d13 setnmr3|13 ;W4 output N2 d8 setnmr3^13. ;;;;;;;stop bubbling;;;;;;;; d14 setnmr3|14 ;W6 output out to release pressure d9 setnmr3^14. ;;;;;;;;; FIRST spectrum ...
A highly versatile automatized setup for quantitative measurements of PHIP enhancements Alexey S. Kiryutin,#1,2 Grit Sauer,#3 Sara Hadjiali,3 Alexandra V. Yurkovskaya,1,2 Hergen Breitzke,3 Gerd Buntkowsky3* 1
International Tomography Center, Institutskaya 3A, Novosibirsk, 630090, Russia. 2
3
Novosibirsk State University, Pirogova 2, Novosibirsk, 630090, Russia
Technische Universität Darmstadt, Eduard-Zintl-Institut für Anorganische und Physikalische Chemie, Alarich-Weiss-Straße 8, Darmstadt, 64287, Germany
Electronic Supplementary Information
Materials and Methods Setup 1 Images Setup 2 Images Magnetic Valves Driver Unit Accessory Parts Quantitative NMR Measurements Implemented Pulse programs
2 3 3 4 6 7 9
Pulse program for PHIP measurement
9
Pulse programs for H2 and N2 dissolution dynamics experiments Pulse program for study reaction kinetics References
11 13 14
S1
Materials and Methods All chemicals were used without additional purification. Catalyst Bis(diphenylphosphino)butane](1,5cyclooctadiene)rhodium(I) tetrafluoroborate was purchased from Strem Chemicals. The substrate Fmoc-O-propargyl-L-tyrosine was purchased from Iris Biotech GmbH. The deuterated solvents (methanol-d4, acetone-d6, and D2O) were purchased from Deutero GmbH. Setup1 and Setup2 were installed in Darmstadt on a 500 MHz NMR spectrometer operated by a Bruker Avance III HD console with the Topspin 3.2 software. In Novosibirsk both setups were installed on 400 MHz and 700 MHz NMR spectrometers operated by Bruker Avance III HD console with the Topspin 3.2 software. NMR spectra were evaluated with MestReNova software (Mestrelab Research).
S2
Setup 1 Images
Fig. S1: Photos of the Setup 1: A) Whole Setup 1 with exhaust pipe on top of the magnet (silver pipe), B) gas switching panel with TTL controlled magnetic valves and connection to the plastic capillary for NMR tube and C) operation and storage panel, the storage bottle for parahydrogen is placed behind the panel (not visible), left-sided the Dewar vessel with the U-tube to generate parahydrogen, the box in front of the operation panel is the magnetic valves driver unit.[1]
Setup 2 Images
Fig. S2: Images of Setup 2, left: operation and storage panel, right: gas switching panel with TTL controlled magnetic valves
S3
Fig. S3: Images of the whole operating and storage unit, the para-H2 generation unit and the power supply and controller for magnetic valves – the magnetic valve driver unit
Magnetic Valves Driver Unit The grey boxes in Figure S3 are the power supply and control unit for the magnetic valves. This magnetic valves driver unit was produced by the workshop of TU Darmstadt (price per unit 150€). The connection diagram is displayed in Figure S4 and the bill of material list is shown in Table S1. Each box can operate 4 magnetic valves. For Setup 2, actually 5 magnetic valves are necessary therefore 2 boxes are needed. The control of magnetic valves can be done either via TTL pulses, programmed from the operation unit of the NMR spectrometer or directly manually by switching or pushing the corresponding button on the front of the grey box.
S4
Fig. S4: Electrical circuit diagram of the magnetic valves driver unit including manual and TTL switches Table S1: Bill of materials for the magnetic valves driver unit Number I1 R1,R2,R3,R4 R9,R10,R11,R12 R5,R6,R7,R8 D2,D3,D4,D5 X7 X9 X8 X2 X3 X1 X4 X5 X6 J4 J3 J2 J1 D7 D8 D6 D9 T1,T2,T3,T4 D1 C2 C1
description
specification
quantity
Controller vertical 1/4 W horizontal R4 1/4 W horizontal R4 1/4 W horizontal R4 1N4007 2-pole connector 2-pole connector 2-pole connector 2-pole connector 2-pole connector BNC connector BNC connector BNC connector BNC connector Jumper 1x2 Jumper 1x2 Jumper 1x2 Jumper 1x2 LED 5mm solder point LED 5mm solder point LED 5mm solder point LED 5mm solder point Power FET N-Chanel MGC-7486/1 Poly RM5 + 7,5 capacitor Poly RM5 + 7,5 capacitor table housing SNT RS 75 24 power supply
7805 1K5 100 4K7 1N4007 MV2 MV4 MV3 UB MV1 TTL1 TTL2 TTL3 TTL4 4 3 2 1 MV2 MV3 MV1 MV4 IRL540 74LS86 330nF 100nF Conrad Electronic Reichelt Electronic
1 4 4 4 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4 1 1 1 1 1
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Accessory Parts Table S2: Bill of materials for Setup 1 and Setup 2 Nr
Name
Quantity
Part number
Vendor or Supplier
Price per item EUR
1
2/2-Way, directly controlled magnetic valves
5
Bored-Through Male Connector, 6 mm Tube OD x 1/8 in. Male ISO Tapered Thread, Stainless Steel (used for the magnetic valves) Pressure regulator 0.05-1 bar
10
GSR Ventiltechnik GmbH&Co Swagelok GmbH
35,50
2
G052.0007 14.010.009 .010 SS-6M0-12RTBT
2
BS-50-1-2
Air Liquide GmbH
211,00
4
S-6M0-16RP
Swagelok GmbH
18,50
1
SSSS6MM PFA-T1015 SS-6P4TMM
Swagelok GmbH Swagelok GmbH Swagelok GmbH
153,90
5
SS-6M0-4
47,55
5
SS-6M0-3
1
SS-200-61LV
Swagelok GmbH Swagelok GmbH Swagelok GmbH
1
Generic
12
Male Connector, 6 mm Tube OD x 3/8 in. Male ISO Parallel Thread, Stainless Steel (used for the pressure regulator) Low-Flow Metering Valve, 6 mm PFA tubing OD 1.56 mm (1/16 inch) 10 meters Hand valves, Plug Valve, 6 mm, Stainless Steel Quarter Turn Instrument Union Cross, 6 mm Tube OD, Stainless Steel Union Tee, 6 mm Tube OD, Stainless Steel Low Dead Volume Reducing Union, 1/8 x 1/16 in. Tube OD, Stainless Steel Copper tube OD=6mm ID=4mm (20m) Manometer (0-15bar)
2
13
Manometer (0-5bar)
1
were taken from on stock material
4,19 (per meter) ~20
14
Manometer (-1–1.5bar)
1
15
Vacuum storage tank 20 L (used in trucks braking system)
1
16
4 channel magnetic valves driver unit – TTL to 24 V controller unit
2
17
Diaphragm vacuum pump
1
3 4
5 6 7
8 9 10
11
Label
1 10
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9,60
17,50 69,25
26,10 42,90
~20 ~20
EG 20 L 206X645 MM Designed and produced by
Amazon by Titgemeyer (Air brake tank) machine shop TU Darmstadt Germany
57,48
150,00
~1000
Quantitative NMR Measurements The basics of quantitative NMR (qNMR) can be found in ref.[2] They are briefly summarized here. The main principle of NMR as a quantitative tool is that the intensities of the resonance lines (M) are directly proportional to the number of spins (N), equation (S.1), 𝑀𝑥 = 𝑘𝐶 ∗ 𝑁𝑥
S.1
where kC is the spectrometer constant and x the considered spin. This means that the amount of molecules can be calculated directly from the absolute integral of a resonance line if the proportional constant kC is known.[3] The constant kC results from the spectrometer parameters, the measurement conditions and the sample properties. For single pulse 1H NMR measurements with certain acquisition parameters this constant C is the same for all resonance lines within the same spectrum, as long as at least 5 times T1 recovery time is given.[4] Therefore, a calibration with an external standard is possible to determine the constant kC. The calibration accuracy depends on the acquisition parameters and as well as the precision of the integration of the signals. The influence of acquisition parameters is described in the literature ref.[4] Note that differences e.g. in pulse power, pre-acquisition delay, pulse angle, acquisition time, sweep width, sample temperature and especially number of scans strongly influence the absolute integral of the resonance lines and thus the calibration accuracy. External standard The amino acid N-Acetyl-alanine (Ac-Ala-OH, purity 98.6%) was chosen as quantitative NMR (qNMR) external standard. For the standard solution 33.25 mg of Ac-Ala-OH were weighted into a 5 ml volumetric flask and filled up with methanol-d4 to yield a 50 mM concentration of the external standard. The 1H NMR recorded at a 500 MHz spectrometer in methanol-d4 shows three signal groups: δ 4.37 ppm (q, J = 7.3 Hz, 1H), 1.97 ppm (s, 3H), 1.38 ppm (d, J = 7.3 Hz, 3H) is shown in Figure S5. The integral at 4.37 ppm for the single proton was integrated and used for comparison and calculation of the spectrometer constant.
Figure S5: 1H NMR Standard Ac-Ala-OH 50 mM solution in methanol-d4; parameters: T = 298 K, LB = 0.5, NS = 1, AQ = 3.2 s The influence of the receiver gain and the applied pulse length on the recorded absolute integral of the CH proton of Ac-Ala-OH in methanol-d4 is evaluated. The results are given in Table S2. The mean value of the normalized magnetization of the CH signal of Ac-Ala-OH, recorded with a 90°pulse, is determined to 130.6±1.6. This equals to a relative error of less than 1.5 %. In case of applying a 45° pulse on the same solution, the mean value of the normalized integral is calculated to 88.5±0.6, which S7
equals a relative error of less than 1 %. The dependency on the receiver gain is linear, and the absolute integral can be normalized by the applied receiver gain. Table S2: Absolute integrals of 1H NMR measurements of CH proton of 50 mM Ac-Ala-OH standard in methanol-d4 in dependence of the receiver gain (RG) shown for different applied pulse length.
90° pulse
45° pulse
RG 128
real RG 119.1
MSt 15680.6
MSt/real RG 131.7
MSt 10480.1
MSt/real RG 88.0
64
62.7
8223.0
131.1
5523.6
88.1
32
30.1
3938.2
130.8
2666.0
88.6
26
15.5
2051.5
132.4
1380.1
89.0
8
7.8
1019.8
130.7
689.0
88.3
4
3.9
509.3
130.6
342.8
87.9
2
2
263.6
131.8
179.5
89.7
1
1
127.0
127.0
87.8
87.8
0.5
0.5
64.8
129.5
44.3
88.6
Mean value
130.6
88.5
1.6
0.6
Spectrometer constant C
0.383 mM
0.565 mM
Standard deviation
0.005 mM
0.004 mM
Standard deviation
T = 298 K, LB = 0.5, NS= 1, AQ = 3.2 s. The real RG is the actual amplification of the signal by the receiver.
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Implemented Pulse programs Pulse program for PHIP measurement Reproducibility measurements – pulse program phip ;phip ;protocol: N2,NMR(NS45),N2,H2,N2,PHIP(45),N2,NMR(NS45) 1 ze ;clear memory ; exchange gas 5 times 2 d11 setnmr3|13 ;W4 output N2 d8 setnmr3^13 d10 setnmr3|14 ;W6 output out to release pressure d9 setnmr3^14 lo to 2 times 5 ;;;;;;;;;;;;;bubbling with N2;;;;;; d13 setnmr3|13 ;W4 output N2 d8 setnmr3^13 ;;;;;;;stop bubbling;;;;;;;; d14 setnmr3|14 ;W6 output out to release pressure d9 setnmr3^14 ;;;;;;;;; FIRST spectrum NMR with NS scans;;;;;;; 3 d1 ; wait for relaxation and bubbles p1*0.5 ph1 go=3 ph31 30m wr #0 if #0 ;;;;;;;;;;;;;bubbling with N2;;;;;; d23 setnmr3|13 ;W4 output N2 d8 setnmr3^13 ;;;;;;;stop bubbling;;;;;;;; d10 setnmr3|14 ;W6 output out to release pressure d9 setnmr3^14 ;;;;;;;exchange gas;;;;;;;;;;; 4 d11 setnmr3|12 ;W2 output H2 d8 setnmr3^12 d10 setnmr3|14 ;W6 output out to release pressure d9 setnmr3^14 lo to 4 times 5 ;;;;;;;;;;;;;bubbling with H2;;;;;; to make PHIP (including time for replacing gas) d12 setnmr3|12 ;W2 output H2 d8 setnmr3^12 ;;;;;;;stop bubbling;;;;;;;; d10 setnmr3|14 ;W6 output out to release pressure d9 setnmr3^14 ;;;;;;;exchange gas;;;;;;;;;;; 5 d11 setnmr3|13 ;W4 output N2 d8 setnmr3^13 d10 setnmr3|14 ;W6 output out to release pressure d9 setnmr3^14 lo to 5 times 5 ;;;;;;;;;;;;;bubbling with N2;;;;;; to replace hydrogen in line only d33 setnmr3|13 ;W4 output N2 d8 setnmr3^13 ;;;;;;;stop bubbling;;;;;;;; S9
d14 setnmr3|14 d9 setnmr3^14
;W6 output out to release pressure
;;;;;;;;SECOND spectrum PHIP 1 scan d2 p1*0.5 ph1 gosc ph31 30m wr #0 if #0 ;;;;;;;;;;;;;bubbling with N2;;;;;; to degas solution d43 setnmr3|13 ;W4 output N2 d8 setnmr3^13 ;;;;;;;stop bubbling;;;;;;;; d14 setnmr3|14 ;W6 output out to release pressure d9 setnmr3^14 ;;;;THIRD spectrum NMR with NS scans 6 d1 p1*0.5 ph1 go=6 ph31 30m wr #0 if #0 ;;;;;;;;;;;;;bubbling with N2;;;;;; to degas solution d43 setnmr3|13 ;W4 output N2 d8 setnmr3^13 ;;;;;;;stop bubbling;;;;;;;; d14 setnmr3|14 ;W6 output out to release pressure d9 setnmr3^14 ;;;;4TH spectrum NMR with NS scans 7 d1 p1*0.5 ph1 go=7 ph31 30m wr #0 if #0 exit ph1=0 ph31=0 ;pl1 : f1 channel - power level for pulse (default) ;p1 : f1 channel - 90 degree high power pulse ;d12 : H2 bubbling time including replace time (20sec) about 50 seconds ;d13 : 1st N2 bubbling time about 180-300 seconds to degas solution out of O2 ;d23 : 2nd N2 bubbling time about 10 seconds to pull out liquid from tip ;d33 : 3rd N2 bubbling time about 10-12 seconds to replace H2 gas only ;d43 : 4th N2 bubbling time about 60 seconds to degas solution out of H2 ;d14 : time to release pressure 0.86 sec before d1 or d2 ;d1 : relaxation delay before NMR spectra ;d2 : relaxation delay before PHIP spectrum ;ns: 1 * 8, number of scans for NMR spectra before and after ;td1: number of experiments must be 3!
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Pulse programs for H2 and N2 dissolution dynamics experiments Dissolving kinetics of hydrogen - n2h2bubl2D ;n2h2bubl2D ;bubbling first with N2 and then with H2 with vacuum step 1 ze 2 500m setnmr3|11 ;U6 Vacuum open 100m setnmr3^11 ;U6 Vacuum close 100m setnmr3|13 ;W4 N2 open d13 setnmr3|10 ;U5 NMR open (start N2 bubbling) 100m setnmr3^13 ;W4 N2 close 200m setnmr3|14 ;W6 OUT open to release pressure 100m setnmr3^14 ;W6 OUT close 100m setnmr3^10 ;U5 NMR close (stop N2 bubbling) 500m setnmr3|11 ;U6 Vacuum open 100m setnmr3^11 ;U6 Vacuum close 100m setnmr3|12 ;W2 H2 open vd setnmr3|10 ;U5 NMR open (start H2 bubbling) 100m setnmr3^12 ;W6 H2 close 200m setnmr3|14 ;W6 OUT open to release pressure 100m setnmr3^14 ;W6 OUT close d1 setnmr3^10 ;U5 NMR close (stop H2 bubbling) ;and wait for relaxation and bubbles p1 ph1 go=2 ph31 30m wr #0 if #0 ivd lo to 1 times td1 exit ph1=0 ph31=0 ;pl1 : f1 channel - power level for pulse (default) ;p1 : f1 channel - 90 degree high power pulse ;d13 : N2 bubbling time about 40-100s seconds ;vd : variable delay, taken from vd-list ;d1 : relaxation delay after bubbling; minimum 1 seconds ;td1: number of experiments = number of delays in vd-list
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Degassing kinetics by nitrogen - h2n2bubl2D ;h2n2bubl2D ;bubbling first with H2 and then with N2 with vacuum step 1 ze 2 500m setnmr3|11 ;U6 Vacuum open 100m setnmr3^11 ;U6 Vacuum close 100m setnmr3|12 ;W2 H2 open d13 setnmr3|10 ;U5 NMR open (start H2 bubbling) 100m setnmr3^12 ;W2 H2 close 200m setnmr3|14 ;W6 OUT open to release pressure 100m setnmr3^14 ;W6 OUT close 100m setnmr3^10 ;U5 NMR close (stop H2 bubbling) 500m setnmr3|11 ;U6 Vacuum open 100m setnmr3^11 ;U6 Vacuum close 100m setnmr3|13 ;W4 N2 open vd setnmr3|10 ;U5 NMR open (start N2 bubbling) 100m setnmr3^13 ;W4 N2 close 200m setnmr3|14 ;W6 OUT open to release pressure 100m setnmr3^14 ;W6 OUT close d1 setnmr3^10 ;U5 NMR close (stop N2 bubbling) ;and wait for relaxation and bubbles p1 ph1 go=2 ph31 30m wr #0 if #0 ivd lo to 1 times td1 exit ph1=0 ph31=0 ;pl1 : f1 channel - power level for pulse (default) ;p1 : f1 channel - 90 degree high power pulse ;d13 : N2 bubbling time about 40-100s seconds ;vd : variable delay, taken from vd-list ;d1 : relaxation delay after bubbling; minimim 1 seconds ;td1: number of experiments = number of delays in vd-list
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Pulse program for study reaction kinetics Pulse program for the PHIP kinetics experiment – phipkinetic ;phipkinetic ;measure PHIP "p2=p1/18" ;5 "p3=p1/18" ;5 "l2=td1-l1-1"
kinetic during and after the bubbling by H2 degree detection degree detection ;number of spectra during the bubbling
1 ze ; first flush by nitrogen 500m setnmr3|11 ;U6 Vacuum open 100m setnmr3^11 ;U6 Vacuum close 100m setnmr3|13 ;W4 N2 open d12 setnmr3|10 ;U5 NMR open (start N2 bubbling) 100m setnmr3^13 ;W4 N2 close 200m setnmr3|14 ;W6 OUT open to release pressure 100m setnmr3^14 ;W6 OUT close 100m setnmr3^10 ;U5 NMR close (stop N2 bubbling) 500m setnmr3|11 ;U6 Vacuum open 100m setnmr3^11 ;U6 Vacuum close ;detect first NMR spectrum 1s p2 ph1 gosc ph31 50m wr #0 if #0 ;bubbling by hydrogen and detection during the bubbling 2 50m setnmr3|12 ;W2 H2 open d13 setnmr3|10 ;NMR open (start H2 bubbling) 50m setnmr3^12 ;W2 H2 close 50m setnmr3|14 ;W6 OUT open to stop bubbling d2 p2 ph1 gosc ph31 50m wr #0 if #0 lo to 2 times l1 ;1st cycle repetition time = AQ+d2+d13+0.2s ; second stage where only NMR detection 3 d3 p3 ph1 gosc ph31 50m wr #0 if #0 lo to 3 times l2 10u setnmr3^14 ;W6 OUT close ;2nd cycle repetition time = AQ+d3+0.05s Exit ph1=0 ph31=0 ;pl1 : f1 channel - power level for pulse (default) ;p1 : f1 channel - 90 degree high power pulse ;d12 : N2 bubbling time ;d13 : repetitive H2 bubbling time ;d2 : delay for repetition in first stage rep=AQ+d2+d13+0.2s ;d3 : delay for repetition in second stage rep=d3+AQ+0.05 S 13
References [1] A.S. Kiryutin, Scientific report of STSM COST TD1103 15.05.2014-15.06.2014 "PC Controlled Apparatus for PHIP experiments", in: STSM COST TD1103, European Cooperation in Science and Technology, 2014, pp. 1-8. [2] U. Holzgrabe, I. Wawer, B. Diehl, NMR spectroscopy in drug development and analysis, WileyVCH, Weinheim u.a., 1999. [3] C. Szántay, High-Field NMR-Spectroscopy as an Analytical Tool for Quantitative-Determinations Pitfalls, Limitations and Possible Solutions, Trac-Trends in Analytical Chemistry, 11 (1992) 332-344. [4] F. Malz, H. Jancke, Validation of quantitative NMR, Journal of Pharmaceutical and Biomedical Analysis, 38 (2005) 813-823.
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