development of an in situfree screening test for the … of an in situfree screening test for the...

Development of an in situ Culture-Free Screening Test for the Rapid Detection of Staphylococcus aureus within Healthcare Environments Alex Sinclair*, Lauren E Mulcahy, Lynsey Geldeard, Samerah Malik, Mark D Fielder* and Adam Le Gresley*

Bacterial Culture

Bacterial isolates were obtained from a clinical setting as a result of both MRSA (MRSA numbers 1-11,13,14,16,17 and 19) and MSSA ( MSSA numbers 58,59.61,62, 63,64, 68, 69,71,72) infection, and were part of a cohort of bacteria taken from UK hospitals. E.coli samples were a combination of clinical isolates and wild type strains (E.coli 1-10), and coagulase negative Staphylococci include strains of S.epidermidis, S. warneri, S.hominus and M. luteus, and were obtained from clinical isolates from Sutton hospital. All bacteria were cultured initially on Brain Heart infusion agar, after which they were subcultured twice onto nutrient agar overnight at 37 °C, under aerobic conditions. Each bacterial isolate possesses individual characteristics, and thus displays a variance in the amount of coagulase produced, and therefore there is a degree of variance in results, lending to a greater amount of error in the results depicted.

Preparation of the LGX solution

A 100 μM and 50 μM solution of compound 3 was prepared by dissolving it in 2.5 % methanol followed by dilution in 1 x PBS. Solutions of Tris Base (0.05 M) and NaCl (0.1 M) in deionised H2O were added to the solution until an optimum pH of 8.5 was achieved. The appropriate amount of human prothrombin in 1 x PBS was then added to produce final prothrombin concentrations of 83.6 nM and 41.8 nM, respectively. The resulting solution was termed “LGX“. The concentrations of LGX, subsequently discussed, refer to the concentration of the active constituent compound 3.

Testing procedure the efficacy of LGX as a selective and sensitive means of detecting S. aureus

In order to detect the presence of staphylocoagulase and thus the efficacy of LGX, varying cell concentrations (106, 105, 103, 102 CFU/mL (50 μM LGX) and 104, 103, 102, 100 CFU/mL (100 μM LGX) were added to a microtitre plate (Nunclon 96 well plates) in a 1:1 ratio with either 50 μM or 100 μM of prepared LGX solution. This provided a final LGX concentration of 25 μM or 50 μM, respectively. The relative fluorescence was then recorded every fifteen minutes over a six hour time period (λex. = 488 nm and λem. = 525 nm). Positive controls used in each experiment were a control strain of MRSA (NCTC 12493), and the negative control was a clinical isolate of E. coli. A 1:1 ratio of both the 100 μM and 50 μΜ of LGX solution with 1 x PBS was used, and termed “LGX alone”. Fluorescence intensity was determined at 15 minute intervals over a 6 hour time period and n=3 samples were assayed in each case. We assayed 15 Strains of MRSA at varying cell concentration (106, 105, 103, 102 CFU/mL (50 μM LGX) and 104, 103, 102, 100 CFU/mL (100 μM) LGX)) with equal volume of both 50 μM and 100 μM LGX, respectively, to give a final LGX concentration of 25 μM or 50 μM, respectively, in addition to a strain of E-coli, which served as a negative control as described above.

1

Electronic Supplementary Material (ESI) for Organic & Biomolecular ChemistryThis journal is © The Royal Society of Chemistry 2013

As can be observed in figure 1, the results of all concentrations of MRSA (average of all 15 samples per data set illustrated) are plotted against a negative control and LGX alone, there is a significantly greater increase in fluorescence intensity in MRSA samples across the 6 hour time period. Taking a lower threshold limit of 20,000 relative fluorescence units (RFU) as a positive result, it can be seen that at a 25µM concentration of LGX, and at all 103 CFU/mL MRSA/MSSA, there is a clear positive result yet, however as expected, this is lower than the effect observed with the 50µM LGX. As with the 50 µM data an instantaneous positive reaction can also be observed. This data shows that the compound harbours the ability to rapidly detect SA at low concentrations.

( )

Figure 1 – The efficacy of 25μM LGX in detecting MRSA concentrations as low as

102 CFU/mL. Average of ten to fifteen bacterial strains (n=3). Error bars represent

standard deviation.

2

0

10000

20000

30000

40000

50000

60000

70000

0 1 2 3 4 5 6

RELA

TIVE

FLU

ORES

CENC

E UN

ITS

(ex

= 48

8 nm

, em

= 5

25 n

m)

TIME (HOURS)

MRSA 10^3 + 25 μM LGX

MSSA 10^3+ 25 μMLGX

E.COLI 10^3+ 25 μM LGX

CNS 10^3+ 25 μM LGX

NEGATIVE CONTROL + 25 μM LGX

LGX ALONE


The data demonstrated in Figure 2 illustrates that, similarly to 50 µM LGX and 500 µM coumarin, there is significantly more fluorescence produced by LGX, when compared to the coumarin system. These results show that at low concentrations, the LGX system demonstrates considerable efficacy in terms of speed of detection, as well sensitivity, and is a vast improvement on the existing coumarin system.

Figure 2 - The efficacy of 25 μM LGX at demonstrating both selectivity and

sensitivity in detecting coagulase positive MRSA and MSSA, compared to the

efficacy 100 μM coumarin, in bacterial concentrations of 103 CFU/mL. Average of

ten to fifteen bacterial strains (n=3). Error bars represent standard deviation.

0

10000

20000

30000

40000

50000

60000

70000

0 1 2 3 4 5 6

RELA

TIVE

FLO

URES

CEN

CE U

NIT

S ( e

x = 4

88 n

m, e

m =

252

nm

(LGX

), ex

= 25

5 nm

, em

= 4

60 n

m (c

oum

arin

))

TIME (HOURS)

MRSA 10^3 +100 μM coumarin

MSSA 10^3 + 100 μM coumarin

MRSA 10^3 + 25 μM LGX

MSSA 10^3+ 25 μMLGX

NEGATIVE CONTROL + 25 μM LGX

LGX ALONE

3


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 0.5 ppm

-0.0

00.

081.

111.

171.

261.

341.

401.

441.

691.

741.

761.

781.

882.

083.

934.

024.

044.

054.

385.

045.

075.

105.

135.

145.

165.

175.

215.

225.

245.

696.

506.

526.

786.

807.

017.

037.

187.

197.

197.

207.

207.

227.

227.

237.

257.

267.

277.

337.

347.

357.

387.

407.

447.

567.

587.

607.

627.

647.

988.

009.

019.

30

0.54

17.6

9

8.50

3.96

1.91

8.06

1.77

1.93

1.85

1.00

22.1

9

1.86

0.92

1.57

3.80

NAME LG1-51 CHCl3 CEXPNO 30PROCNO 1Date_ 20101111Time 20.08INSTRUM av400PROBHD 5 mm PABBO BB-PULPROG zg30TD 65536SOLVENT CDCl3NS 16DS 2SWH 8223.685 HzFIDRES 0.125483 HzAQ 3.9846387 secRG 90.5DW 60.800 usecDE 15.82 usecTE 297.1 KD1 1.00000000 secTD0 1

======== CHANNEL f1 ========NUC1 1HP1 15.00 usecPL1 -3.50 dBPL1W 16.47660065 WSFO1 399.7324685 MHzSI 65536SF 399.7300133 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.00

Rhod-(Arg-(CBz)2-NHBoc)2 (4)


ppm

12345678910 ppm

1

2

3

4

5

6

7

8

9

10

NAME LG1-51 CHCl3 CEXPNO 34PROCNO 1Date_ 20101111Time 20.58INSTRUM av400PROBHD 5 mm PABBO BB-PULPROG cosygpmfqfTD 2048SOLVENT CDCl3NS 2DS 8SWH 3875.969 HzFIDRES 1.892563 HzAQ 0.2642420 secRG 2050DW 129.000 usecDE 6.50 usecTE 297.2 KD0 0.00000300 secD1 1.92750096 secD13 0.00000400 secD16 0.00020000 secIN0 0.00025800 sec

======== CHANNEL f1 ========NUC1 1HP1 15.00 usecPL1 -3.50 dBPL1W 16.47660065 WSFO1 399.7320771 MHz

====== GRADIENT CHANNEL =====GPNAM1 SINE.100GPNAM2 SINE.100GPNAM3 SINE.100GPZ1 16.00 %GPZ2 12.00 %GPZ3 40.00 %P16 1000.00 usecND0 1TD 128SFO1 399.7321 MHzFIDRES 30.281008 HzSW 9.696 ppmFnMODE QFSI 1024SF 399.7300133 MHzWDW SINESSB 0LB 0.00 HzGB 0PC 1.40SI 1024MC2 QFSF 399.7300133 MHzWDW SINESSB 0LB 0.00 HzGB 0

Rhod-(Arg-(CBz)2-NHBoc)2 (4) COSY


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

0.04

25.0

128

.36

28.5

7

44.0

8

54.8

1

67.2

469

.10

76.7

777

.09

77.2

977

.41

80.4

682

.42

107.

9611

4.19

115.

4612

4.05

124.

9812

6.17

128.

0212

8.07

128.

3912

8.40

128.

5112

8.85

128.

9612

9.73

134.

4813

4.50

135.

1113

6.41

139.

6415

1.38

153.

1915

5.76

156.

2116

0.80

163.

5916

9.51

171.

0617

1.09

NAME LG1-51 CHCl3 CEXPNO 32PROCNO 1Date_ 20101111Time 20.40INSTRUM av400PROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536SOLVENT CDCl3NS 256DS 4SWH 24038.461 HzFIDRES 0.366798 HzAQ 1.3631988 secRG 2050DW 20.800 usecDE 9.66 usecTE 297.1 KD1 2.00000000 secD11 0.03000000 secTD0 1

======== CHANNEL f1 ========NUC1 13CP1 9.60 usecPL1 -3.70 dBPL1W 74.19429779 WSFO1 100.5222397 MHz

======== CHANNEL f2 ========CPDPRG2 waltz16NUC2 1HPCPD2 80.00 usecPL2 -3.50 dBPL12 11.04 dBPL13 14.00 dBPL2W 16.47660065 WPL12W 0.57925212 WPL13W 0.29300001 WSFO2 399.7315989 MHzSI 65536SF 100.5121880 MHzWDW EMSSB 0LB 1.00 HzGB 0PC 1.40




ppm

12345678910 ppm

140

120

100

80

60

40

20

0

NAME LG1-51 CHCl3 CEXPNO 31PROCNO 1Date_ 20101111Time 20.10INSTRUM av400PROBHD 5 mm PABBO BB-PULPROG hsqcedetgpsp.3TD 1024SOLVENT CDCl3NS 2DS 16SWH 3875.969 HzFIDRES 3.785126 HzAQ 0.1321460 secRG 2050DW 129.000 usecDE 6.50 usecTE 297.2 KCNST2 145.0000000D0 0.00000300 secD1 1.44777596 secD4 0.00172414 secD11 0.03000000 secD16 0.00020000 secD21 0.00360000 secIN0 0.00003005 secL31 1

======== CHANNEL f1 ========NUC1 1HP1 15.00 usecP2 30.00 usecP28 1.00 usecPL1 -3.50 dBPL1W 16.47660065 WSFO1 399.7320771 MHz

======== CHANNEL f2 ========CPDPRG2 bi_p5m4sp_4sp.2NUC2 13CP3 9.60 usecP14 500.00 usecP31 1900.00 usecP63 1500.00 usecPL0 120.00 dBPL2 -3.70 dBPL12 13.56 dBPL0W 0.00000000 WPL2W 74.19429779 WPL12W 1.39434588 WSFO2 100.5192243 MHzSP3 4.81 dBSP14 6.29 dBSP18 11.02 dBSP31 12.31 dBSPNAM3 Crp60,0.5,20.1SPNAM14 Crp32,1.5,20.2SPNAM18 Crp60_xfilt.2SPNAM31 Crp32,1.5,20.2SPOAL3 0.500SPOAL14 0.500SPOAL18 0.500SPOAL31 0.500SPOFFS3 0.00 HzSPOFFS14 0.00 HzSPOFFS18 0.00 HzSPOFFS31 0.00 Hz

====== GRADIENT CHANNEL =====GPNAM1 SINE.100GPNAM2 SINE.100GPZ1 80.00 %GPZ2 20.10 %P16 1000.00 usecND0 2TD 256SFO1 100.5192 MHzFIDRES 65.046150 HzSW 165.658 ppmFnMODE Echo-AntiechoSI 1024SF 399.7300133 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.40SI 1024MC2 echo-antiechoSF 100.5121880 MHzWDW QSINESSB 2LB 0.00 HzGB 0

Rhod-(Arg-(CBz)2-NHBoc)2 (4) HSQC with DEPT


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 0.5 ppm

0.10

0.10

0.11

0.12

0.12

0.17

1.28

1.28

1.29

1.30

1.48

1.56

1.58

1.60

1.77

1.78

1.79

1.98

2.07

2.20

3.57

3.97

4.01

4.02

4.73

5.11

5.13

5.14

5.15

5.16

5.16

5.18

5.18

5.27

5.33

6.73

6.74

7.07

7.13

7.14

7.29

7.30

7.31

7.33

7.34

7.35

7.36

7.37

7.38

7.39

7.40

7.41

7.63

7.64

7.65

7.66

7.68

7.74

8.05

8.06

9.29

9.43

3.21

3.41

12.4

1

0.13

1.49

2.65

4.68

4.19

0.51

1.38

1.74

27.8

91.

841.

611.

35

2.50

2.38

2.00

NAME LG1-41EXPNO 10PROCNO 1Date_ 20101004Time 12.48INSTRUM av600PROBHD 5 mm PATXI 1H/PULPROG zg30TD 65536SOLVENT CDCl3NS 16DS 2SWH 12345.679 HzFIDRES 0.188380 HzAQ 2.6542580 secRG 228DW 40.500 usecDE 11.34 usecTE 298.1 KD1 1.00000000 secTD0 1

======== CHANNEL f1 ========NUC1 1HP1 7.00 usecPL1 0.25 dBPL1W 16.59961510 WSFO1 600.1337060 MHzSI 65536SF 600.1300000 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.00

Rhodamine-(Arg-CBz)2-NH2)2 (6)


10 9 8 7 6 5 4 3 2 1 0 ppm

0.91

1.33

1.33

1.82

1.83

1.84

1.85

1.86

1.87

1.88

1.89

1.94

1.95

1.96

1.97

1.98

1.99

2.00

3.58

4.06

4.53

5.05

5.05

5.07

5.07

5.16

5.17

5.19

5.20

5.21

5.22

5.27

6.68

6.68

6.69

6.70

7.10

7.10

7.11

7.21

7.23

7.25

7.31

7.31

7.32

7.32

7.36

7.36

7.37

7.41

7.63

7.63

7.64

7.64

7.64

7.65

7.66

7.66

8.04

8.05

8.06

8.82

3.94

1.94

4.88

5.43

0.50

0.43

1.02

0.40

0.44

0.46

0.54

1.06

1.14

0.46

0.52

6.45

1.24

0.21

0.24

0.41

1.00

NAME LG1-57EXPNO 10PROCNO 1Date_ 20101124Time 14.54INSTRUM av600PROBHD 5 mm PATXI 1H/PULPROG zg30TD 65536SOLVENT CDCl3NS 16DS 2SWH 8417.509 HzFIDRES 0.128441 HzAQ 3.8928883 secRG 57DW 59.400 usecDE 6.50 usecTE 308.0 KD1 1.00000000 secTD0 1

======== CHANNEL f1 ========NUC1 1HP1 7.00 usecPL1 0.25 dBPL1W 16.59961510 WSFO1 600.1339008 MHzSI 32768SF 600.1300000 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.00

Rhodamine-(Arg-(Cbz)2-Pro-Val-NHBoc)2 (5)


ppm

12345678910 ppm

1

2

3

4

5

6

7

8

9

10

NAME LG1-57EXPNO 13PROCNO 1Date_ 20101124Time 15.31INSTRUM av600PROBHD 5 mm PATXI 1H/PULPROG cosygpmfqfTD 2048SOLVENT CDCl3NS 2DS 8SWH 6009.615 HzFIDRES 2.934382 HzAQ 0.1704436 secRG 2050DW 83.200 usecDE 6.50 usecTE 308.0 KD0 0.00000300 secD1 2.00000000 secD13 0.00000400 secD16 0.00020000 secIN0 0.00016640 sec

======== CHANNEL f1 ========NUC1 1HP1 7.00 usecPL1 0.25 dBPL1W 16.59961510 WSFO1 600.1330006 MHz

====== GRADIENT CHANNEL =====GPNAM1 SINE.100GPNAM2 SINE.100GPNAM3 SINE.100GPZ1 16.00 %GPZ2 12.00 %GPZ3 40.00 %P16 1000.00 usecND0 1TD 128SFO1 600.133 MHzFIDRES 46.950092 HzSW 10.014 ppmFnMODE QFSI 1024SF 600.1300000 MHzWDW SINESSB 0LB 0.00 HzGB 0PC 1.00SI 1024MC2 QFSF 600.1300000 MHzWDW SINESSB 0LB 0.00 HzGB 0

Rhodamine-(Arg-(Cbz)2-Pro-Val-NHBoc)2 (5) gradient COSY


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

0.02

1.04

14.1

517

.49

17.5

519

.41

25.0

125

.21

28.3

929

.72

31.2

6

44.0

3

47.6

6

54.0

557

.18

57.2

760

.34

60.4

667

.25

69.1

576

.73

77.0

577

.25

77.3

779

.71

82.5

5

108.

1911

4.22

123.

8812

5.06

126.

3212

8.15

128.

1912

8.28

128.

3912

8.51

128.

5312

8.86

128.

9412

9.73

134.

5113

5.04

136.

2413

9.98

151.

5515

5.89

161.

1516

3.49

170.

0217

2.02

NAME LG1-57carbEXPNO 10PROCNO 1Date_ 20101125Time 9.42INSTRUM av400PROBHD 5 mm PABBO BB-PULPROG zgpg30TD 65536SOLVENT CDCl3NS 1024DS 4SWH 24038.461 HzFIDRES 0.366798 HzAQ 1.3631988 secRG 2050DW 20.800 usecDE 9.66 usecTE 296.9 KD1 2.00000000 secD11 0.03000000 secTD0 1

======== CHANNEL f1 ========NUC1 13CP1 9.60 usecPL1 -3.70 dBPL1W 74.19429779 WSFO1 100.5222397 MHz

======== CHANNEL f2 ========CPDPRG2 waltz16NUC2 1HPCPD2 80.00 usecPL2 -3.50 dBPL12 11.04 dBPL13 14.00 dBPL2W 16.47660065 WPL12W 0.57925212 WPL13W 0.29300001 WSFO2 399.7315989 MHzSI 65536SF 100.5121880 MHzWDW EMSSB 0LB 1.00 HzGB 0PC 1.40




ppm

10 9 8 7 6 5 4 3 2 1 ppm

140

120

100

80

60

40

20

0

NAME LG1-57EXPNO 12PROCNO 1Date_ 20101124Time 15.14INSTRUM av600PROBHD 5 mm PATXI 1H/PULPROG hmbcetgpl3ndTD 4096SOLVENT CDCl3NS 4DS 16SWH 6009.615 HzFIDRES 1.467191 HzAQ 0.3408372 secRG 2050DW 83.200 usecDE 6.50 usecTE 307.8 KCNST6 120.0000000CNST7 165.0000000CNST13 10.0000000CNST30 0.5981154D0 0.00000300 secD1 1.50000000 secD6 0.05000000 secD16 0.00020000 secIN0 0.00001490 sec

======== CHANNEL f1 ========NUC1 1HP1 7.00 usecP2 14.00 usecPL1 0.25 dBPL1W 16.59961510 WSFO1 600.1330006 MHz

======== CHANNEL f2 ========NUC2 13CP3 12.00 usecP24 2000.00 usecPL2 -3.30 dBPL2W 191.18331909 WSFO2 150.9178750 MHzSP7 3.28 dBSPNAM7 Crp60comp.4SPOAL7 0.500SPOFFS7 0.00 Hz

====== GRADIENT CHANNEL =====GPNAM1 SINE.100GPNAM3 SINE.100GPNAM4 SINE.100GPNAM5 SINE.100GPNAM6 SINE.100GPZ1 80.00 %GPZ3 14.00 %GPZ4 -8.00 %GPZ5 -4.00 %GPZ6 -2.00 %P16 1000.00 usecND0 2TD 128SFO1 150.9179 MHzFIDRES 261.860260 HzSW 222.095 ppmFnMODE Echo-AntiechoSI 2048SF 600.1300000 MHzWDW SINESSB 2LB 0.00 HzGB 0PC 1.00SI 1024MC2 echo-antiechoSF 150.9028090 MHzWDW SINESSB 2LB 0.00 HzGB 0

Rhodamine-(Arg-(Cbz)2-Pro-Val-NHBoc)2 (5) HMBC


ppm

10 9 8 7 6 5 4 3 2 1 ppm

140

120

100

80

60

40

20

0

NAME LG1-57EXPNO 11PROCNO 1Date_ 20101124Time 15.00INSTRUM av600PROBHD 5 mm PATXI 1H/PULPROG hsqcedetgpsp.3TD 1024SOLVENT CDCl3NS 2DS 16SWH 6009.615 HzFIDRES 5.868765 HzAQ 0.0852468 secRG 2050DW 83.200 usecDE 6.50 usecTE 308.3 KCNST2 145.0000000D0 0.00000300 secD1 1.50000000 secD4 0.00172414 secD11 0.03000000 secD16 0.00020000 secD21 0.00360000 secIN0 0.00002000 secL31 1

======== CHANNEL f1 ========NUC1 1HP1 7.00 usecP2 14.00 usecP28 0.00 usecPL1 0.25 dBPL1W 16.59961510 WSFO1 600.1330006 MHz

======== CHANNEL f2 ========CPDPRG2 bi_p5m4sp_4sp.2NUC2 13CP3 12.00 usecP14 500.00 usecP31 1730.00 usecP63 1500.00 usecPL0 120.00 dBPL2 -3.30 dBPL12 12.02 dBPL0W 0.00000000 WPL2W 191.18331909 WPL12W 5.61629534 WSFO2 150.9133719 MHzSP3 3.28 dBSP14 4.76 dBSP18 8.67 dBSP31 10.78 dBSPNAM3 Crp60,0.5,20.1SPNAM14 Crp32,1.5,20.2SPNAM18 Crp60_xfilt.2SPNAM31 Crp32,1.5,20.2SPOAL3 0.500SPOAL14 0.500SPOAL18 0.500SPOAL31 0.500SPOFFS3 0.00 HzSPOFFS14 0.00 HzSPOFFS18 0.00 HzSPOFFS31 0.00 Hz

====== GRADIENT CHANNEL =====GPNAM1 SINE.100GPNAM2 SINE.100GPZ1 80.00 %GPZ2 20.10 %P16 1000.00 usecND0 2TD 256SFO1 150.9134 MHzFIDRES 97.656288 HzSW 165.658 ppmFnMODE Echo-AntiechoSI 2048SF 600.1300000 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.00SI 1024MC2 echo-antiechoSF 150.9028090 MHzWDW QSINESSB 2LB 0.00 HzGB 0

Rhodamine-(Arg-(Cbz)2-Pro-Val-NHBoc)2 (5) echo/antiecho edited HSQC w/adiabatic bilevel decoupling w/matched adiabatic sweep


10 9 8 7 6 5 4 3 2 1 0 ppm

0.11

0.90

0.91

0.92

1.01

1.31

1.32

1.32

1.33

1.33

1.34

1.35

1.42

1.42

1.43

1.74

1.75

1.76

1.77

1.78

1.86

1.87

1.99

2.00

2.01

2.02

2.03

2.11

2.28

2.29

2.29

3.04

3.24

3.25

3.26

3.28

3.68

3.70

4.16

4.49

4.50

4.51

6.64

6.65

6.73

6.74

6.75

7.18

7.19

7.20

7.20

7.22

7.42

7.74

7.75

7.78

7.80

7.83

7.89

7.99

8.04

8.05

8.18

8.45

8.46

10.1

110

.11

12.3

0

7.79

18.0

0

15.6

7

2.12

4.10

2.16

11.2

6

1.94

1.39

1.73

2.58

1.71

2.59

4.56

1.78

3.77

1.61

0.66

1.61

1.36

NAME ASLG57D2EXPNO 16PROCNO 1Date_ 20110210Time 15.29INSTRUM av600PROBHD 5 mm PATXI 1H/PULPROG noesygppr1dTD 65536SOLVENT MeODNS 16DS 4SWH 12345.679 HzFIDRES 0.188380 HzAQ 2.6542580 secRG 22.6DW 40.500 usecDE 8.36 usecTE 298.4 KD1 4.00000000 secD8 0.01000000 secD12 0.00002000 secD16 0.00020000 secZGOPTNS -DFLAG_BLK

======== CHANNEL f1 ========NUC1 1HP0 7.00 usecP1 7.00 usecSI 65536SF 600.1300000 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.00

Rhodamine-(Arg-Pro-Val-NHBoc)2 (4)



ppm

10 9 8 7 6 5 4 3 2 1 ppm

10

9

8

7

6

5

4

3

2

1

NAME ASLG57D2EXPNO 24PROCNO 1Date_ 20110211Time 10.16INSTRUM av600PROBHD 5 mm PATXI 1H/PULPROG cosygpprqfTD 4096SOLVENT MeODNS 16DS 8SWH 9615.385 HzFIDRES 2.347506 HzAQ 0.2130420 secRG 128DW 52.000 usecDE 6.50 usecTE 298.4 KD0 0.00000300 secD1 2.00000000 secD11 0.03000000 secD12 0.00002000 secD16 0.00020000 secIN0 0.00010395 sec

======== CHANNEL f1 ========NUC1 1HP0 7.00 usecP1 7.00 usecND0 1TD 256SFO1 600.1329 MHzFIDRES 37.576378 HzSW 16.029 ppmFnMODE QFSI 2048SF 600.1300000 MHzWDW SINESSB 0LB 0.00 HzGB 0PC 1.00SI 2048MC2 QFSF 600.1300000 MHzWDW SINESSB 0LB 0.00 HzGB 0

Rhodamine-(Arg-Pro-Val-NHBoc)2 (3) COSY


200 180 160 140 120 100 80 60 40 20 0 ppm

17.25

18.36

22.34

24.75

24.90

27.31

28.85

29.23

29.35

30.23

35.56

40.72

44.10

48.30

48.58

53.70

57.97

60.43

73.31

79.20

82.66

107.38

114.14

115.57

123.72

124.60

126.22

128.03

129.99

135.44

140.71

151.49

156.71

157.44

163.85

169.80

171.04

172.17

173.20

NAME ASLG57D2EXPNO 23PROCNO 1Date_ 20110210Time 20.51INSTRUM av600PROBHD 5 mm PATXI 1H/PULPROG zgpg30TD 65536SOLVENT MeODNS 5120DS 4SWH 36057.691 HzFIDRES 0.550197 HzAQ 0.9088159 secRG 322DW 13.867 usecDE 6.50 usecTE 298.4 KD1 2.00000000 secD11 0.03000000 sec

======== CHANNEL f1 ========NUC1 13CP1 12.00 usecSI 32768SF 150.9028090 MHzWDW EMSSB 0LB 1.00 HzGB 0PC 1.40




ppm

10 9 8 7 6 5 4 3 2 1 ppm

140

120

100

80

60

40

20

NAME ASLG57D2EXPNO 13PROCNO 2Date_ 20110210Time 13.21INSTRUM av600PROBHD 5 mm PATXI 1H/PULPROG hsqcedetgpsisp2.4TD 1024SOLVENT MeODNS 4DS 32SWH 9615.385 HzFIDRES 9.390024 HzAQ 0.0532980 secRG 1DW 52.000 usecDE 6.50 usecTE 299.4 KCNST2 145.0000000CNST17 -0.5000000D0 0.00000300 secD1 1.50000000 secD2 0.00344828 secD4 0.00172414 secD11 0.03000000 secD16 0.00020000 secD21 0.00360000 secD24 0.00089000 secIN0 0.00002000 secL0 0TD0 2

======== CHANNEL f1 ========NUC1 1HP1 7.00 usecP2 14.00 usecP28 0.00 usecND0 2TD 256SFO1 150.9134 MHzFIDRES 97.656288 HzSW 165.658 ppmFnMODE Echo-AntiechoSI 2048SF 600.1300000 MHzWDW QSINESSB 2LB 0.00 HzGB 0PC 1.40SI 1024MC2 echo-antiechoSF 150.9028090 MHzWDW QSINESSB 2LB 0.00 HzGB 0

Rhodamine-(Arg-Pro-Val-NHBoc)2 (3) HSQC


ASLG57D MW=1235? EPSRC National Centre Swansea Alex Sinclair(MeOH)/MeOH + NH4OAc LTQ Orbitrap XL 02/02/2011 12:47:25

KINSIN001-OE-HNESP #18-22 RT: 0.82-0.91 AV: 5 SM: 7G NL: 6.42E6T: FTMS + p NSI Full ms [120.00-2000.00]

200 300 400 500 600 700 800 900 1000 1100 1200

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

618.3317z=2

520.2710z=2

211.0965z=1

391.2844z=1

234.2065z=1

824.4411z=3


ASLG57D MW=1235? EPSRC National Centre Swansea Alex Sinclair(MeOH)/MeOH + NH4OAc LTQ Orbitrap XL 02/02/2011 12:47:25

617 618 619 620 621

m/z

0

20

40

60

80

100

0

20

40

60

80

100

Re

lative

Ab

un

da

nce

Observed Data

618.3317z=2

618.8327z=2

619.3349z=2

619.8374z=2

Theoretical Isotope Model: [M + 2H]2+

618.3322

618.8337

619.3352

619.8366

NL:6.42E6

KINSIN001-OE-HNESP#18-22 RT: 0.82-0.91 AV: 5 T: FTMS + p NSI Full ms [120.00-2000.00]

NL:1.10E4

C 62 H 86 N 14 O 13 HH: C 62 H 88 N 14 O 13

p (gss, s /p:40) Chrg 2R: 100000 Res .Pwr . @FWHM


development of an in situfree screening test for the … of an in situfree screening test for the...

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