From Stars to Life − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – __________________________________________________________________________________________________________ Inauguraldissertation zur Erlangung des Doktorgrades der naturwissenschaftlichen Fachbereiche im Fachgebiet Organische Chemie (Fachbereich 08) der Justus-Liebig-Universität Gießen vorgelegt von Akkad Danho aus Pohlheim angefertigt im Zeitraum von Februar 2022 bis März 2025 am Institut für Organische Chemie der Justus-Liebig-Universität Gießen Betreuer: Prof. Dr. Peter R. Schreiner, PhD i Eidesstattliche Erklärung Hiermit versichere ich, die vorgelegte Dissertation selbständig und ohne unerlaubte fremde Hilfe und nur mit den Hilfen angefertigt zu haben, die ich in der Dissertation angegeben habe. Alle Textstellen, die wörtlich oder sinngemäß aus veröffentlichten Schriften entnommen sind, und alle Angaben, die auf mündlichen Auskünften beruhen, sind als solche kenntlich gemacht. Bei den von mir durchgeführten und in der Dissertation erwähnten Untersuchungen habe ich die Grundsätze guter wissenschaftlicher Praxis, wie sie in der „Satzung der Justus- Liebig-Universität zur Sicherung guter wissenschaftlicher Praxis“ niedergelegt sind, eingehalten. ___________________________ ___________________________ Ort, Datum Unterschrift Dekan: Prof. Dr. Holger Zorn Prodekan: Prof. Dr. Volker Wissemann Studiendekan: Prof. Dr. Reinhard Dammann Erstgutachter: Prof. Dr. Peter R. Schreiner, PhD Zweitgutachter: Prof. Dr. Richard Göttlich ii iii Zusammenfassung Reaktive Intermediate sind kurzlebige, hochreaktive Moleküle, die an verschiedenen chemischen Prozessen beteiligt sind. Solche Zwischenstufen, wie Carbene und Enole, wurden unter anderem im interstellaren Raum nachgewiesen. Es wird vermutet, dass diese Verbindungen bei der Entstehung komplexer, biologisch relevanter Substanzen eine entscheidende Rolle spielen können. Um diese kurzlebigen Moleküle näher zu untersuchen, sind spezielle Methoden wie Matrixisolationsspektroskopie erforderlich, um Intermediate unter kryogenen Bedingungen zu stabilisieren und zu charakterisieren. Unter diesen Umständen beeinflussen Tunneleffekte den Ausgang einer Reaktion maßgeblich. Daher tritt neben der thermodynamischen und kinetischen Kontrolle das Prinzip der Tunnelkontrolle als drittes Prinzip der Reaktivität hervor. In der ersten Veröffentlichung wurde erstmals Prop-1-en-1,1-diol in einer Matrix isoliert und mittels IR- und UV/Vis-Spektroskopie charakterisiert. Das Enol wird durch eine Hochvakuum-Pyrolyse ausgehend von Methylmalonsäure, erhalten. Unter UV- Bestrahlung wandelt sich das Enol in ein Isomer der Propionsäure sowie in Methylketen um. Sowohl Enol als auch Keten und Propionsäure wurden im interstellaren Medium nachgewiesen, wobei Letzteres eine grundlegende Komponente des biologischen Lebens darstellt. 2-Methylprop-1-en-1,1-diol wurde im Rahmen einer zweiten Publikation matrixisoliert und durch IR- und UV/Vis-Spektroskopie charakterisiert. Das Enol wurde ebenfalls durch eine Hochvakuum-Pyrolyse von Dimethylmalonsäure erhalten, wobei in diesem Fall auch Dimethylketen durch denselben Prozess generiert wurde. Dieses Enol weist ähnliche Reaktivität wie das zuvor isolierte Prop-1-en-1,1-diol auf und wandelt sich unter UV-Bestrahlung in Isobuttersäure um. Obwohl 2-Methylprop-1-en-1,1-diol bislang nicht im Weltraum nachgewiesen wurde, spielt die Isobuttersäure eine bedeutsame Rolle in biologischen Prozessen. Eine dritte, bislang unveröffentlichte Studie beschäftigt sich mit der Tunnelkontrolle von Alkylcarbenen. Dabei wurde versucht, mithilfe von Wasserstoffisotopen den Ausgang zweier konkurrierender Tunnelreaktionen zu beeinflussen. Dazu wurden im Verlauf dieser Arbeit unterschiedliche Alkylcarbene auf ihre Tunnelreaktivität untersucht. Dabei wurde ein bis dato unbekanntes, unter kryogenen Bedingungen stabiles Alkylcarben, Pentacyclo[5.4.0.0²,⁶.0³,¹⁰.0⁵,⁹]undecanyliden, in der Matrix isoliert und mittels IR- und UV/Vis-Spektroskopie charakterisiert. Es zeigt eine erhöhte kryogene Stabilität im Vergleich zum bereits bekannten Adamantyliden und wandelt sich erst unter Bestrahlung durch [1,2]-H-Migration zum Homohypostrophen um. Erst die Untersuchung des Protoadamantylidens, dessen Isolierung aufgrund zu kurzer Halbwertszeit nicht gelang, weist zwei konkurrierende Tunnelreaktionen auf. Sowohl das Produkt der [1,2]-H- Migration als auch das der C–H-Insertion wurden beobachtet. Anhand dieser Beobachtungen wurden Wasserstoffisotope gezielt zur Steuerung der Tunnelreaktion eingesetzt. Dieser Ansatz beeinflusste erfolgreich die Produktbildung und stellt den ersten experimentellen Nachweis für eine isotopenkontrollierte selektive Tunnelreaktion dar. iv v Abstract Reactive intermediates are transient, highly reactive molecular species involved in chemical processes. It is proposed that such species might participate in the interstellar synthesis of complex molecules and in atmospheric processes. Enols and carbenes represent a class of reactive species capable of undergoing reactions under certain conditions to form prebiotic compounds. While these species have been detected in interstellar space, knowledge about their origin remains limited. Intermediates can be stabilized with the help of matrix isolation and other specialized techniques and characterized spectroscopically. Under cryogenic conditions, tunneling effects significantly impact the reactivity of molecules. In addition to the principles of thermodynamic and kinetic reaction control, tunneling control has emerged as a third fundamental paradigm of reactivity. In the first publication, prop-1-en-1,1-diol was isolated in a matrix for the first time and characterized using IR and UV/Vis spectroscopy. The enol was obtained via high-vacuum flash pyrolysis starting from methylmalonic acid. Under UV irradiation, the enol is converted into an isomer of propionic acid as well as to methylketene. Enol, ketene, and propionic acid have previously been detected in the interstellar medium, with the latter representing a fundamental prebiotic molecule. In a subsequent publication, 2-methylprop-1-en-1,1-diol was isolated in a matrix and identified by IR and UV/Vis spectroscopy. The enol was generated via high-vacuum flash pyrolysis of dimethylmalonic acid. In this process, dimethylketene formed as well. This enol demonstrates similar reactivity to the previously isolated prop-1-en-1,1-diol and tautomerizes to isobutyric acid under UV irradiation. Although 2-methylprop-1-en- 1,1-diol has not yet been detected in space, isobutyric acid plays a significant role in biological processes. A third, yet unpublished study dealt with quantum mechanical tunneling control of alkyl carbenes. The aim was to influence the outcome of competing tunneling reactions through the appropriate selection of alkyl carbene isotopologs with protium and deuterium. In the course of the study, various alkyl carbenes were examined for their tunneling reactivity. Among these, the previously unknown alkyl carbene pentacyclo[5.4.0.0²,⁶.0³,¹⁰.0⁵,⁹]undecanylidene, was isolated and characterized. This carbene showed increased cryogenic stability compared to the known adamantylidene and only undergoes a [1,2]-H shift to homohypostrophene under irradiation. Only the study of protoadamantylidene, the isolation of which was unsuccessful due to its short half-life, revealed two competing tunneling reactions. Both the product of the [1,2]-H- shift and that of C–H insertion was observed. Based on these observations, hydrogen isotopes were selectively employed to control the tunneling reaction. This approach successfully influenced product formation, marking the first experimental evidence of an isotope-controlled tunneling reaction. vi vii Meiner geliebten Familie in Hochachtung gewidmet. viii ix Table of Contents Eidesstattliche Erklärung................................................................................................ i Zusammenfassung ......................................................................................................... iii Abstract ............................................................................................................................ v 1. Introduction ................................................................................................................. 1 1.1 Motivation and Goals ...................................................................................... 1 1.2 Enol Chemistry ............................................................................................... 2 1.2.1 Enols as Potential Prebiotic Intermediates in Interstellar Space ....... 4 1.2.2 Matrix Isolation Studies on Enols ..................................................... 6 1.3 Carbene Chemistry ........................................................................................ 11 1.3.1 Tunneling Reactions of Alkyl Carbenes ......................................... 13 1.3.2 Controlling Tunneling Reactivity ................................................... 15 1.4 Outlook.......................................................................................................... 17 1.5 Conclusion .................................................................................................... 18 2. Publications ............................................................................................................... 25 2.1 The enol of propionic acid ............................................................................ 25 2.2 The enol of isobutyric acid............................................................................ 31 3. Unpublished Results ................................................................................................. 37 3.1 Cage Alkyl Carbenes Provide Experimental Evidence for Isotope Controlled Selectivity............................................................................................................ 37 3.1.1 Abstract ........................................................................................... 37 3.1.2 Introduction ..................................................................................... 37 3.1.3 Results and Discussion.................................................................... 39 3.1.4 Conclusion ...................................................................................... 44 3.1.5 Experimental Section ...................................................................... 44 4. Acknowledgement ................................................................................................... 115 From Stars to Life ____________________________________________________________________________________ 1 1. Introduction 1.1 Motivation and Goals Since the beginning, humans have wondered how life originated. If life originated on Earth, one fundamental question remains: Where did the organic molecules that formed the first cell come from?1 It is known that roughly 300 tons of organic matter are falling on Earth every year in the form of interplanetary dust.2 In addition, large amounts of organic molecules were detected in carbonaceous meteorites3 such as the Murchison and Murray meteorites4-6 and in comets, for example, 67P/Churyumov-Gerasimenko, consisting of organic matter up to 45%.7 The organic molecules found in space could have seeded primordial Earth and therefore delivered all necessary molecules for the first cell to form.8-12 However, this does not explain how organic molecules are generated in interstellar space: In space, reactions occur under “extreme conditions” such as low temperatures (2.7 K) and at high dilution (1.46 × 10−18 Pa).13 Reactions that occur at ambient temperatures in the laboratory may be infeasible in space. By understanding the formation and interaction of such reactions under interstellar conditions, we gain important insights into the processes that shaped the early chemical platforms of life. Figure 1: Examples of molecules detected in space.14 a) Highly stable molecular building blocks. b) Highly reactive molecular building blocks. c) “Complex” organic molecules. Complex organic molecules in interstellar space may form via small, highly reactive organic molecules. About 300 of these fleeting molecules (Figure 1b) have been detected in the interstellar medium,1 where they remain intact due to the unique conditions of space. However, in regions such as interstellar clouds, organic reactions are thought to take place. It is argued that random energetic events such as gamma-ray bursts convert reactive intermediates into complex organic molecules (Figure 1c).15 Despite their fleeting nature, these reactive intermediates play critical roles in biological,16-18 atmospheric,19-20 and combustion processes.19, 21 Due to their reactivity, 99.9% of these reactive molecules have yet to be generated and characterized.22 − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 2 By simulating the conditions of space, such as extremely low temperatures (2.7 K)23 and low partial pressures (10–7 mbar), the matrix isolation technique is employed to capture and characterize these reactive intermediates. In a typical fashion, a volatile substance is co-deposited with an excess of an inert host gas, such as argon or nitrogen, onto a cold window (Figure 2). The captured molecule is then analyzed with IR and UV/Vis spectroscopy, requiring either a CsI (for IR) or a BaF2 window (for UV/Vis). Depending on how the reactive species are generated in situ, either pyrolysis of a precursor molecule or photolysis following deposition is employed. This technique is used to investigate otherwise unobservable reactions, such as tunneling (vide infra).24 Figure 2: Schematic representation of the matrix isolation apparatus. The subject of this work is the investigation of cryogenic organic reactions with cryo- spectroscopy in combination with density functional theory computations. The thesis is divided into two research topics: The first part deals with the potential formation of complex organic molecules, such as propionic acid, which has been confirmed to exist in space,25 and isobutyric acid under space-like conditions. The publications suggest possible reaction pathways in which the corresponding enols can form the stable carboxylic acids. The second part investigates the formation and reactivity, mainly the tunneling behavior, of alkyl carbenes. Our strategy in this respect is to achieve controlled selective tunneling by implementing hydrogen isotopes as directing groups. 1.2 Enol Chemistry Enols are intermediates including a carbon-carbon double bond (C=C) and at least one vinylic hydroxy group (–OH). Enols rapidly isomerize in solution to their thermodynamically more stable keto tautomer through a bimolecular acid-base reaction, as exemplified by 3-pentanone (1), which is more stable than its enol form 2 (Figure 3).26 Figure 3: Keto-enol tautomerism of 1. Despite their highly reactive nature in solution, enols can be stabilized by changing their physicochemical properties, such as steric hindrance27 or electronic effects,28-29 including From Stars to Life ____________________________________________________________________________________ 3 resonance.30 O'Neill and Hegarty were able to investigate enols of carboxylic acids by hydrating a range of ketenes to form their sterically hindered enols with the general formula of Ar₂C=C(OH)₂, (2,2-bis(2,4,6-trimethylphenyl)-1,1-ethenediol (3) or 1,1- ethenediol, 2,2-bis-(pentamethylphenyl) (4; Figure 4).31-32 Building upon this work, Frey and Rappoport were able to generate stabilized enols of acids by the addition of water and dimethylamine to the corresponding ketenes.33-34 Enols of carboxylic acids are more challenging to generate due to the high reactivity of the double bond and the two hydroxy groups. The authors achieved stabilization of these enols by introducing activated bulky groups, such as the Tip group, which shield the highly reactive double bond while providing additional reactive sites.33 The authors were able to characterize these kinetically stabilized enols through NMR spectroscopy; two representative examples are 5 and 6 (Figure 4). Figure 4: Examples of investigated semi-persistent (isolable) enols. (Mes = 1,3,5- trimethylbenzol, Pmp = 1,2,3,4,5-pentamethylphenyl, Tip = 2,4,6-triisopropylphenyl) Other than changing chemical reactivity, analytical methods, such as time-resolved spectroscopy,35-36 neutralization-reionization mass spectrometry,37-38 NMR, IR spectroscopy,39 and other methods40 can be utilized for characterizing enols. Often, these methods are combined with computational chemistry to better understand structural and energetic properties.41-44 For instance, time-resolved UV spectroscopy in combination with kinetic studies provided valuable insights into the reactivity of benzofulvene-8,8- diol (7),45 fulvene-6,6-diol (8),46 fluoren-9-carboxylic acid enol (9),35 and 2-phenyl-1,1,2- ethenetriol (10) (Figure 4).47 Investigating simpler and smaller enols that lack steric protection remains more difficult and requires different approaches to stabilize them. Schauermann et al. used metal surfaces to stabilize the enol of acetophenone (11). By using a second acetophenone (12) equivalent, the authors achieved stabilization through intermolecular interactions, enabling them to investigate the formation of a keto-enol-dimer (Figure 5a).48 Turecek et al. used a different approach, taking advantage of the higher stability of enols in the gas phase (vide supra). They were able to generate the enol of γ-butyrolactone (13) in the gas phase and characterized it using neutralization-reionization mass spectrometry (Figure 5b).37 − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 4 Figure 5: a) Investigation of a ketone-enol dimer by infrared reflection adsorption spectroscopy in combination with scanning tunneling microscopy. b) Generation of γ-butyrolactone radical cation (13a) by McLafferty rearrangement of a suitable precursor ion. 1.2.1 Enols as Potential Prebiotic Intermediates in Interstellar Space While enols in solution are highly unstable and rapidly isomerize, they are significantly more stable in the gas phase.49 This higher stability arises from the lack of bimolecular reactions in the gas phase and from substantial energy barriers, typically ranging from 40 to 45 kcal mol–1,49-50 which hinder [1,3]H-shifts and prevent tautomerization.37 This stability is also observed in space.51 In 2001, ethenol was discovered in the interstellar cloud Sagittarius B2.51 The enol was detected via microwave emissions. Furthermore, many enols were also identified in cold plasma discharges of alcohols.52 These findings suggest that enols are abundant in the interstellar medium. Typically, they are generated through UV light and cosmic radiation of simpler molecules (Figure 1).52 These observations may hint that enols play an important role in the chemistry of interstellar space by forming amino acids,6 sugars,53 and carboxylic acids,54 which later were delivered to planets by celestial bodies.9, 55-56 For instance, simple amino acids could potentially have originated from amino enols. Like their hydroxy counterparts, enols of simple amides are highly unstable. These elusive molecules have the propensity to undergo fast interconversion to their more thermodynamically favorable amide isomer.57 For example, 1-aminoethenol (14) is suggested as a key intermediate in the formation of acetamide (15),58 which has been identified in many interstellar environments, such as Sagittarius B2 (Sgr B2)59 and Orion KL,60 as well as in comets61 (e.g., 67P/Churyumov–Gerasimenko).7 In addition, it has been suggested that 15 could give rise to dipeptides (Figure 6) or larger peptides.60 Despite their relevance for prebiotic chemistry, no direct spectroscopic evidence of 14 or other enols of amides has been presented,62 and only a few indirect pieces of evidence are known.63-67 From Stars to Life ____________________________________________________________________________________ 5 Figure 6: Possible pathway for the generation of small dipeptides with 15. The first two steps involve neutral-neutral radical reactions, generating methylacetamide (16) and then methylglycinamide (17), with rates of 10–11 cm3 s–1.68 The third step could proceed on meteorite surfaces and generate N-[2-(methylamino)-2-oxoethyl]carbamic acid (18).60 The enol tautomer of glycolaldehyde (19) could be part of the formation of carbohydrates, such as pentoses, under prebiotic conditions on primordial Earth (Figure 7).69 Besides, 19 is the simplest sugar that has been detected in interstellar space.70 1,2-Ethendiol (20) could also be an intermediate for the formose reaction,71 in which formaldehyde (21) is converted into sugars in aqueous environments, catalyzed by organic bases or minerals.72 In 1959, Breslow suggested a series of base-catalyzed aldol reactions, with 19 acting as an autocatalyst. However, the uncatalyzed formation of 19 from formaldehyde remained unclear until 2018,73 when it was shown to occur through a nearly barrierless carbonyl- ene reaction involving nucleophilic hydroxymethylene (22) (Figure 7).74 Figure 7: Gas-phase sugar formation through carbonyl-ene reaction. Decarboxylation of glyoxylic acid (22) forms hydroxycarbene (23), which undergoes a facile carbonyl-ene reaction with formaldehyde to form 19. Photolysis generates 20a and 20b, which can react with formaldehyde to form glyceraldehyde (24) and even larger carbohydrates. − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 6 The reaction progresses through rapid enolization processes that facilitate aldol additions with formaldehyde, generating a variety of aldoses and ketoses. This makes the enols of 19, trans- and especially cis-1,2-ethendiol (20a and 20b), with the former having been detected in space,75 important prebiotic intermediates for the formation of three- to five- carbon sugars.69, 76,77 Moreover, enols with additional hydroxy groups such as 1,1,2-ethentriol (25), could serve as intermediates for the formation of glycolic (26) and glyceric acid (27). These two compounds could potentially serve as a precursor for lipids.78 Both are also involved in glycolysis and play a fundamental role in biochemical processes.78 Despite 26 having been detected in meteorites such as Murchison and Bell,79-81 its origin remains unknown. 1.2.2 Matrix Isolation Studies on Enols Since enols are highly unstable entities, special methods such as matrix isolation are used to capture and characterize them. Notably, two research groups are investigating these small reactive intermediates, with the methods of enol generation differing significantly. The Kaiser group attempts to mimic meteorite surfaces. Typically, enriched ices are used, which are later bombarded with high-energy beams comparable to gamma-ray bursts82 or other highly energetic phenomena in interstellar space. By using carbon dioxide (28) and methane-based ice (29) and subjecting it to high radiation, Kaiser et al. were able to generate 1,1-ethendiol (30) (Figure 8).83-84 Changing the composition of the ice to acetone (31) or methanol (32) and treating it with high-energy radiation produced propen- 2-ol (33) (as well as its isomer, methyl vinyl ether 34) and 20.77, 85 Figure 8: High-energy irradiation of methane- and carbon dioxide-based ice results in the bottom-up generation of 30. During sublimation of the ices, the reactive intermediates were detected, using tunable vacuum ultraviolet photoionization and reflectron time-of-flight mass spectrometry. The experiments were often conducted in conjunction with isotope labeling and isomer- selective photoionization.86 Computational analysis reveals that the presence of water molecules, which are expected on interstellar grains, can reduce the energy barrier for keto-enol tautomerization by fifty percent. The authors demonstrated that these reactive intermediates remain intact on ice-coated nanoparticles, which are also found in molecular clouds and suggest that they persist after sublimation into the gas phase in star- forming regions, thus further exemplifying the role of these enols in interstellar chemistry.87 From Stars to Life ____________________________________________________________________________________ 7 Other methods to generate enols involve the fragmentation of more complex precursors. In 1976, Saito was able to generate ethenol (38) by decarbonylation (pyrolysis) of ethylene glycol (39) (Figure 9).40 It was not until this time that a conjugated enol had ever been produced, and few subsequent studies were conducted. The desired enol was successfully generated and confirmed using microwave spectroscopy in conjunction with isotopic labeling. However, this method produced many side products, and the outcome depended on the temperature used. At lower temperatures, the number of side products increased. Figure 9: Generation of 38 by decarbonylation of 39. At low temperatures, acetaldehyde (40) and ethylene oxide (41) predominantly form. Dicarboxylic acids are more suitable as precursor molecules since their fragmentation can occur under milder conditions and only produces a CO₂. Mardyukov et al. were able to generate and characterize a variety of enols by fragmentation of different precursor molecules: They generated 30 by pyrolysis (decarboxylation) of malonic acid (42) at 400 °C and captured the enol 30 in an argon matrix (Figure 10). Figure 10: Generation of 30 from 42 and subsequent trapping in an argon matrix at 10 K. By implementing the same strategy and choosing the right precursor molecule, they were able to generate 1457 and 25.88 Structures 20a and 20b89 were not generated by decarboxylation, but rather by a retro-Diels-Alder reaction. The pyrolysis of endo,cis- bicyclo[2.2.1]hept-5-ene-2,3-diol (43) at 700 °C resulted in a retro-Diels–Alder reaction, which produced cyclopentadiene (44) and 20a. By pyrolysis of trans-9,10-dihydro-9,10- ethanoanthracene-11,12-diol (45), anthracene (46) and 20b formed (Figure 11). − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 8 Figure 11: Generation of 20a and 20b from 43 and 45 and capturing all formed products in an argon matrix at 10 K. Measuring IR spectra of the trapped molecules indicated the formation of enols and side products such as CO₂ and H₂O. Most enols show similar vibrational bands, with the C=C stretching vibration being the most common and characteristic. This vibration mode appears around 1700 cm⁻¹.90 Attaching an amino group red-shifts the vibrational band to 1682 cm⁻¹, in comparison to a hydroxy group.57 Conversely, attaching a third hydroxy group increases the C=C stretching vibration to 1761 cm⁻¹.88 The IR assignments were confirmed by computed IR signals at the AE-CCSD(T)/cc-pVTZ level of theory.90 These assignments were also confirmed by isotopic labeling experiments. For example, the deuterated 1-aminoethenol showed a significant redshift; for the C=C stretching vibration, the shift was –51 cm⁻¹ (computed –59 cm⁻¹).57 The successful generation of 14,57 30,88 20a and 20b89 was confirmed by UV/Vis spectra, which aligned well with the computed values. The UV/Vis spectra show a strong absorption maximum at 190 nm, which corresponds to a π-π* transition and correlates to a HOMO–LUMO+4 excitation.90 Only 14 shows an absorption of 212 nm due to its less electron-poor character.57 Comparing the reactivity of 14 with 30 reveals that substitution of one OH group with an NH₂ group destabilizes the HOMO, resulting in a smaller HOMO-LUMO gap in 14 (0.195 eV) compared to 30 (0.207 eV). This makes 14 more nucleophilic and prone to oxidation.57 By comparing the energies of the different C2H4O2 isomers, computations at the AE- CCSD(T)/cc-pVTZ level of theory show that 20a and 20b are 8.6 and 12.4 kcal mol−1 higher than 3089: Changing the position of the OH-group also changes the HOMO- LUMO gap by increasing the HOMO-energy, making 20a and 20b more nucleophilic than their isomeric counterpart 30.90 From Stars to Life ____________________________________________________________________________________ 9 Figure 12: Formation of 36 from 30 through a [1,3]H-shift.90 Photolysis experiments resulted in the production of a carboxylic acid through a [1,3]H- shift (Figure 12). Both pathways were confirmed by computations at the AE- CCSD(T)/ccpVTZ level of theory. Quantum mechanical hydrogen tunneling from the enol to the carboxylic acid was not observed, since the enol remained unchanged over the course of several days. This was further corroborated by a computed barrier of 40-50 kcal mol–1. Interestingly, in studies that investigated enols with additional hydroxy groups, the formation of a ketene + water complex was observed. For example, the decarboxylation of malonic acid forms not only 30 but also ketene 47 by water elimination. Ketene was identified by a strong band at 2138 cm⁻¹. This identification was further confirmed by a significant redshift in the deuterated isotopolog and comparison to the calculated IR spectra. These findings could indicate a potential pathway to the formation of the enol in interstellar space by surface-catalyzed hydration of ketene on ice grains.91-92 Additionally, 47 has been observed in space in large quantities.93 Figure 13: Formation of 47 from 30 through water elimination.90 In this thesis, we investigated the enol tautomer of propionic acid (48)94 and isobutyric acid (49).95 The goal of this study was providing spectroscopic data, which are needed for the detection of these molecules in space via radio astronomy, and for studying the reactivity of the enols. Similar to the approaches of Mardyukov et al., we synthesized the enediol by pyrolysis of methylmalonic acid (50) at 500 °C or dimethylmalonic acid (51) at 750 °C and captured all generated products in an argon matrix at 3.5 K (Figure 14). Figure 14: Generation of prop-1-ene-1,1-diol (48) from methylmalonic acid (50) and generation of 2-methyl-prop-1-ene-1,1-diol (49) from dimethylmalonic acid (51). − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 10 Spectroscopic studies revealed characteristic bands that can be attributed to the enols and were confirmed by computational studies at the B3LYP/def2-TZVP level of theory. Furthermore, isotope labeling validated the generation of the enols by displaying characteristic shifts. Both UV/Vis spectra showed absorption maxima at 190 nm, assigned to a π→π* transition, similar to the other characterized enols.57, 88-90 Interestingly, methylketene (52) was also generated by pyrolysis of 50. Compound 52 could also be a key intermediate for prebiotic chemistry as it has been identified in interstellar space96 and can be converted back to its enol form (+35.5 kcal mol–1). In contrast, dimethylketene (53) is only generated after irradiation with 254 nm. Moreover, irradiation experiments show the formation of the carboxylic acids through a [1,3]H-shift via a transition state of 41.9 kcal mol–1 for propionic acid (54) and 51.3 kcal mol–1 for isobutyric acid (55). Furthermore, we reported the formation of propene (56), which is likely to occur via a photochemical mechanism of the 53. This mechanism may involve either a concerted [1,2]H-shift (TS1) or the conversion of the 53 into a singlet carbene (57), which then releases CO upon photoexcitation. Subsequently, 57 undergoes a [1,2]H-shift (TS2), resulting in the formation of 56. Given the small half-life of 57, it is not possible to distinguish between the two pathways (Figure 15). Figure 15: Potential energy profile (ΔH0) in kcal mol−1 of the reactions of 53 at DLPNO- CCSD(T)/cc-pVQZ//B3LYP/def2-TZVP+ZPVE at 0 K. The attempt to detect singlet alkyl carbene 57 was unsuccessful. Carbenes are highly reactive intermediates (see 1.3), and they may contribute to the formation of complex organic molecules in interstellar environments similar to enols. This includes molecules From Stars to Life ____________________________________________________________________________________ 11 crucial for the origins of life, such as amino acids,97 nucleobases,98 and sugars (vide supra).97 The following part focuses on explaining the reactivity of these molecules, with particular emphasis on singlet alkyl carbenes and their unique tunneling reactivity. 1.3 Carbene Chemistry Carbenes are defined by a carbon atom with a formal valence of two with six electrons. This deviation from the octet rule makes this reactive intermediate amphiphilic. The two unpaired valence electrons on the carbon can exist in four states. Either they exist in three spin-paired (singlet state, S = 0, σ1pπ 1, pπ 2, σ2) or in one spin unpaired (triplet state, S = 1, σ1pπ 1) configurations. Depending on their electronic structure, the states show different behavior. Typically singlet carbenes have a bond angle of <120°, which is comparable to typical sp²-hybridization and is caused by the significant orbital overlap, whereas triplet carbenes show bond angles greater than 120°. Triplet carbenes can be considered as diradicals.99 Carbenes can be stabilized by incorporating either electron-donating (EDG) or electron- withdrawing (EWG) groups at the electron-deficient carbene center (Figure 16). Three different types of stabilization are possible: (a) pull-pull stabilization with two EWG 58,100-101 (b) push-pull or captodative stabilization,102 where one substituent donates and the other withdraws electrons, exemplified by 59a-c103-105 and (c) push-push stabilization with two EDG 60.100, 106-107 Stabilization can be enhanced by heteroatoms with a π- donating lone pair, such as nitrogen, attached to the carbon center 61, 64, and 65. This can favor a singlet ground state by p-pπ interaction between the lone pair and the empty pπ orbital of the carbene. Figure 16: Some examples of stabilized carbenes. Due to their fleeting character, it took nearly a century to isolate the first “stable” carbene.72, 108-111 In 1988, Bertrand synthesized the first persistent carbene (63)112 by implementing different substituents and enhancing stability through an electron donating effect. Surprisingly, carbene 63 demonstrated remarkable stability over weeks. The work by Wanzlick113 and Öfele114 on metal-carbene complexes and the equilibrium between monomeric 61 and dimeric imidazolidin-2-ylidenes (62), enabled Arduengo to successfully synthesize the first “bottle-able” carbene 64.100, 115 This carbene is stabilized by two π-electron donating nitrogen atoms and the aromaticity of the imidazole ring.116 The dimerization proposed in the Wanzlick equilibrium was kinetically hindered by the bulky substituents. Eventually, Enders et al. achieved the synthesis of the first commercially available carbene 65 in 1995 (Figure 17).117 − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 12 Figure 17: Historical developments in stable carbene chemistry. To study transient carbenes, techniques such as ultrafast gas-phase spectroscopy or kinetic stabilization at cryogenic temperatures are required. The latter is often achieved through matrix isolation. Notable examples (Figure 18) include dichlorocarbene (58a),101 difluorocarbene (58b),118 di-tert-butylcarbene (58c),119 diadamantylcarbene (58d),120 dicyclopropylcarbene (58e),121 ethynylhydroxycarbene (59a),103 trifluoromethylhydroxycarbene (59b),104 cyanohydroxycarbene (59c),105 cyclopropylidene (66),122 vinylidene carbene (propadienylidene, 67),123 propinylcarbene (propargylene, 68),124 and heterocyclic carbenes such as 2,3-dihydrothiazol-2-ylidene (69a)125 2,3-dihydroimidazol-2-ylidene (69b).126 Figure 18: Several selected reactive carbenes that have been prepared experimentally. Despite multiple attempts, ethylidene, the simplest alkyl carbene besides methylene, has not been spectroscopically characterized due to its tendency to undergo rapid [1,2]H- shift.127-134 By replacing hydrogen atoms with fluorine, the stability was enhanced and observation of 2,2,2-trifluoroethylidene was possible.135 Perhaps unsurprisingly, most research on singlet alkyl carbenes stems from computational work.136 Cyclic alkyl carbenes,137-138 due to their unique structure, are stabilized through through-space interactions, non-classic bonding schemes, and hyperconjugation.139 Some examples are norbornen-7-ylidene (70),140 cyclobutylidene (71),141 and tricyclooct-8-ylidene (72)142 (Figure 19), which have only been investigated theoretically. Adamantylidene (73)143 and pentacyclo[5.4.0.0²,⁶.0³,¹⁰.0⁵,⁹]undecanylidene (PCU-carbene) (74) are among the few singlet alkyl carbenes that have been spectroscopically characterized,119-121 the latter of which was investigated in this work. Figure 19: Examples of cyclic alkyl carbenes. From Stars to Life ____________________________________________________________________________________ 13 1.3.1 Tunneling Reactions of Alkyl Carbenes Quantum mechanical tunneling allows particles to penetrate a potential energy barrier without sufficient kinetic energy.144-146 The tunneling probability depends linearly on the barrier width,147 the square root of the barrier height,147 and the effective mass of the particles.148 This process, even if seemingly insignificant in comparison to thermodynamic or kinetic reactivity, enhances reaction rates by allowing more particles to transition to the product side of a reaction. In very cold environments, such as outer space or matrix isolation setups, tunneling can play a significant role in determining reaction rates. This makes QMT particularly important for long-term processes, such as those occurring on astronomical timescales, and thus must be considered to gain accurate values of rate constants.149-150 Electron or hydrogen tunneling can change the reaction rates or the reaction outcomes entirely; this also holds true for organic chemistry, where QMT is widely regarded as a minor influence on a reaction outcome. Figure 20: Theoretical examples of H-shifts in cyclic singlet alkyl carbenes.141 Carbene 70 can either undergo a [1,2]H-shift to form norbornene (75) or a [1,3]H-shift to form nortricyclene (76). Carbene 71 can either undergo a [1,2]H-shift to form cyclobutene (77) or a [1,3]H-shift to form bicyclo[1.1.0]butane (78). Cyclic singlet alkyl carbenes can undergo different tunneling reactions. Usually, H-shifts are most common (Figure 20); however, when the de Broglie wavelength of a particle approaches the same magnitude as the width of the energy barrier it is penetrating, heavy- atom tunneling can occur.151 This suggests that even carbon tunneling should be considered for accurate reaction rates if the barriers are sufficiently narrow. Usually, ring expansion and insertion- reactions of C–C bonds through heavy atom tunneling can take place (Figure 21).152-153 This makes accurate predictions reliant on precise modeling of the potential energy surface (PES). Accurate computational predictions can be achieved154 by techniques such as Canonical Variational Transition State Theory (CVT)155 corrected for small-curvature tunneling (SCT),156 and combined with density functional theory (DFT).157-158 − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 14 Figure 21: Examples of heavy-atom QMT in cyclic carbenes. Automerization in cyclobutadiene (79)159 and 1,5-dimethylsemibullvalene (80)160-161 as well as ring closure of cyclopentane-1,3-diyl (81)162 to bicyclo[2.1.0]pentane (82).160 As part of this thesis, we aimed to investigate the tunneling behavior of cage alkyl carbenes under cryogenic conditions. We began with adamantylidene (73), a singlet alkyl carbene known to be stable under cryogenic conditions. Bally and Platz only observed the rearrangement of 73 to dehydroadamantane (83) after UV irradiation.143 Although the authors did not report any QMT reactivity,143 in 2014, Kozuch et al., computed a tunneling half-life of 62.2 h for 73 at the CVT/SCT//B3LYP/6-31G(d,p) level of theory.152 We replicated the experiment, but allowed the reaction to proceed overnight in the dark. Interestingly, we noticed diminished IR signals corresponding to 73, and the concomitant formation of 83, indicating a QMT process. We also extended our investigations to pentacyclo[5.4.0.0²,⁶.0³,¹⁰.0⁵,⁹]undecanylidene (PCU-carbene) (75), marking only one of a few full spectroscopic characterization of a singlet alkyl carbene.119-121 Unexpectedly, PCU-carbene did not exhibited QMT reactivity, as it proved to be stable under the cryogenic conditions. Only upon irradiation, a ring-opening reaction to homohypostrophene (84) occurred (Figure 22). We then shifted our focus to a third cage alkyl carbene, protoadamantylidene (85). Figure 22: QMT reactivity of alkyl carbenes 73, 74, and 85. Despite having a half-life too short to capture 85, we could determine two competing QMT reactions: either a [1,2]H-shift forming 86 or a C–H-insertion generating 83. In the From Stars to Life ____________________________________________________________________________________ 15 next section, we will explore how to influence these competing QMT processes through an isotope-controlled selective approach to specifically favor one of these reactions. 1.3.2 Controlling Tunneling Reactivity In 2011, a new paradigm, known as tunneling control, was established.163 This makes it the third principle besides kinetic and thermodynamic control,136, 164 the established cornerstones of mechanistic reasoning. In tunneling control, the consideration of width between species on the potential energy surface is as important as reaction free energies and barrier heights (vide supra).147, 165 In the many examples of tunneling control,144, 166- 167 reactions defy transition state theory (TST), as thermodynamic products can form more rapidly than kinetic ones due to the presence of a higher but narrower energy barrier.144 In the Schreiner group, tunneling control has been uncovered and extensively investigated. In 2017, Schreiner et al. demonstrated that matrix-isolated methylhydroxycarbene (87) does not react as expected to the kinetically favored vinyl alcohol (38) but to the thermodynamically more stable product (acetaldehyde, 88) instead (Figure 23a).144 Lacking external influence due to the cold environment of the matrix setup (11 K), tunneling control provides the only explanation for the observed phenomenon. This was corroborated by high-level computations, employing a focal- point analysis (FPA)168-170 on top of coupled-cluster-optimized geometries.171-172 In 2017, Schreiner et al. could show the exclusive formation of a tunneling product, deviating from classic expectations (Figure 23b).173 Figure 23: a) QMT control in methylhydroxycarbene (87). Relative energies in kcal mol−1 were computed at the FPA//AE-CCSD(T)/cc-pCVQZ level of theory.144 b) The formation of the tunneling product 89 from ketene 90, rather than the expected thermodynamic and kinetic product 91, was observed. Relative energies in kcal mol−1 were computed at the CCSD(T)/cc- pVTZ//MP2/aug-cc-pVDZ level of theory.173 − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 16 In a theoretical study about influencing competing tunneling reactions, Nandi et al. investigated the QMT reactivity of cyclopropylmethylcarbene (92).174 They revealed that, in principle, three QMT reactions can occur: Ring expansion to 1-methylcyclobut- 1-ene (93) and two [1,2]H-shifts to 94 and 95, respectively (Figure 24). Figure 24: Isotope-controlled selectivity via QMT: The isotope significantly alters rate constants and determines whether 92 reacts to form 93 or 94, as shown by B3LYP/6-31G(d,p) computations.174 At high temperatures, ring expansion is favored due to a lower energy barrier. However, at low temperatures, a hydrogen shift by tunneling is enabled. The hydrogen shift is favored despite the activation barrier being higher, making this reaction tunneling- controlled. Computations on the rate constants for both ring expansion and hydrogen shift were performed, which revealed that introducing methoxy groups with different hydrogen isotopes led to distinct outcomes for hydrogen and deuterium tunneling pathways.174 This novel concept was coined isotope-controlled selectivity (ICS). The ICS refers to a reactive system in which the formation of a specific product in a QMT reaction is determined entirely by the isotopic composition of the substrate.174 Although, theoretically investigated, to this date, there are no experimental studies to the concept of ICS. In this thesis, we applied this approach to the newly generated singlet alkyl carbene 85. As mentioned earlier, carbene 85 has two competing tunneling reactions. Computations at the UB3LYP/6-31G(d) level of theory reveal the half-life for the C–H insertion to be 4 × 10–9 h and 3 × 10–4 h for the competing [1,2]H-shift. This makes 83 more favorable and indeed experiments showed a ratio of 2:1 in favor of 83. The short half-life of 85 prevents direct observation of the carbene under matrix isolation conditions, making product analysis necessary for its indirect detection. Replacing hydrogen with deuterium at the α-position changed the ratio to 20:1 in favor of d₂-83. This outcome also aligns with computational results, as there is a greater discrepancy in half-lifes for the deuterated isotopolog compared to the protium isotopolog. This substantial shift in product distribution upon deuteration highlights the significant influence of isotopic substitution, providing strong support for ICS and making it the first experimental evidence. From Stars to Life ____________________________________________________________________________________ 17 Figure 25: Reactivity of matrix isolated of 85 and d2-85 after pyrolysis at 800 °C and ratios of formed products. 1.4 Outlook Despite extensive research on various sulfur-containing compounds, there is a surprising lack of information about the formation of thioacetamide (96). Thioamides, such as 96, are prevalent in different reactions such as prebiotic building block for amino acid synthesis.175 Additionally, thioamides serve as relevant compounds in the atmosphere by forming grain-like structures that facilitate water condensation like other sulfur containing compounds already investigated through matrix isolation (Figure 26).176 Figure 26: Examples of sulfur-containing compounds including the vinylsulfinyl radical (97) alkynyl thiocyanate (98)177 and isomers,178 the phenylsulfinyl radical (99),179 disulfur dioxide (100),180 methoxysulfinyl radical (101),181 hypothiocyanide radical (102)182 and methanethioamide (103).183 As previously discussed, enols could play a significant role in the formation of complex organic molecules in the interstellar medium. Hence, we suggest the formation of thioacetamide (96) through its higher energy enol tautomer 104, which could form in space from hydrogen cyanide (HCN)184 and methanthiole (CH3SH),185 both of which have been detected in interstellar environments. By generating the enol 104 similarly to the previously generated enols,57, 88-90, 94-95 we suggest a pathway to generate 96 from 105, without implying that the transformation of 105 to 104 occurs under interstellar conditions. (Figure 27). − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 18 Figure 27: Generation of 104 and subsequent photorearrangement to 96. 1.5 Conclusion In our first two publications, we successfully isolated two novel enols, 48 and 49 (Figure 28). These reactive intermediates are predicted to be precursor molecules in the interstellar medium.6, 53, 54 These enols were generated by pyrolysis of a suitable precursor and subsequently trapped all products using matrix isolation techniques. Their structures were confirmed through IR and UV/Vis spectroscopy. In addition, the corresponding ketenes, 52 and 53, were also isolated and characterized. The ketenes are considered significant prebiotic molecules as well.91-92 Upon UV irradiation, both enols underwent rearrangement, forming the corresponding carboxylic acids. Quantum mechanical calculations were used to validate both the IR and the UV/Vis – spectra and to further investigate their potential energy surface. Figure 28: Overview of novel reactive intermediates generated in this work. In the third publication, we investigated different cage alkyl carbenes. We successfully generated pentacyclo[5.4.0.0²,⁶.0³,¹⁰.0⁵,⁹]undecanylidene (74) in the matrix and characterized it through IR and UV/Vis spectroscopy (Figure 28). Carbene 75 only underwent [1,2]H-migration to homohypostrophene (84) via irradiation with green light, making it otherwise stable under cryogenic conditions. Isolating protoadamantylidene 85 was unsuccessful due to fast tunneling. Two competing QMT reactions were observed, mainly [1,2]H-migration and C–H insertion. To exploit the tunneling reactions and control product outcomes, hydrogen isotopes were strategically employed as directing groups. This successfully changed the product ratio and demonstrated the ability to perform isotope-controlled selectivity in tunneling reactions. From Stars to Life ____________________________________________________________________________________ 19 Bibliography 1. McGuire, B. A., 2021 Census of Interstellar, Circumstellar, Extragalactic, Protoplanetary Disk, and Exoplanetary Molecules. 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E.; Buhl, D., Observations of radio emission from interstellar hydrogen cyanide. Astrophys. J. 1971, 163, L47. 185. Linke, R. A.; Frerking, M. A.; Thaddeus, P., Interstellar methyl mercaptan. Astrophys. J. 1979, 234, L139- L142. From Stars to Life ____________________________________________________________________________________ 25 2. Publications 2.1 The enol of propionic acid Abstract: We demonstrate the gas-phase synthesis of prop-1-ene-1,1-diol, the hitherto unreported higher energy tautomer of propionic acid. The enol was trapped in an argon matrix and characterized by IR and UV/Vis spectroscopy in combination with density functional theory computations. Upon photolysis, the enol rearranges to propionic acid and methylketene. Reference: Akkad Danho, Artur Mardyukov and Peter R. Schreiner Chem Commun. 2023, 59, 11524-11527. (DOI: 10.1039/d3cc03711h) Reproduced with permission from: © 2024, Royal Society of Chemistry Thomas Graham House (290) Science Park, Milton Read Cambridge (United Kingdom) − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 26 From Stars to Life ____________________________________________________________________________________ 27 − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 28 From Stars to Life ____________________________________________________________________________________ 29 − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 30 From Stars to Life ____________________________________________________________________________________ 31 2.2 The enol of isobutyric acid Abstract: We present the gas-phase synthesis of 2-methyl-prop-1-ene-1,1-diol, an unreported higher energy tautomer of isobutyric acid. The enol was captured in an argon matrix at 3.5 K, characterized spectroscopically and by DFT computations. The enol rearranges likely photochemically to isobutyric acid and dimethylketene. We also identified propene, likely photochemically formed from dimethylketene. Reference: Akkad Danho, Artur Mardyukov and Peter R. Schreiner Chem Commun. 2024, 60, 5161- 5164. (DOI: 10.1039/d4cc01140f) Reproduced with permission from: © 2024, Royal Society of Chemistry Thomas Graham House (290) Science Park, Milton Read Cambridge (United Kingdom) − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 32 From Stars to Life ____________________________________________________________________________________ 33 − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 34 From Stars to Life ____________________________________________________________________________________ 35 − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 36 From Stars to Life ____________________________________________________________________________________ 37 3. Unpublished Results 3.1 Cage Alkyl Carbenes Provide Experimental Evidence for Isotope Controlled Selectivity Akkad Danho, Bastian Bernhardt, Dennis Gerbig, Marija Alešković, and Peter R. Schreiner J. Am. Chem. Soc. 2025, Revision submitted on 10 February 2025. Published in ChemRxiv on 17 December 2024. DOI: 10.26434/chemrxiv-2024-qb4mt. 3.1.1 Abstract We report the gas-phase synthesis and reactivity of adamantylidene (1) and pentacyclo[5.4.0.02,6.03,10.05,9]undecanylidene (2). The latter previously unreported carbene, is persistent under cryogenic conditions and has been characterized spectroscopically. The singlet carbenes were generated through irradiation of their corresponding diazirine precursors followed by trapping the products in argon or nitrogen matrices at 3.5 K. Analyses using IR and UV/Vis spectroscopy, together with density functional theory computations provide strong evidence for the successful preparation of these reactive species. Carbene 1 (∆EST = –3.0 kcal mol–1) undergoes a slow hitherto unreported but theoretically predicted quantum mechanical tunneling (QMT) C–H-bond insertion and ring-closure to 2,4-dehydroadamantane (4). In contrast, 2 (∆EST = –5.2 kcal mol–1) remains unchanged under cryogenic conditions but rearranges to homohypostrophene (9) upon = 627 nm irradiation. Attempts to prepare protoadamantylidene (3) (∆EST = –5.1 kcal mol–1) in a similar fashion did not allow the direct observation of the free carbene, but enabled follow-up QMT reactions, whose selectivities are determined by the 1H and 2H isotopologs, thereby demonstrating isotope- controlled selectivity (ICS). 3.1.2 Introduction Isotope-controlled selectivity (ICS) is defined as a molecular system where one of two conceivable products both resulting from a quantum mechanical tunneling (QMT) reaction from the same starting material forms predominantly, only depending on isotopic − Cold Organic Chemistry and Prebiotic Insights via Matrix Isolation – ____________________________________________________________________________________ 38 composition.1 This novel concept of controlling reactivity has been theoretically predicted1 but not been demonstrated experimentally. Here we investigate the effect of isotopic substitution (hydrogen vs. deuterium) in the reactivity of singlet protoadamantylidene and provide spectroscopic evidence for the formation of different products as a result of ICS. This work is organized to highlight important revelations and unexpected challenges associated with our quest of finding a cage carbene system that would demonstrate ICS, and to underscore that even structurally very similar systems can have quite varying reactivity when QMT operates. Singlet alkyl carbenes are highly unstable, with only a few spectroscopic reports available, including di-tert-butylcarbene,2 diadamantylcarbene,3 dicyclopropylcarbene,4 and adamantylidene5 (1, Figure 1). The majority of research into these carbenes stems from computational and theoretical studies.6 Beyond methylene (which has a triplet ground state), singlet ethylidene represents the simplest alkyl carbene, which, however, undergoes a very facile [1,2]H-shift; despite multiple attempts, the direct spectroscopic characterization of ethylidene has remained elusive.7-14 Analogs such as 2,2,2-trifluo- roethylidene15 have been generated and characterized in noble gas matrices at low temperatures. Carbenes including norbornen-7-ylidene,16-17 cyclobutylidene,17 and tricyclooct-8-ylidene18 have all been investigated theoretically. Such so-called foiled carbenes19-20 display a combination of through-space interactions, non-classical bonding, and hyperconjugative interaction.21 Apart from fast 1,2-shifts, cyclic singlet carbenes may also undergo ring expansion by heavy-atom QMT through C–C or C–H bond insertion reactions.22-23 Heavy-atom QMT is less common,24 owing to the mass dependence of the tunneling rate, which typically results in significantly extended tun- neling half-lives unless the reaction barrier is narrow.25 Examples of reactions involving carbon tunneling include automerizations of cyclobutadiene,26 1,5-dimethyl- semibullvalene,27 and the ring closure of cyclopentane-1,3-diyl.28 As QMT half-lives depend profoundly on particle mass but even more so on barrier height and width,25 computational predictions require a suitable potential energy hypersurface (PES). Canonical Variational Transition State Theory (CVT)29 corrected for small-curvature tunneling (SCT)30 in conjunction with density functional theory (DFT)31-32 provides results in reasonably good agreement with experiment.33 Alkyl carbene 1 was first isolated and spectroscopically characterized using the matrix isolation technique by Bally and Platz et al..5 In 2014, Kozuch et al. studied the tunneling reactivity of 1 and calculated a tunneling half-life of 62.2 h at the CVT/SCT//B3LYP/6- 31G(d,p) level of theory.22 While Bally and Platz et al.5 observed the rearrangement of 1 to dehydroadamantane (4) upon UV irradiation, they (unintentionally) did not wait long enough to also observe its reactivity in the dark. As such, we sought to experimentally examine the predicted QMT ring closure reaction of 1 to 4 in the dark, and to take this QMT reactivity as a model for possibly observing ICS with a carbene. As we will outline, 1 proved not to be suitable for this purpose, even though it does show the predicted QMT reactivity, because alternative rearrangements pathways are not competitive. We then investigated pentacyclo[5.4.0.0²,⁶.0³,¹⁰.0⁵,⁹]-undecanylidene (PCU-carbene) (2), also a cage structure, with the hope that it would undergo competing QMT reactivity.34 Surprisingly, 2 does not display observable QMT reactivity because it is unexpectedly stable under our conditions, most likely due to stabilization through a carbene-carbon- nitrogen complex, which hinders QMT.35-36 We finally arrived at protoadamantylidene From Stars to Life ____________________________________________________________________________________ 39 (3), an isomer of 1 that has rather short QMT half-lives, but does demonstrate H/D-ICS in competing QMT reactions. Figure 1. Outlining the hitherto unreported reactivities of known carbene 1 and the reactivities of novel carbenes 2 and 3. 3.1.3 Results and Discussion Adamantane diazirine (5) and pentacyclo[5.4.0.02,6.03,10.05,9]undecan-8-diazirine (PCU- diazirine 6, Figure 2) were synthesized according to literature procedures from the corresponding ketones 7 and 8.5, 37 Due to their high volatility 5 and 6 could easily be evaporated onto the cold matrix window using an excess of argon as the host gas. Our matrix-IR spectrum of 5 is in excellent agreement with reported d