Metal Activation Produces Different Reaction Environments for Intermediates during Oxidative Addition

Abstract

Commercial zinc metal powder requires activation for consistent and reliable use as a reductant in the formation of organozinc reagents from organohalides, and for the avoidance of supplier and batch-to-batch variability. However, the impact of activation methods on the reaction environments of subsequent intermediates has been unknown. Herein, a fluorescence lifetime imaging microscopy (FLIM) method is developed to bridge this knowledge gap, by imaging and examining reaction intermediates on zinc metal that has been activated by pretreatment through different common methods (i.e., by chemical activation with TMSCl, dibromoethane, or HCl; or by mechanical activation). The group of chemical activating agents, previously thought to act similarly by removing oxide layers, are here shown to produce markedly different reaction environments experienced by subsequent oxidative-addition intermediates from organohalides—data uniquely available through FLIM’s ability to detect small quantities of intermediates in situ coupled with its microenvironmental sensitivity. These different microenvironments potentially give rise to different rates of formation, subsequent solubilization, and reactivity, despite the shared “[RZnX]” molecular structure of these intermediates. This information revises models for methods development for oxidative addition to currently sluggish metals beyond zinc by establishing diverse outcomes for pretreatment activation methods that were previously considered similar.

Graphical Abstract

Widely used metal pretreatment–activation methods create different environments experienced by oxidative-addition intermediates, during the synthesis of organozinc reagents from organohalides and zinc metal. A developed fluorescence lifetime imaging microscopy (FLIM) method reveals these differences.

Introduction

The ability to visualize the reaction environments of intermediates under synthetic organic reaction conditions would be a powerful tool. Environments underlie key behaviors of the chemical reactivity of intermediates–e.g., kinetics of formation and downstream reactivity, and related factors of stability, selectivity, and solubility. Here, a method for fluorescence lifetime imaging microscopy (FLIM) is developed and harnessed as a spatially resolved readout for the environments of molecular reaction intermediates, with an initial focus on environments experienced by organozinc intermediates during oxidative addition of organohalides to zinc metal (Figure 1). To our knowledge, FLIM has not been used previously to determine surface environments of reaction intermediates.

Figure 1.

Knowledge gap addressed by FLIM: determination of how activating agents impact reaction environments of organozinc intermediates arising from oxidative addition to commercial zinc metal powder in solution and under synthetic organic conditions.

Organozinc reagents play key roles in C–C bond forming reactions (e.g., in Negishi cross-coupling reactions).^[1] Oxidative addition of organohalides to commercial zinc metal powder presents the most atom-economical and straightforward approach to synthesize these reagents. Prior to the discovery of metal activation methods, however, this direct route was inconsistent and often unpredictable.^[2] The suite of activation strategies were thought to act in a limited number of ways: reducing passivating surface oxide layers,^[3] mechanical etching,^[4,5] chemical etching,^[6] or, as more recently established, solubilizing otherwise persistent oxidative-addition intermediates.^[7,8] The common chemical activation methods for zinc are HCl, TMSCl, dibromoethane, and LiCl. With the exception of LiCl, these methods are performed as pretreatments to zinc, prior to synthetic steps, to activate zinc in advance of any oxidative addition processes.

Despite the importance of these pretreatment–activation strategies, it has not been possible to characterize the resulting reaction environments of organozinc intermediates after pretreatment–activation—thus providing a significant barrier to the ability to evaluate the central mechanistic hypothesis that mechanical and chemical pretreatment activating methods act in a limited and similar set of ways. This difficulty is due to the current analytical limitations of studying ongoing reactions at solvent–solid interfaces^[9] and of studying the low quantities of intermediates that build up on metals during oxidative addition (necessitating a tool of highest sensitivity). Thus, understanding how these activating agents work on a granular level remains restricted, as does the ability to use this understanding for targeted activation of additional, currently inactive metal powders to oxidative addition beyond zinc.

Prior work in our laboratory leveraged the high sensitivity of fluorescence microscopy to decipher the mechanism of LiCl in accelerating formation of organozinc reagents.^[7,10,11] These studies were foundational for the current studies by establishing that fluorescence microscopy is sufficiently sensitive to detect the small quantities of reaction intermediates that build up on zinc metal surfaces during oxidative addition of organohalides—intermediates which are not detectable by alternative analytical techniques.^[7,10,11] These prior studies, however, were only capable of measuring fluorescence intensity, but not fluorescence lifetime. For this reason, valuable environmental information was not available. The fluorescence lifetimes of small organic fluorophores are exquisitely sensitive to their local microenvironment, enabling their use as environmental sensors. For that reason, FLIM has been used extensively as a method to provide spatially resolved information in biology,^[12,13] but so far only sparingly in chemistry during ongoing reactions.^[8,14–23] Recently, our laboratory investigated the solubilization of organozinc intermediates after TMSCl pretreatment by FLIM,^[8] but did not examine environments of these intermediates on metal surfaces.

We envisioned that the spatially resolved fluorescence lifetime information from FLIM may reveal differences in environments of molecular reaction intermediates during oxidative addition, as here described. FLIM data here show that pretreatment–activation methods induce significant changes to the zinc surface that result in different reaction environments for organozinc intermediates. In these cases, although oxidative-addition intermediates on the surfaces of zinc conceivably have the same “RZnX” chemical bonding structure, their microenvironment and thus plausible propensity for formation, solubility, and reactivity differ markedly.

Results and Discussion

Nine pretreatment–activation processes for zinc were compared in this study (b–j, below). They were comprised of two different batches of commercial zinc (to examine the reported synthetic batch-to-batch variation^[24–26]). After activation, the chemically activated samples were rinsed vigorously with clean solvents to remove residual activating agents, such that the impact of pretreatment could be studied independently of other processes.^[8] A subset of samples were treated with only the associated rinsing steps to control for the effect of these manipulation steps in the absence of the key chemical activating agent:

All samples were manipulated under nitrogen in a glovebox, preventing further oxidation of the zinc surface by oxygen during pretreatment–activation, except that of the HCl pretreatment and its corresponding control, for which manipulation in a glovebox was both impractical and contrary to the published synthetic procedure.^[29,30]

With the pretreatment–activated zinc samples and their rinsing/heating controls in hand, oxidative addition resulting in organozinc intermediates^[7,10,11] was explored by FLIM. An oxidative-addition reactive imaging agent, boron dipyrromethene (BODIPY) aryl iodide 1 (Figure 2; able to produce oxidative-addition intermediate 2 upon reaction with zinc due to the presence of a C–I bond), or control imaging agent 3 (Figure 3; not able to produce oxidative-addition intermediates due to the lack of a C–I bond) in THF was added. These resulting reaction mixtures were heated to 60 °C for 4 h. The BODIPY fluorophore class was selected due to its solubility in organic solvents and the well-established chemical inertness of its core,^[7,32] enabling the BODIPY core to serve as a spectator in the reaction of the C–I bond and in the overall imaging process. This BODIPY core had no advance-known sensitivity to different microenvironments on zinc surfaces, and therefore its environmental response through FLIM formed part of the research question and methods development at hand.

Figure 2. a.

. Reaction schematic: environments of surface organozinc intermediate 2 (b–j). FLIM images of 2 on zinc after different activating pretreatments, in solvent, providing environmental readout. All images were acquired under identical microscopy parameters. Brightness–contrast display settings differ between samples (see SI) to enable all images to be bright enough to display FLIM data; however, the same brightness–contrast settings is fixed within a given treatment method for both regions 1 and 2 and also for both Figures 2 and 3, to enable direct comparison of treatment with 1 or 3.

Figure 3. a.

. Control experiment schematic. b–j. FLIM images physiosorbed 3 on zinc after different activating pretreatments, providing environmental readout. All images were acquired under identical microscopy parameters. Brightness–contrast display settings differ between samples (see SI) to enable all images to be bright enough to display FLIM data; however, the same brightness–contrast settings fixed within a given treatment method for both regions 1 and 2 and also for both Figures 2 and 3, to enable direct comparison of treatment with 1 or 3. Images corresponding to treatment corresponding to h were not obtained because the heating and rinsing steps alone did not change the environments of zinc for intermediate 2 (i.e., in Figure 2).

After 4 h, the solutions of imaging agents were removed, and the residual zinc particles were washed with clean THF to minimize background fluorescence in the sample caused by residual solution-phase 1 or 3. Samples were then transferred to clean solvent for imaging. To account for any spatial heterogeneity in the samples, two or more experimental replicates were performed per treatment and three or more sample regions were examined for each replicate. Two representative regions from different replicate samples are shown in Figure 2. The image consistency with respect to replicates (e.g., region 1 and region 2) clearly establishes that the differences between treatments arise from the pretreatment–activation methods and/or their associated rinsing steps and not from general variation (see SI for additional regions).

FLIM images derived from oxidative-addition reactive aryl iodide 1 are shown in Figure 2b–j with a false-rainbow scale in which shorter fluorescence lifetimes (τ) appear blue shifted and longer fluorescence lifetimes appear red shifted. Immediately noticeable in all images are four features: 1) individual zinc particles of ~1–30 mm length, notable by their generally bright surfaces, caused by buildup of oxidative-addition intermediate 2,^{[7,10,11,27,33]} 2) distinctive microenvironments of 2, as indicated by the different fluorescence lifetimes, displayed as false-colors of 2 on the zinc particles, that were present even in untreated commercial samples, 3) a different “average environment” determined by pretreatment, as qualitatively indicated by color, and 4) a broad “range of environments,” or particle-to-particle heterogeneity in most samples, observable (and assessable quantitatively and qualitatively) as the distinct fluorescence lifetimes among different particles. These differences in environments created by different pretreatment–activation methods and handling methods among different samples, and the spatial heterogeneities within a single sample, reveal markedly distinct reaction environments for intermediate 2. Example histograms and fittings for single-particle regions of interest (ROIs) are available in Figures S10–S12.

Average fluorescence lifetime (τ_{avg int}) provided a quantitative method to evaluate the average environment of 2 in that sample. Individual particles in both region 1 and region 2 were independently analyzed and combined for statistical evaluation (n = 22–81 distinct particles per treatment). The respective standard deviation provided a quantitative method to assess the degree of physiochemical heterogeneity of 2 among different particles containing within the sample and compare this degree among different samples. A limitation of using standard deviation as a measure of heterogeneity is a leveling effect when the values approach their low and high limits (~0.3 ns and ~5.5 ns) and therefore become less sensitive to environmental differences. The standard deviations therefore are considered estimates of quantitative heterogeneity that are most comparable between two samples that possess similar τ_{avg int.}

Using untreated zinc (τ_{avg int zinc batch 1} = 1.7 ± 0.3 ns and τ_{avg int zinc batch 2} = 2.2 ± 0.7 ns) as a baseline for comparison, chemical activation with TMSCl resulted in significantly shorter lifetimes (τ_{avg int} = 1.2 ± 0.3 ns), but with dibromoethane and HCl resulted in significantly longer lifetimes (τ_{avg int} = 3.5 ± 0.4 ns and τ_{avg int} = 3.3 ± 0.4 ns, respectively). This difference in outcomes between the chemical activation methods was fully unexpected, because TMSCl, dibromoethane, and HCl were previously thought to act similarly, by reducing the oxide coating.^[3,5,34] Yet, despite this similarity, their impact on the environments of organometallic intermediates under reaction conditions are clearly different—even “opposite” (e.g., shorter lifetime vs. longer lifetime). Manipulation associated with water, ether, and acetone rinsing and drying for HCl treatment (but in the absence of HCl) proved surprisingly impactful, on its own, with respect to impact on environments of intermediates, resulting in similar increases (τ_{avg int} = 3.6 ± 0.2 ns; Figure 2i). In contrast, the heating and THF rinsing steps (but in the absence of dibromoethane) did not result in a change from the untreated sample. Mechanical activation by stirring resulting in increases in τ_{avg int} and spatial heterogeneity.

With notable differences in reaction environments characterized during oxidative addition caused by pretreatment–activation methods, two main questions arose: (1) Are these environmental differences experienced uniquely by organometallic intermediate 2, or would they be experienced by any organic compound on the post-treatment-activated zinc? (2) What are the root physiochemical causes of the different reaction environments? The remainder of experiments and discussion in the manuscript are dedicated to answering these questions.

Experiments to examine the environments experienced by general organics, i.e., nonspecific physisorption, employed imaging agent 3 (Figure 3), which lacked a C–I bond and therefore was incapable of oxidative addition (instead of 1). This imaging agent 3 produced zinc particles that were considerably less bright (compare brightness of otherwise similar treatments in Figure 2 and Figure 3, under identical acquisition and image display settings). The dimmer samples mean that there is less material (3) on the zinc surface from physisorption than material previously from 1. Thus, the majority of fluorescence signal from treatment with 1 is assigned to chemospecificity arising from reaction of the C–I bond, and thus to organozinc intermediate 2. The exception to this brightness trend was untreated zinc batch 2, which was equally bright with imaging agents 1 and 3 (compare Figure 2e and 3e), a phenomenon assigned to high levels of physisorption in the untreated sample.

Chemical activation by all three reagents, TMSCl, dibromoethane, and HCl, resulted in lower physisorption than occurred on the untreated zinc (e.g., compare Figure 3b with 3d, and 3e with 3f and 3g). In so doing, chemical activation was revealed to increase the selectivity of the surface for oxidative addition over nonspecific physisorption (e.g., compare Figure 2b with 3b, 2e with 3e, 2f with 3f, 2g with 3g).

The impact of pretreatment processes on particle-to-particle spatial heterogeneity was next examined, using both oxidative addition (Figure 2) and physisorption (Figure 3) data to guide analysis. Oxidative addition was examined first. In zinc batch 1, relative to no treatment (±0.3 ns, Figure 2b), heterogeneity increased when zinc was mechanically activated by stirring (±0.8 ns, Figure 2c) and stayed constant when zinc was chemically activated by TMSCl (±0.3 ns, Figure 2d). Surprisingly, although both were commercial samples, zinc batch 2 displayed larger amounts of heterogeneity prior to treatment than did zinc batch 1, as seen by the full span of rainbow colors in the FLIM images and the high standard deviation (±0.7 ns, Figure 2e). Pretreatment of zinc batch 2 with dibromoethane or HCl resulted in decreases to heterogeneities of environments of 2 (±0.4 ns, Figure 2 f, g). A standard deviation of ±0.3 ns was the smallest measured for any sample under these synthetic conditions, apparently representative of a moderate degree of continued heterogeneity inherent to the irregular commercial zinc powder regardless of treatment. For comparison, the standard deviation for a fully homogenous solution measurement of fluorescence lifetime under typical measurement conditions on our instrument is ~±0.1 ns.

Spatial heterogeneity in the physisorption samples was examined next. With the exception of TMSCl pretreatment, which resulted in dark samples without physisorption and therefore could not be evaluated, physisorption resulted in generally similar ranges of distributions of fluorescence lifetimes as occurred with oxidative addition, as indicated by similar standard deviations in Figure 2 and Figure 3. after similar treatments. Thus, the mechanistic origin of the heterogeneity in lifetimes is attributed predominately to environmental factors intrinsic to the zinc and pretreatment–activation method and is independent of the oxidative addition reaction, e.g., the oxidative addition process does not cause the heterogeneity.

Next, the root physiochemical causes of the different reaction environments were examined. Six hypotheses were considered (Figure 4). The hypotheses hinge on the known high starting lifetimes of solution-phase unreacted 1 and 3 in (5.5 ns)^[8] and a drop in their respective lifetimes when in proximity a viable quenching agent.^[8,35,36] Hypothesis 1: Distance from bulk zinc due to different zinc oxide layer thicknesses. The closer the fluorophore is to the bulk metal core, the greater degree of quenching by zinc(0), leading to shorter lifetimes.^[36,37] (Quenching of fluorescence by metal surfaces is a competitive nonradiative decay pathway of the excited state and therefore results in shorter fluorescence lifetimes.^[36,37]) Hypothesis 2: Different ratios of species, physisorption vs. oxidative addition, on each particle. In this hypothesis, the physiosorbed material has a different average fluorescence lifetime than the oxidative-addition intermediate. Specifically, a short fluorescence lifetime arise from oxidative addition intermediates.^[8] Hypothesis 3: Various stages of the oxidative addition and solubilization on the zinc particles at the moment of data acquisition.^[7,8,10,11]

Figure 4.

Hypotheses 1–6: Considered models for different environments of organozinc intermediate 2 (depicted as star), which cause different fluorescence lifetimes.

Previous results from our laboratory showed that solubilization of intermediate 2, and likely also of physisorbed 3, are gradual, ongoing processes that result in higher lifetimes at/near the zinc particles, as intermediate 2 progressively moves further away from the metallic zinc. In this hypothesis, the “red” particles with longer lifetimes would be composed of more organics or organometallics at partial stages of solubilization, perhaps trapped in small local pockets of solvent. Hypothesis 4: Different quantities of iodide. In this hypothesis, the iodide present on the surface of zinc after oxidative addition of aryl iodide 1 quenches the fluorescence.^[32] In this hypothesis, zinc particles with shorter lifetimes would possess a greater concentration of residual iodide (and thus possibly of oxidative- addition product), and thus, shorter lifetimes. Hypothesis 5: Etching of surface from chemical or physical activation and manipulation. The zinc surface is not uniform but rather contains spikes or scratches of zinc metal. These etched particles would therefore have intermediate 2 that was, on average, further away from the bulk core zinc metal that was responsible for most of the quenching. In this hypothesis, longer fluorescence lifetime particles indicate an etched surface with a greater degree of intermediates distributed on “spikes” or “scratches” of zinc. Hypothesis 6: chemical residue of salt byproducts from pretreatment–activation causes different environments and/or increases distance from bulk zinc (e.g., ZnCl₂ from HCl or ZnBr₂ from dibromoethane).

To evaluate these hypotheses, first SEM data was obtained under high vacuum to inform on zinc surface morphology (Figure 5, representative images; additional images in SI). This morphology was helpful for a first-pass assessment of Hypotheses 1 and 5, because these hypotheses involved plausibly observable surface-morphology changes. SEM images established that untreated zinc possessed a highly irregular surface, with substantial differences between regions, and which was generally rough and jagged. Treatment with TMSCl resulted in dramatic smoothing of the surface relative to untreated zinc, consistent with Hypothesis 1. Treatment with HCl resulted in formation of semi-regular surface spikes of ~100 nm, consistent with Hypothesis 5. These spikes arose specifically by action of HCl, and not from the associated soaking, washing, and drying steps, that accompanied activation, as seen in the control (Figure 5b). Interestingly, activation with dibromoethane did not result in noticeable change in surface features by SEM (compare Figure 5a and 5b), despite the marked change in environment experienced by intermediate 2 in situ after activation. FLIM was thus able to detect the impact of dibromoethane activation through its environmental effect on reaction intermediates in situ, which was not visible nor predictable from the ex situ SEM data. In this case, small changes to the metal surface may be masked by the high physical heterogeneity of the surface of commercial zinc, making them undetectable by SEM.

Figure 5.

SEM images with EDS elemental analysis data (derived from mapping) of commercial zinc batch 2 with: a. different chemical activation pretreatments, and b. controls handled similarly but without exposure to the “activating” chemical.

To evaluate Hypothesis 1 more closely, surface elemental composition was analyzed by energy dispersive X-ray spectroscopy (EDS) mapping (Figure 5). Full spatial maps (>3 mm × 3 mm regions) of particles were obtained, and ranges reported in Figure 5 correspond to averages and standard deviations from two different particle maps (e.g., from many measurements on two different particles). EDS data showed that only TMSCl treatment resulted in substantial decrease in surface oxide, from Zn:O 5.8 ± 2.3 before treatment, to 16 ± 14 after TMSCl treatment (from the average of 6.5:1.0 and 26:1.0; higher numbers correspond to more zinc), consistent with Hypothesis 1. All other manipulations produced only marginal differences. This result was unexpected, because HCl treatment is reportedly performed to reduce oxide; ^[29,30] and a reduction in oxide was not measured by EDS. The multiple washing steps in air may provide opportunities for reoxidation or partial reoxidation, in our hands, but also so in the reported synthetic procedures.^[29,30] (As stated earlier, the HCl activation was the only process performed outside of an inert atmosphere and therefore was the only manipulation with a similar opportunity for reoxidation.)

By EDS, zinc batch 2 (5.8 ± 2.3) had marginally more oxide than zinc batch 1 (previously reported, 6.5 ± 2.9^[8]), however, the high standard deviations, reflective of the sample heterogeneity, limited conclusions. A higher average amount of oxide would be consistent with untreated zinc batch 2 displaying higher physisorption of 3 in Figure 3 and displaying less selectivity for oxidative addition over physisorption (compare Figures 2 and 3). Physical features in SEM images were not associated with different elemental compositions in EDS maps (see SI).

The zinc-to-oxygen ratio by EDS did not clearly track with the FLIM trends other than those of TMSCl treatment (Figure 2). This discrepancy suggests that oxide thickness, Hypothesis 1, is the dominant environmental factor only when activation causes substantial removal of the oxide layer; however, the possibility that FLIM is more sensitive than EDS to small differences in oxide ratio and therefore informs on difference invisible to EDS cannot currently be ruled out.

Hypothesis 2 was considered next: a distinct short lifetime that exclusively arose from oxidative-addition intermediate 2. Because the short lifetime components were present with either 1 and with control 3 (especially prominent in zinc batch 2 with control 3; Figure 3e), the shortest lifetimes did not require an organoiodide. Thus, their presence did not arise exclusively from an organozinc intermediate 2. Therefore, Hypothesis 2 was ruled out.

Hypothesis 3 was next considered: the possibility that solubilized intermediates with longer lifetimes (because they are further from the quenching zinc surface) are near the surface and contributing to higher measured lifetimes.¹⁷ Measurements taken over ~15–30 min looked similar to initial measurements (showing no change on this timescale), decreasing but not ruling out the possibility of this contributing factor. The absence of growing “halos” of fluorescent solubilized material around each zinc particle and on the “inside” of the cross-section of particles is further inconsistent with solubilized material contributing significantly.⁸ Thus, Hypothesis 3 was ruled out as a major contributing factor.

Hypothesis 4 was next considered: the possibility that the presence of iodide on the metal surface shortens the average lifetime. Indeed, the τ_{avg int} were shorter when imaging agent 1 was used than when imaging agent 3 is used (except in the case of TMSCl pretreatment, which eliminated physisorption and therefore could not be compared). To allow sample-wide comparison, the lifetimes of many full imaging areas were compared (rather than sets of individual particles). In the case of zinc batch 1 without pretreatment with imaging agent 1 (Figure 2b), τ_{avg int} = 1.7 ± 0.3 ns, which is somewhat lower than the control experiment using 3 (Figure 3b) with τ_{avg int} = 2.2 ± 0.3 ns. These data raised the possibility that iodide could shorten the measured lifetime, but a more definitive test was sought.

In order to test the fundamental ability of iodide to shorten the fluorescence lifetimes of imaging agents on the surfaces of zinc powders, an experiment added soluble iodide to zinc containing physiosorbed 3 (Figure 5). Imaging agent 3 was chosen for these experiments, rather than 1, so that all zinc particles would start with the same amount of surface iodide (i.e., none). It was envisioned that this exogenous iodide would then coordinate to zinc, mimicking the population of surface-coordinated iodide after oxidative addition. Soluble iodide in the form of tetrabutylammonium iodide (final concentration 0.25 M) was added in THF to physiosorbed 3 made with zinc batch 1. A total of n = 6 or n = 10, 157 × 157 μm² imaging areas were analyzed before and after addition, respectively (Figure 6). Standard deviations were calculated from the average of these different imaging areas. Note that in this case, the fluorescence lifetimes and standard deviations refer to the entire imaging areas and not individual particle-to-particle analysis, so as to evaluate sample-wide changes. The dim background (solution-phase) fluorescence was subtracted from these images to avoid convolution of the effect of iodide on zinc-surface species.

Figure 6.

FLIM images; effect of iodide on physiosorbed 3, showing decrease in fluorescence lifetime. Reported lifetimes and standard deviations are derived from the set of the full images in n sample regions, in order to determine the sample-wide effect, and are not separated by particle.

The addition of iodide lead to a measurable decrease in the fluorescence lifetimes of the surface species (from τ_{avg int} = 2.8 ± 0.3 ns to τ_{avg int} = 2.1 ± 0.3 ns), Figure 6. This result shows that iodide is able to produce a quenching effect on the fluorescent material on the zinc particles, leading to lower lifetimes. Thus, it is plausible that oxidative addition—and the iodide or surface organozinc iodide thus produced—does indeed contribute to the overall lower fluorescence lifetimes due to the presence of iodide, though the effect is generally smaller than the range of heterogeneity inherently present in the sample and is thus not the predominant effect.

Hypothesis 5 was considered next: etching of the zinc surface (e.g., in to spikes or scratches), which changes average distance of 2 to the bulk zinc metal. Regarding TMSCl and HCl, data from SEM supported that the treatments produce opposite physical effects: smoothing of the surface after TMSCl treatment, but creation of spikes after HCl treatment (Figure 5a), consistent with the opposite effects observed by FLIM.

The rinsing steps with water, ether, and acetone associated with HCl activation method, alone, produce a similar increase fluorescence lifetime (Figure 2g, j) but produced no corresponding SEM change (Figure 5a, b). Ultimately, the longer fluorescence lifetimes after HCl activation and after control washing steps were assigned to different causes, as described below.

To fully evaluate these data, activation with dibromoethane was also considered. This activation method did not produce a detectable change by SEM. Yet, the FLIM data disclose a dramatic change in microenvironment experienced by 2, consistent with increased distance from bulk zinc—similar to that which occurred with HCl pretreatment or with the multiple manipulations associated with washing in air alone. Key to final analysis, samples stirred under nitrogen exhibited moderately higher lifetimes (Figure 2), although surface changes were also not visible by SEM (Figure 5b), presumably due to high heterogeneity that masked small changes.

Hypothesis 6 was considered next: Treatment with HCl and dibromoethane did not result in buildup of residual halide, e.g., ZnCl₂ or ZnBr₂ (<1% Cl and no detectable Br by EDS, respectively). Thus, salt residue from the byproducts of the activating pretreatments is not the cause of the reaction environment differences. Therefore, Hypothesis 6 was not considered further.

Together, the data point to chemical and/or mechanical etching (Hypothesis 5) as the dominant factor in dictating environments for intermediates after stirring, rinsing with multiple solvents, dibromoethane activation, and HCl activation (i.e., all the manipulations resulting in higher fluorescence lifetime). These pretreatment–activation methods result in reaction environments for 2 with increased distance from bulk zinc by FLIM. In contrast, removal of the oxide layer is the dominant factor after pretreatment–activation with TMSCl, which is the one method resulting in lower lifetimes by FLIM. This pretreatment–activation method results in reaction environments for intermediate 2 that are closer to bulk zinc (Hypothesis 1). These differences may be due to HCl and dibromoethane reacting preferentially with zinc(0), whereas TMSCl may react preferentially with zinc oxide^[8].

Conclusion

Data here shift the prevailing view of zinc metal pretreatment–activation methods from impacts solely prior to the synthetic organic chemistry steps, to continuing to have a lasting impact on differences that occur during the synthetic organic chemistry steps, by producing different reaction environments experienced by organometallic intermediates. FLIM can be used to monitor smaller changes in the environment of the activated surfaces than other techniques. Further, while SEM and EDS can and do provide details about surface physical and chemical features under vacuum/ex situ, they are unable to characterize the way in which these features impact the reaction environments experienced by intermediates in solvent and under synthetic conditions. Chemical activating agents that were previously thought to act similarly instead create different reaction environments for resulting intermediates—information which could not have been known without FLIM (Figure 2d, 2f, 2g). TMSCl is the only pretreatment–activation method examined that results in effective removal of the oxide layer and positions the reaction intermediates in environments in close proximity to bulk zinc metal, mechanistic information that was not previously known.

Chemical activation was revealed to result in enhanced selectivity for oxidative addition over nonspecific physisorption (compare brightness between in Figures 2 and 3 in otherwise identical treatments), suggesting that that chemical activation may overcome part of the reported batch-to-batch variation in the synthetic generation of organozinc reagents^[24,25,38] by decreasing persistent nonspecific physisorption of organics that may otherwise block reactive sites.

The revealed environmental differences may in turn impact reactivity, especially in cases where the electronic or steric effects are determinative, as may be the case for the key synthetic steps of formation and solubilization of these organozinc intermediates.^[7] In cases where residual activating agent is not rinsed away during reported synthetic procedures but rather remains present during the oxidative addition reaction (e.g., TMSCl^[8]), differences in surface environments caused by activating agents may persist beyond the initial reaction stages. These observations may be relevant to understanding other broadly used metal activation processes in organic synthesis (e.g., activation of magnesium metal by stirring,^[39] I₂ or dibromoethane^[40] in the preparation of Grignard reagents). Further, this granular knowledge may aid in the development of methods to generate organometallic reagents by direct insertion to metals beyond zinc, by establishing that there is a range of pathways for activation methods that were previously thought to act similarly.

Table 1.

Zinc samples examined.

Zinc batch	Sample	Pretreatment–Activation
1	b	unactivated, dried commercial, “zinc batch 1”
1	c	mechanically activated by stirring in THF for 15 min,[11,27]
1	d	chemically activated by TMSCl, followed by rinsing with THF;
2	e	unactivated, dried “zinc batch 2”, a different, newly opened commercial bottle,
2	f	activated with dibromoethane at 65 ° C, followed by rinsing with THF,[28]
2	g	activated with HCl followed by rinsing with water, diethyl ether, and acetone,[29,30]
2	h	THF at 65 °C and rinsing with THF only as a control for f,
2	i	rinsing with water, diethyl ether, and acetone only as a control for g
2	j	stirring in THF for 15 min[31]

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.