl ester) 1H NMR [400 MHz, (CD3)2CO]: δ 0.89 (6H, d, J = 6.6 Hz, 2CH3), 1.56 (3H,

s, CH3), 1.59 (3H, s, CH3), 1.85 (1H, m, Me2CHCH2), 2.45 (2H, d, J = 7.1 Hz, CH2), 3.42 (1H, t, J

= 8.0 Hz, CH), 3.55-3.68 (2H, m, CH), 4.01 (1H, d, J = 9.4 Hz, 5´-H), 5.61 (1H, d, J = 8.0 Hz, 1´-

H), 7.12 (2H, d, J = 8.3 Hz, ArH), and 7.33 (2H, d, J = 8.3 Hz, ArH); 13C NMR [100 MHz,

(CD3)2CO] : δ 23.1, 27.2, 27.8, 31.3, 45.8, 47.6, 72.9, 73.7, 77.1, 77.6, 96.1, 126.8, 130.2, 141.1,

143.2 and 206.7; m/z (ESI + ve mode) 419 [M+Na]+. Found: m/z, 419.1693; C20H28O8Na requires

m/z, 419.1682.

4-Bromobenzoyl 1β-acyl glucuronide 2 (Via the PMB ester) 1H NMR [400 MHz, (CD3)2CO]: δ 3.60-3.76 ((3H, 3m, 2´-H, 3´-H and 4´-H),

4.11 (1 H, d, J = 9.3 Hz, 5´-H), 5.83 (1H, d, J = 7.6 Hz, 1´-H), 7.75 and 8.02 (4 H, 2d, ArH); 13C

NMR [100 MHz, (CD3)2CO] : δ 72.9, 73.8, 77.1, 77.4, 96.4, 129.4, 129.9, 132.8, 133.1, 160.5 and

170.2; m/z (ES, -ve ion mode) 375 (M-H)-. Found: m/z, 374.9723. C13H13O879Br requires m/z,

374.9716.

[3-(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-1-yl]acetyl 1β-acyl glucuronide

(Ponolrestat 1β-acyl glucuronide) 3 (Via the allyl ester) This material was finally purified to homogeneity by preparative HPLC. 1H

NMR [400 MHz, (CD3)2SO]: δ 3.45, 3.58, 3.68 (3 H, 3m, 2´-H + 3´-H + 4´-H), 4.06 (1 H, d, J = 9.5

Hz, 5´-H), 4.16 (2 H, s, ArCH2CO), 4.64 (2 H, m, ArCH2N), 5.64 (1 H, d, J = 7.9 Hz, 1´-H), 7.28 (1

H, m, ArH), 7.36 (1 H, m, ArH), 7.45 (1 H, m, ArH), 7.90-8-08 (3 H, m, ArH) and 8.36 (1 H, m,

ArH); m/z (ES –ve mode) 567, 565 [(M-H)- for 81Br, 79Br). Found: m/z, 567.0223 and 565.0237;

C23H19N2O9F81Br requires m/z, 567.0237 and C23H19N2O9F79Br requires m/z, 565.0258.


References 1. D. Bar-Or, R. Bar-Or, L. T. Rael, D. K. Gardner, D. S. Slone and M. L. Craun, Crit Care

Med, 2005, 33, 1638-1641. 2. R. L. Garlick and J. S. Mazer, J Biol Chem, 1983, 258, 6142-6146. 3. N. Iberg and R. Fluckiger, J Biol Chem, 1986, 261, 13542-13545. 4. S. A. Marashi, S. Safarian and A. A. Moosavi-Movahedi, Med Hypotheses, 2005, 64, 881. 5. S. J. Hubbard and J. M. Thornton, Department of Biochemistry and Molecular Biology,

University College London, 1993. 6. S. I. van Kasteren, H. B. Kramer, H. H. Jensen, S. J. Campbell, J. Kirkpatrick, N. J. Oldham,

D. C. Anthony and B. G. Davis, Nature, 2007, 446, 1105-1109. 7. J. T. Gerig and J. D. Reinheimer, J. Am. Chem. Soc., 1975, 97, 168–173. 8. D. C. Carter and J. X. Ho, Adv. Protein Chem., 1994, 45, 153-203.

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