Ocean Medicines Introduction to NMR Spectroscopy Dr Rainer Ebel Aim: To increase understanding of 1H NMR spectroscopy, introduce 13C NMR spectroscopy, with particular emphasis on the analysis of real spectra.

NMR / Structure elucidation: Text books 656 pages, RRP £85.00 OUP USA; 2nd ed., 2009 ISBN-10: 0195336046 ISBN-13: 978-0195336047

NMR / Structure elucidation: Text books 755 pages, RRP £76.99 CRC Press, 2015 ISBN-10: 1498719627 ISBN-13: 978-1498719629

NMR / Structure elucidation: Text books ABOUT THE COVER To the uninitiated, structure elucidation can sometimes feel like a maze of possibilities, with no clear view of how to traverse the gap between the starting point and the solution. In reality however, the analysis will always converge on a single answer, with the correct solution threading a logical line through the data. The cover photograph, taken over Vancouver British Columbia by Aaron O'Dea, represents both the many false avenues that can perplex the structure elucidation scientist, and the single correct route that ultimately leads to each solution. Linington, Roger G. Problems in Organic Structure Determination. CRC Press, 2015.

g-rays x-rays UV VIS IR m-wave radio Information in an NMR Spectrum 10-10 10-8 10-6 10-4 10-2 100 102 wavelength (cm) g-rays x-rays UV VIS IR m-wave radio 1) Energy E = hu h is Planck constant u is NMR resonance frequency Observable Name Quantitative Information Peak position Chemical shifts (d) d(ppm) = uobs –uref/uref (Hz) chemical (electronic) environment of nucleus Peak Splitting Coupling Constant (J) Hz peak separation neighboring nuclei (intensity ratios) (torsion angles) Peak Intensity Integral unitless (ratio) nuclear count (ratio) relative height of T1 dependent integral curve Peak Shape Line width Du = 1/pT2 molecular motion peak half-height chemical exchange uncertainty principle uncertainty in energy

The nuclear magnetic resonance phenomenon the observable in NMR (the free induction decay, FID) is the energy that is released when the spins return to the ground state (relaxation) the underlying frequencies are extracted by a mathematical process called Fourier transformation

Fourier Transformation The 90o pulse simultaneously excites all 1H frequencies. Result is data in time domain – Free Induction Decay (FID). After Fourier transformation (FT) the data is in the frequency domain. Main advantage is improved signal to noise (S/N) by acquisition of large number of FIDs and adding them together. S/N  √n where n is the number of FIDs added. FT Intensity envelope  e-t/T2 Transformed FID gives Spectrum in frequency domain FID in time domain – sum of 16 acquisitions

The chemical shift scale deshielded shielded Chemical shift in ppm (d) = Shift from a standard (Hz) Spectrometer frequency (MHz) 150Hz/100MHz = 1.5 ppm 300Hz/200MHz = 1.5 ppm

Comparison of 1H and 13C NMR spectroscopy 13C NMR spectroscopy is much less sensitive (lower abundance and smaller magnetogyric ratio)* 1H NMR spectra display (in principle) three parameters for each signal, i.e. chemical shift, integral and coupling pattern 13C NMR spectra display (in principle) only one parameter for each signal, i.e. chemical shift** 13C NMR spectra normally show much less signal overlap 13C NMR spectra give signals for quaternary carbons (not visible in 1H) both techniques are complimentary explanations / footnotes: * 1H 99.985%, 13C 1.1%; g is approx. 4 times smaller for 13C, but contributes by the power of 3 ** integrals are not defined due to measuring process, coupling to 13C is too “rare” to be visible (10-4), coupling to 1H is suppressed through broadband decoupling

Each NMR Observable Nucleus Yields a Peak in the Spectra Information in an NMR Spectrum Each NMR Observable Nucleus Yields a Peak in the Spectra “fingerprint” of the structure 2-phenyl-1,3-dioxep-5-ene 1H NMR spectrum 13C NMR spectrum

NMR and sensitivity 1H NMR spectrum of caffeine 8 scans ~12 secs 13C NMR spectrum of caffeine 8 scans ~12 secs 13C NMR spectrum of caffeine 10,000 scans ~4.2 hours

The chemical shift scale We use a relative scale, and refer all signals in the spectrum to the signal of a particular compound The good thing is that since it is a relative scale, the d in a 100 MHz magnet (2.35 T) is the same as that obtained for the same sample in a 600 MHz magnet (14.1 T) Tetramethyl silane (TMS) is used as reference because it is soluble in most organic solvents, is inert, cheap, non-toxic, volatile, and has 12 equivalent 1Hs and 4 equivalent 13Cs (intense signals): nowadays, we mainly reference to the solvent signal! w - wref d = ppm (parts per million) wref H 3 C S i

1H NMR Chemical Shifts in Organic Compounds

Chemical shifts of substituted aromatic compounds 1H NMR spectrum of aniline in CDCl3 (250 MHz) similar to their effects on eletrophilic aromatic substitutions, substituents exhibiting a +M effect increase electron density ortho and para to their own (= ipso-) position, which thus resonate more upfield (1H + 13C NMR)

Chemical shifts of substituted aromatic compounds 1H NMR spectrum of nitrobenzene in CDCl3 (250 MHz) similar to their effects on eletrophilic aromatic substitutions, substituents exhibiting a -M effect decrease electron density ortho and para to their own (= ipso-) position, which thus resonate more downfield (1H + 13C NMR)

NMR solvents routine NMR spectra are acquired in deuterated solvents deuterated solvents always have a small amount of residual (mono-) non-deuterated solvent (< 0.5% ) which gives a signal in 1H NMR chemical shift referencing is normally achieved by referencing to this residual solvent signal (which by itself has been referenced to TMS) common NMR solvents also give a characteristic water (HDO) peak solvents can be divided into protic and aprotic solvents protic solvents include D2O, CD3OD aprotic solvents include DMSO-d6, C6D6 XH (X = O, N) signals are only detected in aprotic solvents (in protic solvents, they exchange with D and disappear) XH protons (when sharp lines = little water in the solvent) couple to their CH neighbours according to n+1 rule, or may just be broad (in the presence of water)

NMR solvents (in C6D6) (in CDCl3) changing the solvent can give a better signal spread or reveal overlapping signals 1H NMR spectra can appear quite different in different solvents (d ± 0.3 ppm) changing the solvent can make a non-first order spectrum a first order one changing between protic and aprotic solvents can identify XH protons

Coupling patterns

Couplings to more than one coupling partner if a proton A is coupled to more than one coupling partner (M and X), its signal will become more complex, if JAM ≠ JAX for each coupling, the n+1 rule should be applied separately (by drawing a splitting diagram)

Iterative application of the n + 1 rule Karplus curve if a proton A is coupled to more than one coupling partner (A, B and C), its signal will become more complex, if JAB ≠ JAC if two coupling constants are identical (or at least of similar magnitude), they may fall together (JAB ⋲ JAD, so the coupling to A becomes a triplet)

Complex 1st order spin systems dd dt ddd dq

Coupling patterns for disubstituted aromatic systems dd td dd td dt t t dt “d” “d” 3JAB(ortho) = 6 – 10 Hz 4JAB(meta) = 1 – 3 Hz 5JAB(para) = 0 – 1 Hz (normally not visible)

Magnitudes of coupling constants common vicinal aliphatic coupling: 3JH,H = 6.5 – 7 Hz [this is only true for flexible alkyl chains!] in reality (= more generally!), the magnitude of the vicinal coupling constant depends on the dihedral angle θ (Karplus relationship)

Magnitudes of coupling constants 3JAB(cis) = 6 – 11 Hz 3JAC(trans) = 12 – 19 Hz the geometry of double bonds can normally be inferred from the magnitude of the vicinal coupling constants 3JAB(cis) = 6 – 11 Hz 3JAC(trans) = 12 – 19 Hz 2JBC(gem) = 0 – 3 Hz the two protons B and C are in different chemical environments, appear at different chemical shifts and therefore also couple: geminal coupling 2JH,H = 0 – 3 Hz

Nomenclature of spin systems: examples 1H NMR spectrum of styrene in CDCl3 (250 MHz) AMX 3JAM = 17.6 Hz 3JAX = 10.9 Hz 2JMX = 1.0 Hz all dd

Magnitudes of coupling constants 3JAB(ortho) = 6 – 10 Hz 4JAB(meta) = 1 – 3 Hz 5JAB(para) = 0 – 1.5 Hz in aromatic systems, protons ortho to one another show a large coupling (typically 8 Hz) meta-couplings are much smaller (1 – 3 Hz) and thus may or may not appear in the 1H NMR spectrum (signals may simply be broadened) para-couplings are very small (0 – 1 Hz) and normally do not appear in the 1H NMR spectrum

Magnitudes of coupling constants 2JAB = 10 - 16 Hz in optically active molecules, any methylene group is diastereotopic* the two protons A and B are in different chemical environments, appear at different chemical shifts and therefore also couple: geminal coupling 2JH,H = 10 – 16 Hz * covered later in this lecture

Long range couplings in aliphatic systems, the most common coupling is the vicinal coupling over three bonds (3JH,H ) – longer couplings are rarely observed when double bonds are involved, long range couplings can occur (examples already seen: aromatic rings): 4JAB(meta) = 1 – 3 Hz and 5JAB(para) = 0 – 1.5 Hz further examples are allylic and the homoallylic couplings (sometimes resolved in the 1H NMR spectrum, sometimes only evident through signal broadening) 4JAB(allylic) = 0 – 3 Hz 5JAB(homoallylic) = 0 – 3 Hz

Spectral order: first order and non-first order transition from AX to AB system: 500 MHz spectra (simulated) 3JAX = 12 Hz, dA = 1.0 ppm dX = dB variable important criterion: D / JAX (first order: > 6 – 10) the smaller the difference in chemical shift, the more likely the spectrum will become non-first order roof effect

Spectral order: first order and non-first order transition from AX to AB system: 500 MHz spectra (simulated) 3JAX = 12 Hz, dA = 1.0 ppm dX = dB variable “quartet” important criterion: D / JAX (first order: > 6 – 10) the smaller the difference in chemical shift, the more likely the spectrum will become non-first order

Spectral order: first order and non-first order transition from AX to AB system: 500 MHz spectra (simulated) 3JAX = 12 Hz, dA = 1.0 ppm dX = dB variable A2 if the chemical shifts become identical (isochronous), the multiplet will become a singlet (no mattter whether chemically equivalent or accidentally isochronous) non-first order spin system can sometimes be resolved at higher field strength (e.g. measuring at 400 MHz instead of 100 MHz)

Non-first order spectra: 1,4-disubstituted aromatic rings 4-hydroxybenzoic acid 1,4-disubstituted aromatic rings will give 2 doublets in the aromatic region, integrating for 2 protons each and a coupling constant of 8 Hz even though this again is a “higher order” (AA’BB’ spin system) spectrum, it is easy to recognize and can be analysed the usual way

Chemical and magnetic equivalence chemical equivalence: two nuclei i and k are chemically equivalent if they have the same resonance frequency or chemical shift, i.e. di = dk in first order spectra, we do not observe coupling between chemically equivalent protons example: CH3 protons always appear as one signal (integral 3H) magnetic equivalence: two nuclei i and k are magnetically equivalent if they are chemically equivalent (di = dk), and for all coupling to other nuclei such as l in the molecule, the relationship Jil = Jkl is satisfied

Chemical and magnetic equivalence magnetic equivalence: two nuclei i and k are magnetically equivalent if they are chemically equivalent (di = dk), and for all coupling to other nuclei such as l in the molecule, the relationship Jil = Jkl is satisfied 3JH4/H5 = 3JH5/H6 H-4 and H-6 are chemically and magnetically equivalent A2B spin system 3JH2/H3 ≠ 5JH6/H3 and 3JH6/H5 ≠ 5JH2/H5 H-2 and H-6 are chemically, but not magnetically equivalent (the same for H-3 and H-5) AA'BB' spin system

Non-first order spectra: monosubstituted aromatic rings bromobenzene monosubstituted aromatic rings give characteristic multiplet signals in the aromatic region, integrating for 5 protons (often 2 + 2 + 1) these signals cannot be analysed the usual way, since they represent so-called “higher order” spectra

Spectral order: first order and non-first order 1H NMR spectrum of 1,2-dichlorobenzene (90 MHz) A: experimental B: calculated non first-order spectra can become extremely complex and impossible to analyse by hand iterative algorithms (based on quantum mechanical description of the coupling processes) allow to simulate spectra, and to compare them to the experimental data

Homotopic, enantiotopic and diastereotopic groups homotopic protons are equivalent they give one signal in the 1H NMR spectrum example: methylene chloride (CH2Cl2) two-fold (C2) axis of symmetry enantiotopic protons occur in prochiral compounds example: bromochloromethane: Ha is pro-R, Hb is pro-S plane of symmetry further examples: monosusbstituted allenes exchanging one of the two H (for D) would give enantiomers they are indistinguishable in the 1H NMR spectrum

Homotopic, enantiotopic and diastereotopic groups diastereotopic protons are nonequivalent there is no symmetry operation that can convert one into the other one (also no rotation!) example: 1,2-propandiol replacing one of them (by D) would give diastereomers they will always be in different chemical environments they will have different chemical shifts, unless they are accidentally isochronous if they appear at different chemical shifts, they will mutually couple (2JH,H)

Homotopic, enantiotopic and diastereotopic groups diastereotopic protons are nonequivalent there is no symmetry operation that can convert one into the other one (also no rotation!) example: 1,2-propanediol replacing one of them (by D) would give diastereomers they will always be in different chemical environments they will have different chemical shifts, unless they are accidentally isochronous da avg = x1 × d1 + x2 × d2 + x3 × d3 db avg = x1 × d4 + x2 × d5 + x3 × d6 xi = weighting or mole fraction of each rotamer normally, da ≠ db, even if x1 = x2 = x3 = 1/3

Diastereotopic methylene and methyl groups 1H NMR spectrum of citric acid in D2O (250 MHz) if Ha and Hb appear at different chemical shifts, they will mutually couple (2JH,H) however, the further away the CH2 from the chiral centre, the more likely the two shifts will be (accidentally) isochronous

For CaHbOc: dbe = [(2a + 2) – b]/2 Double bond equivalents (dbe) Double bond equivalents* give the total number of double bonds and rings. For CaHbOc: dbe = [(2a + 2) – b]/2 For CaHbOcNd : dbe = [(2a + 2) – (b – d)]/2 Halogens count as hydrogen for this purpose. The number of oxygen atoms is immaterial. *synonyms: degrees of unsaturation (e.g. Chem3), index of hydrogen deficiency (e.g. Blackman)

Double bond equivalents (dbe) If you can identify securely by spectroscopic or other means the number of multiply bonded functional groups, e.g. carbonyl, present, the remainder will be rings. It is therefore possible to decide if the compound is acyclic, monocyclic, bicyclic, etc. and this is extremely helpful in imagining a structure. Once you have obtained the molecular formula, the first step in a structure determination is to work out the dbe.

for all examples, the molecular formula is C7H5BrO2, and dbe thus is 5 Double bond equivalents (dbe): examples # of double bonds: 4 4 3 # of rings: 1 1 2 for all examples, the molecular formula is C7H5BrO2, and dbe thus is 5

Coupling between NMR active nuclei homonuclear coupling: e.g. 1H-1H (very frequent), 13C-13C (difficult to observe) 2D INADEQUATE heteronuclear coupling: e.g. 1H-13C in 1H NMR: evident as “13C satellite” peaks symmetrical around the solvent signal, separated by the 1JC,H coupling constant, and showing 0.55% of the intensity of the central signal in 13C NMR: deliberately supressed (1H broadband decoupled) CHCl3 (solvent peak in CDCl3)

Coupling to deuterium: solvent signals e.g. 1H-D in 1H NMR CD2HOD (solvent peak in deuterated methanol: quintet) “CD3OD” is ≈ 99.5% CD3OD, and ≈ 0.5% CD2HOD e.g. 13C-D in 13C NMR CD3OD (solvent peak in deuterated methanol: septet) not observed, as no H present not observed, as amount too small Explanation: nuclear spin quantum number I of 1H and 13C is ½, but for 2H, it is 1 (allowed states -1, 0 and +1) general rule for coupling: coupling with n neighbours gives (2×I×n+1) lines for I = ½, this becomes the n+1 rule further information including simulator: http://fluorine.ch.man.ac.uk/research/nmr_terms.php

Coupling to other NMR active nuclei heteronuclear coupling: e.g. 1H-31P, 31P-13C or 1H-19F, 19F-13C (300 MHz in DMSO-d6)

Coupling to other NMR active nuclei heteronuclear coupling: 1H-19F (19F: 100% abundance, I=1/2) dd (8.5, 4.7) ddd (9.8, 8.5, 2.7) dd (10.2, 2.7)

Coupling to other NMR active nuclei heteronuclear coupling: 1H-19F (19F: 100% abundance, I=1/2) 7 dd (8.5, 4.7) 6 4 ddd (9.8, 8.5, 2.7) dd (10.2, 2.7)

Coupling to other NMR active nuclei heteronuclear coupling: 13C-19F (19F: 100% abundance, I=1/2)

Coupling to other NMR active nuclei heteronuclear coupling: 13C-19F (19F: 100% abundance, I=1/2) 2JF,C-6 = 26 Hz* 3JF,C-7 = 10 Hz 2JF,C-4 = 24 Hz* 1JF,C-5 = 231 Hz 3JF,C-3a = 11 Hz *assignment C-4/C-6 may be interchanged