Editing Kinetic isotope effect (section)

=== Kinetic isotope effect measurement at natural abundance ===
'''KIE measurement at natural abundance''' is a simple general method for measuring KIEs for [[chemical reaction]]s performed with materials of [[natural abundance]]. This technique for measuring KIEs overcomes many limitations of previous KIE measurement methods. KIE measurements from isotopically labeled materials require a new synthesis for each isotopically labeled material (a process often prohibitively difficult), a competition reaction, and an analysis.<ref name="Simmons_2012" /> The KIE measurement at [[natural abundance]] avoids these issues by taking advantage of high precision quantitative techniques ([[nuclear magnetic resonance spectroscopy]], [[isotope-ratio mass spectrometry]]) to site selectively measure [[kinetic fractionation]] of [[isotope]]s, in either product or starting material for a given [[chemical reaction]].

==== Single-pulse NMR ====
Quantitative single-pulse [[nuclear magnetic resonance spectroscopy]] (NMR) is a method amenable for measuring [[kinetic fractionation]] of [[isotope]]s for natural abundance KIE measurements. Pascal et al. were inspired by studies demonstrating dramatic variations of deuterium within identical compounds from different sources and hypothesized that NMR could be used to measure {{sup|2}}H KIEs at natural abundance.<ref>{{Cite journal| vauthors = Martin GJ, Martin ML |title=Deuterium labelling at the natural abundance level as studied by high field quantitative 2H NMR |journal=Tetrahedron Letters|language=en|year=1984|volume=22|issue=36|pages=3525–3528|doi=10.1016/s0040-4039(01)81948-1}}</ref><ref name=":0" /> Pascal and coworkers tested their hypothesis by studying the [[insertion reaction]] of dimethyl diazomalonate into [[cyclohexane]]. Pascal et al. measured a KIE of 2.2 using {{sup|2}}H NMR for materials of natural abundance.<ref name=":0">{{cite journal| vauthors = Pascal Jr RA, Baum MW, Wagner CK, Rodgers LR |title=Measurement of deuterium kinetic isotope effects in organic reactions by natural-abundance deuterium NMR spectroscopy|journal=Journal of the American Chemical Society|date=September 1984|volume=106|issue=18|pages=5377–5378|doi=10.1021/ja00330a071 |bibcode=1984JAChS.106.5377P }}</ref>

[[File:Chemical reaction 1.png|frameless|none|500px]]

Singleton and coworkers demonstrated the capacity of {{sup|13}}C NMR based natural abundance KIE measurements for studying the mechanism of the [4 + 2] [[cycloaddition]] of [[isoprene]] with [[maleic anhydride]].<ref name="Singleton_1995" /> Previous studies by Gajewski on isotopically enrich materials observed KIE results that suggested an asynchronous transition state, but were always consistent, within error, for a perfectly synchronous [[reaction mechanism]].<ref>{{cite journal| vauthors = Gajewski JJ, Peterson KB, Kagel JR, Huang YJ |date=December 1989|title=Transition-state structure variation in the Diels-Alder reaction from secondary deuterium kinetic isotope effects. The reaction of nearly symmetrical dienes and dienophiles is nearly synchronous|journal=Journal of the American Chemical Society|volume=111|issue=25|pages=9078–9081|doi=10.1021/ja00207a013 |bibcode=1989JAChS.111.9078G }}</ref>

[[File:Chemical reaction 2.png|frameless|none|500px]]

This work by Singleton et al. established the measurement of multiple {{sup|13}}C KIEs within the design of a single experiment. These {{sup|2}}H and {{sup|13}}C KIE measurements determined at natural abundance found the "inside" hydrogens of the diene experience a more pronounced {{sup|2}}H KIE than the "outside" hydrogens and the C1 and C4 experience a significant KIE. These key observations suggest an asynchronous [[reaction mechanism]] for the [[cycloaddition]] of [[isoprene]] with [[maleic anhydride]].

[[File:KIE measurements.png|frameless|274x274px]]

The limitations for determining KIEs at natural abundance using NMR are that the recovered material must have a suitable amount and purity for NMR analysis (the signal of interest should be distinct from other signals), the reaction of interest must be irreversible, and the [[reaction mechanism]] must not change for the duration of the [[chemical reaction]].

Experimental details for using quantitative single pulse NMR to measure KIE at natural abundance as follows: the experiment needs to be performed under quantitative conditions including a relaxation time of 5 T{{sub|1}}, measured 90° flip angle, a digital resolution of at least 5 points across a peak, and a signal:noise greater than 250. The raw FID is zero-filled to at least 256K points before the Fourier transform. NMR spectra are phased and then treated with a zeroth order baseline correction without any tilt correction. Signal integrations are determined numerically with a minimal tolerance for each integrated signal.<ref name="Singleton_1995" />{{clarify|date=November 2018}}

==== Organometallic reaction mechanism elucidation examples ====
Colletto et al. developed a regioselective β-arylation of benzo[b]thiophenes at room temperature with aryl iodides as coupling partners and sought to understand the mechanism of this reaction by performing natural abundance KIE measurements via single pulse NMR.<ref name=":1">{{cite journal | vauthors = Colletto C, Islam S, Juliá-Hernández F, Larrosa I | title = Room-Temperature Direct β-Arylation of Thiophenes and Benzo[b]thiophenes and Kinetic Evidence for a Heck-type Pathway | journal = Journal of the American Chemical Society | volume = 138 | issue = 5 | pages = 1677–83 | date = February 2016 | pmid = 26788885 | pmc = 4774971 | doi = 10.1021/jacs.5b12242 | bibcode = 2016JAChS.138.1677C }}</ref>
[[File:Reaction scheme for the beta arylation of benzo b thiophenes.png|none|thumb|553x553px|Regioselective β-arylation of benzo[b]thiophenes. HFiP (usually HFIP) refers to [[hexafluoroisopropanol]], or CF<small>3</small>-CHOH-CF<sub>3</sub>]]

[[File:1H KIEs for beta arylation of benzo b thiophenes.png|none|thumb|200x200px|{{sup|2}}H KIEs measured at natural abundance]]

[[File:13C KIEs for beta arylation of benzo b thiophenes.png|none|thumb|200x200px|{{sup|13}}C KIEs measured at natural abundance]]

The observation of a primary {{sup|13}}C isotope effect at C3, an inverse {{sup|2}}H isotope effect, a secondary {{sup|13}}C isotope effect at C2, and the lack of a {{sup|2}}H isotope effect at C2; led Colletto ''et al.'' to suggest a Heck-type reaction mechanism for the regioselective {{math|β}}-arylation of benzo[b]thiophenes at room temperature with aryl iodides as coupling partners.<ref name=":1" />

Frost ''et al.'' sought to understand the effects of [[Lewis acid]] additives on the mechanism of enantioselective [[palladium]]-catalyzed C-N bond activation using natural abundance KIE measurements via single pulse NMR.<ref name=":2">{{cite journal | vauthors = Frost GB, Serratore NA, Ogilvie JM, Douglas CJ | title = Mechanistic Model for Enantioselective Intramolecular Alkene Cyanoamidation via Palladium-Catalyzed C-CN Bond Activation | journal = The Journal of Organic Chemistry | volume = 82 | issue = 7 | pages = 3721–3726 | date = April 2017 | pmid = 28294618 | doi = 10.1021/acs.joc.7b00196 | pmc = 5535300 }}</ref>
[[File:Enantioselective intramolecular alkene cyanoamidation reaction scheme.png|none|thumb|513x513px|Enantioselective intramolecular alkene cyanoamidation]]
[[File:13C KIEs for enantioselective intramolecular alkene cyanoamidation reaction scheme.png|none|thumb|542x542px|{{sup|13}}C KIEs for enantioselective intramolecular alkene cyanoamidation reaction (left no additive, right add BPh{{sub|3}})]]
The primary {{sup|13}}C KIE observed in the absence of BPh{{sub|3}} suggests a reaction mechanism with rate limiting cis oxidation into the C–CN bond of the [[cyanoformamide]]. The addition of BPh{{sub|3}} causes a relative decrease in the observed {{sup|13}}C KIE which led Frost et al. to suggest a change in the rate limiting step from cis oxidation to coordination of palladium to the cyanoformamide.<ref name=":2" />

==== DEPT-55 NMR ====
Though KIE measurements at natural abundance are a powerful tool for understanding reaction mechanisms, the amounts of material needed for analysis can make this technique inaccessible for reactions that use expensive reagents or unstable starting materials. To mitigate these limitations, Jacobsen and coworkers developed {{sup|1}}H to {{sup|13}}C polarization transfer as a means to reduce the time and material required for KIE measurements at natural abundance. The [[distortionless enhancement by polarization transfer]] (DEPT) takes advantage of the larger [[gyromagnetic ratio]] of {{sup|1}}H over {{sup|13}}C, to theoretically improve measurement sensitivity by a factor of 4 or decrease experiment time by a factor of 16. This method for natural abundance kinetic isotope measurement is favorable for analysis for reactions containing unstable starting materials, and catalysts or products that are relatively costly.<ref>{{cite journal | vauthors = Kwan EE, Park Y, Besser HA, Anderson TL, Jacobsen EN | title = 13C Kinetic Isotope Effect Measurements Enabled by Polarization Transfer | journal = Journal of the American Chemical Society | volume = 139 | issue = 1 | pages = 43–46 | date = January 2017 | pmid = 28005341 | doi = 10.1021/jacs.6b10621 | pmc = 5674980 }}</ref>

Jacobsen and coworkers identified the [[thiourea]]-catalyzed glycosylation of galactose as a reaction that met both of the aforementioned criteria (expensive materials and unstable substrates) and was a reaction with a poorly understood mechanism.<ref>{{cite journal | vauthors = Park Y, Harper KC, Kuhl N, Kwan EE, Liu RY, Jacobsen EN | title = Macrocyclic bis-thioureas catalyze stereospecific glycosylation reactions | journal = Science | volume = 355 | issue = 6321 | pages = 162–166 | date = January 2017 | pmid = 28082586 | pmc = 5671764 | doi = 10.1126/science.aal1875 | bibcode = 2017Sci...355..162P }}</ref> Glycosylation is a special case of nucleophilic substitution that lacks clear definition between S{{sub|N}}1 and S{{sub|N}}2 mechanistic character. The presence of the oxygen adjacent to the site of displacement (i.e., C1) can stabilize positive charge. This charge stabilization can cause any potential concerted pathway to become asynchronous and approaches intermediates with oxocarbenium character of the S{{sub|N}}1 mechanism for glycosylation.

[[File:Reaction Scheme for Thiourea catalyzed glycosylation of galactose.png|alt=Reaction scheme for thiourea catalyzed glycosylation of galactose|none|thumb|492x492px|Reaction scheme for thiourea catalyzed glycosylation of galactose]]
[[File:Kinetic Isotope Effect Measurements for Thiourea catalyzed glycosylation of galactose.png|alt=13C kinetic isotope effect measurements for thiourea catalyzed glycosylation of galactose|none|thumb|350x350px|{{sup|13}}C kinetic isotope effect measurements for thiourea catalyzed glycosylation of galactose]]

Jacobsen and coworkers observed small normal KIEs at C1, C2, and C5 which suggests significant oxocarbenium character in the transition state and an asynchronous reaction mechanism with a large degree of charge separation.

==== Isotope-ratio mass spectrometry ====

High precision [[isotope-ratio mass spectrometry]] (IRMS) is another method for measuring [[kinetic fractionation]] of [[isotope]]s for natural abundance KIE measurements. Widlanski and coworkers demonstrated {{sup|34}}S KIE at natural abundance measurements for the [[hydrolysis]] of [[sulfate]] monoesters. Their observation of a large KIE suggests S-O bond cleavage is rate controlling and likely rules out an associate [[reaction mechanism]].<ref>{{cite journal | vauthors = Burlingham BT, Pratt LM, Davidson ER, Shiner VJ, Fong J, Widlanski TS | title = 34S isotope effect on sulfate ester hydrolysis: mechanistic implications | journal = Journal of the American Chemical Society | volume = 125 | issue = 43 | pages = 13036–7 | date = October 2003 | pmid = 14570471 | doi = 10.1021/ja0279747 | bibcode = 2003JAChS.12513036B }}</ref>
[[File:34S isotope effect on sulfate ester hydrolysis reaction scheme.png|none|thumb|525x525px|34S isotope effect on sulfate ester hydrolysis reaction]]

The major limitation for determining KIEs at natural abundance using IRMS is the required site selective degradation without isotopic fractionation into an analyzable small molecule, a non-trivial task.<ref name="Singleton_1995" />