Laura Castañar, 2 Manuela Garcia, 1 Erich Hellemann, 1 Pau Nolis 2 Roberto R. Gil, 1, * and Teodor Parella 2, *

Transcription

1 One-Shot Determination of Residual Dipolar Couplings: Application to the Structural Discrimination of Small Molecules Containing Multiple Stereocenters. Laura Castañar, 2 Manuela Garcia, 1 Erich Hellemann, 1 Pau Nolis 2 Roberto R. Gil, 1, * and Teodor Parella 2, * 1 Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA; 2 Servei de Ressonància Magnètica Nuclear, Universitat Autònoma de Barcelona, Facultat de Ciències, E Bellaterra (Barcelona), Catalonia, Spain. 1

2 Table of contents: Figure S1: Pulse sequence scheme for real-time BIRD-based homodecoupled J- scaled BIRD HSQC (HD-JSB-HSQC) experiment 4 Figure S2: Comparison between the A) conventional JSB-HSQC and B) HD-JSB- HSQC spectra of estradiol.5 Figure S3: Comparison between the A) conventional J-HSQC and B) HD-J-HSQC spectra of estradiol.6 Figure S4: Comparison between several A-D) HD-JSB-HSQC (J-scaling factors of 1,2,4 and 8 respectively) and E) the analog HD-J-HSQC spectra of estradiol. 7 Figure S5: Effect of nonuniform sampling (NUS) in the experimental time of the HD-J-HSQC experiment Figure S6: Effect of non-uniform sampling (NUS) in the signal resolution of HD-J- HSQC experiment.. 9 Figure S7: a) Conventional J-HSQC, b) equivalent HD-J-HSQC with real-time homodecoupling during acquisition and C) HD-J-HSQC spectra using 25% NUS of strychnine.. 10 Figure S8: Edited CH/CH 2 internal F 1 projections extracted from B) J-HSQC with INEPT, C) HD-J-HSQC with INEPT and D) HD-J-HSQC with PEP transfer spectra of strychnine Figure S9: (left) 2D J-HSQC and (right) 2D HD-J-HSQC spectra of an anisotropic sample of strychnine dissolved in PMMA/CDCl 3 12 Figure S10: Expansions extracted from spectra of Fig. S9 that show the multiplet J pattern simplification achieved in the HD-J-HSQC spectrum by real-time BIRD homodecoupling Figure S11: Effect of NUS in the HD-J-HSQC experiment of strychnine in PPMA/CDCl 3 gel Figure S12: Expansion of the HD-J-HSQC spectrum of strychnine in PMMA/CDCl 3 ( Fig. S11B) corresponding to the diastereotopic CH 2 protons Table S1: Comparison between the experimental 1 J CH, 1 T CH and 1 D CH values extracted from a single HD-J-HSQC spectrum of strychnine in PMMA/CDCl 3 ( 2 H quadrupolar splitting of Q = 33.4 Hz) (this work) with those published previously with a similar sample ( 2 H quadrupolar splitting of Q = 36 Hz) using two separate JSB-HSQC measurements. 16 2

3 Table S2: RDC-based stereochemical analysis of the 8 possible diastereoisomeric structures of strychnine minimized by Molecular Mechanics and the correct one at a DFT level.. 17 Table S3: Experimental and calculated 1 D CH coupling constants of strychnine as a function of the source data and structure calculation (Molecular Mechanics and DFT). 18 Figure S13: A-B) HD-JSB-HSQC and C-D) HD-J-HSQC spectra of ludartin in isotropic CDCl 3 (A and C) and anisotropic PMMA/CDCl 3 (B and D) sample conditions. 19 Table S4. Comparison of RDC values for ludartin extracted from the corresponding HD-JSB-HSQC (Fig. S11B) and HD-J-HSQC (Fig. S11D) spectra. 20 Automated quantitative extraction of 1 J CH coupling values from J-resolved HSQC spectra 21 Pulse program for Bruker AVANCE spectrometers 23 3

4 Figure S1: Pulse sequence scheme for real-time BIRD-based homodecoupled J-scaled BIRD HSQC (HD-JSB-HSQC) experiment. Narrow and wide filled bars correspond to 90 and 180 pulses, respectively, with phase x unless indicated otherwise. All inversion and refocusing 180º 13 C pulses can be applied as adiabatic shaped pulses. The interpulse delay in INEPT and BIRD elements are set to = 1/(2* 1 J CH ). Coherence order selection and echo antiecho phase sensitive detection in the 13 C-dimension are achieved with gradient pulses G1 and G2 in the ratio of 80:20.1. Purging gradient pulses G3 and G4 are used with typically of 1 ms duration followed by a recovery delay of 100 μs. Each J- refocusing block applied during acquisition consists of a BIRD element, a hard 180 proton pulse, and a data acquisition window. The first and last chunks are half the duration (= AQ/2n) of the remaining chunks (AQ/n). A basic two-step phase cycle is used: 1 = x,-x and rec = x,-x. 4

5 Figure S2: Comparison between the A) conventional JSB-HSQC and B) homodecoupled JSB-HSQC (HD-JSB-HSQC) spectra of an isotropic sample of estradiol (3) acquired with the basic pulse scheme of Fig. S1 (normal FID acquisition vs BIRD-based homodecoupled acquisition, respectively). Right 1D slices from some selected CH and CH 2 correlations are shown to visualize the homodecoupling signal collapsing. Both experiments were acquired and processed under the same experimental conditions: 140 ppm in the 13 C dimension, 2 scans per t 1 increment, 128 t 1 increments and a J-scaling factor of 4. The experimental time for each 2D spectrum was 5 min 32 s for A) and 6 min 12 s for B). 5

6 Figure S3: Comparison between the A) conventional J-HSQC and B) HD-J-HSQC spectra of an isotropic sample of estradiol (3) acquired with the basic pulse scheme of Fig. 1 (normal FID acquisition vs BIRD-based homodecoupled acquisition, respectively). Right 1D slices from some selected CH and CH 2 correlations are shown to visualize the homodecoupling signal collapsing. Both experiments were acquired and processed under the same experimental conditions: 4 ppm in the 13 C dimension, 2 scans per t 1 increment, and 128 t 1 increments. The experimental time for each 2D spectrum was 5 min 46 s for A) and 6 min 26 s for B). These spectra are equivalent to those of Fig. S2. 6

7 Figure S4: Comparison between several A-D) HD-JSB-HSQC (J-scaling factors of k = 1,2,4 and 8 respectively) and E) the analog HD-J-HSQC spectra of an isotropic sample of estradiol (3). All spectra have been acquired under the same experimental conditions (128 t 1 increments) and with similar experimental times. Below are the corresponding F 1 projections at the specific C11H11b and C9H9 correlations to compare experimental splittings, spectral resolution and linewidths. Note that improved resolution and linewidths resolution by a factor of 40 is achieved in the HD-J-HSQC spectrum, rendering a much more accurate determination of 1 J CH. 7

8 Figure S5: Effect of nonuniform sampling (NUS) on the experimental time of the HD- J-HSQC experiment. Comparison between several HD-J-HSQC spectra of an isotropic sample of estradiol (3) acquired with (left) conventional uniform sampling (128 t 1 increments) and with (center) 50% and (right) 25% of NUS. Below are some F 1 columns extracted at specific proton resonances. It shows that similar spectral quality, signal resolution and linewidths are achieved in all spectra, allowing an important spectrometer time saving factor of 4 when using 25% of NUS. Also note that signal sensitivity is retained in all equivalent spectra. 8

9 Figure S6: Effect of NUS on the signal resolution of HD-J-HSQC experiment. Comparison between several HD-J-HSQC spectra of an isotropic sample of estradiol (3) acquired with (left) conventional uniform sampling (128 t 1 increments) and with (center) 50% (of 256 t 1 increments) and (right) 25% (of 512 t 1 increments) of NUS. All experiments were collected in the same experimental time of 6 min 3 s. Below are some F 1 columns extracted at specific proton resonances. It is shown that improved signal resolution, sharper linewidths and better signal sensitivity are achieved with increased use of NUS. 9

10 Figure S7: A) Conventional J-HSQC, B) equivalent HD-J-HSQC (with real-time homodecoupling during acquisition) and C) HD-J-HSQC spectra using 25% NUS of an isotropic sample of strychnine (1) in CDCl 3. All spectra have been acquired under the same conditions and experimental time. 10

11 Figure S8: Edited CH/CH 2 internal F 1 projections extracted from B) J-HSQC with INEPT, C) HD-J-HSQC with INEPT and D) HD-J-HSQC with PEP transfer spectra of strychnine in CDCl 3. The corresponding averaged SNR values for each edited CH (red) and CH 2 (blue) subgroups of protons are shown in B-D). 11

12 Figure S9: (A) 2D J-HSQC and (B) 2D HD-J-HSQC spectra of an anisotropic sample of strychnine (1) dissolved in PMMA/CDCl 3. Experimental details: spectral width (F 1 ) = 4 ppm, number of complex data points in F 2 = 2048, number of t 1 increments = 512, FID resolution = 2.35 Hz, number of scans = 4, relaxation delay = 1 s, experimental time = 53 min 13 s, spectral resolution after zero filling to 2k in F 2 = 0.29 Hz. Data acquisition was divided into 8 chunks with = 10.3 ms each one. 12

13 Figure S10: Expansions extracted from spectra of Fig. S9 that show the multiplet J pattern simplification achieved in the HD-J-HSQC spectrum by real-time BIRD homodecoupling. All CH cross-peaks (red) are converted to singlet resonances whereas diastereotopic CH 2 protons (black) are simplified to doublets. 13

14 Figure S11: Effect of NUS in the HD-J-HSQC experiment of strychnine (1) in PMMA/CDCl 3 gel. Both experiments were recorded under the same experimental conditions: A) Conventional sampling in t 1 (512 t 1 increments, experimental time = 53 min 13 s and B) with 25% of NUS (512 t 1 increments, experimental time 11 min 34 s). Note that very accurate 1 D CH values can be measured directly from each CH resonance. Expanded region corresponding to the upfield components of the CH protons are shown in the spectra below. 14

15 Figure S12: Expanded region corresponding to the upfield components of the diastereotopic CH 2 protons in the HD-J-HSQC spectrum of strychnine in PMMA/CDCl 3 (see Fig. S11B). Red and blue boxes represent the isotropic (provides 1 J CH ) and anisotropic (provides 1 T CH ) components for each individual proton resonance. Note that very accurate signs and magnitudes of ( 1 D Cha + 1 D CHb )/2 values can be measured directly from each pair of iso/aniso components. 15

16 Table S1: Comparison between the experimental 1 J CH, 1 T CH and 1 D CH values extracted from a single HD-J-HSQC spectrum of strychnine in PMMA/CDCl 3 ( 2 H quadrupolar splitting of Q = 33.4 Hz) (this work) with those published previously with a similar sample ( 2 H quadrupolar splitting of Q = 36 Hz) b using two separate JSB-HSQC measurements. JSB-HSQC b HD-J-HSQC Isotropic Anisotropic Anisotropic (this work) CH pairs 1 J CH Hz 1 D CH Hz 1 J CH Hz 1 T CH Hz 1 D CH Hz C1H C2H C3H C4H C8H C11H11a+C11H11b a a C12H C13H C14H C15H15a+C15H15b a a C16H C17H17a+C17H17b a a C18H18a+C18H18b ,89 a a C20H20a+C20H20b a a C22H c c c C23H23a+C23H23b a a a These values correspond to ( 1 D Cha + 1 D CHb )/2 to be used in MSpin. b Experimental data obtained from J. D. Snider et al. Magn. Reson. Chem. 50 (2012) S86. c Not measured 16

17 Table S2: RDC-based stereochemical analysis of the 8 possible diastereoisomeric structures of strychnine minimized by Molecular Mechanics (MMFFs) and the correct one at a DFT level: Isomer From ref. 1 a,c This work from data of ref. 1 a,c Q factors This work from HD- J-HSQC data b,c This work from data of ref. 1 a,d This work from HD- J-HSQC data b,d 1 7R,8R,12R,13R,14R,16S R,8R,12S,13R,14R,16S R,8R,12R,13S,14R,16S R,8R,12S,13S,14R,16S R,8S,12R,13R,14R,16S R,8S,12S,13R,14R,16S R,8S, 12R,13S,14R,16S R,8S,12S,13S,14R,16S a Experimental data obtained from [1] J. D. Snider et al. Magn. Reson. Chem. 50 (2012) S86 ( Q = 36 Hz) b Experimental data obtained from HD-J-HSQC data ( Q = 33.6 Hz) c Structures calculated by Molecular Mechanics (MMFFs) d Structure calculated at a DFT level (B3LYP/6-31G*) 17

18 Table S3: Experimental and calculated 1 D CH coupling constants of strychnine as a function of the source data and structure calculation (Molecular Mechanics and DFT) This work from data of ref. 1 a This work from HD-J-HSQC data b Exp 1 D CH (Hz) a Calc 1 D CH (Hz) c Calc 1 D CH (Hz) d Exp 1 D CH (Hz) b Calc 1 D CH (Hz) c Calc 1 D CH (Hz) d C1H C2H C3H C4H C8H C11H11a C11H11b C12H C13H C14H C15H15a C15H15b C16H C17H17a C17H17b C18H18a C18H18b C20H20a C20H20b C22H *** C23H23a C23H23b a Experimental data obtained from [1] J. D. Snider et al. Magn. Reson. Chem. 50 (2012) S86 ( Q = 36 Hz) b Experimental data obtained from HD-J-HSQC data ( Q = 33.6 Hz) c Structures calculated by Molecular Mechanics d Structure calculated at a DFT (B3LYP/6-31G*) level 18

19 Figure S13: A-B) HD-JSB-HSQC and C-D) HD-J-HSQC spectra of ludartin (2) in isotropic CDCl 3 (A and C) and anisotropic PMMA/CDCl 3 (B and D) sample conditions. 19

20 Table S4. Comparison of RDCs values (in Hz) for ludartin (2) extracted from the corresponding HD-JSB-HSQC (Fig. S13B) and HD-J-HSQC (Fig. S13D) spectra. 1 D CH JSB-HSQC (Hz) 1 D CH HD-J-HSQC (Hz)

21 Automated extraction of 1 J CH coupling values from J-resolved HSQC spectra: The simplified multiplet pattern feature of cross-peaks in the HD-J-HSQC spectrum offers a new approach toward an automated analysis and extraction of 1 J CH values. An algorithm has been developed to automatically extract these 1 J CH values thanks to the symmetry of each correlation with respect to F 1 = 0. Tutorial on automatic peak picking: 1) Perform automatic peak piking analysis of both positive and negative peaks in Bruker software. 2) Order peaks in ascendant F 2 frequency values (optional) and export to.csv file. 21

22 3) Open the Jmeasurement applet and browse the.csv file, click measure to perform the automatic analysis of 1 J CH. Use the print button for external printing. Use the clear button to perform a new analysis. (1/2) (2/2) Notice that the 1 J CH measurement is reported separately for CH 3, CH 2 and CH spins systems. Important note: In order to launch the java applet Jmeasurement.jar is a requirement to have previosuly installed in your system the JRE (Java Runtime Environtment). Any questions to pau.nolis@uab.cat. 22

23 Pulse program for Bruker spectrometers: ;J-resolved HSQC ;Shift Correlation version s> cnst10= 0 ;J-resolved version =>cnst10= 1 ;F1-heterocoupled version==> cnst14= 0 ;F1-heterodecoupled version==> cnst14= 1 ;with J scaling in t1 ;J-scaled in shift correlation experiments==> cnst12= 1-10 ;J-scaled in J-resolved experiments==> cnst12= 1 ;Conventional acquisition==> cnst15= 0 ;Pure shift acquisition==> cnst15= 1 ;phase sensitive using Echo/Antiecho-TPPI gradient selection ;with decoupling during acquisition ;using shaped pulses for inversion and refocussing on f2 - channel ;using G_BIRD(r) to remove long range couplings in t1 ;Avance III ;Topspin3.1 #include <Avance.incl> #include <Grad.incl> #include <Delay.incl> #include <De.incl> "p2=p1*2" "p4=p3*2" "d2=1s/(cnst2*2)" "d4=1s/(cnst2*4)" "d11=30m" "d0=3u" "d20=3u" "in0=inf1/2" "in20=in0*cnst12" "DELTA=d0*2+p2+p16+d16-p3*4/PI" "DELTA1=p16+d16-p3*4/PI" "DELTA2=d4-larger(p2,p14)/2" "DELTA3=d2-larger(p2,p39)/2" "DELTA4=d4-p16-d16-larger(p2,p14)/2" "DELTA5=d2-larger(p2,p14)/2" 23

24 "DELTA6=d24-cnst17*p24/2-d16-4u" "acqt0=0" baseopt_echo #define homodec #ifdef homodec dwellmode explicit "d22=aq/2*l2" #else #endif 1 ze 2 d11 d1 do:f2 d12 pl2:f2 UNBLKGRAD (p3 ph1):f2 4u p16:gp4 d16 } (p1 ph1) DELTA2 (center (p2 ph1) (p14:sp3 ph6):f2 ) DELTA2 pl2:f2 p28 ph1 4u (p1 ph8) p16:gp3 d16 (p3 ph3):f2 if "cnst10==0" { if "cnst14==0" { d20 (p1 ph1) DELTA3 (center (p2 ph1) (p39:sp4 ph1):f2 ) DELTA3 (p1 ph1) d20 d0 (p2 ph1) d0 p16:gp1*ea d16 pl0:f2 24

25 (p39:sp4 ph14):f2 DELTA pl2:f2 } else { d0 (p2 ph1) d0 p16:gp1*ea d16 pl0:f2 (p39:sp4 ph14):f2 DELTA pl2:f2 } } else { d0 (p1 ph1) DELTA3 (center (p2 ph1) (p39:sp4 ph1):f2 ) DELTA3 (p1 ph1) d0 p16:gp1*ea d16 pl0:f2 (p39:sp4 ph14):f2 DELTA1 pl2:f2 } (ralign (p3 ph4):f2 (p1 ph1):f1 ) DELTA6 pl0:f2 (center (p2 ph1) (p24:sp7 ph1):f2 ) DELTA6 pl2:f2 (center (p1 ph2) (p3 ph5):f2 ) DELTA2 pl0:f2 (center (p2 ph1) (p14:sp3 ph1):f2 ) DELTA2 pl12:f2 (p1 ph1) DELTA1 (p2 ph1) p16:gp2 d16 BLKGRAD if "cnst15==1" { ACQ_START(ph30,ph31) 0.05u DWL_CLK_ON cpd2:f2 0.1u REC_UNBLK d u DWL_CLK_OFF 0.1u REC_BLK do:f2 5 ;bird y 25

26 (p1 ph15) DELTA5 pl0:f2 (center (p2 ph16) (p14:sp3 ph16):f2 ) DELTA5 pl12:f2 (p1 ph17) ;chem. shift refoc. 20u (p2 ph15):f1 20u 0.05u DWL_CLK_ON cpd2:f2 0.1u REC_UNBLK d u DWL_CLK_OFF 0.1u REC_BLK do:f2 ;bird -x (p1 ph25) DELTA5 pl0:f2 (center (p2 ph26) (p14:sp3 ph26):f2 ) DELTA5 pl12:f2 (p1 ph27) ;chem. shift refoc. 20u (p2 ph25):f1 20u 0.05u DWL_CLK_ON cpd2:f2 0.1u REC_UNBLK d u DWL_CLK_OFF 0.1u REC_BLK do:f2 lo to 5 times l2 10 rcyc=2 } else { go=2 ph31 cpd2:f2 } d11 do:f2 mc #0 to 2 F1EA(calgrad(EA) & calph(ph5, +180), caldel(d0, +in0) & caldel(d20, +in20) & calph(ph3, +180) & calph(ph6, +180) & calph(ph31, +180)) exit ph1=0 26

27 ph2=1 ph3=0 2 ph4=0 ph5=1 ph6=0 ph8=1 ph14=0 ph15=1 ph16=2 ph17=3 ph25=2 ph26=3 ph27=0 ph30 = 0 ph31=0 2 ;pl0 : 120dB ;pl1 : f1 channel - power level for pulse (default) ;pl2 : f2 channel - power level for pulse (default) ;pl12: f2 channel - power level for CPD/BB decoupling ;sp3: f2 channel - shaped pulse 180 degree for inversion ;sp4: f2 channel - shaped pulse 180 degree for refocussing ;p1 : f1 channel - 90 degree high power pulse ;p2 : f1 channel degree high power pulse ;p3 : f2 channel - 90 degree high power pulse ;p14: f2 channel degree shaped pulse for inversion ;p16: homospoil/gradient pulse ;p28: f1 channel - trim pulse ;p39: f2 channel degree shaped pulse for refocussing ; Bip720,100,10.1 (160us at MHz) ;d0 : incremented delay (2D) [3 usec] ;d1 : INEPT (1s) / iinept (3s) ;d2 : 1/(2J)XH ;d4 : 1/(4J)XH ;d11: delay for disk I/O [30 msec] ;d16: delay for homospoil/gradient recovery ;inf1: 1/SW(X) = 2 * DW(X) ;in0: 1/(2 * SW(X)) = DW(X) ;nd0: 2 ;NS: 1 * n ;DS: >= 16 ;td1: number of experiments ;FnMODE: echo-antiecho ;cpd2: decoupling according to sequence defined by cpdprg2 ;pcpd2: f2 channel - 90 degree pulse for decoupling sequence ;cnst2: = J(XH) ;cnst10: correlation (0)/J-resolved (1) ;cnst12: J(scale) factor = correlation (1-10) / J-resolved (1) ;cnst14: F1-heterocoupled (0) / F1-heterodecoupled (1) 27

28 ;cnst15: Conventional acquisition (0) / Pure shift acquisition (1) ;use gradient ratio: gp 1 : gp 2 ; 80 : 20.1 for C-13 ; 80 : 8.1 for N-15 ;for z-only gradients: ;gpz1: 80% ;gpz2: 20.1% for C-13, 8.1% for N-15 ;gpz3: 33% ;gpz4: 17% ;use gradient files: ;gpnam1: SINE.100 ;gpnam2: SINE.100 ;gpnam3: SINE.100 ;gpnam4: SINE