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S1
Supporting Information
for
Effective one-pot multienzyme (OPME) synthesis of monotreme milk
oligosaccharides and other sialosides containing a 4-O-acetyl sialic acid
Hai Yu,‡a Jie Zeng,‡
a,b Yanhong Li,
a Vireak Thon,§
a Baojun Shi,
a,c and Xi Chen*
a
aDepartment of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA.
E-mail: [email protected]; Fax: +1 530 752-8995; Tel: +1 530 754-6037
bSchool of Food Science, Henan Institute of Science and Technology, Xinxiang, Henan 453003,
China
cCollege of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China
‡These authors contributed equally to this work
§Current address: Laboratory of Bacterial Polysaccharides, Food and Drug Administration,
Bethesda, MD 20892, USA.
*Corresponding author. E-mail: [email protected]; Fax: +1 530 752-8995; Tel: +1 530 754-
6037
Table of Contents
Fig. S1 Mass spectrometry (MS) chromatograms of reactions catalyzed by various bacterial
CMP-sialic acid synthetases (CSSs) and mutants...................................................................S2–S4
Fig. S2 Mass spectrometry (MS) chromatograms of human influenza virus neuraminidase-
catalyzed hydrolysis of Neu4,5Ac23Lac...............................................................................S5–S6
1H and
13C NMR spectra of Neu4,5Ac2 (1)........................….....................……..........................S7
1H and
13C NMR spectra of Neu4Ac5Gc (2) ........................….....................…….......................S8
1H and
13C NMR spectra of GlcNAc3LacProN3 (Lc3).............................................................S9
1H and
13C NMR spectra of LNnTProN3 (23)...........................................................................S10
1H and
13C NMR spectra of sialosides 24–41......................................................................S11–S28
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2016
S2
Fig. S1 Mass spectrometry (MS) chromatograms of reactions catalyzed by various bacterial
CMP-sialic acid synthetases (CSSs) and mutants. Reactions were performed in a total volume of
10 L containing Tris-HCl buffer (100 mM, pH 7.1), MgCl2 (20 mM), Neu4,5Ac2 (10 mM), and
CTP (10 mM). After the addition of a CMP-sialic acid synthetase, the reaction was allowed to
proceed for 6 h at 37 C. The precipitates in the reaction mixture were removed by centrifugation
and aliquots of 1 L clear solution were diluted 100 times and injected for MS analysis.
Molecular ion m/z: Neu5Ac, 308.0982; Neu4,5Ac2, 350.1087;
CMP-Neu5Ac, 613.1394; CMP-Neu4,5Ac2, 655.1500;
CTP, 481.9767; CDP, 402.0104; CMP, 322.0440
Simon Neu45Ac Nm #23-61 RT: 0.25-0.66 AV: 39 NL: 4.38E6T: FTMS - p ESI Full ms [290.00-850.00]
300 350 400 450 500 550 600 650 700
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
655.1484
315.1080
402.0096481.9751
677.1291
322.0441348.2304 613.1374503.9568380.2198 459.9677431.1820 549.9495
409.0250
701.5653635.1197
568.1416
NmCSS CMP-Neu4,5Ac2
S3
Simon Neu45Ac NmS81R #16-66 RT: 0.17-0.72 AV: 51 NL: 3.26E6T: FTMS - p ESI Full ms [290.00-850.00]
300 350 400 450 500 550 600 650 700
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
655.1475
315.1076481.9745
402.0092
677.1283322.0438 503.9562
613.1364348.2300 374.2532 549.9489423.9907 459.9672 571.9301525.9379 701.5797635.1183
Simon Neu45Ac NmQ164A #16-45 RT: 0.17-0.48 AV: 30 NL: 3.92E6T: FTMS - p ESI Full ms [290.00-850.00]
300 350 400 450 500 550 600 650 700
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
655.1464
402.0086481.9739
315.1072
677.1271322.0433
503.9555358.2580613.1354380.2186 412.9638 549.9481459.9664 525.9375 571.9297 701.5877
635.1175
NmCSS_Q164A CMP-Neu4,5Ac2
NmCSS_S81R
CMP-Neu4,5Ac2
S4
Simon Neu45Ac HdCSS #16-45 RT: 0.17-0.48 AV: 30 NL: 2.96E6T: FTMS - p ESI Full ms [290.00-850.00]
300 350 400 450 500 550 600 650 700
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
655.1471
315.1071
402.0088 481.9741
322.0434358.2581 677.1278
503.9559 613.1359374.2528 412.9638459.9667 549.9484
396.2137
521.9668 623.9984579.5485 697.1559
647.0740
Simon Neu45Ac PmCSS #17-48 RT: 0.18-0.52 AV: 32 NL: 3.19E6T: FTMS - p ESI Full ms [290.00-850.00]
300 350 400 450 500 550 600 650 700
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
655.1467
402.0087
315.1070 481.9740
677.1273322.0433 503.9557
613.1355412.9637358.2579 459.9664 549.9482380.2186 525.9374 571.9297 640.0880 701.5939
HdCSS CMP-Neu4,5Ac2
PmCSS CMP-Neu4,5Ac2
S5
Fig. S2 Mass spectrometry (MS) chromatograms of human influenza virus neuraminidase-
catalyzed hydrolysis of Neu4,5Ac23Lac
A/Udorn/307/72 H3N2 (A/Udorn72)
A/Memphis/71 H3N1 (A/Mem71)
Ud 10mM 1 #19-147 RT: 0.18-1.39 AV: 129 NL: 1.25E6T: FTMS - p ESI Full ms [195.00-1000.00]
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
Re
lative
Ab
un
da
nce
411.0858
545.1630
674.2120
350.1086
536.1630
628.1232
308.0412 330.0231427.0595 567.1449479.0726
464.0996 584.1593503.1524 622.1059377.0843 644.0970319.9945 704.1187
686.0812358.2583
531.0445
398.0095342.2272
665.1221
553.0048
Mem 10mM 1 #21-133 RT: 0.19-1.26 AV: 113 NL: 7.69E5T: FTMS - p ESI Full ms [195.00-1000.00]
280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
52
54
56
58
Re
lative
Ab
un
da
nce
411.0857
674.2118
545.1629
350.1081
628.1228330.0231 427.0594308.0411 536.1625358.2583
479.0724
374.2533
622.1057507.3081473.0556 584.1590567.1448 638.0777 696.1097437.1936 606.1426
679.3660
660.0711
Neu4,5Ac23Lac
Neu4,5Ac2
Neu5Ac
Neu4,5Ac23Lac
Neu4,5Ac2
Neu5Ac
S6
A/Puerto Rico/34/8 H1N1 (A/PR8)
A/Philippines/2/82/X-79 H3N2 (A/Philips)
PR 10mM 1 #17-135 RT: 0.15-1.27 AV: 119 NL: 1.47E6T: FTMS - p ESI Full ms [195.00-1000.00]
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
Re
lative
Ab
un
da
nce
674.2121
545.1629
350.1082
628.1230
427.0596308.0411330.0231
536.1626479.0725 584.1591
622.1059469.0440 638.0779567.1449358.2583 503.1524
374.2533
707.2150445.2111
690.0932
660.0711528.2039
Phi 10mM 1 #22-130 RT: 0.20-1.23 AV: 109 NL: 1.33E6T: FTMS - p ESI Full ms [195.00-1000.00]
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
m/z
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
52
54
56
Re
lative
Ab
un
da
nce
674.2123411.0857
545.1629
350.1083
628.1230
330.0231308.0412
479.0725 536.1626427.0596567.1448469.0440 622.1058584.1590512.1593374.2534
358.2583319.9945
707.2147644.0968485.1418
398.0095
445.2166
688.0773
553.0051
660.0708
343.3265
Neu4,5Ac23Lac
Neu4,5Ac2
Neu5Ac
Neu4,5Ac23Lac
Neu4,5Ac2
Neu5Ac
S7
1H and
13C NMR spectra of Neu4,5Ac2 (1)
S8
1H and
13C NMR spectra of Neu4Ac5Gc (2)
S9
1H and
13C NMR spectra of GlcNAc3LacProN3 (Lc3)
S10
1H and
13C NMR spectra of LNnTProN3 (23)
S11
1H and
13C NMR spectra of Neu4,5Ac23Lac (24)
S12
1H and
13C NMR spectra of Neu4,5Ac23LNnT (25)
S13
1H and
13C NMR spectra of Neu4,5Ac23LNT (26)
S14
1H and
13C NMR spectra of Neu4,5Ac23GalpNP (27)
S15
1H and
13C NMR spectra of Neu4,5Ac23LacProN3 (28)
S16
1H and
13C NMR spectra of Neu4,5Ac23LacNAcProN3 (29)
S17
1H and
13C NMR spectra of Neu4,5Ac23Gal3GlcNAcProN3 (30)
S18
1H and
13C NMR spectra of Neu4,5Ac23Gal3GlcNAcProN3 (31)
S19
1H and
13C NMR spectra of Neu4,5Ac23Gal3GalNAcProN3 (32)
S20
1H and
13C NMR spectra of Neu4,5Ac23Gal3GalNAcProN3 (33)
S21
1H and
13C NMR spectra of Neu4,5Ac23LNnTProN3 (34)
S22
1H and
13C NMR spectra of Neu4Ac5Gc3LacProN3 (35)
S23
1H and
13C NMR spectra of Neu4Ac5Gc3LacNAcProN3 (36)
S24
1H and
13C NMR spectra of Neu4Ac5Gc3Gal3GlcNAcProN3 (37)
S25
1H and
13C NMR spectra of Neu4Ac5Gc3Gal3GlcNAcProN3 (38)
S26
1H and
13C NMR spectra of Neu4Ac5Gc3Gal3GalNAcProN3 (39)
S27
1H and
13C NMR spectra of Neu4Ac5Gc3Gal3GalNAcProN3 (40)
S28
1H and
13C NMR spectra of Neu4Ac5Gc3LNnTProN3 (41)