Glycomimetic ligands for the human asialoglycoprotein receptor

Article abstract of DOI:10.1021/ja2104679

The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.

Full text of DOI:10.1021/ja2104679

Glycomimetic Ligands for the Human Asialoglycoprotein Receptor
Sreeman K. Mamidyalaa‡, Sanjay Duttaa‡, Boris A. Chrunykb, Cathy Previlleb, Hong Wangb, Jane M.
Withkab, Alexander McCollb, Timothy A. Subashib, Steven J. Hawrylikb, Matthew C. Grifforb, Sung Kimb,
Jeffrey A. Pfefferkorn, David A. Priceb, Elnaz Menhaji-Klotzb, Vincent Mascittib*, and M.G. Finna*
aDepartment of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines. Rd., La Jolla, CA
92037, USA
bPfizer Global Research & Development, Eastern Point Road, Groton, CT 06340, USA
Supporting Information
Contents of This File
A. Complete SPR Results ……………………………………………………………………..………………… S2
B. General ……………………………………………………………………………………………………………… S8
C. Synthesis of 2-Azido-D-Galactose Derivatives ……………………………………………………. S8
D. Synthesis of Allyl-D-Galactosamine Derivatives …………………………………………………. S10
E. Synthesis of N-Trifluoroacetamide-D-Galactosamine Derivatives ……………………… S12
F. General Procedure for Click Chemistry ………..……………………………………………………. S14
G. General Procedure for O-Benzyl Deprotection...………………………………………………….S14
H. Surface Plasmon Resonance Measurements ……………………………………………………… S14
I. Characterization Data for Selected High-Affinity Ligands …………………………………. S17
Accompanying File (Supporting Information, part b)
J. NMR Data for Other Ligands ………………………………………………………………………S27-S103
page S1

A. Complete SPR Results. Table S1 shows the structures and binding constants of all
compounds contributing to this study, from surface plasmon resonance measurements as
described in the text and below. Values denoted as “ca. XXX µM” and “> 1 mM” are
approximate or given as a lower bound either because binding behavior was not well-resolved
or because only one or two replicate measurements were performed, since these compounds
were of lesser interest. In some instances, slower kinetic behavior was observed, but this could
not be fit to simple kinetic models and usually exhibited Ru values above the maximum for 1:1
interaction, suggesting either nonspecific interactions or multisite binding. No KD values were
attributed to these compounds, denoted by “slow kinetics” in the “notes” column.
Table S1. Complete results from SPR studies, organized by compound type and in order of
decreasing affinity within each type, except for reference compounds listed at the beginning of
each section. The blue background marks those structures included in Figure 2 in the main
paper.
R1
R1
R1
HRMS
R2
OH
R2
Kd (n = # SPR
replicate
measurements)
R2
EIMS m/z
calc; obs a
O
calc; obs
O
O
notes
Cpd.
ID
(M+H)+ unless
otherwise noted
R3
R3
OH
R3
OH
OH
OH
OH
N
N
N
1a
1b
R
CH2OH
CH2N3
20-40 µM b
IC50 = 46 µM c
IC50 = 48 µM c
H
O
CH3
NH2
H
N
β-OMe
β-OMe
O
F3
C
CH3
NH2
H
N
N
N
1c
N
CH2OH
CH2OH
CH2OH
O
40.4 ± 9.5 µM
GalNAc
OH
NHCOCH3
OH
(n=45)
880 ± 200 µM
OMe
(n=12)
257 ± 23 µM
β-D-glucose
β-Me
lactose
OH
(n=6)
178.1074; 178.107
200.0893; 200.0892
(M+Na)+
ca. 570 µM
NH2
CH2OH
177.1; 178.1
219.1; 220.1
(n=2)
220.1179; 220.1182
242.0999; 242.0996
(M+Na)+
41.0 ± 7.0 µM
β-Me
β-Me
β-Me
3a1
3a6
NHCOCH3
NHCOCF3
CH2OH
CH2OH
CH2OH
(n=3)
14.8 ± 2.0 µM
273.1; 296.0
(M+Na)+
274.0897; 274.0894
(n=8)
288.1053; 288.105
310.0873; 310.0867
(M+Na)+
33.1 ± 1.4 µM
3a10
NHCOCH2CF3
NHCOnPr
287.1; 288.1
(n=8)
248.1492; 248.1493
517.3; 518.3 270.1312; 270.1314
(M+Na)+
EIMS on
tris(benzyl
ether)
175 ± 9.5 µM
β-Me
β-Me
3a2
3a3
CH2OH
CH2OH
(n=4)
248.1492; 248.1490
517.3; 518.3 270.1312; 270.1309
(M+Na)+
EIMS on
tris(benzyl
ether)
189 ± 22.0 µM
NHCOiPr
(n=4)
page S2

R1
R1
R1
OH
HRMS
R2
OH
R2
Kd (n = # SPR
replicate
measurements)
R2
EIMS m/z
calc; obs a
O
calc; obs
O
O
notes
Cpd.
ID
(M+H)+ unless
otherwise noted
R3
R3
OH
R3
OH
OH
OH
262.1649; 262.1647
531.3; 532.3 284.1468; 284.1470
(M+Na)+
EIMS on
tris(benzyl
ether)
254 ± 4.0 µM
β-Me
β-Me
3a4
3a8
NHCOtBu
CH2OH
CH2OH
(n=4)
286.0897; 286.0902
263.1; 264.1
199 ± 102 µM
NHCOCH2CO2H
(M+Na)+
(n=4)
compare to 1b
and 1c, all with
β-
aminoacetamide
substituents
235.1288; 235.1298
257.1108; 257.1109
(M+Na)+
β-Me
3a9
NHCOCH2NH2
CH2OH
>500 µM (n=4)
234.1; 235.1
H
N
N
490 ± 210 µM
β-Me
β-Me
3a17
3a11
CH2OH
CH2OH
316.1; 317.1 317.1456; 317.1453
N
(n=4)
O
OH
N
O
276.1442; 276.1442
541.2; 542.3 298.1261; 298.1261
(M+Na)+
EIMS on
tris(benzyl
ether)
O
> 1 mM (n=2)
> 1 mM (n=2)
HN
O
312.1442; 312.1438
581.3; 582.3 334.1261; 334.1260
(M+Na)+
EIMS on
tris(benzyl
ether)
OMe
β-Me
HN
3a14
CH2OH
270.1336; 270.1332
269.1; 270.1 292.1155; 292.1157
(M+Na)+
β-4-OMePh
β-4-OMePh
β-4-OMePh
β-4-OMePh
β-4-OMePh
β-4-OMePh
NH2
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
> 1 mM (n=2)
312.1442; 312.1445
334.1261; 334.1265
(M+Na)+
202 ± 58.5 µM
3b1
3b6
3b11
3b4
3b7
NHCOCH3
(n=3)
366.1159; 366.1157
9.8 ± 0.7 µM
NHCOCF3
365.1; 366.1
388.0978; 388.0979
(M+Na)+
(n=4)
O
368.1704; 368.1708
367.2; 368.1 390.1523; 390.1528
(M+Na)+
181 ± 4.0 µM
O
HN
(n=4)
354.1911; 354.1910
353.2; 354.2 376.1731; 376.1732
(M+Na)+
182 ± 27.5 µM
NHCOtBu
(n=4)
370.1860; 370.1868
369.2; 392.2
ca. 250 µM
NHBoc
392.1680; 392.1678
(M+Na)+
(n=2)
(M+Na)+
O
347 ± 113 µM
β-4-OMePh
β-4-OMePh
HN
N
3b16
3b15
CH2OH
CH2OH
(n=4)
O
ca. 360 µM
HN
(n=2)
CN
374.1598; 374.1601
373.2; 374.1 396.1418; 396.1421
(M+Na)+
ca. 380 µM
β-4-OMePh
β-4-OMePh
β-4-OMePh
3b12
3b2
NHCOPh
NHCOnPr
NHCOiPr
CH2OH
CH2OH
CH2OH
(n=2)
340.1755; 340.1759
339.2; 340.1 362.1574; 362.1578
(M+Na)+
ca. 730 µM
(n=2)
340.1755; 340.1752
339.2; 340.1 362.1574; 362.1574
(M+Na)+
3b3
>1 mM (n=2)
page S3

R1
R1
R1
OH
HRMS
R2
OH
R2
Kd (n = # SPR
replicate
measurements)
R2
EIMS m/z
calc; obs a
O
calc; obs
O
O
notes
Cpd.
ID
(M+H)+ unless
otherwise noted
R3
R3
OH
R3
OH
OH
OH
O
404.1704; 404.1709
426.1523; 426.1526
(M+Na)+
HN
β-4-OMePh
β-4-OMePh
3b13
3b14
CH2OH
CH2OH
>1 mM (n=2)
>1 mM (n=2)
OMe
O
OMe
HN
274.1034; 274.1033
296.0853; 296.0860
(M+Na)+
N
N
24.2 ± 4.8 µM
β-Me
β-Me
β-Me
N
4a19
4a14
4a15
CH2OH
CH2OH
CH2OH
273.1; 274.1
CO2H
(n=4)
422.1769; 422.1779
421.2; 422.1 444.1589; 444.1594
(M+Na)+
N
N
N
O
OH
OH
O
113 ± 6.5 µM
HO
(n=4)
OH
260.1241; 260.1238
282.1060; 282.1058
(M+Na)+
N
N
134 ± 26.3 µM
OH
N
N
N
(n=8)
α-Ar analogue
4b12 binds
better (40 µM)
N
N
N
N
ca. 200 µM
β-Me
β-Me
β-Me
β-Me
N
4a12
4a4
CH2OH
CH2OH
CH2OH
CH2OH
(n=2)
NH2
ca. 212 µM
N
320.2; 321.1
N
(n=2)
O
378.1772; 378.1771
400.1591; 400.1596
(M+Na)+
ca. 300 µM
(n=2) (slow
kinetics?)
HN
N
N
4a13
4a16
N
N
Me
N
N
276.1666; 276.1667
(M + NH4)+
ca. 600 µM
N
(n=2)
NH2
N
363.1663; 363.1675
385.1482; 385.1490
(M+Na)+
EIMS on
tris(benzyl
ether)
N
ca. 620 µM
N
Ph
β-Me
β-Me
β-Me
4a20
4a17
4a21
CH2OH
CH2OH
CH2OH
632.3; 633.3
HN
(n=2)
O
N
N
N
> 0.7 mM (n=2)
NHMe
α-Ar analogue
binds better (93
µM)
N
N
323.1326; 323.1327
(M+Na)+
ca. 840 µM
N
NHAc
(n=2)
287.1714; 287.1712
309.1533; 309.1533
(M+Na)+
α-Ar analogue
binds much
better (95 µM)
N
N
N
286.2; 287.1
β-Me
β-Me
β-Me
β-Me
β-Me
β-Me
4a18
4a2
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
> 1 mM (n=2)
> 1 mM (n=2)
> 1 mM (n=2)
> 1 mM (n=2)
> 1 mM (n=2)
> 1 mM (n=2)
NMe2
336.1554; 336.1554
358.1373; 358.1374
(M+Na)+
N
N
N
OMe
335.2; 336.1
386.1710; 386.1714
385.2; 386.1 408.1530; 408.1532
(M+Na)+
N
N
N
4a7
OMe
398.1710; 398.1714
397.2; 398.1 420.1530; 420.1532
(M+Na)+
N
N
Ph
4a8
N
O
N
O
N
N
4a9
417.1769; 417.1774
N
MeO
N
310.1510; 310.1508
309.1; 310.1 332.1329; 332.1323
(M+Na)+
N
N
N
4a11
N
Me
page S4

R1
R1
R1
OH
HRMS
R2
OH
R2
Kd (n = # SPR
replicate
measurements)
R2
EIMS m/z
calc; obs a
O
calc; obs
O
O
notes
Cpd.
ID
(M+H)+ unless
otherwise noted
R3
R3
OH
R3
OH
OH
OH
306.1448; 306.1450
305.1; 306.1 328.1268; 328.1268
(M+Na)+
N
N
N
β-Me
β-Me
4a1
4a3
CH2OH
CH2OH
slow kinetics
slow kinetics
336.1554; 336.1542
335.2; 336.1 358.1373; 358.1376
(M+Na)+
OMe
N
N
N
N
N
N
β-Me
β-Me
4a5
CH2OH
CH2OH
slow kinetics
slow kinetics
348.2; 349.1
NMe2
HO
N
N
4a10
N
453.1881; 453.1886
452.2; 453.1 475.1700; 475.1702
(M+Na)+
N
N
N
N
β-4-OMePh
β-4-OMePh
β-4-OMePh
β-4-OMePh
β-4-OMePh
β-4-OMePh
β-4-OMePh
β-4-OMePh
4b12
4b14
4b21
4b18
4b15
4b16
4b20
4b6
N
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
39.7 ± 5.4 (n=8)
42.3 ± 5.2 (n=6)
93.3 ± 4.4 (n=6)
N
514.2031; 514.2033
N
N
O
OH
OH
O
N
513.2; 514.2
392.2; 393.1
536.1851; 536.1855
(M+Na)+
HO
OH
393.1769; 393.1769
415.1588; 415.1588
(M+Na)+
H
N
N
N
N
O
379.1976; 379.1979
378.2; 379.2 401.1795; 401.1799
(M+Na)+
N
N
94.5 ± 24.5
(n=4)
NMe2
N
352.1503; 352.1506
351.2; 352.1 374.1323; 374.1320
(M+Na)+
α-Ar analogue
binds better
(134 µM)
N
N
ca. 300 µM
OH
N
(n=4)
EIMS on
tris(benzyl
ether)
N
N
ca. 600 µM
NH2
N
620.3; 621.3
(n=2)
O
455.1925; 455.1930
454.2; 455.1 477.1745; 477.1748
(M+Na)+
HN
ca. 640 µM
N
N
(n=2)
N
399.1663; 399.1666
N
N
N
N
> 1 mM (n=2)
398.2; 399.1
397.2; 398.2
421.1482; 421.1485
(M+Na)+
398.1710; 398.1721
420.1530; 420.1530
(M+Na)+
N
N
N
β-4-OMePh
β-4-OMePh
β-4-OMePh
4b1
4b3
CH2OH
CH2OH
CH2OH
> 1 mM (n=2)
> 1 mM (n=2)
> 1 mM (n=2)
poor solubility
poor solubility
OMe
N
N
N
N
402.1772; 402.1777
401.2; 402.1 424.1591; 424.1593
(M+Na)+
N
N
N
N
4b11
Me
α-Me analogue
binds much
better (24 µM);
EIMS on
tris(benzyl
ether)
N
N
β-4-OMePh
β-4-OMePh
N
4b19
4b7
CH2OH
CH2OH
>1 mM
635.3; 636.2 366.1296; 366.1303
477.2; 478.2 478.1973; 478.1975
CO2H
N
N
N
slow kinetics
poor solubility
OMe
page S5

R1
R1
R1
OH
HRMS
R2
OH
R2
Kd (n = # SPR
replicate
measurements)
R2
EIMS m/z
calc; obs a
O
calc; obs
O
O
notes
Cpd.
ID
(M+H)+ unless
otherwise noted
R3
R3
OH
R3
OH
OH
OH
262.1285; 262.1285
301.2; 302.2 284.1105; 284.1109
(M+Na)+
EIMS on
C3,C4-
acetonide
10.7 ± 1.9 µM
α-O-allyl
α-O-allyl
α-O-allyl
9
NHCOCH3
NHCOCH3
NHCOCH3
CH2OH
CH2N3
CCH
(n=6)
1.6 ± 0.60 µM
11
10
286.1; 287.2 287.1350 287.1354
(n=6)
256.1179; 256.1182
295.1; 296.4 278.0999; 278.1000
(M+Na)+
EIMS on
C3,C4-
acetonide
44.0 ± 4.3 µM
(n=4)
kon = 4.3 x 104
MeO
O
NHBn
1.2 ± 0.6 µM
M-1s-1
!-
!-
17
16
NHCOCF3
NHCOCH3
581.2; 582.2 582.2170; 582.2179
334.2; 335.2 335.1561; 335.1569
N
N
(n=6)
koff = 0.05 s-1
N
OH
O
OMe
6.0 ± 0.45 µM
CH2N3
(n=4)
OH
414.1772; 414.1768
453.2; 454.2 436.1591; 436.1590
(M+Na)+
405.1769; 405.1771
404.2; 405.2 427.1588; 427.1592
(M+Na)+
EIMS on
C3,C4-
acetonide
15.8 ± 0.6 µM
N
N
α-O-allyl
α-O-allyl
α-O-allyl
α-O-allyl
6e
6d
6f
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
NC
N
(n=4)
MeO
22.0 ± 9.9 µM
N
(n=8)
N
N
F
485.0830; 485.0835
524.1;
EIMS on
C3,C4-
acetonide
25.0 ± 1.0 µM
507.0650; 507.0654
525.1/527.1
N
(n=4)
N
Br
N
(M+Na)+
EIMS on
C3,C4-
acetonide
EtO2
C
25.9 ± 2.3 µM
6c
N
486.2; 487.2
(n=4)
N
N
MeO
O
OMe
29.3 ± 12.1 µM
!-
13
6g
6b
NHCOCH3
NHCOCH3
NHCOCH3
452.2; 453.3 451.1834; 451.1822
N
(n=4)
N
N
OH
403.1976; 403.1982
402.2; 403.2 425.1795; 425.1802
(M+Na)+
34.2 ± 6.3 µM
N
α-O-allyl
α-O-allyl
Me
N
N
(n=8)
453.0768; 453.0767
492.1;
EIMS on
C3,C4-
acetonide
Br
34.4 ± 1.6 µM
N
475.0588; 475.0586
493.1/495.1
(n=4)
N
(M+Na)+
N
Cl
N
N
41.5 ± 3.0 µM
N
α-O-allyl
6j
NHCOCH3
477.2; 478.1 478.1600; 478.1593
(n=4)
N
N
N
Me
457.1693; 457.1693
496.2; 497.2 479.1513; 479.1515
(M+Na)+
EIMS on
C3,C4-
acetonide
41.3 ± 0.8 µM
N
N
α-O-allyl
α-O-allyl
6h
6i
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
F3C
(n=4)
N
390.1772; 390.1778
N
61.0 ± 14.0 µM
N
389.2; 390.1
412.1591; 412.1597
(M+Na)+
N
N
(n=4)
528.2453; 528.2451
527.2; 528.3 550.2272; 550.2267
(M+Na)+
MeO
O
NHBn
66.2 ± 29.9 µM
!-
14
6a
N
(n=4)
N
N
OH
EIMS on
C3,C4-
acetonide
104.7 ± 12.4
µM (n=6)
Me
α-O-allyl
N
352.2; 353.1 313.1506; 313.1519
N
N
page S6

R1
R1
R1
OH
HRMS
R2
OH
R2
Kd (n = # SPR
replicate
measurements)
R2
EIMS m/z
calc; obs a
O
calc; obs
O
O
notes
Cpd.
ID
(M+H)+ unless
otherwise noted
R3
R3
OH
R3
OH
OH
OH
data suggests
additional weak
binding mode at 541.3; 542.3 564.2429; 564.2431
MeO
542.2609; 542.2611
O
NHBn
N
0.69 ± 0.15 µM
N
N
!-
15
NHCOCH3
(n=5)
(M+Na)+
high
OH
concentration
O
N
N
3.2 ± 2.0 µM
N
α-O-allyl
α-O-allyl
8i
8j
NHCOCH3
NHCOCH3
483.2; 484.2 484.2191; 484.2202
N
(n=14)
O
NH
N
6.6 ± 2.5 µM
N
N
451.2; 452.2 452.1598; 452.1606
S
(n=12)
MeO
N
8.2 ± 0.7 µM
N
N
α-O-allyl
α-O-allyl
α-O-allyl
8e
8b
8h
NHCOCH3
NHCOCH3
NHCOCH3
418.2; 419.2 419.1925; 419.1935
388.2; 389.2 389.1819; 389.1829
(n=12)
N
N
8.5 ± 1.4 µM
N
(n=6)
Me
N
N
N
9.0 ± 4.1 µM
392.2; 393.2 393.1881; 393.1890
419.1925; 419.1928
N
N
(n=14)
N
N
11.2 ± 1.0 µM
MeO
α-O-allyl
α-O-allyl
8c
NHCOCH3
NHCOCH3
418.2; 419.2
N
441.1745; 441.1747
(M+Na)+
(n=6)
evidence of
nonspecific
binding at high
concentration
N
N
N
Me2
N
8d
approx. 12 µM
431.2; 432.2
390.1772; 390.1779
412.1591; 412.1595
(M+Na)+
N
N
N
12.3 ± 3.5 µM
α-O-allyl
α-O-allyl
α-O-allyl
8g
8a
8f
NHCOCH3
NHCOCH3
NHCOCH3
389.2; 390.1
342.2; 343.1
480.2; 481.2
N
(n=10)
343.1612; 343.1616
365.1432; 365.1431
(M+Na)+
HO
N
N
14.9 ± 6.9 µM
N
(n=10)
481.2082; 481.2686
503.1901; 503.1903
(M+Na)+
N
N
N
18.6 ± 7.1 µM
Ph
O
(n=10)
(a) Electrospray ionization mass spectrometry; calculated values for the uncharged compound; parent observed
ions were [M+H]+ unless otherwise indicated. (b) Stokmaier, D.; Khorev, O.; Cutting, B.; Born, R.; Ricklin, D.; Ernst,
T. O. G.; Boni, F.; Schwingruber, K.; Gentner, M.; Wittwer, M.; Spreafico, M.; Vedani, A.; Rabbani, S.; Schwardt, O.;
Ernst, B. Bioorg. Med. Chem. 2009, 17, 7254-7264. (c) Riva, C., Targeting the liver via the asialoglycopro-tein-
receptor: synthesis of directed small molecule libraries for the H1-CRD. Ph.D. Thesis, University of Basel, 2006.
page S7

B. General. Solvents THF, acetonitrile, diethyl ether, dichloromethane, and toluene were dried
by passage through activated alumina columns.1 Chromatography was performed using SINGLE
StEP pre-packed MPLC columns. Reaction progress was monitored by TLC using silica gel 60 F-
254 with UV detection. Silica gel 60 (40–63 μm) was used for column chromatography.
Solutions of phosphomolybdic acid or anisaldehyde/EtOH were used in addition to UV light with
fluorescent TLC plates. Reactions requiring anhydrous conditions were performed under
nitrogen. 1H and 13C NMR spectra were recorded on Bruker DRX-600 equipped with a 5mm DCH
cryoprobe, Bruker DRX-500, Varian Mercury-300 or Varian Mercury-200 MHz spectrometers in
CDCl3, CD3OD, or DMSO-d6 solvent, as indicated. NMR data are presented as follows: chemical
shift (ppm), multiplicity (s = singlet, d = doublet, t= triplet, q = quartet, quin = quintet, sep =
septet, m = multiplet, b = broad), coupling constant (Hz) and integration. Routine mass spectra
(LCMS) were obtained using an Agilent 1100 (ESI MSD) with mobile phase composed of 9:1
CH3CN:H2O containing 0.1% CF3CO2H.
High resolution mass spectra data were gathered on an Agilent 1100 LC with MSD TOF
(Agilent model G1969A) mass spec detector running with an electrospray ionization source. The
instrument acquisition and data handling was done with Agilent MassHunter TOF/Q-TOF B.02
(B11285) Patches 1.2.3 software. Samples were analyzed using direct flow injection (no
chromatography column), with the following parameters: injection volume = 1.0 µL; flow rate =
0.5 mL/min, run time = 1.0 min, solvent = methanol containing 0.1% formic acid and 0.05%
ammonium formate. The time-of-flight analyzer used electrospray ionization in positive mode,
gas temperature = 325 °C, drying gas = 6 L/min, nebulizer = 50 psg, VCap = 3500 V, mass range
was set to a width of 100 units centered on the expected m/z value, acquisition rate = 0.99
spectra/s, acquisition time = 1012.8 ms/spectrum.
C. Synthesis of 2-Azido-D-galactose Derivatives
To a solution of tri-O-acetyl-D-galactal (A, 5.0 g, 18.3 mmol) in MeOH (50 mL) was added
NH3/MeOH (8N, 3 mL). The reaction mixture was stirred at room temperature for 5 h. After
completion of the reaction, the solvent was removed under reduced pressure to give D-galactal2
as a solid (2.6 g, 97%). This material (2.5 g, 17.1 mmol) was taken up in dry DMF (20 mL), cooled
to 0 °C, and solid NaH was added (4 equiv). BnBr (6.75 mL, 56.5 mmol) was then added slowly
to the cooled solution. The reaction mixture was allowed to warm to room temperature with
stirring over 30 min. After reaching completion (assayed by TLC), cold water was added and the
mixture was extracted with diethyl ether. The organic layer was separated, dried (Na2SO4), and
the solvent was removed by rotary evaporation. Purification of the crude product by column
chromatography gave the tri-O-benzyl-D-galactal2 B as a colorless syrup (6.2-7.0 g, 87-98%).
1. (a) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.; Timmers, F. J. Organometallics 1996, 15, 1518-
1520. (b) Alaimo, P. J.; Peters, D. W.; Arnold, J.; Bergman, R. G. J. Chem. Ed. 2001, 78, 64.
2. Bovin, N. V.; Zurabyan, S. E.; Khorlin, A. Y. Carbohydr. Res. 1981, 98, 25-35
page S8

OH
1) NH3/MeOH
RT/5 h
THF-H2O (5:1)
Conc.HCl
O
O
PCC, CH2Cl2
O
AcO
BnO
OAc
BnO
OBn
RT, 16 h
85%
RT, 20 h
90%
2) BnBr/NaH
0 oC -RT/ 30 min
OBn
OAc
OBn
OBn
A
B
C
92% 2 steps
R1
O
O
1) RLi or RMgBr, toluene,
-78 oC, 1 h
KHMDS, TIBSN3
-78 oC, 3 h
N3
N3
O
O
O
BnO
BnO
BnO
2) Et3SiH, BF3.Et2O
MeCN, -40 oC
OBn
OBn
OBn
OBn
OBn
OBn
D
E
F
1) R1 = Me,
2) R1 = 4-OMePh
A solution of B (6.0 g, 14.4 mmol) in THF-H2O (2:1, 60 mL) was treated with 2N aq. HCl
(3.0 mL) and stirred at room temperature for 16 h. After completion of the reaction, CH2Cl2 (50
mL) was added and the mixture was washed with saturated aqueous solution of NaHCO3 and
water. The organic layer was separated, dried (Na2SO4), and evaporated to give tri-O-benzyl-D-
galactopyranoside3 (C) as a solid (5.7g, 92%).
To a solution of C (5.0 g, 11.5 mmol ) in CH2Cl2 was added pyridinium chlorochromate
(4.9 g, 23.0 mmol) in two portions. The reaction mixture was stirred at room temperature for
12 h, diluted with CH2Cl2 (50 mL), and filtered through a pad of celite. The solvent was removed
by rotary evaporation and the residue was purified by column chromatography
(EtOAc/hexanes) to give tri-O-benzyl-2-deoxy-D-galactono-1,5-lactone3 (D) as a colorless syrup
(4.2 g, 85%).
A solution of D (1.0 g, 2.3 mmol) in THF (15 mL) was cooled to -78 °C and a 15% w/w
solution of potassium hexamethyldisilazane (KHMDS) in toluene (6.0 mL, 4.6 mmol) was added
dropwise with vigorous stirring. After 15 min, a precooled solution of trisyl azide (1.4 g, 4.6
mmol) in THF (5.0 mL) was added dropwise. After another 5 min, AcOH (0.27 mL, 4.6 mmol)
was added. The cooling bath was then removed and the mixture was stirred at room
temperature overnight. After addition of H2O (30 mL), the mixture was extracted with CH2Cl2 (3
x 30 mL). The combined CH2Cl2 layers were dried and concentrated to give a crude product that
was purified by flash column chromatography (silica gel, 6:1 hexanes:EtOAc) to isolate Tri-O-
benzyl-2-azido-2-deoxy-D-galactono-1,5-lactone4 (E, 0.65 g, 60%) as a colorless oil.
A solution of E (0.6 g, 1.6 mmol) in toluene (15 mL) was cooled to -78 °C and 1.5 M MeLi
in ether (1.6 mL, 2.2 mmol) was added dropwise with vigorous stirring. The reaction mixture
was stirred for 1 h, and quenched by addition of saturated NH4Cl solution. The mixture was
extracted with ether (30 mL), and the organic layer was dried (Na2SO4) and evaporated to give
o
the crude lactol. This material was dissolved in dry MeCN (15 mL) and cooled to -40 C.
Triethylsilane (1.0 mL, 8.0 mmol) was added slowly, the resulting mixture was stirred for 5 min
o
at -40 C, and then BF3•Et2O (1.0 mL, 9.6 mmol) was added slowly. The mixture was stirred
while warming to room temperature. Saturated NaHCO3 was added and the mixture was
3. Dupradeau, F.-Y.; Hakornoriar, S.-I.; Toyokuni, T. J. Chem. Soc. Chem. Commun. 1995, 221-222.
4. Dileep Kumar, J. S.; Dupradeau, F. -Y.; Strouse, M. J.; Phelps, M. E.; Tatsushi Toyokuni, T. J. Org. Chem. 2001, 66,
3220-3223.
page S9

extracted into CH2Cl2 (30 mL). The organic layer was separated, washed with water, dried
(Na2SO4), and evaporated. Purification by flash column chromatography afforded methyl-tri-O-
benzyl-2-azido-2-deoxy-D-galactopyranoside5 (F1, 0.39 g, 65%) as a syrup.
The analogous procedure was performed with E (0.6 g, 1.6 mmol) in THF (20 mL) and 1.0
M p-OMePhMgBr in THF (2.5 mL, 2.2 mmol), followed by triethylsilane reduction as above. The
product p-methoxyphenyl-tri-O-benzyl-2-azido-2-deoxy-D-galactopyranoside (F2, 0.5 g, 72%)
was obtained as a syrup.
Following CuAAC reactions as described below to give precursors to triazoles 4a and 4b,
O-benzyl deprotection was accomplished by the following general procedure. To a solution of
tri-O-benzyl-D-galactose derivative (0.1 mmol) in MeOH (10 mL) was added 20% Pd(OH)2 on
carbon (350 mg/mmol, 0.1 g) and ammonium formate (100 mg/0.2 mmol, 0.15 g). The reaction
mixture was stirred at reflux temperature for 3 h. After completion of the reaction, the mixture
was filtered through a pad of Celite, and the fitrate was concentrated under vacuum to isolate
the desired product.
D. Synthesis of Allyl-D-Galactosamine Derivatives
OH
OH
OPiv
OPiv
OPiv
Pivaloyl chloride
Pyridine, 0 o
1) Tf2O, pyridine
2) H2O, 90 o
C
1) Ac2O, NaOMe,
MeOH, RT
O
OH
O
O
O
O
O
O
O
O
C
+
2) allyl alcohol,
BF3.Et2O
reflux
HO
NH3Cl
HO
NHAc
PivO
NHAc
HO
NHAc
HO
NHAc
OH
OH
OH
OPiv
OPiv
I
G
H
J1
J2
MeO
OH
N3
O
OH
O
TsCl, CH2Cl2
Et3N, RT, 3h
2,2-dimethoxy-
propane
O
O
O
O
O
O
NaOMe, MeOH
12 h
O
O
O
O
+
p-toluene-
sulfonic acid (cat)
O
NHAc
NaN3, DMF
100 oC, 5 days
NHAc
HO
NHAc
O
NHAc
OH
O
7
5
K
L1
L2
i) Swern
ii) Bestmann-Ohira
coupling
O
O
50% aq. AcOH:H2O
RT, 1 h
O
NHAc
O
[Adapted from Carbohydr. Res. 1999, 321, 176]
To a slurry of D-glucosamine hydrochloride (10 g, 46.3 mmol) in MeOH (100 mL) was
added 1.2 equiv. of NaOMe (solid). The mixture was filtered to remove solid NaCl and acetic
anhydride (1.1 equiv.) was added. The reaction was stirred at room temperature for 2 h,
monitored by TLC (15% MeOH/CH2Cl2). After completion, the solvent was removed under
reduced pressure to give N-acetyl-D-glucosamine6 as an off-white solid (8.5 g, 85%)
A suspension of this compound (10.0 g, 45.2 mmol) in 100 mL allyl alcohol was treated
with BF3•Et2O (2.0 mL, 15.9 mmol) and the mixture was heated at reflux under a drying tube.
After 2 h a clear solution was obtained, and the mixture was cooled and the solvent removed
under vacuum. The residue was recrystallized in EtOH/ether to give allyl-N-acetyl-α-D-
glucosamine7 (H) as a solid (9.0-10.0 g, 76-85%).
5. Ayadi, E.; Stanislas Czernecki, S.; Xie, J. Chem. Commun. 1996, 347-348.
6. Liang, H.; Grindley, T. B. J. Carbohydr. Chem. 2004, 23, 71-82
7. Wong, T. C.; Townsend, R. R.; Lee, Y. C. Carbohydr. Res. 1987, 170, 27-46
page S10

A solution of compound H (10.0 g, 38.3 mmol) in pyridine (100 mL) was cooled to -15 °C
and treated with pivaloyl chloride (11.2 mL, 91.9 mmol). The reaction mixture was stirred at
room temperature for 12h. After completion of the reaction (monitored by TLC), solvent was
removed and the residue was dissolved in CH2Cl2 (200 mL). The solution was washed with 1N
HCl (50 mL), H2O (100 mL), saturated NaHCO3 (100 mL), and H2O (100 mL). The organic layer
was separated, dried (Na2SO4) and concentrated to give a crude product which was purified by
flash column chromatography (EtOAc/hexanes) to give allyl-N-acetyl-3,6-O-pivaloyl-α-D-
glucosamine8,9 (I) as a white solid (12 g, 73%).
A solution of I (10.0 g, 23.3 mmol) in CH2Cl2/pyridine (1:1) was cooled to -15 °C and
treated with triflic anhydride (4.7 mL, 27.9 mmol). The reaction mixture was stirred at room
temperature for 1 h. After completion of the reaction (TLC), H2O (4.2 mL) was added and the
mixture was refluxed for 2 h. The mixture was then cooled, diluted with CH2Cl2, and washed
with 1N HCl, sat. NaHCO3, and H2O in succession. The organic layer was separated, dried
(Na2SO4) and removed by rotary evaporation. The crude product was purified by flash column
chromatography to give an uncharacterized mixture of J1 and J2 as an off-white solid (6.0-7.0 g,
60-70%).
A solution of J (6.0 g, 13.9 mmol) in MeOH (30 mL) was treated with NaOMe (1.8 g, 34.7
mmol) and was stirred at room temperature for 12 h. After completion, the reaction was
neutralized with IR-120 (H+) resin, filtered, and the resin washed with MeOH (30 mL). The
solvent was removed under reduced pressure to give allyl-N-acetyl-α-D-galactosamine9 (K) as a
white solid, which was further purified by silica gel chromatography (elution with
MeOH/CH2Cl2) (3.6 g, 99%).
To a mixture of K (3.0 g, 11.5 mmol) and 2,2-dimethoxypropane (20 mL) was added p-
toluenesulfonic acid (PTSA) (0.2 g). The reaction mixture was stirred at room temperature for 5
h. After the starting material was consumed, the reaction was neutralized with triethylamine
and the solvent was removed. The crude mixture was purified by flash column chromatography
(EtOAc/hexanes/CH2Cl2) to give a mixture of compounds L1 and L2. To this mixture in MeOH (20
mL) was added 50% aq. AcOH (10 mL) and the mixture was stirred at room temperature for 1 h.
After disappearance of the top spot on TLC, the reaction was neutralized with Na2CO3 and the
solvent was removed by rotary evaporation. The residue was dissolved in EtOAc, washed with
water, and the organic layer was separated, dried (Na2SO4) and concentrated. The crude
product was purified by flash column chromatography (EtOAc/hexanes/CH2Cl2) to give allyl-N-
acetyl-3,6-isopropylidene-α-D-galactosamine9 (L2) as an off-white solid (2.5, 73%).
In an alternative procedure, compound K (1.63 mmol) was suspended in 7.5 mL of
anhydrous DMF and treated with 2,2-dimethoxypropane (5 mL, 40.6 mmol) and
camphorsulfonic acid (0.38 mmol, 0.23 equiv). The mixture was stirred for 3 h at room
temperature. After completion of reaction as monitored by TLC, the reaction mixture was
neutralized with triethylamine, filtered and concentrated under vacuum at 60°C. The product
was purified by column chromatography as described above to obtain L2 (43 % yield).
A solution of oxalyl chloride (0.23 mL, 2.64 mmol) in dry CH2Cl2 (10 mL) was cooled to
-78 °C under a nitrogen atmosphere. Dry DMSO (0.37 mL, 5.28 mmol) was slowly added, and
8. Ljevakovi, D.; Tomic, S.; Tomasic, J. Carbohydr. Res. 1988, 182, 197-205
9. a) Rye, C. S.; Withers, S. G. J. Am. Chem. Soc. 2002, 124, 9756-9767. b) Cipolla, L.; Ferla, B. L.; Lay, L.; Peri, F.;
Nicotra, F. Tetrahedron: Asymmetry 2000, 11, 295–303
page S11

the reaction mixture was stirred for 10 min. A solution of L2 (0.4 g, 1.32 mmol) in CH2Cl2 was
then added by syringe to the cooled solution. After stirring for 15 min, Et3N (0.74 mL, 5.28
mmol) was added and the reaction mixture was allowed to warm slowly to room temperature
and was stirred for 30 min. The reaction was quenched by dilution with CH2Cl2 (20 mL) and
washing with water (20 mL) . The organic layer was separated, dried (Na2SO4) and concentrated
to give the crude intermediate aldehyde (0.38 g). This compound was dissolved in MeOH (10
mL), treated with K2CO3 (0.35 g, 2.5 mmol), and stirred for 10 min. at RT. The Bestmann-Ohira10
reagent (dimethyl-1-diazo-2-oxopropylphosphonate, 0.36 g, 1.9 mmol) was then added and the
reaction was stirred for 2h. After completion, the mixture was dried (Na2SO4), concentrated and
the residue purified by flash column chromatography to give 5 as a white solid (0.27 g, 73%).
To a solution of compound L2 (0.3 g, 1.0 mmol) in dry CH2Cl2 (10 mL) was added
triethylamine (0.2 mL, 1.5 mmol), and the reaction mixture was cooled to 0 °C. A solution of p-
toluenesulfonyl chloride (TsCl, 0.2 g, 1.1 mmol, in CH2Cl2) was added; the reaction mixture was
allowed to warm to room temperature and was stirred for 3 h. After completion of the
reaction, it was diluted with CH2Cl2 and washed with water. The organic layer was separated,
dried, and concentrated to give the 6-tosylated intermediate (0.40 g, 90%). The tosylate (0.14
mmol) was dissolved in 1.3 mL of DMSO, and NaN3 (10 equiv) dissolved in 0.2 mL of water was
added. The reaction mixture was stirred at 100 °C for 4 days, and was then diluted with EtOAc
(30 mL) and washed with water (20 mL). The organic layer was separated, dried (Na2SO4), and
concentrated. The residue was purified by column chromatography (EtOAc/hexanes) to give
allyl-N-acetyl-6-azido-3,6-O-isopropylidene-α-D-galactosamine (81% yield).
E. Synthesis of N-Trifluoroacetamide-D-Galactosamine Derivatives
OMe
O
O
O
P(OMe)2
O
O
O
O
HO
O
O
O
N
Swern
H
mCPBA
N2
CuAAC
N
O
O
O
O
NHCbz
O
NHCbz
N
O
NHCbz
0-4 °C
O
O
K2CO3
CH2Cl2, 20 h
O
NHCbz
MeOH, 2h
O
P
Q
R
OMe
OMe
OMe
O
O
N
N
N
BnNH2
LiClO4
(Boc)2O
OH
O
OH
10% Pd/C
O
N
N
N
O
O
O
O
NHCH2Ph
O
O
O
N
Ph
N
N
N
H2, 5h
MeOH
Et3N
20 h
MeCN, 70 °C
O
NHCbz
O
NHCbz
O
NHCbz
O
S
T
U
OMe
OMe
OMe
CF3
1) 1 equiv KOH/ THF
0 C, 15 min
N
N
N
15 equiv (F3CCO)2O
N
N
OH Boc
N
O
O
Boc
N
OH
H
N
N
N
O
O
Ph
O
O
O
Ph
O
O
Ph
N
N
10 equiv Et3N
20h
2) 2M HCl/Et2O/MeOH
0 °C, 1 hr
O
NH2
O
NH
HO
NH
CH2Cl2
O
HO
O
CF3
O
CF3
V
W
17
OMe
OMe
N
N
N
MeOH, acidic Dowex
65 °C, 16 h
N
OH
O
O
O
O
N
O
O
OMe
N
O
NHCOCH3
HO
NHCOCH3
OH
X
Y
10. (a) Roth, G. J.; Liepold, B.; Stephan G. Müller, S. G.; Bestmann, H, J. Synlett. 1996, 521-522. (b) Roth, G. J.;
Liepold, B.; Stephan G. Müller, S. G.; Bestmann, H, J. Synthesis 2004, 59-61
page S12

[Note: the procedures described here for epoxidation and ring opening are
representative of those used for the preparation of 14-16, as well as 17.] N-Cbz protected
alcohol O11 was converted to aldehyde P by Swern oxidation (isolated yield of 83%). Aldehyde P
was converted to alkyne Q using the Bestmann-Ohira procedure as described in the preceding
section (see the preparation of 5) in 66% yield. Alkyne Q was then clicked with 3-ethynylanisole
by standard CuAAC reaction conditions as described below to generate the triazole R.
Compound R (130 mg, 0.24 mmol) was dissolved in dry CH2Cl2 and kept on ice. To this
solution was added 3-chloroperoxybenzoic acid (70-75%, 2.6 equiv, 150 mg) and the mixture
was stirred at 0-4 ⁰C for 20 h. The reaction was judged to have gone to completion by TLC, and
the mixture was washed with saturated aqueous NaHCO3. The combined organic layers were
dried with MgSO4, and the solvent was removed by rotary evaporation. The resulting epoxide
was purified by column chromatography, eluting with EtOAc/hexane/CH2Cl2) to give the
diastereomeric mixture of epoxides S in 65% yield.
For epoxide opening by methanol (for the synthesis of 13 and 16), the following
procedure is representative. Epoxide X (25 mg, 0.042 mmol) was dissolved in 1.5 mL anhydrous
methanol containing 20 mg of Dowex 50W X8 -200 mesh (ion exchange resin, acidic) and the
mixture was heated at 65 °C for 16 h. Filtration, washing, and concentration provided the
acetonide-deprotected intermediate Y in quantitative yield.
For epoxide opening by benzylamine: comound S (35 mg, 0.06 mmol) was dissolved in 2
mL dry acetonitrile. To this solution was added LiClO4 (1 equiv) and benzylamine (10 equiv, 0.08
mL), and the mixture was heated at reflux (70 ⁰C bath temperature) for 20 h. The reaction
mixture was cooled, evaporated to dryness, redissolved in CH2Cl2, and washed with 0.1 N HCl.
The organic layers were dried and purified by column chromatography (eluting with
CH2Cl2/MeOH, to give amine T as a diastereomic mixture in 92% yield.
Compound T (0.06 mmol) was dissolved in 5 mL of dry CH2Cl2 and was treated with di-
tert-butyldicarbonate (0.36 mmol) and triethylamine (0.3 mmol). The mixture was stirred for 20
h, and then was washed with water and back-extracted with CH2Cl2. The combined organic
layers were dried and the product purified by column chromatography (gradient elution with
CH2Cl2/MeOH to give U in 98% yield. (The crude material was occasionally contaminated with
small amounts of what appears to be the O-tert-butyl ether.) Removal of the
benzyloxycarbamate group in compound U (0.07 mmol) was accomplished by hydrogenation (1
atm) in the presence of catalytic (15% w/w) 10% Pd/C in methanol for 5 h. Compuond V (80%
yield) was isolated by filtration of the reaction mixture through Celite and evaporation of the
solvent, and was used without further purification.
Compound V was converted to the bis(trifluroacetamide) derivative by reacting with 15
equiv trifluoroacetic anhydride and 10 equiv triethylamine in CH2Cl2 for 20 h. After completion
of the reaction the products were extracted from water with CH2Cl2, and the organic layer was
dried, evaporated, and the product purified by column chromatography (elution with MeOH
and CH2Cl2) in 95% isolated yield. Hydrolysis of the trifluoroacetamide ester to the
corresponding monotrifluroacetamide was performed by treatment with 1 equiv. KOH on ice
for 15 min, giving quantitative yield. The resulting intermediate was deprotected (both the tert-
11. See Fukuda, Y.; Sasai, H.; Suami, T. Bull. Chem. Soc. Jpn.1982, 55, 1574-1578, for preparation of the analogous
OMe glycoside. The same procedures were used to prepare compound O.
page S13

butylcarbamate and acetonide) by treatment with 2 M HCl in 1:1 Et2O:MeOH to give the final
trifluoroacetamide derivative 17 in 95 % yield.
F. General Procedure for Click Chemistry
Using solution-phase catalyst. To a solution of alkyne (0.58 mmol, 1 equiv) and azide (1.2
equiv) in MeOH (2.0 mL) was added THPTA12 or TBTA13 (5 mol%), CuSO4 (1 mol %) in a small
amount of water, and sodium ascorbate (10 mol%) in a small amount of water. The reaction
was vigorously stirred for 5 h at room temperature, and was then diluted with CH2Cl2 (10 mL)
and water (10 mL). The organic phase was separated and the aqueous phase was further
extracted with CH2Cl2 (2 x 10 mL). The combined organic phases were dried over sodium
sulfate, concentrated by rotary evaporation, and the residue was purified by chromatography
over a short silica gel column to isolate the desired product.
Using a resin-bound catalyst. The strongly-binding BimPy2 ligand reported earlier14 was
immobilized on a flexible tether to a NovaPEG resin. The preparation and properties of this
ligand are described separately.15 To the NovaPEG-BimPy2 resin (5 mol% ligand with respect to
limiting azide or alkyne reagent) was added MeoH, 5 mol% aq. CuSO4, and alkyne or azide (0.05
mmol). The mixture was shaken for a minute, followed by addition of azide or alkyne (0.06
mmol) and 10 mol% aqueous sodium ascorbate solution. The mixture was sealed and was
tumbled gently at room temperature throughout the reaction period. The reaction was
monitored by TLC and after completion; the resin was filtered, washed with MeOH. The solvent
was removed under reduced pressure to give the crude compound. The crude residue was
purified by column chromatography to afford the triazole derivatives (90-95% yields).
G. General procedure for O-benzyl deprotection10
To a solution of tri-O-benzyl-D-galactose derivatives (1 mmol) in MeOH (10 mL) was
added 20% Pd(OH)2 (10% w/w) and ammonium formate (0.3 g). The reaction mixture was
stirred at reflux temperature for 2 h. After completion of the reaction, the mixture was filtered
through a pad of Celite, and the fitrate was concentrated under vacuum to isolate the desired
product.
H. Surface Plasmon Resonance measurements
Preparation of biotinylated ASGPR. ASGPr H1 wild type was expressed as an insoluble inclusion
body (IB) in E. coli. The cell pellet was re-suspended in TBS buffer pH 8.0 with 0.1 mg/mL
lysozyme + 25 µg/mL DNase + 10 µg/mL RNase + 10 mM MgCl2 and stirred on ice until well
blended. The mixture was passed through a microfluidizer twice and centrifuged 30 minutes at
30,000 x g. The resultant IB pellet was washed with TBS buffer pH 8.0 + 3M urea and
centrifuged and this process was repeated three additional times with water. The IB pellet was
12. Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Angew. Chem. Int. Ed, 2009, 48, 9879-9883.
13. Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett. 2004, 6, 2853-2855.
14. Presolski, S.I.; Hong, V.; Cho, S.-H.; Finn, M.G. J. Am. Chem. Soc. 2010, 132, 14570-14576.
15. Presolski, S.I.; Manzenrieder, F.; Mamidyala, S.K.; Finn, M.G. 2011, submitted.
page S14

solubilized in 10 mM Tris + 8M urea + 100 mM β-mercaptoethanol (pH 8.5), stirred for
approximately 20 minutes, then centrifuged to discard any precipitate. For the following
manipulations, “buffer T” refers to 20 mM Tris + 0.5 M NaCl + 25 mM CaCl2. The supernatant
was diluted to approximately 0.5 mg/mL in buffer T + 2mM β-mercaptoethanol + 8M urea (pH
8.0), then dialysed vs. 8-10x excess volume of buffer T + 2mM β-mercaptoethanol + 2 M urea
(pH 8.0) overnight at 4°C. Dialysis was repeated in buffer T + 1mM β-mercaptoethanol + 1 M
urea (pH 8.0) for approximately 24 h at 4°C with several volume changes with a final dialysis vs.
buffer T + 5 mM GSH + 1 mM GSSG (pH 7.5) with 3 volume changes. The resulting sample was
centrifuged and the supernatant was loaded onto an N-acetyl-D-galactosamine agarose packed
Pharmacia XK 26 column, equilibrated in buffer T + 2mM TCEP (pH 8.0). The column was
washed with this buffer until baseline was re-established. The bound protein was eluted with
20 mM Tris + 0.5 M NaCl + 2 mM TCEP + 2 mM EDTA (pH 8.0). The final yield was approximately
50 mg from 5 L (70 wgm.) E.coli cell pellet.
While BSA has one cysteine (Cys34) not engaged in disulfide bonding, this thiol group is
often blocked as a mixed disulfide with small thiols such as cysteine, or as the sulfenic acid by
aerobic oxidation.16 Disulfide reduction and labeling were found to be the cleanest method for
derivatization of the ASGPR binding domain among those that we tested. Mild disulfide
reduction conditions were chosen so as not to disrupt buried intra-strand (solvent inaccessible)
crosslinks. Thus, immediately prior to biotinylation, the ASGPR sample was first incubated with
1 mM TCEP to ensure that the sample contained a single free thiol. The protein was reacted
with a 19-fold molar excess of Pierce Maleimide-PEG2-biotin reagent in PBS overnight at 4°C.
Excess biotin was removed from the sample using either PD-10 columns (GE Healthcare) or
Zeba Spin Desalting columns (Thermo/Pierce) using manufacturer protocols. LC-MS analysis of
the product verified the presence of a single mass with addition of 525 amu consistent with the
molecular weight of the maleimide adduct. Since TCEP was present throughout the maleimide
reaction, if the protein had been fully reduced (making other cysteine thiols available), the
result would have been a heterogeneous product containing multiple adducts, and probably
misfolding or precipitation upon removal of key structural disulfide linkages. No such outcomes
were observed. Furthermore, the observation of standard binding specificity (GalNAc vs. methyl
galactoside vs. lactose) strongly suggests that enough of the protein on the SPR chip was
properly folded and displayed.
SPR binding measurements. All SPR measurements were performed using a Biacore 3000
instrument (GE Healthcare) at 25°C. Biotinylated ASGPR was immobilized typically at 2000-
3500 resonance units (Ru) using either SA sensor chips (GE Healthcare) or custom sensor chips
with Neutravidin (Pierce Biochemical) immobilized by standard amine coupling to CM5 sensor
chips (GE Healthcare). The running buffer was HBS (10 mM HEPES, 150 mM NaCl), 50 mM
CaCl2, 0.01% p20, 3% DMSO pH 7.5 or 50 mM Tris, 150 mM NaCl, 50 mM CaCl2, 0.01% p20, 3%
DMSO pH 7.5. [Some of the initial data sets were collected in HEPES but Tris buffer appeared to
produce data with better signal-to-noise ratios. The affinity values were unchanged by this
difference in buffer.] Compounds were serially diluted 3 fold from 900 µM to 3.7 µM for initial
evaluation. Compound solutions were injected at 50 µL/min for 1 min followed by a 1 min
dissociation in duplicate for each concentration. Data was processed using Scrubber2 (Biologic
16. Kettenhofen, N. J.; Wood, M. J. Chem. Res. Toxicol. 2010, 23, 1633-1646.
page S15

Software, Inc.) to zero, align, double reference and correct the data for excluded volume
effects. Values of 1/Kads (the immobilized-phase analogue to solution-phase Kd) for were
determined by fitting the steady state binding responses for the six concentrations to a single
site binding model in Scrubber2. Typical signal levels (Rmax) achieved for GalNAc were on the
order of 15 Ru consistent with approximately 50% of the surface bound protein showing
activity.
In the course of these experiments, there were observed shifts in the baselines after
some compound injections at the highest concentrations used (900 µM) and were often
excluded from the fits of the data. Also many of the compounds failed to approach saturation
and a 1/Kads value could not be obtained. Decreasing signals can often be observed at high
concentrations of analyte as the sample plug may not be uniform. Note that minor “negative”
signals of the type previously reported for carbohydrates binding to maltose binding protein17
were observed in the initial development of the ASGPR assay, but were largely eliminated upon
optimization of the assay, save for occasional aberrant injections.
17. Gestwicki, J.E.; Hsieh, H.V.; Pitner, J.B. Anal. Chem. 2001, 23, 5732-5737.
page S16

I. Characterization data for selected high-affinity ligands
Table S2. High-resolution electrospray ionization mass spectrometry and optical rotation data.
calcd mass obsvd mass
23
[α]D
Cpd. ID
Structure
Kd
(M+H)+
(M+H)+
O
CH3
NH
O
8b
8.5 ± 1.4 µM
389.1819
389.1829
+174.4°
N
O
N
N
OH
OH
O
CH3
O
MeO
NH
OH
8e
8.2 ± 0.7 µM
9.0 ± 4.1 µM
419.1925
393.1881
419.1935
393.1890
+86.5°
N
O
N
N
OH
O
CH3
NH
O
Me
8h
+126.7°
N
O
N
N
N
OH
N
OH
O
CH3
NH
O
O
N
O
8i
3.2 ± 2.0 µM
484.2191
484.2202
+58.0°
N
N
OH
N
OH
O
CH3
O
O
NH
OH
NH
8j
11
16
6.6 ± 2.5 µM
1.6 ± 0.60 µM
6.0 ± 0.45 µM
452.1598
287.1350
335.1561
452.1606
287.1354
335.1569
+94.2°
+82.3°
n/a
N
O
N
N
S
OH
O
CH3
O
NH
OH
O
N3
OH
OH
MeO
O
CH3
O
NH
OH
O
N3
OH
OH
Ph
N
H
O
CF3
O
MeO
NH
OH
17
1.2 ± 0.6 µM
582.2170
582.2179
n/a
N
O
N
N
OH
page S17

1H NMR, 500MHz, DMSO-d6, (Compound 8b)
13C NMR, 125 MHz, DMSO-d6, (Compound 8b)
page S18

1H NMR, 500MHz, CDCl3, (Compound 8e, acetonide protected)
1H NMR, 500MHz, DMSO-d6, (Compound 8e)
page S19

13C NMR, 125 MHz, DMSO-d6, (Compound 8e)
1H NMR, 600 MHz, CD3OD, (Compound 8h)
page S20

13C NMR, 167 MHz, CD3OD, (Compound 8h)
1H NMR, 600 MHz, DMSO-d6, (Compound 8i)
page S21

13C NMR, 167 MHz, DMSO-d6, (Compound 8i)
1H NMR, 500 MHz, DMSO-d6, (Compound 8j)
page S22

13C NMR, 125 MHz, DMSO-d6, (Compound 8j)
1H NMR, 500 MHz, CDCl3, (Compound 11)
page S23

13C NMR, 125 MHz, CDCl3, (Compound 11)
1H NMR, 600 MHz, CD3OD, (Compound 16)
page S24

13C NMR, 167 MHz, CD3OD, (Compound 16)
1H NMR, 600 MHz, CD3OD, (Compound 17)
page S25

13C NMR, 167 MHz, CD3OD, (Compound 17)
page S26