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NMR Spectroscopy, Applications of 3H
NMR Spectroscopy, Applications of 3H John R Jones, University of Surrey, Guildford, UK ã 2017 Elsevier Ltd. All rights reserved.
It is undoubtedly true that if tritium were not radioactive it would be one of the most widely used of all NMR nuclei. Its favourable properties – an isotope of hydrogen, one of the most important of all the elements, with a nuclear spin I ¼ 12 and the most sensitive of all NMR nuclei – counts but little with those who quake at the mention of the word ‘radioactivity’, let alone think of spinning radioactive samples. However, there are those, increasing in number, who believe that tritium is the most favoured of all the nuclei, combining the advantages of favourable radioactive properties weak b emitter (Eavg6 keV), convenient half-life (12.3 years), ready detection by liquid scintillation counting with good efficiencies (typically >50%) with these positive NMR characteristics. Furthermore the technology for synthesizing and handling tritiated compounds has been in place for many years whilst the development of spectrometers operating at ever increasing fields means that less tritium is required for NMR detection. In addition there is virtually no natural abundance tritium concentration, unlike the situation that exists for stable isotopes, so that the dynamic range is enormous. It is this factor above all others that will lead to an expansion in the use of tritium and tritium NMR spectroscopy in the life sciences. Recent publications show that such possibilities are being increasingly appreciated. The whole subject of tritium NMR spectroscopy was reviewed in 2005, see Further Reading.
Properties of the Nucleus As well as having a nuclear spin I ¼ 12 tritium has a high nuclear magnetic moment which is responsible for the magnetogyric constant being larger than for any other nucleus, as also is its sensitivity to NMR detection, 21% higher than that for 1H. At 11.7 T, at which field the 1H NMR frequency is 500 MHz, the 3 H NMR frequency will be 533.3 MHz.
Sample Preparation and Spectrum Measurement Before embarking on any 3H NMR work the personnel must become designated radiation workers, have the appropriate radiochemical facilities and become familiar with tritiation procedures. In this respect it is frequently useful for initial training to be given in appropriate deuteration studies although the corresponding tritium work will usually be carried out on a much smaller scale and the purification procedures will depend greatly on appropriate radio-chromatographic methods, as distinct from chromatographic methods. With appropriate rules and regulations in place a radiochemical This article is reproduced from the previous edition, Copyright 1999, Elsevier Ltd.
Encyclopedia of Spectroscopy and Spectrometry, Third Edition
laboratory need not be any more hazardous than an ordinary chemistry laboratory, particularly if the rule ‘that only the minimum amount of radioactivity consistent with the requirements of the project’ is used. There are two separate units of radioactivity in use, the first being the curie (Ci) which is defined as an activity of 3.71010 disintegrations per second. This is a large unit, hence the frequent use of smaller subunits, the millicurie (103 Ci) and the microcurie (106 Ci). The second, and more recently introduced unit, is the becquerel (Bq). At one disintegration per second this is an extremely small amount of radioactivity. The conversions are 1 Bq ¼ 2:703 1011 Ci ð27:03 p CiÞ, 1 Ci ¼ 3:7 1010 Bq or 37 GBq Although there are a large number of methods available for preparing tritiated compounds, the most widely used stem from the following categories:
• • • • •
catalytic hydrogenation of an unsaturated precursor using 3 H2 gas; catalytic halogen–tritium replacement reactions; hydrogen isotope exchange reactions catalysed by acids, bases or metals; reductions using reagents such as sodium borotritide; methylation reactions using reagents such as tritiated methyl iodide.
Microwaves have been used to greatly accelerate the rates of many of these reactions whilst the development of microwaveenhanced solid state tritiation procedures offers considerable potential. For 3H NMR analysis 1–10 mCi of material dissolved in 50–100 ml of a deuterated solvent is usually sufficient to obtain a spectrum of good signal-to-noise in a matter of 1–10 h, depending on whether the radioactivity is located at one site (a specifically labelled compound) or in several positions (a general labelled compound) – this assumes a spectrometer operating at 300 MHz for 1H and 320 MHz for 3H. For reasons of safety the radioactive samples are placed in narrow cylindrical tubes, sealed at the top, which themselves are placed in standard NMR tubes – this double containment procedure, initially introduced when much higher levels of radioactivity were required, provides a measure of safety as well as reassurance. Experience shows that 3H NMR spectra are of two kinds. Firstly there are those in which the specific activity of the compound is less than 1 Ci mmol1 so that 3 H–3H couplings are absent and the 3H NMR (1H decoupled) spectra consist of a series of single lines, which on integration give the relative incorporation of 3H at each site. Nuclear Overhauser effects (NOEs) are small and differential effects even smaller so that there is no need to obtain NOE-supressed spectra. It should also be mentioned that there is no need to synthesize a tritiated organic standard – all the 3H chemical
http://dx.doi.org/10.1016/B978-0-12-803224-4.00158-8
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NMR Spectroscopy, Applications of 3H
shifts are obtained via the 1H chemical shifts and the Larmor frequency ratio. For compounds at high specific activity, e.g. prepared by the addition of 3H2 gas across the unsaturated group of a precursor, there will be tritium–tritium couplings, the magnitude of which are similar to those of hydrogen, i.e. J(1H–3H)¼J (1H–1H)1.066. Small isotope effects are present but these do not complicate the interpretation of the spectra; on the contrary they can aid the analysis of the relative proportions of isotopomers present, e.g. RC 3H3, RC 1H3H2 and RC 1 H2 3H. Tritium couplings to boron, deuterium, carbon, fluorine and phosphorus are also in agreement with theory.
Applications of Tritium NMR New Tritium Labelling Reagents The development of 3H NMR spectroscopy has made possible many new applications and in the process has stimulated research into the development of new labelling reagents and hence new/improved labelling procedures. One such area is that of tritide reagents. Essentially carrier-free LiB3H4 can now be obtained via the two-step sequence: BuLiþ3 H2 ! Li3 H þ Bu3 H 4Li3 H þ BBr3 ! LiB3 H4 þ 3LiBr Similarly, carrier-free sodium triethylborotritide, a useful reagent for the stereo- and regiospecific reduction of carbonyl-containing compounds, can be synthesized in the following manner:
The well-known iridium catalyst [Ir(COD)(Cy3P)(Py)]PF6, where COD¼1,5-cyclooctadiene and Py¼ pyridine, demonstrates excellent regioselectivity in isotopic exchange reactions of acetanilides and other substituted aromatic substrates. 3H NMR spectroscopy is invaluable in identifying the site(s) of tritium incorporation – there are many instances where the broad signals in the corresponding 2H NMR spectra are much less informative. Another iridium catalyst that exhibits good regioselectivity in hydrogen isotope exchange reactions is the complex [IrH2(acetone)2(PPh3)2]BF4. As in the previous studies the transient existence of a metallocyclic intermediate is indicated. Considerable interest has also been shown in the development of the high temperature solid state catalytic isotopic exchange (HSCIE) method developed by Myasoedev and colleagues. Although labelling is uniform in most instances, some regiocontrol can be exerted by careful temperature control.
Chiral Methyl, Stereochemistry and Biosynthesis The analysis of stereochemical problems in both chemistry and biochemistry has benefited greatly from the use of compounds that contain a methyl group with one atom each of 1H, 2H and 3 H. Such compounds exist as a pair of enantiomers, identified by R and S, and early work in this area will always be associated with the names of Arigoni and Cornforth. Recently a very efficient five-stage synthesis of chiral acetate has been reported (in which the penultimate reaction uses supertritide) with an enantiomeric purity of 100%.
BuLi þ NaOBut þ3 H2 ! Na3 H þ LiOBut þ Bu3 H Na3 H þ Et3 B ! NaEt3 B3 H Tri-n-butyltin and lithium tri-s-butylborotritide are other useful reagents. Increasingly sophisticated tritium labelling technology is being developed as an alternative to the more traditional hydrogenation and catalytic dehalogenation reactions. The procedures will find wide application in the tritiation of molecules of biological importance. Thus N-tritioacetoxyphthalimide, a high specific activity tritioacetylating reagent, has been used to label a number of acetylenes, ketones and alcohols whilst radical-induced tritiodeoxygenation reactions can lead to the synthesis of important heterocyclic compounds.
New More Selective Tritiation Procedures Hydrogen isotope exchange reactions are widely used not only to study reaction mechanisms but also for labelling compounds with either deuterium or tritium. The reactions may be catalysed by acids or bases under both homogeneous or heterogeneous conditions and frequently lead to generally labelled compounds. The same is true for transition metals. Considerable effort has been directed at developing more selective procedures – homogeneous rhodium trichloride has been shown to be very effective in introducing both deuterium and tritium into the ortho-aromatic positions of a wide range of pharmaceutically important compounds.
In the past the determination of whether an unknown sample contained an excess of an (R)- or (S)-configured chiral methyl group relied on using a reaction in which one hydrogen is removed to generate a methylene group in which tritium is now unevenly distributed between the two methylene hydrogens. The condensation of acetyl coenzyme A with glyoxylic acid catalysed by the enzyme malate synthase, which exhibits a primary kinetic isotope effect kH/kD of 3.8, was the chosen reaction. Analysis of the tritium distribution, together with a knowledge of kH/kD and the steric course of the reaction, yields the required information – the configuration of the original chiral methyl group and an estimate of the enantiomeric excess. 3 H NMR spectroscopy can provide the necessary information directly; whether 3H has 1H or 2H as a neighbour can be determined directly from the 1H–3H coupling and the 2H isotope shift on the 3H signal. The only problem with the 3H NMR method is that it requires a few mCi of tritiated material, at least with current-day NMR spectrometers. With improvements in spectrometer design and the absence of ‘natural
NMR Spectroscopy, Applications of 3H
abundance’ tritium signals this may not always be the case. As it is, the method is direct, does not require any knowledge of the primary isotope effect and no chemical degradations are required. There are many examples of enzymatic methyl-transfer reactions in biochemistry to which the chiral methyl/3H NMR technology can be applied. One such example involves the important biological methyl donor S-adenosylmethionine. Combined with other studies the results show that the transfer of a methyl group to a variety of different nucleophiles all operate with inversion of methyl group configuration.
Substrate–Receptor Interactions Most NMR studies in this area have used 13C- or 15N-labelled ligands, the synthesis of which is frequently more demanding than is the case for 3H ligands. Furthermore, the sensitivity of both 13C and 15N nuclei to NMR detection is considerably less favourable than is the case for tritium. It is therefore somewhat surprising and at the same time disappointing that there are still relatively few examples of protein–ligand interaction studies based on the use of 3H-labelled ligands. In an early study 3H NMR spectroscopy was used to monitor the anomeric binding specificity of a- and b-maltodextrins binding to a maltosebinding protein whilst in another study 3H NMR spectroscopy was used to measure the dynamic properties of tosyl groups in specifically 3H-labelled tosylchymotrypsin. Preliminary details of a 3H NMR binding study of a tritium-labelled phospholipase A2 inhibitor to bovine pancreatic PLA2 suggest that the tritium atoms are located within the hydrophobic pocket of the protein. In a more extensive study a number of high specific activity tritiated folic acids and methotrexates have been prepared and their complexes with Lactobacillus casei dihydrofolate reductase (DHFR) investigated. The 3H NMR results confirm the presence of three pH-dependent different conformational forms in the complex DHFRNADPþfolate, whereas both the binary and ternary methotrexate complexes (DHFRMTX, DHFRNADPþMTX) were shown to exist as a single conformational state. An interesting 3H NMR study of the complex formed by [4-3H]benzenesulfonamide and human carbonic anhydrase 1 reveals details that are widely different from those obtained when using a fluorinated inhibitor, highlighting the dangers of using fluorine as a ‘substitute’ isotope for one of the hydrogen isotopes.
305
Macromolecules The methods that have been developed for tritiating small organic molecules do not lend themselves very readily to the tritiation of large macromolecules such as proteins although there are a few examples where Myasoedov’s HSCIE procedure proved successful. It is not surprising therefore that very little work has been reported on, for example, the 3H NMR of proteins. The polymer area, however, has seen more activity, mainly because it has been much easier to tritiate such compounds – hydrogenation with 3H2 gas of a suitable monomer followed by polymerization leads to a specifically tritiated product. Many polymers are difficult to solubilize and the question has been asked several times whether in view of its good NMR characteristics it is possible to obtain satisfactory solid state spectra. The potential problems have been overcome partly by the development of zirconia rotors and partly by enclosing the tritium probe in a Perspex shield so that, in the event of an accident, radioactivity would be retained on a suitable filter. Magic-angle spinning at 17 kHz rotation provides spectra with line widths at half-height of the order of 120 Hz. This has been achieved without 1 H decoupling, this being a more difficult task than for solution studies. It is too early to say at this stage whether 3H NMR spectroscopy of solids will develop into as widely used a technique as 13C NMR spectroscopy. The main factor will undoubtedly be how far improvements in NMR sensitivity can be extended.
See also: Labelling Studies in Biochemistry Using NMR; Natural Abundance Deuterium NMR Spectroscopy.
Further Reading Andres H, Morimoto H, and Williams PG (1990) Preparation and use of LiEt3BT and LiAlT4 at maximum specific activity. Journal of the Chemical Society, Chemical Communications 627. Evans EA, Warrell DC, Elvidge JA, and Jones JR (1985) Handbook of Tritium NMR Spectroscopy and Applications. Chichester: Wiley. Filer CN (2006) Progress in tritium NMR: 1990–2005. Journal of Radioanalytical and Nuclear Chemistry 268: 663–669. Floss HG and Lee S (1993) Chiral methyl groups: Small is beautiful. Accounts of Chemical Research 26: 116–122. Junk T and Catallo WJ (1997) Hydrogen isotope exchange reactions involving C-H(D,T) bonds. Chemical Society Reviews 401–406. Kubinec MG and Williams PG (1996) Tritium NMR. In: Grant DM and Harris RK (eds.) Encyclopedia of NMR, vol. 8, pp. 4819–4839. Chichester: Wiley. Saljoughian M, Morimoto H, Williams PG, Than C, and Seligman SN (1996) Journal of Organic Chemistry 61: 9625–9628.
It is undoubtedly true that if tritium were not radioactive it would be one of the most widely used of all NMR nuclei. Its favourable properties – an isotope of hydrogen, one of the most important of all the elements, with a nuclear spin I ¼ 12 and the most sensitive of all NMR nuclei – counts but little with those who quake at the mention of the word ‘radioactivity’, let alone think of spinning radioactive samples. However, there are those, increasing in number, who believe that tritium is the most favoured of all the nuclei, combining the advantages of favourable radioactive properties weak b emitter (Eavg6 keV), convenient half-life (12.3 years), ready detection by liquid scintillation counting with good efficiencies (typically >50%) with these positive NMR characteristics. Furthermore the technology for synthesizing and handling tritiated compounds has been in place for many years whilst the development of spectrometers operating at ever increasing fields means that less tritium is required for NMR detection. In addition there is virtually no natural abundance tritium concentration, unlike the situation that exists for stable isotopes, so that the dynamic range is enormous. It is this factor above all others that will lead to an expansion in the use of tritium and tritium NMR spectroscopy in the life sciences. Recent publications show that such possibilities are being increasingly appreciated. The whole subject of tritium NMR spectroscopy was reviewed in 2005, see Further Reading.
Properties of the Nucleus As well as having a nuclear spin I ¼ 12 tritium has a high nuclear magnetic moment which is responsible for the magnetogyric constant being larger than for any other nucleus, as also is its sensitivity to NMR detection, 21% higher than that for 1H. At 11.7 T, at which field the 1H NMR frequency is 500 MHz, the 3 H NMR frequency will be 533.3 MHz.
Sample Preparation and Spectrum Measurement Before embarking on any 3H NMR work the personnel must become designated radiation workers, have the appropriate radiochemical facilities and become familiar with tritiation procedures. In this respect it is frequently useful for initial training to be given in appropriate deuteration studies although the corresponding tritium work will usually be carried out on a much smaller scale and the purification procedures will depend greatly on appropriate radio-chromatographic methods, as distinct from chromatographic methods. With appropriate rules and regulations in place a radiochemical This article is reproduced from the previous edition, Copyright 1999, Elsevier Ltd.
Encyclopedia of Spectroscopy and Spectrometry, Third Edition
laboratory need not be any more hazardous than an ordinary chemistry laboratory, particularly if the rule ‘that only the minimum amount of radioactivity consistent with the requirements of the project’ is used. There are two separate units of radioactivity in use, the first being the curie (Ci) which is defined as an activity of 3.71010 disintegrations per second. This is a large unit, hence the frequent use of smaller subunits, the millicurie (103 Ci) and the microcurie (106 Ci). The second, and more recently introduced unit, is the becquerel (Bq). At one disintegration per second this is an extremely small amount of radioactivity. The conversions are 1 Bq ¼ 2:703 1011 Ci ð27:03 p CiÞ, 1 Ci ¼ 3:7 1010 Bq or 37 GBq Although there are a large number of methods available for preparing tritiated compounds, the most widely used stem from the following categories:
• • • • •
catalytic hydrogenation of an unsaturated precursor using 3 H2 gas; catalytic halogen–tritium replacement reactions; hydrogen isotope exchange reactions catalysed by acids, bases or metals; reductions using reagents such as sodium borotritide; methylation reactions using reagents such as tritiated methyl iodide.
Microwaves have been used to greatly accelerate the rates of many of these reactions whilst the development of microwaveenhanced solid state tritiation procedures offers considerable potential. For 3H NMR analysis 1–10 mCi of material dissolved in 50–100 ml of a deuterated solvent is usually sufficient to obtain a spectrum of good signal-to-noise in a matter of 1–10 h, depending on whether the radioactivity is located at one site (a specifically labelled compound) or in several positions (a general labelled compound) – this assumes a spectrometer operating at 300 MHz for 1H and 320 MHz for 3H. For reasons of safety the radioactive samples are placed in narrow cylindrical tubes, sealed at the top, which themselves are placed in standard NMR tubes – this double containment procedure, initially introduced when much higher levels of radioactivity were required, provides a measure of safety as well as reassurance. Experience shows that 3H NMR spectra are of two kinds. Firstly there are those in which the specific activity of the compound is less than 1 Ci mmol1 so that 3 H–3H couplings are absent and the 3H NMR (1H decoupled) spectra consist of a series of single lines, which on integration give the relative incorporation of 3H at each site. Nuclear Overhauser effects (NOEs) are small and differential effects even smaller so that there is no need to obtain NOE-supressed spectra. It should also be mentioned that there is no need to synthesize a tritiated organic standard – all the 3H chemical
http://dx.doi.org/10.1016/B978-0-12-803224-4.00158-8
303
304
NMR Spectroscopy, Applications of 3H
shifts are obtained via the 1H chemical shifts and the Larmor frequency ratio. For compounds at high specific activity, e.g. prepared by the addition of 3H2 gas across the unsaturated group of a precursor, there will be tritium–tritium couplings, the magnitude of which are similar to those of hydrogen, i.e. J(1H–3H)¼J (1H–1H)1.066. Small isotope effects are present but these do not complicate the interpretation of the spectra; on the contrary they can aid the analysis of the relative proportions of isotopomers present, e.g. RC 3H3, RC 1H3H2 and RC 1 H2 3H. Tritium couplings to boron, deuterium, carbon, fluorine and phosphorus are also in agreement with theory.
Applications of Tritium NMR New Tritium Labelling Reagents The development of 3H NMR spectroscopy has made possible many new applications and in the process has stimulated research into the development of new labelling reagents and hence new/improved labelling procedures. One such area is that of tritide reagents. Essentially carrier-free LiB3H4 can now be obtained via the two-step sequence: BuLiþ3 H2 ! Li3 H þ Bu3 H 4Li3 H þ BBr3 ! LiB3 H4 þ 3LiBr Similarly, carrier-free sodium triethylborotritide, a useful reagent for the stereo- and regiospecific reduction of carbonyl-containing compounds, can be synthesized in the following manner:
The well-known iridium catalyst [Ir(COD)(Cy3P)(Py)]PF6, where COD¼1,5-cyclooctadiene and Py¼ pyridine, demonstrates excellent regioselectivity in isotopic exchange reactions of acetanilides and other substituted aromatic substrates. 3H NMR spectroscopy is invaluable in identifying the site(s) of tritium incorporation – there are many instances where the broad signals in the corresponding 2H NMR spectra are much less informative. Another iridium catalyst that exhibits good regioselectivity in hydrogen isotope exchange reactions is the complex [IrH2(acetone)2(PPh3)2]BF4. As in the previous studies the transient existence of a metallocyclic intermediate is indicated. Considerable interest has also been shown in the development of the high temperature solid state catalytic isotopic exchange (HSCIE) method developed by Myasoedev and colleagues. Although labelling is uniform in most instances, some regiocontrol can be exerted by careful temperature control.
Chiral Methyl, Stereochemistry and Biosynthesis The analysis of stereochemical problems in both chemistry and biochemistry has benefited greatly from the use of compounds that contain a methyl group with one atom each of 1H, 2H and 3 H. Such compounds exist as a pair of enantiomers, identified by R and S, and early work in this area will always be associated with the names of Arigoni and Cornforth. Recently a very efficient five-stage synthesis of chiral acetate has been reported (in which the penultimate reaction uses supertritide) with an enantiomeric purity of 100%.
BuLi þ NaOBut þ3 H2 ! Na3 H þ LiOBut þ Bu3 H Na3 H þ Et3 B ! NaEt3 B3 H Tri-n-butyltin and lithium tri-s-butylborotritide are other useful reagents. Increasingly sophisticated tritium labelling technology is being developed as an alternative to the more traditional hydrogenation and catalytic dehalogenation reactions. The procedures will find wide application in the tritiation of molecules of biological importance. Thus N-tritioacetoxyphthalimide, a high specific activity tritioacetylating reagent, has been used to label a number of acetylenes, ketones and alcohols whilst radical-induced tritiodeoxygenation reactions can lead to the synthesis of important heterocyclic compounds.
New More Selective Tritiation Procedures Hydrogen isotope exchange reactions are widely used not only to study reaction mechanisms but also for labelling compounds with either deuterium or tritium. The reactions may be catalysed by acids or bases under both homogeneous or heterogeneous conditions and frequently lead to generally labelled compounds. The same is true for transition metals. Considerable effort has been directed at developing more selective procedures – homogeneous rhodium trichloride has been shown to be very effective in introducing both deuterium and tritium into the ortho-aromatic positions of a wide range of pharmaceutically important compounds.
In the past the determination of whether an unknown sample contained an excess of an (R)- or (S)-configured chiral methyl group relied on using a reaction in which one hydrogen is removed to generate a methylene group in which tritium is now unevenly distributed between the two methylene hydrogens. The condensation of acetyl coenzyme A with glyoxylic acid catalysed by the enzyme malate synthase, which exhibits a primary kinetic isotope effect kH/kD of 3.8, was the chosen reaction. Analysis of the tritium distribution, together with a knowledge of kH/kD and the steric course of the reaction, yields the required information – the configuration of the original chiral methyl group and an estimate of the enantiomeric excess. 3 H NMR spectroscopy can provide the necessary information directly; whether 3H has 1H or 2H as a neighbour can be determined directly from the 1H–3H coupling and the 2H isotope shift on the 3H signal. The only problem with the 3H NMR method is that it requires a few mCi of tritiated material, at least with current-day NMR spectrometers. With improvements in spectrometer design and the absence of ‘natural
NMR Spectroscopy, Applications of 3H
abundance’ tritium signals this may not always be the case. As it is, the method is direct, does not require any knowledge of the primary isotope effect and no chemical degradations are required. There are many examples of enzymatic methyl-transfer reactions in biochemistry to which the chiral methyl/3H NMR technology can be applied. One such example involves the important biological methyl donor S-adenosylmethionine. Combined with other studies the results show that the transfer of a methyl group to a variety of different nucleophiles all operate with inversion of methyl group configuration.
Substrate–Receptor Interactions Most NMR studies in this area have used 13C- or 15N-labelled ligands, the synthesis of which is frequently more demanding than is the case for 3H ligands. Furthermore, the sensitivity of both 13C and 15N nuclei to NMR detection is considerably less favourable than is the case for tritium. It is therefore somewhat surprising and at the same time disappointing that there are still relatively few examples of protein–ligand interaction studies based on the use of 3H-labelled ligands. In an early study 3H NMR spectroscopy was used to monitor the anomeric binding specificity of a- and b-maltodextrins binding to a maltosebinding protein whilst in another study 3H NMR spectroscopy was used to measure the dynamic properties of tosyl groups in specifically 3H-labelled tosylchymotrypsin. Preliminary details of a 3H NMR binding study of a tritium-labelled phospholipase A2 inhibitor to bovine pancreatic PLA2 suggest that the tritium atoms are located within the hydrophobic pocket of the protein. In a more extensive study a number of high specific activity tritiated folic acids and methotrexates have been prepared and their complexes with Lactobacillus casei dihydrofolate reductase (DHFR) investigated. The 3H NMR results confirm the presence of three pH-dependent different conformational forms in the complex DHFRNADPþfolate, whereas both the binary and ternary methotrexate complexes (DHFRMTX, DHFRNADPþMTX) were shown to exist as a single conformational state. An interesting 3H NMR study of the complex formed by [4-3H]benzenesulfonamide and human carbonic anhydrase 1 reveals details that are widely different from those obtained when using a fluorinated inhibitor, highlighting the dangers of using fluorine as a ‘substitute’ isotope for one of the hydrogen isotopes.
305
Macromolecules The methods that have been developed for tritiating small organic molecules do not lend themselves very readily to the tritiation of large macromolecules such as proteins although there are a few examples where Myasoedov’s HSCIE procedure proved successful. It is not surprising therefore that very little work has been reported on, for example, the 3H NMR of proteins. The polymer area, however, has seen more activity, mainly because it has been much easier to tritiate such compounds – hydrogenation with 3H2 gas of a suitable monomer followed by polymerization leads to a specifically tritiated product. Many polymers are difficult to solubilize and the question has been asked several times whether in view of its good NMR characteristics it is possible to obtain satisfactory solid state spectra. The potential problems have been overcome partly by the development of zirconia rotors and partly by enclosing the tritium probe in a Perspex shield so that, in the event of an accident, radioactivity would be retained on a suitable filter. Magic-angle spinning at 17 kHz rotation provides spectra with line widths at half-height of the order of 120 Hz. This has been achieved without 1 H decoupling, this being a more difficult task than for solution studies. It is too early to say at this stage whether 3H NMR spectroscopy of solids will develop into as widely used a technique as 13C NMR spectroscopy. The main factor will undoubtedly be how far improvements in NMR sensitivity can be extended.
See also: Labelling Studies in Biochemistry Using NMR; Natural Abundance Deuterium NMR Spectroscopy.
Further Reading Andres H, Morimoto H, and Williams PG (1990) Preparation and use of LiEt3BT and LiAlT4 at maximum specific activity. Journal of the Chemical Society, Chemical Communications 627. Evans EA, Warrell DC, Elvidge JA, and Jones JR (1985) Handbook of Tritium NMR Spectroscopy and Applications. Chichester: Wiley. Filer CN (2006) Progress in tritium NMR: 1990–2005. Journal of Radioanalytical and Nuclear Chemistry 268: 663–669. Floss HG and Lee S (1993) Chiral methyl groups: Small is beautiful. Accounts of Chemical Research 26: 116–122. Junk T and Catallo WJ (1997) Hydrogen isotope exchange reactions involving C-H(D,T) bonds. Chemical Society Reviews 401–406. Kubinec MG and Williams PG (1996) Tritium NMR. In: Grant DM and Harris RK (eds.) Encyclopedia of NMR, vol. 8, pp. 4819–4839. Chichester: Wiley. Saljoughian M, Morimoto H, Williams PG, Than C, and Seligman SN (1996) Journal of Organic Chemistry 61: 9625–9628.