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1

Supplementary Information

Interception of teicoplanin oxidation intermediates yields new antimicrobial

scaffolds

Yu-Chen Liu1,2,5

, Yi-Shan Li1, Syue-Yi Lyu

1,4, Li-Jen Hsu

1, Yu-Hou Chen

1,3, Yu-Ting

Huang1, Hsiu-Chien Chan

1, Chuen-Jiuan Huang

1, Gan-Hong Chen

1, Chia-Cheng

Chou6, Ming-Daw Tsai

1,3,5 & Tsung-Lin Li

1,7*

1Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.

2Chemical Biology and Molecular Biophysics Program, Taiwan International

Graduate Program, Institute of Biochemistry, Academia Sinica, Taipei 115, Taiwan. 3Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.

4Department of Microbiology and Immunology, National Yang-Ming University,

Taipei 112, Taiwan. 5Institute of Biochemical Science, National Taiwan University, Taipei 115, Taiwan.

6National Synchrotron Radiation Research Center, Hsinchu 300, Taiwan.

7Biotechnology Center, National Chung Hsing Univerisity, Taichung 402, Taiwan.

* To whom correspondence should be sent. E-mail: [email protected]

This PDF file includes:

Supplementary Methods

Supplementary Results

(Supplementary Figures 1-19, Supplementary Tables 1-4)

Supplementary References

Nature Chemical Biology: doi:10.1038/nchembio.556

mailto:[email protected]

2

Supplementary Methods

Cloning and protein purification. Protein expression, purification and confirmation

of purity were performed as described previously1-4

. The dbv29 gene was amplified

from genomic DNA by PCR using the first two primers shown in the Primer list

below. The products were each ligated according to the manufacturers' instruction

(Novagene) into the pET-28a(+) expression vector which provides an N-terminal

His6-tagged protein. E. coli cells were grown at 37°C in 1 L of LB medium until an

A600 of about 0.7 was reached. Protein expression was induced with 0.2 mM IPTG at

16°C, and, after 12 h, the cells were harvested by centrifugation, resuspended in 10

mM imidazole-HCl buffer, pH 7.8 (binding buffer), and ruptured using a

microfluidizer. The cell-free extract prepared by centrifugation was applied to an

Ni2+

-NTA resin column, which was then washed successively with binding buffer and

wash buffer before eluting the target protein with 200 mM imidazole-HCl. Gel

filtration was performed using an Ä kta FPLC system equipped with an S-200

Superdex column (Amersham Bioscience) under isocratic conditions (20 mM Tris,

pH 7.6, 100 mM KCl). The buffer was exchanged for 50 mM HEPES buffer, pH 7.2

using Millipore centrifugal filters. Protein concentrations were estimated using the

Bradford assay. The purified protein was confirmed by SDS-PAGE, Western blotting,

and electrospray mass spectrometry (ESI-MS).

Primer list (primers used in this study)

Mutants Primer

Dbv29-f AAA TTT CAT ATG ACC GGC GGC ACC GGA GCG

Dbv29-r GGG CCC CTC GAG TCA GGG CCG GAT CGA CAA CGC

Dbv29-C26A-f CCG GAG CTG CGG GGC GAG CGC GCC TTA CCG CCG GCC GGC CCG

GTC Dbv29-C26A-r GAC CGG GCC GGC CGG CGG TAA GGC GCG CTC GCC CCG CAG CTC

CGG Dbv29-C92A-f GCC GTC CGC AGC GGT GGG CAC GCT TTC GAG GAC TTC GTC GAC

AAC Dbv29-C92A-r GTT GTC GAC GAA GTC CTC GAA AGC GTG CCC ACC GCT GCG GAC

GGC Dbv29-C151A-f GTG ACC ATA CCG GGT GGG GTC GCC GGC GGG GTC GGC GTC GGC

GGA Dbv29-C151A-r TCC GCC GAC GCC GAC CCC GCC GGC GAC CCC ACC CGG TAT GGT

CAC Dbv29-C161A-f GTC GGC GTC GGC GGA CAC ATC GCC GGA GGC GGG TAC GGC CCG

CTG Dbv29-C161A-r CAG CGG GCC GTA CCC GCC TCC GGC GAT GTG TCC GCC GAC GCC

GTC Dbv29-H91A-f GTC CGC AGC GGT GGG GCC TGT TTC GAG GAC TTC

Dbv29-H91A-r GAA GTC CTC GAA ACA GGC CCC ACC GCT GCG GAC

Dbv29-I401A-f GGC GCC GTC TGG CTG GCC GGC TAC GGC GGG AAG


3

Dbv29-I401A-r CTT CCC GCC GTA GCC GGC CAG CCA GAC GGC GCC

Dbv29-I401W-f GGC GCC GTC TGG CTG TGG GGC TAC GGC GGG AAG

Dbv29-I401W-r CTT CCC GCC GTA GCC CCA CAG CCA GAC GGC GCC

Dbv29-N44A-f GAC CCG CGC TAC CTC GCC CTG AAG CTG CGT GGC

Dbv29-N44A-r GCC ACG CAG CTT CAG GGC GAG GTA GCG CGG GTC

Dbv29-R41A-f AACCTT CCCCGG GGAACC GGAACC CCCCGG GGCCTT TTAACC CCTTCC AAAACC CCTTGG AAAAGG

Dbv29-R41A-r CCTTTT CCAAGG GGTTTT GGAAGG GGTTAA AAGGCC CCGGGG GGTTCC GGTTCC CCGGGG AAGGTT

Dbv29-R360E-f CCG GGG CGA GGA GGC GAA GGC CCG GCG TCG AAG

Dbv29-R360E-r CTT CGA CGC CGG GCC TTC GCC TCC TCG CCC CGG

Dbv29-R360L-f CCG GGG CGA GGA GGC CTG GGC CCG GCG TCG AAG

Dbv29-R360L-r CTT CGA CGC CGG GCC CAG GCC TCC TCG CCC CGG

Dbv29-S364K-f GGC AGG GGC CCG GCG AAA AAG ACG AAA GCC GGC

Dbv29-S364K-r GCC GGC TTT CGT CTT TTT CGC CGG GCC CCT GCC

Dbv29-S364R-f GGC AGG GGC CCG GCG AGG AAG ACG AAA GCC GGC

Dbv29-S364R-r GCC GGC TTT CGT CTT CCT CGC CGG GCC CCT GCC

Dbv29-T366A-f GGC CCG GCG TCG AAG GCC AAA GCC GGC TAC CTG

Dbv29-T366A-r CAG GTA GCC GGC TTT GGC CTT CGA CGC CGG GCC

Dbv29-T366E-f GGC CCG GCG TCG AAG GAA AAA GCC GGC TAC CTG

Dbv29-T366E-r CAG GTA GCC GGC TTT TTC CTT CGA CGC CGG GCC

Dbv29-T366L-f GGC CCG GCG TCG AAG CTG AAA GCC GGC TAC CTG

Dbv29-T366L-r CAG GTA GCC GGC TTT CAG CTT CGA CGC CGG GCC

Dbv29-W399A-f GAC TAC GGC GCC GTC GCC CTG ATC GGC TAC GGC

Dbv29-W399A-r GCC GTA GCC GAT CAG GGC GAC GGC GCC GTA GTC

Dbv29-W399F-f GAC TAC GGC GCC GTC TTC CTG ATC GGC TAC GGC

Dbv29-W399F-r GCC GTA GCC GAT CAG GAA GAC GGC GCC GTA GTC

Dbv29-Y135F-f ACG CTC TCA GAG GTG TTC GAA AAG CTC TAC CTG

Dbv29-Y135F-r CAG GTA GAG CTT TTC GAA CAC CTC TGA GAG CGT

Dbv29-Y165F-f TGC GGA GGC GGG TTC GGC CCG CTG TCA

Dbv29-Y165F-r TGA CAG CGG GCC GAA CCC GCC TCC GCA

Dbv29-Y165W-f ATC TGC GGA GGC GGG TGG GGC CCG CTG TCA CGG

Dbv29-Y165W-r CCG TGA CAG CGG GCC CCA CCC GCC TCC GCA GAT

Dbv29-Y370F-f ACG AAA GCC GGC TTC CTG CGC AAG CGG CTG

Dbv29-Y370F-r CAG CCG CTT GCG CAG GAA GCC GGC TTT CGT


4

Dbv29-Y403F-f GTC TGG CTG ATC GGC TTC GGC GGG AAG GTG AAC

Dbv29-Y403F-r GTT CAC CTT CCC GCC GAA GCC GAT CAG CCA GAC

Dbv29-Y428F-f ATA CTC AAG GTG AAC TTC ATC ACC GGT TGG GCG

Dbv29-Y428F-r CGC CCA ACC GGT GAT GAA GTT CAC CTT GAG TAT

Dbv29-Y453F-f CTC TAT GCC GAT GTG TTC GCC GAG ACC GGC GGG

Dbv29-Y453F-r CCC GCC GGT CTC GGC GAA CAC ATC GGC ATA GAG

Dbv29-Y470F-f GTC AGC GAT GGG GCG TTC ATC AAT TAC CCC GAC

Dbv29-Y470F-r GTC GGG GTA ATT GAT GAACGC CCC ATC GCT GAC

Dbv29-Y473F-f GGG GCG TAC ATC AAT TTC CCC GAC AGC GAC CTC

Dbv29-Y473F-r GAG GTC GCT GTC GGG GAA ATT GAT GTA CGC CCC

Dbv29-Y473E-f GGG GCG TAC ATC AAT GAA CCC GAC AGC GAC CTC

Dbv29-Y473E-r GAG GTC GCT GTC GGG TTC ATT GAT GTA CGC CCC

Synthetic conditions for new analogs. Teicoplanin (a mixture of five analogs) was

purchased from AAPIN Chemicals Ltd (UK). The mixture was subjected to HPLC

purification to obtain C10-Tecicoplanin with purity >95%. 5-Azide pentylamine (99%)

was obtained from Chung-Yi Wu (Academia Sinica) as a gift. All other chemicals

were obtained from Sigma/Aldrich Chemical Co. (St. Louis, MO, USA) without

further purification unless otherwise stated. Teicoplanin analogs (2-6) were

enzymatically synthesized as described previously1-4

. In brief, compound 2 was

prepared by adding Dbv21 or Orf2* (10 g) into a typical buffer solution (50 mM

HEPES, pH 7.2, 100 mM NaCl, 1 mM DTT) containing Tei (1, 1 mM) for 5 h at 37C;

compounds 3-6 were prepared by adding Dbv8 (10 g) into the same buffer solution

but containing compound 2 and butanoyl-, hexanoyl-, octanoyl-, or decanoyl-CoA for

compounds 3-6, respectively, at 25C overnight. For the final reaction, Teicoplanin

analogs (9, 10, 11, 25 and 32, which showed relatively higher yields from initial tests)

were enzymatically synthesized using the optimized conditions described below.

Enzyme stability (and, indirectly, the extent of exposure of the aldehyde for

functionalization) was tested in an array of organic solvents (MeCN, MeOH, EtOH,

DHF, DMF, DCM, DMSO etc.); DMSO turned out to be the most appropriate solvent

as Dbv29 is highly stable in up to 90% DMSO. 50% DMSO, however, is included in

the current protocol as it gives relatively higher yields. Other conditions, such as the

amounts of alkylamine and reductant5, were also optimized one at a time against the

fixed concentration of Tei (0.5 mM) for a better yield (below). In general, 10-fold of

alkylamine and the reducing agent versus Tei was determined to be most appropriate

for the current protocol, which is summarized as follows: Tei (0.5 mM), alkylamine (5

mM; carbon number >6), and Na(CN)BH3 (5 mM) in 50% DMSO buffer solution at


5

37C overnight incubation.

0

200

400

600

800

1000

0 5 10 15 20

mA

U

octylamine (mM)

(10x Tei)

Fixed NaCNBH3 (5 mM) and Tei (0.5 mM)

0

200

400

600

800

0 5 10 15 20

mA

U

NaCNBH3 (mM)

Fixed octylamine (5 mM) and Tei (0.5 mM)

(10x Tei)

0

500

1000

1500

2000

0 5 10 15 20 25 30

mA

U

benzylamine (mM)

0

500

1000

1500

2000

0 5 10 15 20 25 30

mA

U

NaCNBH3 (mM)

Fixed NaCNBH3 (5 mM) and Tei (0.5 mM)

Fixed benzylamine (5 mM) and Tei (0.5 mM)

(10x Tei)

(10x Tei)

A 10-fold ratio of alkylamine (5 mM) and NaCNBH3 (5 mM) to Tei (0.5 mM) proved sufficiently

optimized for the octylamine-Tei derivative (left) and the benzylamine-Tei derivative (right), although

higher the concentrations of either reagent provided marginally higher yields.

Compound characterization. All compounds were characterized by MS and HPLC,

which can be found in Supplementary Figure 11. For select compounds, NMR

analyses were performed on a Bruker Avance 600 spectrometer equipped with

CryoProbe™, with tetramethylsilane (TMS) as an internal standard. Compounds were

dissolved in deuterated dimethyl sulfoxide (DMSO-d6) if not otherwise stated, and

spectra were recorded at room temperature. NMR peaks are listed below; original

spectra can be found in Supplementary Figures 12-18.

Teicoplanin (1). HRMS ES(+): 950.7935 [M+H+Na]2+

, calc. for C88H97Cl2N9O33Na

950.7771 [M+H+Na]2+

(Supplementary Figure 19). 1H-NMR (600 MHz, DMSO-d6)

(Supplementary Figure 12): 8.43 (s, 1H), 7.75 (m, 1H), 7.50 (m, 1H), 7.34 (m, 2H),

7.21 (m, 1H), 7.13 (d, J 8.3, 1H), 7.05 (d, J 8.3, 1H), 6.91 (m, 2H), 6.84 (m, 1H), 6.77

(m, 1H), 6.68 (m, 2H), 6.43 (s, 1H), 6.23 (s, 1H), 5.70 (s, 1H), 5.55 (m, 1H), 5.46 (m,

2H), 5.36 (m, 1H), 5.28 (d, J 8.4, 1H), 5.21 (s, 1H), 4.50 (m, 2H), 4.20 (s, 1H), 4.05

(m, 1H), 3.90 (m, 2H), 3.70 (m, 8H), 3.52 (m, 2H), 3.41 (m, 4H), 3.07 (m, 1H), 2.17

(m, 2H), 2.00 (s, 2H), 1.55 (m, 2H), 1.42 (m, 1H), 1.15 (m, 8H), 0.77 (m, 6H). 13

C

NMR (600 MHz, Methanol-d4): 178.10, 177.90, 175.13, 172.17, 171.86, 171.25,


6

171.09, 170.93, 168.65, 160.77, 159.51, 158.05, 155.81, 155.04, 153.78, 153.09,

151.99, 150.99, 150.14, 145.25, 139.74, 139.39, 136.62, 136.51, 134.74, 134.58,

132.87, 130.94, 129.79, 129.05, 128.70, 127.89, 127.41, 126.50, 125.72, 125.16,

123.02, 121.55, 121.00, 119.42, 118.53, 110.98, 110.34, 109.31, 107.11, 105.82,

104.25, 102.97, 101.25, 97.82, 78.92, 77.48, 77.37, 74.81, 74.24, 71.66, 71.50, 71.06,

67.41, 63.63, 62.09, 61.71, 60.60, 58.78, 57.53, 57.27, 56.66, 55.33, 49.71, 49.58,

49.44, 49.30, 49.15, 49.01, 48.87, 48.72, 46.83, 39.80, 37.59, 37.10, 30.21, 30.09,

28.74, 27.98, 26.55, 23.40, 23.21, 23.18. An expanded version of HSQC and HMBC

spectra of the reference compound is shown in Supplementary Figure 12.

Compound 2. HRMS ES+: 828.7479 [M+2H]

2+, calc. for C80H83N9O26Cl2 828.7491

[M+2H]2+


(Supplementary Figure 13): 9.66 (m, 2H), 9.32 (m, 1H), 8.70 (m, 1H), 7.91 (m, 2H),

7.77 (m, 2H), 7.60 (m, 1H), 7.31 (m, 1H), 7.15 (m, 6H), 6.97 (m, 2H), 6.71 (m, 4H),

6.45 (m, 3H), 6.31 (m, 1H), 5.26 (m, 6H), 5.08 (m, 4H), 4.91 (m, 1H), 4.64 (m, 1H),

4.38 (m, 7H), 3.78 (m, 4H), 3.04 (m, 5H), 2.87 (m, 4H), 1.89 (m, 3H), 1.26 (m, 1H),

1.15 (m, 1H). 13

C NMR (600 MHz, DMSO-d6): 169.81, 169.78, 169.63, 169.25,

169.23, 168.90, 168.37, 167.73, 167.21, 158.80, 158.63, 158.60, 157.97, 157.77,

157.36, 157.24, 155.53, 155.39, 152.84, 151.95, 151.76, 151.38, 150.10, 149.89,

149.02, 148.12, 143.42, 135.48, 127.23, 127.19, 126.20, 125.07, 124.85, 120.88,

120.84, 120.22, 120.05, 118.38, 116.38, 106.06, 98.80, 97.46, 96.58, 78.10, 77.90,

77.01, 76.92, 75.71, 73.77, 73.62, 72.99, 72.90, 72.53, 70.71, 70.64, 70.00, 69.90,

69.81, 66.00, 65.94, 63.10, 61.23, 61.17, 60.98, 60.85, 60.76, 59.32, 57.66, 57.42,

56.89, 56.30, 56.10, 55.99, 54.74, 45.57, 23.24, 23.14. An expanded version of HSQC,

HMBC and 1H COSY spectra of the reference compound is shown in Supplementary

Figure 13.

Compound 9. HRMS ES (+): 957.7779 [M+H+Na]2+

, calc. for C88H96Cl2N9O34Na

957.7667 [M+H+Na]2+


(Supplementary Figure 14): 8.45 (s, 1H), 7.72 (s, 1H), 7.51 (m, 1H), 7.33 (m, 2H),

7.21 (m, 2H), 7.07 (m, 2H), 6.93 (m, 3H), 6.84 (d, J 8.4, 1H), 6.77 (m, 2H), 6.62 (m,

3H), 6.42 (s, 1H), 6.19 (s, 1H), 5.71 (s, 1H), 5.55 (s, 1H), 5.46 (m, 4H), 5.30 (s, 1H),

5.18 (s, 2H), 4.47 (m, 4H), 4.20 (s, 1H), 4.09 (m, 1H), 3.91 (m, 1H), 3.70 (m, 10H),

3.51 (m, 2H), 3.41 (m, 3H), 3.11 (m, 1H), 2.22 (m, 2H), 2.03 (m, 3H), 1.55 (m, 2H),

1.41 (m, 1H), 1.27 (m, 3H), 1.14 (m, 5H), 1.04 (m, 2H), 0.87 (m, 1H), 0.77 (m, 6H). 13

C NMR (600 MHz, Methanol-d4): 220.76, 220.02, 217.63, 217.32, 177.88, 175.22,

172.20, 171.82, 171.09, 171.03, 170.87, 168.72, 160.82, 159.48, 158.02, 155.77,

155.02, 153.32, 152.92, 152.30, 150.97, 150.47, 145.43, 139.82, 139.36, 139.20,


7

136.50, 136.34, 134.49, 134.21, 132.80, 130.84, 129.83, 129.06, 128.88, 127.90,

126.55, 125.76, 125.03, 124.47, 123.01, 121.54, 121.26, 119.51, 118.51, 116.20,

111.08, 110.31, 109.87, 107.17, 105.77, 103.38, 102.95, 101.17, 97.80, 78.83, 77.35,

74.85, 74.22, 73.75, 71.62, 71.51, 71.10, 67.41, 63.59, 61.73, 58.79, 57.68, 57.59,

57.32, 57.13, 56.80, 56.56, 55.29, 39.81, 37.59, 37.03, 30.27, 30.15, 30.10, 29.99,

28.77, 28.05, 26.67, 23.42, 23.21, 23.18, 23.10. An expanded version of HSQC and

HMBC spectra of the reference compound is shown in Supplementary Figure 14.

Compound 10. HRMS ES (+):1009.3786 [M+2H]2+

, calc. for C98H120O32N10Cl2

1009.37235 [M+2H]2+

(Supplementary Figure 19). 1H NMR (600 MHz, DMSO-d6):

7.71 (m, 1H), 7.16 (m, 3H), 5.32 (m, 1H), 4.90 (m, 1H), 4.26 (m, 1H), 4.13 (m, 1H),

3.67 (m, 1H), 3.57 (m, 1H), 3.43 (m, 1H), 3.33 (m, 3H), 3.06 (m, 3H), 2.88 (m, 1H),

2.52 (m, 4H), 2.01 (m, 2H), 1.88 (m, 1H), 1.26 (m, 23H), 0.82 (m, 9H). 13

C NMR

(600 MHz, DMSO-d6): 174.35, 158.08, 157.87, 157.67, 157.47, 131.73, 131.66,

130.13, 130.06, 129.69, 128.71, 120.37, 118.38, 116.38, 114.56, 96.92, 92.25, 76.87,

76.78, 74.85, 73.79, 73.10, 72.52, 72.39, 72.00, 70.69, 70.60, 70.31, 69.80, 67.44,

63.10, 61.23, 61.05, 60.96, 56.06, 38.10, 35.14, 31.31, 31.25, 30.93, 30.01, 29.82,

29.15, 29.12, 29.06, 28.91, 28.86, 28.75, 28.72, 28.61, 28.59, 28.40, 27.41, 26.72,

26.63, 26.59, 25.14, 25.07, 22.59, 22.44, 22.13, 18.59, 14.00, 13.95. An expanded

version of HSQC and HMBC spectra of the reference compound is shown in

Supplementary Figure 15.

Compound 25. HRMS ES(+): 984.3033 [M+2H]2+

, calc. for C95H104O32N10Cl2

984.3098 [M+2H]2+


(Supplementary Figure 16): 9.67 (m, 2H), 8.61 (m, 1H), 8.40 (m, 1H), 7.93 (m, 2H),

7.77 (m, 2H), 7.18 (m, 14H), 6.71 (m, 3H), 6.48 (m, 2H), 6.23 (m, 1H), 5.24 (m, 7H),

4.96 (m, 1H), 4.62 (m, 1H), 4.39 (m, 5H), 4.08 (m, 1H), 3.70 (m, 2H), 3.00 (m, 4H),

2.14 (m, 1H), 1.99 (m, 1H), 1.86 (m, 3H), 1.48 (m, 2H), 1.37 (m, 1H), 1.15 (m, 12H),

0.84 (d, J 6.5, 6H). 13

C NMR (600 MHz, DMSO-d6): 172.87, 172.79, 172.41, 172.36,

169.94, 169.81, 169.39, 169.24, 168.88, 168.76, 168.35, 167.28, 158.54, 158.41,

158.21, 158.00, 157.80, 157.67, 157.48, 157.41, 155.59, 148.27, 135.73, 135.62,

134.68, 131.88, 131.82, 130.21, 130.09, 129.96, 129.88, 129.20, 129.12, 128.86,

128.80, 128.38, 125.02, 120.79, 120.74, 120.20, 118.28, 116.29, 114.62, 109.20,

98.91, 96.62, 76.95, 76.83, 73.80, 73.73, 72.85, 72.80, 72.71, 72.56, 72.44, 72.20,

70.76, 70.68, 70.09, 69.81, 66.02, 63.13, 61.30, 61.20, 61.08, 60.76, 56.58, 56.38,

56.19, 55.77, 54.57, 51.74, 51.00, 50.16, 35.98, 31.37, 29.23, 29.10, 29.02, 28.97,

28.92, 28.87, 28.80, 27.47, 27.40, 26.76, 26.71, 26.67, 25.11, 24.97, 23.25, 23.17,

22.65, 22.18,14.09. An expanded version of HSQC, HMBC and 1H COSY spectra of


8

the compound 25 is shown in Supplementary Figure 16. For selected 1H NMR

assignment see Supplementary Figure 18 and Supplementary Table 4.

Compound 32 (de-r4-Tei). HRMS ES(-): 780.1677 [M-2H]-2

, calc. for

C72H68O28N8Cl2 780.1760 [M-2H]-2

(Supplementary Figure 19). 1H-NMR (600

MHz, DMSO-d6) (Supplementary Figure 17): 9.68 (m, 2H), 9.27 (m, 1H), 8.65 (m,

1H), 8.53 (s, 1H), 8.42 (s, 1H), 7.92 (m, 2H), 7.80 (m, 1H), 7.26 (m, 3H), 7.18 (m,

2H), 7.10 (m, 4H), 6.97 (m, 1H), 6.75 (m, 3H), 6.46 (m, 2H), 6.23 (m, 1H), 5.27 (m,

2H), 5.13 (m, 2H), 4.42 (m, 2H), 4.34 (m, 1H), 4.28 (d, J 11.5, 1H), 4.12 (m, 1H),

3.06 (m, 2H), 2.95 (m, 1H), 1.88 (m, 2H), 1.26 (m, 1H). 13

C NMR (600 MHz,

DMSO-d6): 172.79, 172.72, 169.87, 169.63, 169.32, 168.59, 168.44, 167.36, 167.27,

167.19, 158.76, 158.67, 158.56, 158.49, 158.29, 158.08, 157.87, 157.46, 157.38,

155.52, 155.09, 150.27, 150.23, 149.06, 148.56, 148.49, 147.63, 143.51, 135.58,

134.67, 134.41, 128.26, 128.19, 127.13, 126.20, 125.66, 125.25, 123.43, 120.71,

120.19, 120.08, 118.29, 116.30, 105.66, 99.26, 99.13, 97.52, 96.65, 76.91, 76.81,

73.80, 73.66, 73.00, 72.55, 70.59, 70.00, 69.92, 69.81, 65.99, 65.92, 63.11, 61.13,

60.75, 59.25, 56.12, 56.07, 55.86, 55.79, 55.17, 54.59, 23.21, 23.12. An expanded

version of HSQC, HMBC and 1H COSY spectra of the reference compound is shown

in Supplementary Figure 17.

Antibiotic assays. MIC assays were performed as described previously1.

In vivo study.6-8

ICR female mice were purchased from the National Laboratory

Animal Breeding and Research Center, Taipei, Taiwan. Mice with average body

weight of 27 to 30 g were subjected to infection via intravenous (i.v.) injection with

1.3 × 105 cfu/mouse of E. faecalis (ATCC 51559) at day 0. For treatment study, mice

were randomized into four groups at the start of the experiment and administered 10

mg/kg of either vancomycin, teicoplanin, benzylamine-teicoplanin (25) or saline

(control) by i.v. twice a day for three days (from day 1 to day 3, a total of 6 doses).

Mice were subjected to anesthesia; whole blood was then sampled from orbital sinus

on day 1, day 2, and day 3. The whole blood underwent serial dilutions with PBS,

which were plated on Brain Heart infusion agar (BHI agar; Difico, Detroit, MI, USA)

for enumeration of cfu (colony formation unit). The graphs and statistical analyses

were performed using SigmaPlot® and SigmaStat

®. Differences were considered

significant if the P value was < 0.05.


9

Supplementary Results

Supplementary table 1. Data collection and refinement statistics.

Dbv29 Dbv29/Teicoplanin

Data collection

Space group P6122 P21

Cell dimensions

a, b, c (Å ) 66.09, 66.09, 790.46 64.75, 152.23, 109.77

() 90.00, 90.00, 120.00 90.00, 91.03, 90.00

Resolution (Å ) 30.00-3.21 (3.32-3.21) * 30.00-2.30 (2.38-2.30)

Rmerge 8.3 (16.0) 8.8 (63.0)

I / I 24.8 (17.0) 22.5 (2.5)

Completeness (%) 95.7 (92.0) 98.9 (96.7)

Redundancy 9.7 (9.8) 7.3 (6.4)

Refinement

Resolution (Å ) 3.21 2.30

No. reflections 16838 88256

Rwork / Rfree 0.237/0.291 0.197/0.252

No. atoms

Protein 7710 15420

Ligand/ion 106 345

Water 69 830

B-factors

Protein 29.2 38.3

Ligand/ion 24.3 47.7

Water 9.0 38.4

R.m.s. deviations

Bond lengths (Å ) 0.005 0.015

Bond angles () 0.837 1.679

The number of crystals is indicated in parentheses for the following data sets: Dbv29 (2) and

Dbv29/Teicoplanin (1). *Values in parentheses are for highest-resolution shell.


10

91

151

473

165


11

Supplementary figure 1. Multiple sequence alignments for Dbv29 and homologues.

Residues that covalently link to FAD are highly conserved; H91 and C151 of Dbv29

are marked on the top of sequences. Residues corresponding to Tyr-473 are conserved

in Dbv29, AknOx (Tyr-450) and GOOX (Tyr-429). Notes: 1) In GOOX9, Tyr-429

initiates the catalytic activity by proton abstracton that is also facilitated by Asp-355.

In AknOx10

, this second residue is Tyr-144 that may coordinate with Tyr-450. In

BBE11

, the proton abstraction step is carried out by Glu-417). 2) Accession codes for

Dbv29 (Q7WZ62; Nonomuraea sp. ATCC39727), AknOx (Q0PCD7;

Streptomyces galilaeus), GOOX (Q6PW77; Acremonium strictum) and BBE (P30986;

Eschscholzia califomica) are designated by the UniProtKB databank

(http://www.uniprot.org/). 3) Identical residues are colored in red, similar residues are

colored in yellow.


http://www.uniprot.org/

12

F-domain S-domain

Subdomain 1 Subdomain 2

B1

B2

B3B4

B5B6

B7

B8B9

B10B11

B12

B13

B14 B15B16

H1

H2 H3

H4

H5

H6H7

H8

H9

H10

H11

H12H13

H14

H15

H16

H17

H18

H19

H20

33

35

41

47

60

64

66

81

83

87

103

108 113

117122

126

130

143

155

163

168

173

174

177

178

188192

200

206

213

223

230

238

242

248

257

258

262

263

281

286

289

290

299 308

317

321

335

346

350

363

371

376

388

395

403

404

410

423

432

434

437

438

453454

457

475

480

489

496

498

510

a


13

b

B1B2B3

B4

B5B6

B7 B8B9

B10

B11

B12

B13

B14

B15B16

H1

H2

H3

H4

H5H6

H7

H8

H9

H10

H11

H12

H13

H14

H15

H16

H17

H18

H19

H20


14

c

FAD

C151

H91


15

d


16

e


17

399401

165

473

f


18

g

Dbv29AknOx

C RMSD 0.734

Supplementary figure 2. Overall structure of Dbv29 and comparisons to AknOx. (a)

Dbv29 can be dissected into two domains, F and S. The F and S domains consist of

residues 1–230 and 235–523, respectively, in which the F domain can be further

divided into two subdomains. (b) For the F domain, the N-terminal subdomain

(subdomain 1, residues 1-105) consists of a central four-stranded -sheet (B1-B4 with

the strand order of 1, 2, 4, 3, wherein B1 runs antiparallel to others), sandwiched on

each side by one -helix (H1 and H2); the second subdomain (subdomain 2, resides

113-163 and 165-230) consists of a -sheet of five antiparallel strands (B5-B9 with

the strand order of 5, 6, 9, 7, 8) that faces against five irregular -helices (H3, H4, H6,

H7 and H20). For the S domain, this domain is composed of an extended antiparallel

seven-stranded -sheet (B10-B16 with the strand order of 13, 10, 12, 11, 15, 16, 14),

faced by four major -helices in a row (H12, H10, H13, and H16). These two

domains are joined by two long loop regions (residues 231–241 and 454–474). (c)

The 2Fo-Fc electron density map of FAD contoured at 1.0 . (d) The isoalloxazine

ring of FAD is situated at the interface of the flavin- and substrate-binding domains,

while the ADP-ribosyl part is packed into a deep pocket between the two subdomains


19

of the F-domain. (e) The lipid cavity mainly is formed by residues Phe-93, Ser-364,

Tyr-395, Trp-399 and Ile-429. (f) The residues for FAD binding are highly conserved

(framed in blue), while the residues for substrate binding account for the major

variations between the proteins (framed in black). The tyrosine pair (Y165/Y473) is

conserved in both Dbv29 and AknOx, while AknOx also need a second general base

pair (W399 and I401). (g) Dbv29 and AknOx were superimposed with the rmsd of

0.734 Å for 480 Cα, which suggests both tertiary structures are similar but with

variations in the substrate-binding domain framed in red.


20

Dimeric form

a


21

Monomeric form

b


22

c

Dbv29 (dimer)

FAD


23

d

FAD

diol-Tei (8)

Tei-bound Dbv29 (monomer)


24

e

nativecomplex

Ca RMSD 0.291


25

f

3.02 Å

3.72 Å

R41Y139

L140

2.82 Å

3.81 Å

3.11 Å

2.80 Å

N44

R349

L351

Supplementary figure 3. Interfaces for and structural analysis of dimeric and

monomeric Dbv29. Analyses of native (a) and ligand-bound (b) structures of Dbv29

with PISA12

showed that the interfaces for the native and complex structures are 1350

and 300 Å , respectively. These results agree with that the native and complex

structures are dimeric and monomeric, respectively. The analysis statistics for each

protein is demonstrated on bottom panel. (c) The dimer of Dbv29 in a P6122 space

group (two polypeptide chains in an asymmetric unit); FAD cofactors are represented

as stick models. (d) Monomer of Dbv29 in complex with a water-coordinated-diol

intermediate (also see Fig. 2c) in a P21 space group (an asymmetric unit consists of


26

four polypeptide chains in which there are no typical protein-protein interactions with

one another); the FAD cofactors and the Tei intermediate are represented as stick

models in orange and blue, respectively. (e) Superposition of the ligand-free and

ligand-bound structures. Substrate-free and -bound structures were superimposed with

the rmsd of 0.291 Å for 498 Cα, which suggests binding of ligand does not induce

substantial conformational changes. (f) Residues Arg41 (forming two hydrogen bonds)

and Asn44 (four hydrogen bonds) were identified as potentially significant residues in

the dimer interface, and were investigated further by site-directed mutagenesis

(Supplementary Table 2) and AUC analysis (Supplementary figure 7 m, n).

(Protein interfaces, surfaces and assemblies were analyzed using the PISA server at

the European Bioinformatics Institute

(http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html)).


http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html)

27

a


28

b

diol-Tei (8)


29

c

Y395W399

M304

G305

E306

Q294

Q314

Q348

W350

L351

S353P355

R357


30

d

R357

M304

G356P355

S353

L351

Q348

W350

L168

Q314

P167

W292

Q294

Y165Y473

W399

F93

S364

Y395

G149

V150

C151


31

e

Y165

Y473

F93

T366

S364I429

Y395

W399

I401

FAD


32

f

Y135

Y165

Y473

Y470

Y403

Y370

g

Y403

Y370

4.15 Å

h

Y428

Y453

2.82 Å

i

R360

T366

Supplementary figure 4. Substrate binding site. (a) Residues that line the

ligand-binding site are located in different loops highlighted in orange (residues

148-152, 165-168, 300-308, 350-362, and 470-473). (b) The 2Fo-Fc electron density

map of the diol intermediate contoured at 1.0 . (c,d) Surface (c) and ball-and-stick (d)

illustrations of hydrophobic interactions (residues and surface are colored according

to their hydrophobicity; blue: positive charge; red: negative charge; white: neutral)

and hydrogen bonds (yellow dashed line) with the heptapeptide backbone, indicating

the major interacting forces between the aglycone and substrate-binding-site residues.

(e) View of the lipid tunnel in Dbv29, in which the N-acyl moiety of the aminosugar

at r4 is inserted into a hydrophobic cavity that is mainly composed of residues Phe93,

Ser364, Thr366, Tyr395, Trp399 and Ile429 (electrostatic surface potentials of

negative potential (78 KT) and positive potential (78 KT) are colored in red and blue,

respectively; neutral surface potential regions are displayed in white). In particular,

Trp399 is nearby the reducing end of the sugar, and, together with Ile401, stands

above the shoulder of the r4 sugar head and poises the head facing the isoalloxazine

ring. Ile401 stands on the other side of the sugar head, nearby the C6 carbon. Thr366,


33

situated opposite Trp399 at the entry of the lipid cavity, may help the lipid chain

insert into the lipid cavity (panel d). Ser364, which lines the bottom of the cavity, may

force the lipid to fold in a spiral manner. (f-h) Tyrosine residues near the active site

that could play a role in enzyme structure or function. (f) Tyr135, Tyr370, Tyr403 and

Tyr470 are shown in addition to the previously identified Tyr165 and Tyr473. (g,h).

Close up view of two tyrosine pairs, Tyr403/Tyr370 (4.1 Å ) (g) and Tyr428/Tyr453

(2.8 Å ) (h), that are positioned within approximate hydrogen-bond distances of each

other. (i) Close up view of two additional residues (Arg360 and Thr366) postulated to

play a role in Dbv29 function. Thr366 gates the lipid cavity along with bulky and

charged Arg360 that sits on the same long loop. Specific residues in proximity to the

ligand, as shown in panels c-i, were subject to mutagenesis to explore these

interactions further (see Supplementary Table 2).


34

Supplementary Table 2. Relative enzymatic activities of Dbv29 mutants

Mutants Expression Flavinylation/

FAD binding

Relative

activitya

Rationale Outcome Source of result

C26A + + 1.11 Putative point of active

site general base

Role disproven Ref. 2


site general base


C151A + + 0.23

Putative point of active

site general base/

Putative point of

covalent attachment

Role disproven/

Role proven

Ref. 2


site general base


H91A + + 0.11 Putative point of

covalent attachment

Role proven Ref. 2

I401A + + 0.09 Potential

ligand-binding residue

Role proven This work

I401W + + 0.02 Potential



N44A + + 0.87 Potential interface

interacting residue


R41A + + 0.72 Potential interface

interacting residue


R360E + + 0.76 Putative point affecting

FAD attachment

Role disproven This work

R360L + + 0.23 Putative point affecting

FAD attachment


S364K + + 0.04

Potential

ligand-binding residue/

Putative point affecting

FAD attachment

Role proven/

Role disproven

This work

S364R + + 0.08

Potential



FAD attachment

Role proven/

Role disproven

This work

T366A + + 0.11

Potential



FAD attachment

Role proven/

Role disproven

This work

T366E + + 0.05

Potential



FAD attachment

Role proven/

Role disproven

This work

T366L + + 0.13

Potential



FAD attachment

Role proven/

Role disproven

This work

W399A + + － Potential



W399F + + 0.16 Potential



Y135F －－－ Putative point affecting

FAD attachment

Not conclusive This work

Y165F + + 0.70 Putative point of active

site general base

Role proven Ref. 2

Y165W + + 0.03 Putative point of active

site general base



35

Y370F + + 0.10 Putative point affecting

FAD attachment



FAD attachment



FAD attachment



FAD attachment



FAD attachment


Y473F + + 0.23 Putative point of active

site general base

Role proven Ref. 2

Y473E + + － Putative point of active

site general base


H91A/C151A + + 0.05 Putative point of

covalent attachment

Role proven Ref. 2

R41A/N44A + + 0.59 Potential interface

interacting residue


R360E/T366E + + 0.03 Putative point affecting

FAD attachment


R360L/T366A + + 0.03 Putative point affecting

FAD attachment


R360L/T366E + + 0.01 Putative point affecting

FAD attachment


Y165F/Y473F + －－ Putative point of active

site general base

Role proven Ref. 2

Y428F/Y453F + + 1.08 Putative point affecting

FAD attachment


Y370F/Y403F + + 0.05 Putative point affecting

FAD attachment


a. The products of mutants were analyzed by HPLC; peak areas were integrated and

averaged for 3 triplicate points in 1.5 hours; the reaction rates were calculated using

linear regression equation; relative activities were determined by dividing individual

reaction rate with that of WT; the relative activity of WT is 1.0.


36

a

b 14 16 18 20 22 min

3 16

919162 i

ii

iii

iv

v

vi

vii

viii

ix

x

9*

1*6*


37

c

kcat (s-1

) Km (M-1

) kcat/ Km (s-1M

-1)

C2-pseudo-Tei 5.66 15 0.38

C10-pseudo-Tei 4.7 1.3 3.62

Model: OneSitesChi^2/DoF = 1.634E7N 1.05 0.0128 SitesK 9.42E5 2.24E5 M-1DH -7.476E4 1492 cal/molDS -219 cal/mol/deg

0.0 0.5 1.0 1.5 2.0

-80.00

-70.00

-60.00

-50.00

-40.00

-30.00

-20.00

-10.00

0.00

-2.50

-2.00

-1.50

-1.00

-0.50

0.00

0.50-10 0 10 20 30 40 50 60 70 80

Time (min)

µcal/

sec

Molar Ratio

KCal/

Mole

of I

njecta

ntca

l/se

c

d

Supplementary figure 5. Substrate preferences, kinetics, and thermodynamics for

Dbv21, Dbv29, and Orf2* (a Dbv21 ortholog in Tei (1) producing strain3,13,14

). (a)

The reactions catalyzed by each protein. Compounds 9, 16, and 19 are products of

Dbv29 when compounds 1, 3, and 6 serves as substrates, respectively. (b) HPLC

traces for enzymatic reactions of Dbv21 and/or Dbv29 in given conditions (described

below; note that compounds 1/6 and 9/19 have the same retention time).

i) LC trace of C4-Tei (3) that was enzymatically synthesized by Dbv8.

ii) LC trace of the reaction catalyzed by Dbv29 in the presence of C4-Tei (3).

iii) LC trace of the reaction catalyzed by Dbv21 in the presence of 3.

iv) LC trace of C10-Tei (6) that was enzymatically synthesized by Dbv8.

v) LC trace of the reaction catalyzed by Dbv29 in the presence of C10-Tei (6).

vi) LC trace of the reaction catalyzed by Dbv21 in the presence of C10-Tei (6).

vii) LC trace of the reaction catalyzed by both Dbv21 and 29 in the presence C10-Tei

(6); the ratio of products 19 to 2 is about 10, indicating Dbv29 is more efficient than


38

Dbv21 when both are included in the same reaction.

viii) LC trace of the reaction catalyzed by Dbv29 in the presence of substrates C4-Tei

(3) and C10-Tei (6); the ratio of products 19 to 16 is about 10.

ix) LC trace for compound 1 in a solution with no acid* added.

x) LC trace for compound 9 and compound 1 in a solution with no acid.

Comparing traces ii and v, we see that Dbv29 favors a substrate with a longer acyl

side chain (6, C10) to that with a shorter chain (3, C4) by 10-fold. Similarly, comparing

traces iii and vi, and the data in panel c, we conclude that Dbv21 also favors longer

acyl substrates. The data in traces ix and x explore the role of glycosylation in

increasing compound solubility. In acidic conditions (which will protonate carboxylic

acid), Tei (1) elutes later than when no acid is added (20.7 min vs. 20.4 min). The

solubility of oxo-Tei (9) changes more drastically in the absence of acid, eluting at

21.2 minutes with acid (after Tei) and 16.8 minutes with no acid (before Tei). (c)

Kinetic data of Orf2* against short (C2) and long (C10) lipid chain substrates. The

results show C10 is about 10-fold higher than C2 in terms of enzyme specificity

(kcat/Km), in which kcat is about the same for both while C10 has higher substrate

affinity. Thus Orf2* also prefers a longer chain. (d) ITC thermogram for Dbv29 and

Teicoplanin. Each exothermic heat pulse corresponds to injection of 2 l of Tei (1 mM)

into the protein solution (0.22 mM); integrated heat areas constituted a differential

binding curve that was fit to a standard single-site binding model (Origin 7.0,

MicroCal iTC200) to give the stoichiometry of binding15

, N = 1.05, binding affinity,

KA = 9.42 × 105

M-1

(KD = 1 M) and enthalpy of binding, DH = -75 kcal mol-1

.


39

Supplementary Table 3. Product yields of oxidized, aminated or amidated teicoplanin analogs synthesized by Dbv29.

Product

Coenzyme A

derivative

(Carbon No.)1

Amine added

Product type:

amide or

amine

Yield

(%)2

－－－ 100

－

Amine 25

－

Amide 10

－

Amide <10

－

Amide 10

－

Amine 25

－

Amine 17

C4 －－ 100


40

C6 －－ 100

C8 －－ 100

C10 －－ 100

C6

Amide 10

C8

Amide 10

C6

Amine 30

C8

Amine 30

－

Amide <10

－

Amine 46


41

－

Amine <5

－

Amine <5

－

Amine <5

－

Amide <5

－

Amine <10

－

Amine

25

1. Dbv8 was used to add various lengths of acyl side chains from corresponding

coenzyme A derivatives to the C2 amine group. If no CoA derivative is listed,

Teicoplanin was used directly.

The product yields were determined by HPLC; yield = (peak area of aminated or

amidated product)/(peak areas of aminated and amidated and oxidized products) ×

100%.


42

a

b

Supplementary figure 6. Mice infection test for analog 25 in blood bacterial

clearance. (a) The plot shows the whole blood bacterial counts of mice infected by E.

faecalis (ATCC 51559) and treated with vancomycin (Van, in black) or teicoplanin (1,

in red) or analog 25 (in green) or saline (control, in blue) for three days. The bacterial

counts for the control are steadily increased over time, while the counts for groups

treated with drugs are all suppressed but in various extents. Teicoplanin and analog 25

are more prominent than vancomycin in terms of overall drug efficacy. (b) The bar

chart shows the bacterial counts at day 3 (end of treatment). In general, these three

drugs all show positive effects against the infection, while only analog 25 displays

significant. The asterisk (*) indicates significant difference (P < 0.05); data were

expressed as mean ± SD for a group of three mice)

0 1 2 3

3

4

5

6

7

8

9

Van

1

25

Saline

Lo

g E

.faec

ium

cfu

/ml

wh

ole

blo

od

Day

Van 1 25 Saline

0

2

4

6

8

10

Log E

.faec

ium

cfu

/ml

wh

ole

blo

od


43

WT

Dimers=5.97Mcal=103.9 (kDa)

fr=1.33

Y165F


fr=1.44

Y473F


fr=1.00

Y165F/Y473F

Monomers=3.44, 4.12 Mcal=68.9, 64.1 (kDa)

fr=1.66


44

0

H91A/C151A


fr=1.44

10

WT + Tei (1)

Monomers=4.17Mcal=79.7 (kDa)

R41A/N44A

Mix of monomer and dimers=3.9, 4.4 Mcal=67.7, 58.8 (kDa)s=6.4Mcal=95.7 (kDa)

m n

fr=1.22

Supplementary figure 7. Analytical ultracentrifugation (AUC) analyses for Dbv29

and mutants16

. (a-n) The raw experimental data were analyzed by Sedfit

(http://www.analyticalultracentrifugation.com/default.htm) and the plots were

generated by MATLAB (MathWork, Inc.). The spectra in left panels (a, c, e, g, i, k, m)

are the calculated c(s, fr) distributions that are shown as two-dimensional distribution

with grid lines representing the s and fr grid in the thermograph. Below this c(s, fr)

surface a contour plot of the distribution is projected into the s-fr plane, where the

magnitude of c(s, fr) is indicated by contour lines at constant c(s, fr) in equidistant

intervals of c. Contour plots in the right panels (b, d, f, h, j, l, n) are transformed from


http://www.analyticalultracentrifugation.com/default.htm

45

the calculated c(s, fr) distribution and are shown as c(s, M) distribution. The dotted

lines (colored in blue) indicate lines of fr (frictional ratio). The signal of the c(s, M)

distribution is indicated by the color temperature. The insert grayscale bars in the right

panels indicate the residuals bitmap of each fit. (a, b) Dbv29 WT (dimer, s=5.97 (red

dashed line), fr=1.33 (blue dashed line)); (c, d) Dbv29 Y165F (dimer, s=5.92, fr=1.44);

(e, f) Dbv29 Y473F (dimer, s=6, fr=1.00); (g, h) Dbv29 Y165F/Y473F (monomer,

s=3.44, fr=1.66); (i, j) Dbv29 H91A/C151A (dimer, s=6.28, fr=1.44); (k, l) Dbv29 WT

in the presence of 10 mM Tei (1) (monomer, s=4.17, fr=1.44); (m, n) Dbv29

R41A/N44A (monomer, s=3.9, fr=1.22). The wild-type enzyme and the two single

mutants (Y165F and Y473F) (a-f) remain associated as dimers as opposed to the

double mutant that dissociates into monomers (g, h). The mutant also has a larger

frictional ratio (fr), suggesting its molecular shape has been altered considerably.


46

a

0 20 40 60 80 100 120 ml1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 3637

Dbv29 WT

b

0 20 40 60 80 100 120 ml1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 3637

Dbv29 Y165F

c 0 20 40 60 80 100 120 ml1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 3637

Dbv29 Y473F

d

e f 0 20 40 60 80 100 120 ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 3637

Dbv29 Y165F/Y473F

Supplementary figure 8. Gel filtration analyses for wild-type and mutant Dbv29.

The wild-type enzyme (a), single mutations Y165F (b) and Y473F (c), and the double

mutant H91A/C151A (d) all display sharp and symmetric peaks. Mutations at the

interface (R41A/N44A) cause a destabilization of dimeric structure, yielding a

bimodal distribution (e). The broadened and distorted peak for the Y165F/Y473F

double mutant (f) suggests structural heterogeneity.


47

-8000

-6000

-4000

-2000

0

2000

4000

190 200 210 220 230 240

Wavelength (nm)

Mea

n R

esid

ue

Ell

ipti

city

WT

Y165F

Y473F

Y165F/Y473F

H91A/C151A

R41A/N44A

Helix

(%)

Strand

(%)

Turns

(%)

Unordered

(%)

WT 8 38 11 43

Y165F 9 36 11 43

Y473F 8 37 12 43

Y165F/Y473F 16 31 13 40

H91A/C151A 8 35 11 44

R41A/N44A 16 26 20 39

Supplementary figure 9. Circular dichroism (CD) analyses for Dbv29 and mutants.

CD spectra of WT and mutants (Y165F, Y473F, Y165F/Y473F, H91A/C151A and

R41A/N44A), in which Y165F/Y473F and R41A/N44A double-mutations are

somewhat deviated from others as shown in secondary structure distributions.


48

a

b

Supplementary figure 10. CD analysis of protein stability. (a) The denaturation

temperature (Tm) for WT was determined by CD to be 84.7C, meaning Dbv29 is

unusually stable. (b) The denaturation temperature (Tm) for the Y165F/Y473F mutant

was determined to be 50.4C; the protein was significantly destabilized by lacking the

two oxygen atoms on the tyrosine pair. Therefore, residues Y165 and Y473 have

considerable influences on protein stability. (Tm = 34.3C)


49

RT: 0.00 - 42.00

0 5 10 15 20 25 30 35 40Time (min)

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

uA

U

13.31

14.0012.822.79 39.8224.67 28.3122.0716.41 33.374.34 35.677.86

NL:2.03E6Channel B UV 080612-orf2-P

(2)

O

NH

HN

HN

HN

NH

ONH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

O OH

OHHO

H2N

HO O

HO

O

OH

HOHO

NH

O

O

OH

OH

OHOH

O

Chemical Formula: C78H79Cl2N9O32

Exact Mass: 1723.4208

i

080612-orf2-P #4305-4699 RT: 12.53-13.63 AV: 395 NL: 3.25E5T: ITMS + c ESI Full ms [500.00-2000.00]

1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bu

nd

an

ce

1726.24

1744.401521.18 1691.28 1806.931645.87 1888.37 1945.38 1999.391565.12

(2)

1724.40

RT: 9.96 - 25.14

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25Time (min)

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

uAU

14.03

13.79 15.2012.55 15.49 23.6122.00 24.7522.4811.7410.72 19.6918.46

NL:1.40E6Channel B UV 080619-4CP

C4-Tei (3)


50

ii

080619-4CP #4826-4894 RT: 14.05-14.23 AV: 69 NL: 2.08E6T: ITMS + c ESI Full ms [500.00-2000.00]

1740 1760 1780 1800 1820 1840 1860 1880 1900 1920 1940m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

1795.53

1807.08 1822.051739.29 1841.13 1850.61 1877.151779.35 1896.36 1905.491756.20 1937.281926.29

C4-Tei (3)

1794.43

RT: 9.89 - 25.05

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25Time (min)

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

uAU

15.44

15.19

15.8716.1514.7714.12 23.6212.69 21.9311.75 22.43 24.7517.4911.00 19.7118.46


C6-Tei (4)

iii


1740 1760 1780 1800 1820 1840 1860 1880 1900 1920 1940m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

1823.90

1837.441759.19 1860.55 1878.94 1933.121910.111772.64 1819.60 1889.421804.961783.49

C6-Tei (4)

1822.30


51

RT: 9.89 - 25.04

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25Time (min)

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

uA

U17.10

15.19 17.4816.73 21.85 23.6114.23 18.81 24.7612.69 22.5710.83 11.73 19.61


C8-Tei (5)

iv


1740 1760 1780 1800 1820 1840 1860 1880 1900 1920 1940m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

1852.15

1756.83 1862.23 1874.59 1890.491780.22 1933.861907.48 1919.271834.191789.94 1811.90

C8-Tei (5)

1850.07

RT: 9.93 - 25.06

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25Time (min)

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

uA

U

18.79

19.1315.19 18.45 21.79 23.6122.2114.23 24.7412.69 17.6815.31 19.4110.83 11.25

NL:5.88E5Channel B UV 080619-10cp

C10-Tei (6)


52

v

080619-10cp #6473-6530 RT: 18.85-19.00 AV: 58 NL: 2.82E6T: ITMS + c ESI Full ms [500.00-2000.00]

1740 1760 1780 1800 1820 1840 1860 1880 1900 1920 1940m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

danc

e

1880.26

1775.53 1888.62 1907.971802.08 1918.65 1936.101811.981740.95 1863.031787.41 1827.75 1839.781754.97

C10-Tei (6)

1878.36

RT: 0.00 - 50.00

0 5 10 15 20 25 30 35 40 45Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

ance

17.50

20.7716.82 37.4721.6716.14 34.7313.30 29.655.01

NL:3.46E6Base Peak m/z= 1199.50-1200.50 MS 090327-teicohydrolysis-test3

aglycone (7)

vi

090327-teicohydrolysis-test3 #5333-5540 RT: 17.01-17.64 AV: 208 NL: 1.60E6T: ITMS + c ESI Full ms [200.00-2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

dan

ce

1200.05

1798.47608.83 1999.441360.001181.07 1599.00789.55533.12303.19 877.60

aglycone (7)

1198.20


53

RT: 16.98 - 23.06

17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5 23.0Time (min)

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

uA

U20.23

20.62

20.0419.85 23.0222.4722.2921.15 21.6919.4917.22 19.0517.75 18.7718.23

NL:5.56E5Channel B UV 090714-tei+dbv29+dmso+butylamine

12

090714-tei+dbv29+dmso+nacnbh3+butylamine #3554-3569 RT: 20.81-20.93 AV: 8 SB: 179 21.12-23.08 , 19.55-20.64 NL: 4.15E4

F: ITMS + c ESI Full ms [100.00-2000.00]

1920 1930 1940 1950 1960 1970 1980 1990 2000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

dan

ce

1949.41

1948.38

1971.771950.44

1970.83

1972.721947.311969.80

1951.43

1973.721961.35

1952.44

1962.23 1990.091980.131944.61

1957.681939.811922.73 1963.68 1992.261932.62

1933.721923.60

1937.49

(1947 + Na)

vii

090714-tei+dbv29+dmso+butylamine #3557-3575 RT: 20.84-20.98 AV: 9 SB: 178 21.12-23.07 , 19.55-20.64 NL: 9.78E3F: ITMS + c ESI Full ms [100.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

da

nce

385.33

367.38

321.36 459.55407.40248.31 475.53265.26 515.91349.38282.50 425.75 539.40488.07


54

RT: 14.96 ‐ 25.08 SM: 15B

15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

20.83

19.71

19.70

19.77

20.97

20.11

21.11 24.2523.0623.8919.30 22.81 24.6121.24

19.21 22.7821.5416.57 18.4115.98 16.70 17.8315.16

090707‐Tei+Dbv29+DMSO+NaCNBH3+anline

13

090707-tei+dbv29+dmso+nacnbh3+anline #6635-6683 RT: 20.96-21.12 AV: 49 NL: 1.28E4T: ITMS + c ESI Full ms [200.00-2000.00]

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

1969.23

1967.151991.68

1970.22

1992.68

1989.611971.23

1993.71

1972.31

1994.731980.67

1999.511973.321954.371908.451984.861894.24 1909.47

1950.20 1955.401919.451895.36 1938.311931.84

13

(1967 + Na)

viii

090707-tei+dbv29+dmso+nacnbh3+anline #6635-6683 RT: 20.96-21.12 AV: 49 NL: 6.86E3T: ITMS + c ESI Full ms [200.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bu

nda

nce

405.29

373.35

387.32

316.31

330.27298.31 528.39447.14341.34 431.12369.35287.33266.31 543.10465.05 488.79 509.10


55

RT: 16.97 - 23.01

17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5 23.0Time (min)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

60000

65000

70000

75000

80000u

AU

19.84

20.21

20.75

18.9721.73 22.14 22.7121.0119.65

17.33 18.8118.39

NL:8.29E4Channel B UV 090511-tei+dbv29+dmso+nacnbh3+octyalmine-10mm-025mm

14

090612-Tei+Dbv29+DMSO+NaCNBH3+octylamine-10mM-ms #3566-3589 RT: 20.88-21.05 AV: 12 SB: 235 18.65-20.73 , 21.55-23.48 NL: 2.85E5F: ITMS + c ESI Full ms [100.00-2000.00]

1920 1930 1940 1950 1960 1970 1980 1990 2000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

1991.60

1990.591992.59

1989.55

1993.58

1994.56

1999.53

1976.061966.331954.271936.07 1947.07 1962.22 1984.081924.92 1929.87

14

ix

090612-Tei+Dbv29+DMSO+NaCNBH3+octylamine-10mM-ms #3567-3596 RT: 20.88-21.11 AV: 15 SB: 235 18.65-20.73 , 21.55-23.48 NL: 7.63E3F: ITMS + c ESI Full ms2 [email protected] [50.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

danc

e

409.39

427.31

391.41255.22 381.54337.45268.26 349.45293.25 312.28 443.08 470.43


56

RT: 14.94 ‐ 25.05 SM: 7G

15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

23.48

23.69 24.9819.70

19.80

22.57 24.5021.9421.0920.3118.19 19.5218.0716.4116.22

090707‐Tei+Dbv29+DMSO+NaCNBH3+dodecylamine

15

090707-tei+dbv29+dmso+nacnbh3+dodecylamine #2777-2797 RT: 23.46-23.81 AV: 21 SB: 77 22.64-23.34 , 24.07-24.68 NL: 4.45E3T: ITMS + c ESI Full ms [200.00-2500.00]

2000 2010 2020 2030 2040 2050 2060 2070 2080 2090m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

danc

e

2047.11

2046.082048.13

2045.08

2070.08

2049.07

2068.41 2084.022050.16

2082.752071.022059.48 2088.512007.73 2058.192032.24 2080.50

2013.73 2052.962001.41 2075.012063.482018.64 2024.89 2036.45

15

x

090707-tei+dbv29+dmso+nacnbh3+dodecylamine #7523-7581 RT: 23.82-24.00 AV: 59 NL: 2.10E4T: ITMS + c ESI Full ms [200.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

da

nce

465.50

483.38

528.29

447.39266.23 316.18293.33 415.25395.37 431.10 545.31348.16 487.67364.25 505.18

O

OHHO

NH

O

NH

Chemical Formula: C28H55N2O4•

Exact Mass: 483.42


57

Time (min)

0

6 8 10 12 14 16 18 20 22 24 26

500000

uAU

14.03

14.56

13.77

16

xi

080619-4CP+Dbv29 #4933-5092 RT: 14.33-14.77 AV: 160 NL: 3.15E5T: ITMS + c ESI Full ms [500.00-2000.00]

1700 1750 1800 1850 1900m/z

20

40

60

80

100

Re

lativ

e A

bu

nd

an

ce

1810.26

1796.221830.28

1848.401688.12 1876.48 1911.251708.16 1729.07 1786.581759.48

1808.25

16

0

6 8 10 12 14 16 18 20 22 24 26Time (min)

1000000

uA

U

16.10

15.22 17


58

xii

080619-6cp+dbv29 #5461-5655 RT: 15.83-16.35 AV: 195 NL: 3.16E6T: ITMS + c ESI Full ms [500.00-2000.00]

1700 1750 1800 1850 1900m/z

20

40

60

80

100

Re

lativ

e A

bu

nd

an

ce

1837.88

1701.47 1851.511736.85 1823.32 1876.51 1920.061770.66 1902.971804.40

1836.40

O

NH

HN

HN

HN

NH

O

NH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

ONH

HO O

HO

O

OH

HOHO

NH

O

O

OH

OH

OH

OH

O

OHOH

O

OH

O


Exact Mass: 1835.47

17

0

500000

uA

U

17.75

6 8 10 12 14 16 18 20 22 24 26Time (min)

18

xiii


1700 1750 1800 1850 1900m/z

20

40

60

80

100

Re

lativ

e A

bu

nd

an

ce

1866.29

1888.541715.44 1904.471765.62 1853.511830.371798.621702.15

1864.3918


59

6 8 10 12 14 16 18 20 22 24 26Time (min)

0

400000

uAU

19.40

19

xiv


1700 1750 1800 1850 1900m/z

20

40

60

80

100

Re

lativ

e A

bu

nda

nce

1894.38

1689.24 1916.551730.21 1792.96 1880.441858.381813.27 1827.561748.43

1892.2919

RT: 14.92 ‐ 25.08 SM: 5G

15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

19.40

23.33

23.52 24.9117.62 23.0517.7816.60 22.8919.7816.8816.44 20.59 22.3018.6515.75

090721‐6C‐Tei+Dbv29+DMSO+NaCNBH3+hexylamine

20


60

090721-6C-Tei+Dbv29+DMSO+NaCNBH3+hexylamine #3103-3114 RT: 19.40-19.49 AV: 4 NL: 1.03E4F: ITMS + c ESI Full ms [100.00-2000.00]

1880 1890 1900 1910 1920 1930 1940 1950 1960m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bund

anc

e

1921.13

1920.04

1919.04 1922.26

1942.63

1943.67

1944.681941.13

1924.70

1906.85 1932.78 1945.671960.271904.87 1956.711930.83 1938.051887.76 1897.23 1913.56

1885.74

20

xv

090721-6C-Tei+Dbv29+DMSO+NaCNBH3+hexylamine #3100-3111 RT: 19.37-19.46 AV: 4 SB: 110 19.91-21.38 , 17.26-19.21 NL: 3.15E3F: ITMS + c ESI Full ms [100.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

357.24321.22

339.39447.05335.82

532.96273.23315.72279.05 443.66367.09 519.42414.50 502.27471.71308.55 387.26355.20254.05 542.25433.13294.18

RT: 14.94 ‐ 25.11 SM: 5G

15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

20.46

23.82

23.3923.2922.11

18.68 23.14 24.5118.37 18.83 22.4521.2119.6816.8416.53 17.0915.22

090721‐8C‐Tei+Dbv29+DMSO+NaCNBH3+hexylamine

21


61


1910 1920 1930 1940 1950 1960 1970 1980 1990m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bund

anc

e

1948.22

1949.16

1950.09

1969.71

1951.21

1972.621952.26 1982.74

1964.381933.66 1973.581911.59 1986.551938.74

1942.88 1989.651917.97 1974.591955.921925.56

21

1947.20

xvi


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

385.37

442.83481.50 508.08

383.53 430.25334.91 522.17365.28254.59 465.92390.53305.27274.69 545.36406.85 500.63

RT: 14.90 ‐ 25.04 SM: 5G

15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

22.21

19.28

24.3523.7322.7420.5319.6517.00 18.9616.66 17.9315.09 21.06 21.8315.81

090721‐6C‐Tei+Dbv29+DMSO+NaCNBH3+octylamine22


62

090721-6c-tei+dbv29+dmso+nacnbh3+octylamine #3082-3107 RT: 19.18-19.40 AV: 8 SB: 110 19.90-21.38 , 17.26-19.21 NL: 9.66E3F: ITMS + c ESI Full ms [100.00-2000.00]

1900 1910 1920 1930 1940 1950 1960 1970 1980m/z

0

10

20

30

40

50

60

70

80

90

100R

elat

ive

Abu

ndan

ce1935.35

1934.40

1936.331933.45

1937.301957.57

1955.421958.57

1938.41 1974.641961.70

1900.271948.551939.45 1977.741931.481922.52 1973.361940.31

22

xvii


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

dan

ce

353.36

371.30

321.19

291.28

260.27538.36337.12277.19 313.38 471.35 517.38431.06377.95 483.93446.96405.46 542.45

RT: 14.91 ‐ 25.04 SM: 5G

15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

23.22

19.96

23.5421.36 24.2221.0218.55 22.1419.7416.15 18.3317.4015.50 16.46

090721‐8C‐Tei+Dbv29+DMSO+NaCNBH3+octylamine23


63


1920 1930 1940 1950 1960 1970 1980 1990 2000m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bund

ance

1963.52

1962.44

1961.40

1964.46

1985.57

1965.37

1983.401986.58 1999.281946.66 1955.01 1966.34

1934.83 1987.531967.40 1993.861931.63 1973.69

1995.561957.68 1982.281935.77 1970.041924.03 1944.34

23

xviii

090723-8C-Tei+Dbv29+DMSO+NaCNBH3+octylamine #3150-3160 RT: 19.86-19.95 AV: 4 SB: 117 20.31-21.95 , 17.65-19.66 NL: 1.19E3F: ITMS + c ESI Full ms [100.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bu

nd

an

ce

381.43

399.28

328.20

457.22

498.89418.83 463.98454.50 504.98 520.53253.87 275.35 298.56 431.02369.93345.29 483.29 540.86316.38

269.36

RT: 16.98 - 23.07

17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5 23.0Time (min)

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

uA

U

19.85

20.22

19.30

21.1921.0320.68

22.9721.63 21.83 22.6119.1518.9617.29 18.4917.95

NL:2.33E5Channel B UV 090723-Tei+Dbv29+DMSO+NaCNBH3+benzylamine

24


64

090723-tei+dbv29+dmso+nacnbh3+benzylamine #6667-6673 RT: 21.01-21.03 AV: 7 SB: 641 21.27-22.60 , 20.10-20.79 NL: 1.35E4T: ITMS + c ESI Full ms [200.00-2000.00]

1920 1930 1940 1950 1960 1970 1980 1990 2000m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bund

anc

e

1983.14

1982.14

1981.141984.15

1985.07

1977.43 1986.11 1994.31

1932.741955.90

O

NH

HN

HN

HN

NH

O

NH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

ONH

HO O

HO

O

OH

HOHO

NH

O

O

OH

OH

OH

OH

O

OHOH

O

HN

O


Exact Mass: 1980.6024

xix

090723-tei+dbv29+dmso+nacnbh3+benzylamine #6645-6684 RT: 20.94-21.07 AV: 40 NL: 5.00E3T: ITMS + c ESI Full ms [200.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

419.29

316.29

403.39

431.09387.38

528.34312.21

RT: 16.98 - 23.07

17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5 23.0Time (min)

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

uA

U

19.85

20.22

19.30

21.1921.0320.68

22.9721.63 21.83 22.6119.1518.9617.29 18.4917.95

NL:2.33E5Channel B UV 090723-Tei+Dbv29+DMSO+NaCNBH3+benzylamine

25


65


1920 1930 1940 1950 1960 1970 1980 1990 2000m/z

0

10

20

30

40

50

60

70

80

90

100R

ela

tive

Ab

un

da

nce

1969.32

1970.33

1968.40

1971.28

1967.33

1991.531982.041972.33

1992.5225

xx


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bun

dan

ce

387.34

405.23

419.42369.38

O

OHHO

NH

O

HN

C23H37N2O4•

405.275

RT: 14.96 ‐ 25.09 SM: 7G

15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

19.99

22.9922.76 23.4521.14 24.9824.0119.15 22.0215.40 18.1817.8117.0215.72

091109‐Tei+Dbv29+DMSO+NaCNBH3+1‐Admantanemethylamine

26


66

091109-tei+dbv29+dmso+nacnbh3+1-admantanemethylamine #2683-2697 RT: 19.87-20.05 AV: 5 SB: 72 20.42-22.30 , 18.46-19.90 NL: 9.21E2F: ITMS + c ESI Full ms2 [email protected] [50.00-2000.00]

850 900 950 1000 1050 1100 1150m/z

0

10

20

30

40

50

60

70

80

90

100R

ela

tive

Ab

un

da

nce

1013.67

932.59

924.06

1005.12

1153.21

912.07

26 2+

xxi

091109-tei+dbv29+dmso+nacnbh3+1-admantanemethylamine #2683-2697 RT: 19.87-20.05 AV: 5 SB: 72 20.42-22.30 , 18.46-19.90 NL: 3.30E3F: ITMS + c ESI Full ms2 [email protected] [50.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bund

anc

e

463.33

445.37

427.41

RT: 14.97 ‐ 25.09 SM: 5G

15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

20.08

23.00

19.8922.63 23.32

24.0122.21 24.5719.71 21.8420.5917.16 19.3815.03 17.02 17.85 18.4115.77

091109‐Tei+Dbv29+DMSO+NaCNBH3+1‐Admantylamine

27


67

091109-tei+dbv29+dmso+nacnbh3+1-admantylamine #2694-2699 RT: 20.05-20.10 AV: 2 NL: 3.88E2F: ITMS + c ESI Full ms2 [email protected] [50.00-2000.00]

850 900 950 1000 1050 1100 1150m/z

0

10

20

30

40

50

60

70

80

90

100R

ela

tive

Ab

un

da

nce

1006.56

925.51

997.90

1153.29

1125.34

27

27 2+

xxii

091109-tei+dbv29+dmso+nacnbh3+1-admantylamine #2693-2704 RT: 20.05-20.19 AV: 4 NL: 8.83E2F: ITMS + c ESI Full ms2 [email protected] [50.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bu

nda

nce

449.35

431.41

RT: 14.96 ‐ 25.09 SM: 5G

15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

19.99

23.5022.81 23.69 24.7022.6221.6519.34 20.5915.07 17.3915.77 18.2316.88

091109‐Tei+Dbv29+DMSO+NaCNBH3+2‐Admantyiamine

28


68

091109-tei+dbv29+dmso+nacnbh3+2-admantyiamine #2682-2703 RT: 19.87-20.14 AV: 7 SB: 72 20.42-22.30 , 18.46-19.90 NL: 8.89E2F: ITMS + c ESI Full ms2 [email protected] [50.00-2000.00]

850 900 950 1000 1050 1100 1150m/z

0

10

20

30

40

50

60

70

80

90

100R

ela

tive

Ab

un

da

nce

1006.55

925.59917.05

998.01

905.16

1125.32992.57896.82 1108.30

28 2+

28

xxiii

091109-tei+dbv29+dmso+nacnbh3+2-admantyiamine #2686-2699 RT: 19.91-20.10 AV: 5 SB: 72 20.42-22.30 , 18.46-19.90 NL: 2.78E3F: ITMS + c ESI Full ms2 [email protected] [50.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nda

nce

449.35

431.38

447.33413.34

RT: 14.93 ‐ 25.06 SM: 7G

15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

19.10

20.30

18.79

24.9222.6120.64 22.95 23.8421.9318.0916.63 17.1915.62

100301‐Tei+Dbv29+DMSO+NaCNBH3+propargylamine

29


69

100301-tei+dbv29+dmso+nacnbh3+propargylamine #3200-3210 RT: 20.21-20.27 AV: 3 SB: 121 20.73-22.97 , 18.50-19.98 NL: 1.54E5F: ITMS + c ESI Full ms [100.00-2000.00]

1800 1820 1840 1860 1880 1900 1920 1940 1960 1980 2000m/z

0

10

20

30

40

50

60

70

80

90

100R

ela

tive

Ab

un

da

nce

1931.29

1894.35

1892.40

1914.18

1942.171905.901924.01

1887.08

1929.30

xxiv

100301-tei+dbv29+dmso+nacnbh3+propargylamine #3198-3207 RT: 20.18-20.24 AV: 3 SB: 121 20.73-22.97 , 18.50-19.98 NL: 5.87E3F: ITMS + c ESI Full ms [100.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bu

nda

nce

367.29

316.30 330.26

389.42 431.44

RT: 14.93 ‐ 25.06 SM: 7G

15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

19.10

20.30

18.79

24.9222.6120.64 22.95 23.8421.9318.0916.63 17.1915.62

100301‐Tei+Dbv29+DMSO+NaCNBH3+propargylamine

30


70

100301-tei+dbv29+dmso+nacnbh3+propargylamine #3092-3100 RT: 19.10-19.16 AV: 3 NL: 2.82E5F: ITMS + c ESI Full ms [100.00-2000.00]

1840 1860 1880 1900 1920 1940 1960 1980 2000m/z

0

10

20

30

40

50

60

70

80

90

100R

ela

tive

Ab

unda

nce

1917.42

1928.251938.641899.861892.251864.32

1915.42

xxv

100301-tei+dbv29+dmso+nacnbh3+propargylamine #3076-3112 RT: 18.92-19.29 AV: 13 NL: 3.08E4F: ITMS + c ESI Full ms [100.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bu

ndan

ce

335.35

353.19

O HN

OHHO

NH

O


Exact Mass: 353.24

RT: 14.95 ‐ 25.04 SM: 7G

15 16 17 18 19 20 21 22 23 24 25

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

19.54

21.7923.9922.25 22.43 24.41

20.6918.6216.51 18.3015.22 17.0115.86

100301‐Tei+Dbv29+DMSO+NaCNBH3+azidepentylamine

31


71

100301-tei+dbv29+dmso+nacnbh3+azidepentylamine #2667-2678 RT: 19.50-19.63 AV: 4 NL: 6.59E4F: ITMS + c ESI Full ms [100.00-2500.00]

1850 1900 1950 2000 2050 2100 2150m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bund

ance

1990.25

1877.91 1933.38

1988.25

xxvi

100301-tei+dbv29+dmso+nacnbh3+azidepentylamine #2667-2682 RT: 19.52-19.70 AV: 5 NL: 2.01E2F: ITMS + c ESI Full ms2 [email protected] [50.00-2000.00]

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bun

dan

ce

408.39

426.23

406.40

314.32 442.23

O HN

OHHO

NH

O

N3


Exact Mass: 426.31

RT: 7.98 - 24.40

8 10 12 14 16 18 20 22 24Time (min)

0

100000

200000

300000

400000

500000

600000

700000

800000

uA

U

16.21

17.3020.1516.0012.16 18.0414.02 19.0110.98 20.558.17 9.29 23.37

NL:8.48E5Channel B UV 090703-R6+mannose-teico

32


72

xxvii

090703-R6+mannose-teico #4906-5066 RT: 15.94-16.46 AV: 161 SB: 2490 7.77-10.07 , 16.66-22.44 NL: 8.59E4T: ITMS + c ESI Full ms [200.00-2000.00]

1450 1500 1550 1600 1650 1700m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

1565.12

1585.331685.131463.13 1726.201603.391547.09 1646.371529.061483.441433.27

1562.22

Supplementary figure 11. LC traces and mass spectra for Tei analogs. (i) LC trace

and ESI/MS spectrum of deacyl-Tei (2). (ii) LC trace and ESI/MS spectrum of C4-Tei

(3). (iii) LC trace and ESI/MS spectrum of C6-Tei (4). (iv) LC trace and ESI/MS

spectrum of C8-Tei (5). (v) LC trace and ESI/MS spectrum of C10-Tei (6). (vi) LC

trace and ESI/MS spectrum of Tei aglycone (7). (vii) LC, MS and MSMS spectra of

the butylamine-oxo-Tei amidated analog (12). (viii) LC, MS and MSMS spectra of the

aniline-oxo-Tei amidated analog (13). (ix) LC, MS and MSMS spectra of the

octylamine-Tei aminated analog (14). (x) LC, MS and MSMS spectra of the

dodecylamine-Tei aminated analog (15). (xi) LC trace and ESI/MS spectrum of

(C4)-oxo-Tei (16). (xii) LC trace and ESI/MS spectrum of (C6)-oxo-Tei (17). (xiii) LC

trace and ESI/MS spectrum of (C8)-oxo-Tei (18). (xiv) LC trace and ESI/MS

spectrum of (C10)-oxo-Tei (19). (xv) LC, MS and MSMS spectra of the

hexcylamine-(C6)-oxo-Tei amidated analog (20). (xvi) LC, MS and MSMS spectra of

the hexcylamine-(C8)-oxo-Tei amidated analog (21). (xvii) LC, MS and MSMS

spectra of the octylamine (C6)-oxo-Tei aminated analog (22). (xviii) LC, MS and

MSMS spectra of the octylamine (C8)-Tei aminated analog (23). (xix) LC, MS and

MSMS spectra of the benzylamine-Tei amidated analog (24) (as a minor product in

the given condition). (xx) LC, MS and MSMS spectra of the benzylamine-Tei

aminated analog (25) (as a major product in the given condition). (xxi) LC, MS and

MSMS spectra of the 1-admantanemetylamine-Tei aminated analog (26). (xxii) LC,

MS and MSMS spectra of the 1-admantylamine-Tei aminated analog (27). (xxiii) LC,

MS and MSMS spectra of the 2-admantylamine-Tei aminated analog (28). (xxiv) LC,

MS and MSMS spectra of the propargylamine-Tei amidated analog (29). (xxv) LC,

MS and MSMS spectra of the propargylamine-Tei aminated analog (30). (xxvi) LC,

MS and MSMS spectra of the 5-azide-pentylamine-Tei aminated analog (31). (xxvii)

LC and MS spectra of the r6-GlcNac,r7-Man pseudo-Tei (32, de-r4-Tei).


73

Notes: 1. The diol substrate (8) was undetectable; likely it has higher affinity than Tei

(1 M, Supplementary Fig. 5d); 2. teicoplanin aglycone (7) was prepared by

hydrolysis of teicoplanin in trifluoroacetic acid (TFA) solution17; C4-10-Tei were

enzymatically synthesized using Dbv21 and Dbv8, of which Dbv21 deacetylates r4

GlcNac of the Tei precursor and Dbv8 acylates the Glm resultant with corresponding

coenzyme A derivatives1-4. 3. NMR information for representative compounds is

shown in Supplementary Fig. 12-18 and Supplementary Table 4. 4. HRMS for

representative compounds is shown in Supplementary Fig. 19.


74

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1H NMR spectrum for compound 1

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13C NMR spectrum for compound 1


75

ppm

9 8 7 6 5 4 3 2 1 ppm

160

140

120

100

80

60

40

20

0

1H-13C HSQC spectrum for compound 1

ppm

11 10 9 8 7 6 5 4 3 2 1 ppm

180

160

140

120

100

80

60

40

20

1H-13C HMBC spectrum for compound 1

Supplementary figure 12. NMR information for compound 1. NMR spectra include 1H, 13C, HSQC, and HMBC.


76

10 9 8 7 6 5 4 3 2 1 ppm


180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm


O

NH

HN

HN

HN

NH

ONH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

O OH

OHHO

H2N

HO O

HO

O

OH

HOHO

NH

O

O

OH

OH

OHOH

O


77

ppm

9 8 7 6 5 4 3 2 1 ppm

160

140

120

100

80

60

40

20

0


O

NH

HN

HN

HN

NH

ONH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

O OH

OHHO

H2N

HO O

HO

O

OH

HOHO

NH

O

O

OH

OH

OHOH

O

ppm

11 10 9 8 7 6 5 4 3 2 1 ppm

180

160

140

120

100

80

60

40

20


O

NH

HN

HN

HN

NH

ONH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

O OH

OHHO

H2N

HO O

HO

O

OH

HOHO

NH

O

O

OH

OH

OHOH

O


78

ppm

10 9 8 7 6 5 4 3 2 1 ppm10

9

8

7

6

5

4

3

2

1

1H COSY spectrum for compound 2

O

NH

HN

HN

HN

NH

ONH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

O OH

OHHO

H2N

HO O

HO

O

OH

HOHO

NH

O

O

OH

OH

OHOH

O

Supplementary figure 13. NMR information for compound 2. NMR spectra include 1H, 13C, HSQC, HMBC, and COSY.


79

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm


180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm



80

ppm

9 8 7 6 5 4 3 2 1 ppm

160

140

120

100

80

60

40

20

0


ppm

11 10 9 8 7 6 5 4 3 2 1 ppm

180

160

140

120

100

80

60

40

20


Supplementary figure 14. NMR information for compound 9. NMR spectra include 1H, 13C, HSQC, and HMBC.


81

10 9 8 7 6 5 4 3 2 1 ppm


180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm


O

NH

HN

HN

HN

NH

ONH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

O NH

OHHO

NH

HO O

HO

O

OH

HOHO

NH

O

O

OH

OH

OHOH

O

O


82

ppm

9 8 7 6 5 4 3 2 1 ppm

160

140

120

100

80

60

40

20

0


ppm

11 10 9 8 7 6 5 4 3 2 1 ppm

180

160

140

120

100

80

60

40

20



83

ppm

10 9 8 7 6 5 4 3 2 1 ppm10

9

8

7

6

5

4

3

2

1


O

NH

HN

HN

HN

NH

ONH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

O NH

OHHO

NH

HO O

HO

O

OH

HOHO

NH

O

O

OH

OH

OHOH

O

O

Supplementary figure 15. NMR information for compound 10. NMR spectra include 1H, 13C, HSQC, HMBC and COSY.


84

10 9 8 7 6 5 4 3 2 1 ppm


180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm



85

ppm

9 8 7 6 5 4 3 2 1 ppm

160

140

120

100

80

60

40

20

0


O

NH

HN

HN

HN

NH

ONH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

O HN

OHHO

NH

HO O

HO

O

OH

HOHO

NH

O

O

OHOH

OHOH

O

O

ppm

11 10 9 8 7 6 5 4 3 2 1 ppm

180

160

140

120

100

80

60

40

20



86

ppm

10 9 8 7 6 5 4 3 2 1 ppm10

9

8

7

6

5

4

3

2

1


Supplementary figure 16. NMR information for analog 25. NMR spectra include 1H, 13C, HSQC, HMBC, and COSY.


87

10 9 8 7 6 5 4 3 2 1 ppm

1H NMR spectrum for compound 32 (de-r4-Tei)

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13C NMR spectrum for compound 32 (de-r4-Tei)


88

ppm

9 8 7 6 5 4 3 2 1 ppm

160

140

120

100

80

60

40

20

0

1H-13C HSQC spectrum for compound 32 (de-r4-Tei)

OH

NH

HN

HN

HN

NH

ONH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

HO O

HO

O

OH

HOHO

NH

O

O

OH

OH

OHOH

O

ppm

11 10 9 8 7 6 5 4 3 2 1 ppm

180

160

140

120

100

80

60

40

20

1H-13C HMBC spectrum for compound 32 (de-r4-Tei)


89

ppm

10 9 8 7 6 5 4 3 2 1 ppm10

9

8

7

6

5

4

3

2

1

1H COSY spectrum for compound 32 (de-r4-Tei)

Supplementary figure 17. NMR information for 32 (de-r4-Tei). NMR spectra

include 1H, 13C, HSQC, HMBC, and COSY.


90

Supplementary Table 4. NMR assignments for compound 25 as determined from

spectra shown in Supplementary Figures 16, 17, and 18.

O

HOOH

NH

NH

O

21

3

4

5

6

7

8

9

101'

2'

3'

4'

5'6'

7'

1"

2" 3"

4"

5"6"

O

NH

HN

HN

HN

NH

ONH2

O

O

O

O

O

O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

HO O

HO

O

OH

HOHO

NH

O

O

OH

OH

OHOH

r4

r6

r7 25 Reference: 32 (de-r4-Tei)

Position 13C-NMR (δ ppm) 1H-NMR (δ ppm)

C-1 172.4 —

C-2 28.9 1.18, 1.18 m (multiplicity)

C-3 28. 1.08, .08 m

C-4 26.8 1.0 , 1.08 m

C-5 26.7 1.17, 1.17 m

C-6 25.1 1.46, 1.46 m

C-7 25.0 1.38, 1.38 m

C-8 27.4 1.46 m

C-9/10 22.7 0.84, 0.84, 0.84 d (J 6.5)

C-1' 102.8 5.27 m

C-2' 72.4 3.28 m

C-3' 72.8 3.67 m

C-4' 72.2 3.28 m

C-5' 75.5 5.42 m

C-6' 51.7 3.85, 3.85 m

C-1" 133.3 —

C-2“/C-6" 129.2 7.27 m

C-3“/C-5" 130.1 7.48 m

C-4" 128.9 7.32 m

C-7" 48.1 4.83, 4.83 m


91

12

5.

62

12

5.

93

12

6.

25

12

7.

80

12

8.

38

12

8.

62

12

8.

80

12

8.

86

12

9.

04

12

9.

13

12

9.

20

12

9.

88

12

9.

96

13

0.

09

13

0.

21

13

0.

47

13

0.

63

13

1.

07

13

1.

14

126127128129130131132 ppm

DEPT 135

25

32 (de-r4-Tei)

*

*

*

*

*

C-5’’C-3’’

C-6’’

C-2’’

C-4’’

72

.15

72

.20

72

.2

6

72

.44

72

.5

6

72

.71

72

.80

72

.8

5

73

.1

3

73

.73

73

.80

74

.1

7

75

.5

4

75

.8

6

75

.9

6

72.573.073.574.074.575.075.576.0 ppm

DEPT 135

25

32 (de-r4-Tei)

*

* **

C-5’

C-3’ C-2’C-4’


92

10

1.

38

10

1.

64

10

1.

78

10

2.

20

10

2.

75

10

3.

72

10

3.

96

10

4.

63

10

5.

04

100.5101.0101.5102.0102.5103.0103.5104.0104.5105.0 ppm

DEPT 135

25

32 (de-r4-Tei)

*C-1’

17

1.

75

17

2.

36

17

2.

41

17

2.

79

17

2.

87

171.2171.4171.6171.8172.0172.2172.4172.6172.8173.0173.2173.4 ppm

13C

*C-1

25

32 (de-r4-Tei)


93

22

.6

5

23

.1

62

3.

25

27

.4

02

7.

47

23.023.524.024.525.025.526.026.527.027.528.028.529.029.5 ppm

DEPT 135

25

32 (de-r4-Tei)

**C-2 C-3

*C-8

*** *

*

C-4 C-5C-6 C-7

C-9C-10

Supplementary figure 18. NMR information for analog 25. While the structure of

analog 25 is big and complicated, the H1 and C13 chemical shifts (listed in

Supplementary Table 4) for the r4 (2)N-decsyl-(6)N-benzyl Glm moiety (bracketed)

were assigned. To achieve this, teicoplanin was subjected to partial degradation.

r7-Mannose (Man) pseudo-teicoplanin was identified and isolated from the

degradation mixtures. r6-GlcNac,r7-Man pseudo-teicoplanin (32, de-r4-Tei) (that

served as a reference) was enzymatically synthesized using r7-Man

pseudo-teicoplanin as a substrate and Orf1 (from Tei biosynthetic pathway) as a

catalyst, which (3 mg) was purified from reaction mixtures and subjected to NMR

analysis. Target peaks were identified by subtracting the spectra of analog 25 with

those of 32. Spectra of 13C and DEPT are shown here as both are useful to

differentiate primary, secondary and tertiary carbons. Peak assignments were also

assisted from spectra of 1H, COSY, HSQC, HMBC (Supplementary Fig. 16, 17, see

also Supplementary Table 4).


94

a

961.7654

962.2925

962.7728

963.2820

+MS, 0.3-0.5min #(23-36), Smoothed (0.00,2,GA)

0

1

2

3

4

Intens.

960 961 962 963 964 965 966 m/z

Compound 1HRMS ES(+): 961.7654 [M+2Na]2+, calc. for C88H97Cl2N9O33Na2 961.7680 [M+2Na]2+

b

873.7280

874.2443

874.7433 875.2475

875.7393

+MS, 0.5-0.7min #(39-54), Smoothed (0.0,2, GA)

0

1

2

3

4

5

6

Intens.

872 873 874 875 876 877 878 m/z

O

NH

HN

HN

HN

NH

ONH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

O OH

OHHO

H2N

HO O

HO

O

OH

HOHO

NH

O

O

OH

OH

OHOH

O

Compound 2HRMS ES(+): 873.7280 [M+H+Na]2+, calc. for 873.7092 C78H80Cl2N9O32Na [M+H+Na]2+


95

c

957.7779

958.2754

958.7741

959.2989

959.7795

960.2555

+MS, 0.6-1.2min #(46-94), Smoothed (0.00,2,GA)

0

1

2

3

4

Intens.

955 956 957 958 959 960 961 962 963 m/z

Compound 9HRMS ES(+): 957.7779 [M+H+Na]2+, calc. for C88H96Cl2N9O34Na 957.7667 [M+H+Na]2+

d

1009.3786

1009.8808

1010.3791

1010.8801

1011.3976

1011.8808

1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 m/z0

100

200

300

400

Intens.

Compound 10HRMS ES(+): 1009.3786 [M+2H]2+, calc. for C98H120O32N10Cl2 1009.37235 [M+2H]2+


96

e

984.3033

984.7898

985.2953

985.8031

986.2985

986.7818

982 983 984 985 986 987 988 989 m/z0

50

100

150

200

Intens.

Compound 25HRMS ES(+): 984.3033 [M+2H]2+, calc. for C95H104O32N10Cl2984.3098 [M+2H]2+

f

1006.3450

1006.8514

1007.3505

1007.8441

1008.3488

1008.8258

+MS, 0.1-0.6min #(10-49), Smoothed (0.0,2, GA)

0

2

4

6

Intens.

1004 1005 1006 1007 1008 1009 1010 1011 1012 m/z

Compound 28HRMS ES(+): 1006.3450 [M+2H]2+, calc. for C98H114Cl2N10O32 1006.3411 [M+2H]2+


97

g

958.2853

958.7712

959.2653

959.7758

960.2661

960.7763

961.2732

4p-tei.d: +MS, Smoothed (0.0,3, GA)

0

50

100

150

200

250

300

Intens.

957 958 959 960 961 962 963 m/z

O

NH

HN

HN

HN

NH

ONH2

O

O

O

O O

O

H

HO

Cl

Cl

HHN

O

HO2CH

O

HO

O HN

OHHO

NH

HOO

HO

O

OH

HOHO

NH

O

O

OH

OH

OHOH

O

O

Compound 31HRMS ES(+): 958.2853 [M+2H]2+, calc. for C91H102Cl2N10O32

958.2941 [M+2H]2+

h

780.1677

780.6697

781.1707

781.6668

782.1841

782.6713

-MS, 0.0-0.5min #(4-51), Smoothed (0.0,2, GA)

0.5

1.0

1.5

2.0

Intens.

777 778 779 780 781 782 783 784 785 m/z

32 de-r4-TeiHRMS ES(-): 780.1677 [M-2H]-2, calc. for C72H68O28N8Cl2780.1760 [M-2H]-2

Supplementary figure 19. HRMS data for representative compounds. High

resolution mass spectra were obtained for compounds 1 (a), 2 (b), 9 (c), 10 (d), 25 (e),

28 (f), 31 (g), and 32 (h), in which experimental and theoretical values are designated.


98

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4 Chan, H. C. et al. Regioselective deacetylation based on

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99

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