Investigations of Acetonitrile Solvent Cluster Formation inSupercritical Carbon Dioxide, and Its Impact on Microscale

Syntheses of Carbon-11-Labeled Radiotracers for PETRichard A. Ferrieri, Ivonne Garcia, Joanna S. Fowler and Alfred P. Wolf

DEPARTMENT OF CHEMISTRY, BROOKHAVEN NATIONAL LABORATORY, UPTON, NEW YORK, USA

ABSTRACT. A new strategy has been developed for synthesizing positron emission tomography (PET)radiotracers using [11C]methyl iodide. This strategy relies on the ability of organic co-solvents to clusterwithin mixtures of supercritical fluids resulting in localized regions of high density which can serve asmicroscopic pockets for reaction. We’ve shown that acetonitrile will cluster about dilute solutes whenmixtures of this co-solvent with carbon dioxide are forced to behave as a homogeneous fluid at the criticalpoint. We applied this strategy in a systematic investigation of the conditions for optimized reaction betweenmethyl iodide and L-a-methyl-N-2-propynyl phenethylamine (nordeprenyl) to yield L-deprenyl. Variablessuch as temperature, ultraviolet light exposure, co-solvent concentration, system pressure, and methyl iodideconcentration were explored. The synthesis of radioactive [11C]-L-deprenyl using no-carrier-added concen-trations of [11C]methyl iodide was also tested. Results showed that greater than 90% radiochemical yield ofthe desired product could be attained using 40 times less labeling substrate than in conventional PET tracersyntheses. NUCL MED BIOL 26;4:443–454, 1999. © 1999 Elsevier Science Inc. All rights reserved.

KEY WORDS. Supercritical fluids, [11C]Methyl iodide, Solvent cage

INTRODUCTION

The incorporation of carbon-11 into biomolecules of interest forpositron emission tomography (PET) has presented a particularchallenge to synthetic chemists over the years for two reasons (17).On the one hand, its short half-life places an enormous constrainton the time needed to complete a synthesis (typically less than40 min). Thus a reaction between some labeling precursor and alarger substrate molecule must be fast and in high yield. Thesequalities typically do not lend themselves well to the fact that theamount of material one has to react in the form of the labelingprecursor is usually extremely small, on the order of micrograms.Conventional practices demand that milligram amounts of a label-ing substrate be added in order to sustain chemistry with any degreeof efficiency. However, this presents an additional challenge ofseparating microgram amounts of radiolabeled product from milli-gram amounts of starting material. For this reason it would beadvantageous to develop synthetic strategies where the amount oflabeling substrate could be reduced perhaps even to stoichiometriclevels commensurate with the labeled precursor. Not only could thepurification of the labeled radiotracer be simplified, but also thequality of the final formulation greatly improved.

One way we found to meet this challenge is to create microscopicreactors from transient molecular cages. The general strategy herecapitalizes on the ability of some organic solvents to cluster aboutdilute solute molecules in mixtures of supercritical fluids andpressurized liquids, and confine the space through which these“trapped” molecules can diffuse. This action could increase the oddsthat “trapped” molecules might encounter a reactive collision.

Under these circumstances, it might be possible to maintain highreaction efficiency with minute, and possibly stoichiometricamounts of reactants.

Solvent clustering creating localized regions of high density isknown to occur within mixtures of pressurized liquids, as well assupercritical fluids. Part of the reason for this behavior is that in andaround the critical region, a fluid or fluid mixture becomes highlycompressible. For example, the density of carbon dioxide fluidincreases almost 40-fold at its critical point (Tc 5 31°C, Pc 5 79bar) (22). Because of differences in size and energy between soluteand solvent molecules, solute–solvent interactions can becomestrong within this region resulting in large negative partial molarvolumes (13), and partial molar enthalpies (46) for dilute solutes.

Several methodologies have evolved to investigate this phenom-enon. The first relies on a photochemical paradigm that is based onthe well established mechanism for a-cleavage of photoexcitedtriplet ketones yielding geminate radicals (44, 47). Recombinationof these radicals can then provide a probe to evaluate the localizeddensity augmentation brought on by co-solvent clustering. Asecond method relies on solvatochromic waveshifts of a suitablechromophoric solute that is sensitive to the nature of the solventcage around it (5, 27–29, 31). Additional spectroscopic techniquesinclude time-resolved electron spin resonance as a direct probe ofthe magnetic features of geminate radicals (9, 30), as well astime-resolved optical laser spectroscopy to probe the photochemicaldynamics within the cage (25, 30, 53).

Despite clear evidence for these aggregates, it is still questionablewhether there is sufficient structure, as well as lifetime, of thesesolvent clusters in and around the critical region to have ameasurable impact on diffusional processes of dilute solutes (4, 34).For example, recent studies on the photolysis of dibenzylketones inpure supercritical ethane and carbon dioxide showed no change inthe radical recombination reaction as a function of pressure (36).Interestingly enough, the addition of small amounts of liquid

Address correspondence to: Dr. Richard A. Ferrieri, Department of Chem-istry, Brookhaven National Laboratory, Upton, NY 11973-5000, USA;e-mail rferrieri@bnl.gov.

Received 14 August 1998.Accepted 24 January 1999.

Nuclear Medicine & Biology, Vol. 26, pp. 443–454, 1999 ISSN 0969-8051/99/$–see front matterCopyright © 1999 Elsevier Science Inc. All rights reserved. PII S0969-8051(99)00011-6

organic co-solvent to supercritical carbon dioxide fluid can stronglyinfluence its solvating power of many substances (12, 50, 52). Thisaction is believed to be due to enhanced solvent–solvent interac-tions between the organic additive. In fact, local density augmen-tation of about an 6.5-fold increase has been measured using thesolvatochromic waveshift of phenol blue in low concentrations ofmethanol in supercritical CO2 fluid (31). This suggests thatmixtures of organic co-solvents in supercritical fluids might be aviable course to generate molecular cage effects that can impact onchemistry.

Within the scope of this work, we investigated the potential foracetonitrile to cluster in CO2 above and below the critical point forthe mixture, thus creating localized regions with density augmen-tation. Basic investigations were carried out measuring solventclustering over a broad pressure range using the caged reaction ofultraviolet (UV)-generated benzyl radicals as a probe to measurediffusional processes. The ability to perform useful microscalechemistry within such pockets of high density organic solvent, as itapplies to producing useful quantities of radiotracers for PET, wasalso investigated using the model alkylation reaction betweenmethyl iodide and L-a-methyl-N-2-propynyl phenethylamine (nor-deprenyl) to yield L-deprenyl. Systematic studies measuring productyield were carried out as a function of temperature, UV lightexposure, co-solvent concentration, system pressure and methyliodide concentration. We chose this reaction for two reasons. Wehad previously demonstrated that it was possible to carry out thisN-methylation reaction in pure supercritical CO2 (16). Othermethylation reactions using methyl iodide have been observed in

pure supercritical CO2 as well (6, 10). In addition, [11C)-L-deprenylis an important tracer in PET used in measuring concentrations ofmonoamine oxidase B in the brain (18).

EXPERIMENTALSupercritical Fluid System

We have designed and constructed an optical cell (see Fig. 1) thatis capable of sustaining fluid pressures up to 350 bar and tempera-tures to 125°C. The cell was equipped with both entrance and exitsapphire windows that were sealed onto a stainless steel reactorhousing through Teflon gaskets, which yielded an active volume of16 mL. UV light was allowed to enter as well as exit the cell so thatlight input to the cell could be maximized by adjusting the focusinglens position. The ultraviolet light source consisted of an unfilteredhigh pressure mercury arc lamp (Osram Inc., 500-watt lamp) whoselight output was focused through reflection from a parabolic mirroronto an additional focusing lens which maximized the photon fluxinto a 2-mm diameter fiber optic bundle (Oriel, Inc.). The light wasagain refocused to a 1-mm spot just prior to entering the cell. Celltemperatures were maintained to within 2°C through four resistivecartridge heaters (Omega, Inc.) mounted within an outer plastichousing and controlled through feedback from a platinum resistanceelectrode.

System pressures were maintained through an Isco syringe pump(model 260D) designed for operation with supercritical fluid extrac-tors. The pump can operate in either constant pressure mode orconstant flow mode. All studies carried out in this work were static,

FIG. 1. Schematic of high-pressure supercritical fluid optical cell for carrying out molecular cage studies.

444 R. A. Ferrieri et al.

thus the pump was always used to maintain a constant source offluid pressure. Sample introduction to the cell was accomplished inone of two ways; either through a high pressure Rheodyne injectionvalve (model 7010) with a 20-mL volume sample loop, or throughtwo Valco 3-way high-pressure valves. In the case of the lattermethod, the cell was completely filled with sample and excessallowed to flow out through a cell waste line.

Investigations of Acetonitrile Cluster Formation inSubcritical and Supercritical CO2:Acetonitrile Mixtures

Our general method for investigating cage formation relied on usingphotochemical systems to generate radicals. For example, radicalrecoupling reactions induced by the photochemistry of certainsubstituted benzyl ketones are ideal for investigating distant-depen-dent interactions between radical fragments (36, 48).

A stock solution was prepared of dibenzyl ketone (DBK) inacetonitrile solvent to yield a concentration of 50 mg DBK/mL. TheDBK and acetonitrile were purchased from Aldrich, Inc. Theacetonitrile was anhydrous grade, and shipped in 1-mL sure-sealbottles to maintain solvent integrity.

Cage experiments were conducted as a function of systempressure from 50 to 310 bar. The supercritical fluid source consistedof pure carbon dioxide, and was purchased from Scott SpecialtyGases, Inc. The fluid was used without additional drying orprocessing. The Isco model 260 D syringe pump was preloaded withthe source fluid to capacity then the pump’s cylinder head waspre-pressurized to the desired operating pressure of the experiment.The cylinder head was equipped with a water jacket to maintainfluid temperatures within it at 35°C. The pump outlet valve leadingto the sample injector and optical cell was kept closed during thisprocedure. Thus the sample injector and cell were at atmosphericpressure initially.

In typical cage studies, approximately 5-mL samples of DBK stocksolution were introduced to the system via the Rheodyne injectionvalve. More or less volume was used depending on the operatingpressure. The valve loop had a capacity to hold up to 20 mL of liquidsample. The total acetonitrile concentration was adjusted to main-tain the bulk loading of co-solvent at a constant 15% throughoutthe measurements. Once loaded, the injection valve was actuatedand the pump outlet valve opened to allow the system to pressure.Sample was displaced from the loop into the cell for photolysis.Equilibration to the operating pressure was usually rapid, involvinga matter of seconds, because the total volume of the reaction systemwas only 36 mL. During the course of photolysis of the sample, thesyringe pump maintained constant pressure through pressure feed-back at the cylinder head. A check valve located at the inlet to theinjector valve prevented sample back-flow. Photolyzes were typi-cally carried out for 20 min, after which samples were extracted bydepressurization of the cell through a 0.5 m 3 50 mm internaldiameter silica capillary tube into 1 mL of acetone solvent.

Samples were concentrated to 200-mL volumes, and 1-mL ali-quots were analyzed by capillary gas chromatography (GC) using aflame ionization detector (FID) on a Hewlett Packard 5890 instru-ment. A 30 m 3 0.52 mm i.d. HP-5 column was used in productseparation. The GC method included the following: the injectorwas maintained at 275°C; the FID was maintained at 350°C; thecolumn head pressure with helium carrier gas was maintained at 7psi; the injection split ratio was 40-to-1; the column was maintainedat 90°C initially with a 1-min hold time, then temperatureprogrammed to 250°C at a rate of 10°C/min. An identical columnand method were used on a Finnegan GC-MS (gas chromatogra-

phy-mass spectroscopy) to correlate compound identity with elutiontime. Product yields were measured using the FID response, andpeak areas (PAU) were calculated using Vision 4 chromatographysoftware (Scientific Systems, Inc.) on a personal computer. Noattempt was made to correlate detector response to actual amountsof product because the cage magnitude is only a relative number.The following formula was used to calculate the relative magnitudeof the cage effect:

Relative Cage Magnitude 5 PAUTol/(PAUDB 1 PAUTol).

The peak areas for dibenzyl (DB) and toluene (Tol) were integrated(PAU) and a relative toluene yield calculated from the ratio ofPAUTol/(PAUDB 1 PAUTol).

Synthesis of L-Deprenyl Using Methyl Iodideand Nordeprenyl

Studies were conducted to determine the optimized reaction con-ditions for maximized L-deprenyl product yield from the N-methyl-ation reaction between methyl iodide and nordeprenyl. Stocksolutions of methyl iodide were prepared daily owing to thepropensity for this compound to hydrolyze to methanol. Also, stocksolutions of methyl iodide in acetonitrile were prepared at variousconcentrations ranging from 5 to 90 mg/mL depending on thenature of the study. A stock solution of nordeprenyl substrate wasalso prepared in acetonitrile solvent. We found that both thesubstrate and methylated product were stable in this solvent whensubjected to the high-pressure environment under UV light expo-sure.

Reactions were carried out as a function of reaction time, UVlight stimulation, temperature, CO2:acetonitrile composition, sys-tem pressure, and the amount of methyl iodide. With the exceptionof the last study varying the amount of methyl iodide, all studieswere carried out using 0.38 mmol of methyl iodide and 0.90 mmol ofnordeprenyl.

Mechanistic Studies using [D3]Methyl Iodide anda,a-[D2]-Nordeprenyl

Early in our studies we observed a second reaction product that weidentified as N,N,a-trimethyl benzylethyl amine (TMBEA). Todetermine whether this product was the result of some internalrearrangement and decomposition pathway or simple dialkylation,we decided to track its formation using combinations of [D3]methyliodide with nordeprenyl in one set of reactions, and methyl iodidewith a,a-[D2]-nordeprenyl (34) in a second set of reactions, lookingfor the extent of deuterium incorporation in the TMBEA productusing GC-MS. In all mechanistic studies, we kept the reactionconditions the same using: 0.38 mmol methyl iodide and 0.90 mmolnordeprenyl; 10-min reactions at 95°C with the UV light on; andsystem pressures were maintained at 285 bar using a fluid feed of5% acetonitrile in CO2.

Synthesis of [11C]-L-Deprenyl using [11C]Methyl Iodideand Nordeprenyl

[11C]Carbon dioxide was produced at the Brookhaven NationalLaboratory PET Center using a Japan Steel Works 41-inch cyclo-tron by irradiation of a target containing 99.9995% nitrogen gas(MG Industries, scientific grade) with a 17.4-MeV proton beam,producing carbon-11 by the 14N(p,a)11C nuclear reaction. The

Solvent Cages in PET Radiotracer Synthesis 445

[11C]carbon dioxide was extracted from the nitrogen gas stream asthe target was depressurized after bombardment using 200 mL of 1 Nlithium aluminum hydride dissolved in tetrahydrofuran (Aldrich).The [11C]carbon dioxide was reduced, then iodinated using 57%hydroiodic acid (Aldrich) to yield [11C]methyl iodide quantita-tively. The [11C]methyl iodide was distilled from the HI solution inan argon gas stream, and collected in 250 mL of acetonitrile cooledto 220°C, and containing 60 nmol/mL of nordeprenyl. The opticalcell was loaded to capacity with this solution. The total volumeresulted in a reaction between 2 mmol of nordeprenyl substrate, anda no-carrier-added concentration of [11C]methyl iodide. The totalamount of radioactivity in the cell was approximately 5 mCi, decaycorrected to the end-of-bombardment. The total radioactivity wasstandardized by taking known volume aliquots and counting themon a well-type NaI(Tl) scintillation counter (Picker, Inc.). Correc-tions were made for sample fraction, radioactive decay, and detectorefficiency. The reaction was carried out under the followingconditions: 10 min of reaction at 95°C with UV light stimulation;system pressure at 83 bar using 5% acetonitrile in CO2. (Theco-solvent concentration was boosted to 20% after reagents wereintroduced into the cell.) After reaction, the contents of the cellwere emptied into 0.5 mL of methanol solvent cooled to 220°C.Samples were analyzed by radio high performance liquid chroma-tography (HPLC) using a C-18 reversed phase column ( 4.6 3 250mm) for product separation, and a mobile phase consisting of(60:40) methanol:0.05 N ammonium formate. The LC system wascomprised of a Knauer reciprocating pump, and a Beckman variablewavelength UV detector connected in series with a flow-throughb1 detector. In the LC analyses of the [11C]-L-deprenyl, unlabelledreference compounds were added, and the UV and radiochromato-grams were compared. The authentic L-deprenyl was purchasedfrom RBI, Inc.

Comparisons were made with [11C]-L-deprenyl prepared by con-ventional alkylation (34). This method involved dissolving 4 mg ofnordeprenyl in 200 mL of 3:1 dimethyl formamide:dimethyl sulfox-ide along with 300 mL of acetonitrile placed in a 1-mL volume glass

vessel. [11C]Methyl iodide was prepared in the same way asdescribed above, and cryogenically trapped in the reaction solventcontaining the substrate. The reaction vessel was then closed offand heated to 130°C for 5 min using a hot oil bath. After reaction,aliquots of the reaction vessel were analyzed by radio-HPLC analysisusing the same system as previously described.

RESULTS AND DISCUSSIONInvestigations of Acetonitrile Cluster Formation inSubcritical and Supercritical Carbon Dioxide:Acetonitrile Mixtures

The UV photolysis of dibenzyl ketone at between 250 and 350 nmwavelengths will generate a benzyl radical and the phenylacetylradical as geminate radicals through a three-step process involvingintersystem crossing (ISC) of the excited singlet state to tripletDBK prior to undergoing homolytic a-cleavage (Fig. 2).

Solution-phase laser flash photolysis experiments have demon-strated that this process occurs on a very short subnanosecond timescale (1). The phenylacetyl radical will subsequently undergounimolecular decarbonylation with reported rate constants in therange of k-CO 5 5–9 3 1026 s21 at room temperature (note thatthis process requires a significantly longer time, about 100 ns, thanthe expected lifetime of the co-solvent cage, Fig. 3) (33, 49).

Molecular dynamics simulations suggest that solvent cages onlypossess limited lifetimes of less than 10 ns in duration (37), andtherefore, should not impact on the initial photodissociation step.This has been verified through studies on the spectroscopic prop-erties of DBKs, in which no measurable solvatochromic shifts in theUV absorption maxima were detected in various supercritical fluids(36). Therefore, solvent clustering should only impact on the aboveprocesses in two ways. On the one hand, it can stabilize the benzylradical formed during the initial homolytic cleavage of DBK byproviding a labile hydrogen source on the co-solvent. The carbon-hydrogen bond strength in acetonitrile is relatively weak at 86kcal/mol (21). Hydrogen abstraction would then yield toluene as an

FIG. 2. Photolysis scheme ofdibenzyl ketone.

FIG. 3. Unimolecular decarbonylation ofthe phenylacetyl radical to the benzyl rad-ical will occur at room temperature with arate constant k-CO 5 529 3 1026 s21.

446 R. A. Ferrieri et al.

end product. Parallel studies on the decomposition of acetylperoxide in various solvent clusters has shown that methyl radicalswill abstract hydrogen to yield methane in about 10 ns, similar tothe cluster lifetime proposed by simulations (20). By analogy,hydrogen abstraction by the benzyl radical might occur on a similartime frame as the acetonitrile cluster lifetime in the present study(Fig. 4).

Likewise, solvent clustering might slow the decarbonylation rateand even stabilize the phenylacetyl radical through hydrogenabstraction to yield phenylacetaldehyde. Again, a comparison withparallel studies on acetyl peroxide decomposition under similarcircumstances indicated that the acetate radical did not abstracthydrogen from the solvent. However, the decarboxylation rate inthis instance was measured to be between 109 and 1010 s21, nearlythree orders of magnitude faster than the decarbonylation rate forthe phenylacetyl radical. Therefore, the increased stability of thephenylacetyl radical may allow it to encounter a labile hydrogensource, or at least escape the confines of the high-density regionintact. There is some validity to this later statement in that previousstudies using the reactive free radical trap 2,2,6,6-tetramethylpip-eridine-1-oxyl demonstrated that the phenylacetyl radical could besuccessfully trapped outside this region (41) (Fig. 5).

Therefore, with minimal caging effects imposed by the solventcluster, the benzyl radical produced from the initial a-cleavage ofDBK would have a greater opportunity to recombine with a benzylradical produced outside the cage to yield dibenzyl. Given thispremise, ratios of toluene-to-dibenzyl should then reflect changes inthe degree to which the acetonitrile clustering is impacting on theabove processes (Fig. 6).

Figure 7 shows a plot of the relative cage effect as a function ofsystem pressure. Our observations in the pressure studies indicatedthat at certain pressures, localized density augmentation caused byco-solvent cluster formation could have a significant impact on thechemical processes. We observed an exponential rise in the relativecage effect from zero at about 50 bar to a maximum value of 0.47 at160 bar pressure. We also observed an exponential fall-off in theeffect at pressures above 160 bar with an apparent leveling atabout 0.25.

It was necessary to ascertain whether or not the binary mixture ofacetonitrile and carbon dioxide behaved as a single-phase liquid atthe temperature and pressures used for these studies or existed insome vapor-liquid equilibrium. In addition, we needed to knowwhether any experimental conditions satisfied the critical parame-ters of the binary mixture such that it behaved as a supercriticalfluid. Estimations of the liquid-vapor critical loci for acetonitrileand carbon dioxide mixtures have been reported using a peak-shapemethod which has proven to be reasonably accurate (7, 56). Thecritical mixture curve is the locus of mixture critical points

spanning the entire range of composition of the binary mixture(35). Zeigler et al. (56) reported continuous phase behavior betweenthe critical points of the pure components for acetonitrile:carbondioxide, and concluded that it followed Type I phase behavior asdescribed by van Konynenburg and Scott (51). A type I mixture isformed when two solvents are miscible as liquids. Reighard et al.also verified this behavior using visual observation of the vapor-liquid phase boundary in a variable-volume view cell (40). Theywere able to construct a three-dimensional surface describing thephase behavior between acetonitrile and carbon dioxide as afunction of pressure, temperature, and composition. A conclusiondrawn from this work suggests that the binary mixture would notbehave as a single-phase liquid or supercritical fluid below 100°Cuntil a pressure of about 150 bar or more was attained. This wouldmean that for our cage studies carried out at 95°C, and below thispressure limit we were most likely experiencing some vapor-liquidequilibrium and not a homogeneous fluid.

Several numerical methods have also been discussed for calcu-lating critical parameters. Yonker and Smith used the methodoutlined by Prausnitz and co-workers to estimate critical loci (8, 39,55). We reconstructed part of this curve using the same method andpresented the separated critical parameters plotted as a function ofacetonitrile mole fraction in Figure 8A and 8B. The endpoints ofeach plot depict the critical pressure and critical temperature of thepure components. Pure carbon dioxide has a critical temperatureand pressure of 31.3°C and 79 bar, whereas pure acetonitrile has acritical temperature and pressure of 274.7°C and 47.7 bar (32, 54).From these plots we determined that the critical pressure andcritical temperature were of the order of 150 bar and 97°C. Thiswould mean that the sharp rise in the cage effect reflects a region ofpressure and temperature in our studies where the binary mixture isin transformation, but quickly becoming supercritical, and highlycompressible. At its critical point, the cage effect appears to bemaximized. In the supercritical region well above this point,however, we observed a fairly constant effect, although certainlynot optimal relative to the region of pressure about the criticalpoint.

To determine whether the behavior observed in the present studyis typical of a cage effect, we needed to look at what has been donepreviously. There is an abundance of experimental data (5, 13–15,23, 26–29, 31, 46) and theoretical evidence (4, 11, 37, 38) verifyingthe fact that many organic co-solvents will aggregate in and aroundthe supercritical region. For example, Kim and Johnston (31) testedacetone, methanol, ethanol, and n-octane as co-solvents in super-critical CO2 at concentrations ranging from 1% to 5.25%, fluidpressures ranging from 80 bar to 300 bar and fluid temperature at35°C. They utilized solvatochromic shifts in the UV absorptionmaxima of phenol blue, which was sensitive to the localized

FIG. 4. Formation of toluene through hy-drogen abstraction by the benzyl radical.

FIG. 5. Possible stabilization of the phen-ylacetyl radical through hydrogen abstrac-tion to yield phenylacetaldehyde.

Solvent Cages in PET Radiotracer Synthesis 447

co-solvent density about it. Typical behavior found was that localcompositions of co-solvents were substantially elevated in andaround the critical point (80 bar), averaging about a 6.5% concen-tration for a 1% bulk loading concentration. This effect decreasedsignificantly with increasing pressure, but was still measurable evenat 300 bar. They also reported a slight decrease in this overall effectas the initial bulk loading concentrations were increased, and beganto see subtle differences in local composition depending on thenature of the co-solvent. This observation may have been due todifferences in the nature through which these co-solvent moleculeswere aggregating (i.e., hydrogen bonding versus Van der Waalsattraction).

The trend we observed in our pressure study, at least in the rangeof pressure extending up from the critical point, correlates well withgeneral co-solvent cage behavior. It is difficult to say exactly whatis happening in the subcritical region of our study, because we areunsure how such large changes in the vapor-liquid equilibriumwithin this region impact on diffusional processes. The highcompressibility of the fluid near the critical region resulting insolvent clustering about solute molecules can also have drasticsecondary effects on the local solvent polarity, and dielectricstrength that can impact on the macroscopic thermodynamicproperties of the system, as well as on the rates of any chemicalreactions occurring (36). It is impossible to discern whether thechanges we observed reflect changes solely in the diffusionalprocesses, or changes in some other factors influencing chemistry.

The question remains whether the behavior seen in the presentstudy is typical for supercritical CO2/acetonitrile mixtures. Insurveying the literature, we found that only the work of Roberts etal. (42, 43) addressed the topic of diffusion controlled reactions insupercritical CO2/acetonitrile mixtures. Two processes were inves-tigated in this instance including the triplet-triplet annihilation

process of benzophenone (3BP), as well as the self-terminationreaction of benzyl radicals produced from the photocleavage ofDBK. Reaction kinetics were measured by laser flash photolysis atvarious pressures and temperatures above the critical point. Inter-estingly enough the authors reported that there was no evidence ofenhanced cage effects due to increased local density about thediffusive encounter pairs probed. This was not to imply that solventcages were altogether absent. Rather, the co-solvent environmentwas only able to maintain its integrity as a cage or aggregate on atime scale (possibly nanoseconds) shorter than the diffusion processunder investigation. The authors did suggest, however, that theirinability to observe enhanced solute-solute or solute-solvent effectsmay have been due to the extreme dilution of the 3BP or benzylradicals in their studies.

There is one obvious difference in the work of Roberts et al. (42)and the present work in that we used considerably higher concen-trations of acetonitrile. This difference might affect the size orlifetime of the co-solvent cluster to an extent that it could provideobservable changes in the diffusional processes as reflected by theimpact on the radical recombination reaction. Whether thesechanges reflect changes in solute–solute or solute–solvent interac-

FIG. 6. Formation of dibenzylthrough benzyl radical recombi-nation.

FIG. 7. Relative magnitude of acetonitrile cage effect mea-sured as a function of system pressure. The dashed linerepresents the critical pressure zone, calculated for the sol-vent composition and temperature, above which the solventmixture behaves as a homogeneous supercritical fluid.

FIG. 8. Calculated critical pressure (A) and critical tempera-ture (B) as a function of acetonitrile mole fraction for binarymixtures of acetonitrile in carbon dioxide.

448 R. A. Ferrieri et al.

tions are beyond the scope of this work, but certainly warrantfurther investigation.

Synthesis of L-Deprenyl Using Methyl Iodideand Nordeprenyl

The ability to control the diffusional processes of two dilutereactants in a bimolecular reaction can have enormous impact onmicroscale chemistry related to organic synthesis with carbon-11.For this reason we tested the efficacy of carrying out microscalereactions between methyl iodide and nordeprenyl, where thereaction being investigated involved methylation at the nitrogenatom site of the substrate (Fig. 9).

Systematic studies were carried out where a number of conditionsof reaction were varied. These conditions included, temperatureand UV exposure, solvent composition, system pressure, and methyliodide concentration. Results from these studies are shown inFigures 10–12, respectively.

Effect of UV Light and Reaction Temperature

In the light exposure/temperature studies (Fig. 10), all otherreaction conditions were kept constant. These fixed parametersincluded: maintaining a constant pressure of 275 bar; using fixedamounts of reactants, including 0.38 mmol of methyl iodide and0.9 mmol of nordeprenyl; using a fixed concentration of acetonitrileco-solvent set at 21%; and keeping the reaction time constant at10 min. Three key observations were made from these first studies.First, no significant levels of product until a reaction temperature(.95°C) near the critical temperature of the fluid mixture was

reached. The operating pressure of the system was in excess of thecritical pressure of the mixture so that we can assume this chemistryoccurred within the supercritical regime at or above 95°C. Sec-ondly, it is clear that UV light enhanced the methylation reactionrate. This rate increased fourfold under the influence of UV lightwhen reactions were carried out at 95°C. However, UV light wasnot the only factor driving the reaction. In the absence of UV light,deprenyl product yields were still substantial, and showed continuedgrowth with temperature. Thirdly, we observed a second reactionproduct identified as TMBEA using GCMS.

Mechanistic Considerations

The observation of UV enhancement of the methylation reactionrate, and a second reaction product in the spectrum raised questionsregarding reaction mechanism. On the one hand, was the UV lightpromoting radical reactions? Evidence in the literature suggests thatthe wavelength of light emitted by a mercury arc lamp is sufficientlylow to break the carbon-iodine bond, and produce methyl radicals(2, 3, 45). We tested this premise in our system by exposing a traceof methyl iodide and toluene to the same reaction conditions. Ifmethyl radicals were produced, then they would abstract the labilehydrogen from the toluene’s methyl group, thereby creating a sourceof benzyl radicals. Recombination of these radicals would then leadto dibenzyl product, a good signature for the formation of methylradicals since dibenzyl is not seen as an impurity constituent intoluene. We observed levels of dibenzyl commensurate with theamounts of methyl iodide introduced into the cell, suggesting thatmost of the methyl iodide is dissociated under our reaction condi-tions. Additional evidence supporting a radical mechanism is the

FIG. 9. L-Deprenyl can besynthesized through the N-methylation reaction ofmethyl iodide with L-a-meth-yl-N-2-propynyl phenethyl-amine (nordeprenyl).

FIG. 10. Effect of ultraviolet(UV) light and reaction celltemperature on the deprenylproduct yield arising from theN-methylation reaction be-tween 0.38 mmol methyl io-dide and 0.9 mmol of norde-prenyl. System pressure was250 bar with 21% bulk load-ing of acetonitrile co-solventin supercritical carbon diox-ide.

Solvent Cages in PET Radiotracer Synthesis 449

observation that the deprenyl product yield was essentially the sameat 95°C and 125°C with the UV light on, but without light, showeda substantial increase from 12% to 42%. Typically, radical reactionsare not temperature dependent, whereas nucleophilic substitutionsare (19, 24, 25).

A second mechanistic question regards the nature of TMBEAformation. Clearly, scission of the nordeprenyl carbon skeleton

must occur at some stage of this product’s formation. However,when we subjected both nordeprenyl and deprenyl samples to thesame reaction conditions without methyl iodide, we were able toquantitatively recover all of the samples suggesting that thesecompounds were not sensitive to any photolytic processes withinthe wavelength range emitted by the UV source. Additionally, asample of L-deprenyl and methyl iodide subjected to the sameconditions failed to yield TMBEA. Therefore, bond scission mostlikely occurred as a consequence of the initial methyl attack at thenitrogen atom site of the substrate, and was likely due to internalenergy transfer as a result of this reaction. What we were unsure ofwas whether this scission involved the carbon-nitrogen bond(channel A), or the carbon-carbon bond (channel B) adjacent tothe acetylenic group (Fig. 13).

To answer this question, we performed two deuterium-labelingexperiments. In the first, [D3]methyl iodide was reacted withnordeprenyl at 95°C and 300 bar pressure using a 21% concentra-tion of acetonitrile in CO2. The TMBEA product was analyzed byGC-MS to measure the extent of deuterium incorporation, as wellas the labeling position within the molecule. Results showed thatboth methyl groups attached to the nitrogen atom were completelydeuterated, suggesting that TMBEA arises from double methylation.Subsequent to this, we carried out a parallel experiment under thesame reaction conditions using methyl iodide and a,a-[D2]-norde-prenyl. Results from this experiment showed no deuterium incor-poration in the TMBEA product. Therefore, TMBEA arises from adouble methylation reaction involving carbon-nitrogen bond scis-sion, which occurs at some point after the first methylation step.Interestingly, TMBEA is also observed in those high-pressurereactions carried out in the absence of UV light. Therefore, it is notessential that the mechanism for TMBEA formation involve radicalspecies. For example, 1,2-hydrogen migration across the carbon-nitrogen bond prior to scission of that bond would yield N,a-

FIG. 11. (A) The effect of system pressure on individualproduct yields: L-deprenyl (●); and N,N,a-trimethyl benzyl-ethyl amine (TMBEA) (Œ). Product yields were only relativeand were measured as the ratio of product peak areas tonordeprenyl peak area. (B) The ratio of deprenyl-to-TMBEAor single-to-double methylation as a function of system pres-sure. Reactions were carried out for 10 min at 95&C, with theultraviolet light on and using 0.38 mmol methyl iodide and0.90 mmol nordeprenyl in a 21% concentration of acetonitrileco-solvent.

FIG. 12. Effect of the amount of methyl iodide reacted on thesingle-to-double methylation product ratio. Reactions werecarried out for 10 min at 95&C, with the ultraviolet lamp onusing 0.9 mmol nordeprenyl in a 21% concentration ofacetonitrile co-solvent in supercritical carbon dioxide at 275bar.

450 R. A. Ferrieri et al.

dimethyl benzylethyl amine, a stable molecule under normal cir-cumstances. However, in the presence of methyl iodide it should bejust as susceptible to methylation as the starting nordeprenylcompound was.

Effect of Solvent Composition

In the co-solvent composition study the acetonitrile concentrationwas varied from 10% to 72%, while maintaining all other reactionconditions constant. Both L-deprenyl and TMBEA products peakswere integrated on the GC chromatogram, and reported as a ratioof the product peak area to nordeprenyl peak area in Table 1. Wealso included a peak area ratio of L-deprenyl-to-TMBEA to monitorthe degree of the single-to-double methylation reaction. Error barsrepresented standard deviations on at least three separate reactions.Results showed little or no change in the deprenyl product yieldwith 10% and 21% acetonitrile loading, but then increased some-what from 0.28 to 0.46 with 72% acetonitrile. Contrary to this,TMBEA increased substantially from about 0.05 to 0.30 over thesame co-solvent concentration range giving a net effect where thedouble methylation reaction appeared to benefit more with thehigher co-solvent concentration. This is interesting in view of thefact that at 72% acetonitrile concentration, the system was sub-critical. Referring back to Figures 8A and 8B, the reaction condi-tions satisfied the critical pressure requirement of 120 bar, but werewell below the critical temperature requirement of 250°C for themedium to behave as a supercritical fluid. Using our pressure data inFigure 7 for guidance, we observed that the cage effect fell-offsharply once the reaction conditions of that study could no longersustain the supercritical state of the binary mixture. Regardless ofwhether the reaction variable is pressure, as it happened to be inthat instance, or some other variable like co-solvent concentrationis unimportant. The outcome should be the same. The net cage

effect should diminish as the solvent environment falls below itscritical point. Therefore, increased diffusion of constituents wouldbe expected, with increased concentrations of acetonitrile co-solvent, resulting in a net decrease in the single-to-double methyl-ation product ratio.

Effect of Pressure

In subsequent studies, the operating pressure of the system wasvaried from 62 to 310 bar, while maintaining all other conditions ofreaction constant. Figure11A illustrates the pressure dependence ofthe deprenyl and TMBEA relative product yields. A very slight risein both products’ yields was noticed above 150 bar, although therewas not sufficient data to say whether this rise was significant.However, as the pressure decreased below this limit, we observedlarge increases in both yields. Interestingly, the double methylationchannel exhibited greater pressure dependence, increasing nearlyfivefold relative to the single methylation channel’s increase of 1.5.This difference in dependence becomes even more obvious inFigure 11B, where a plot of the single-to-double methylationproduct as a function of pressure exhibited a maximum at about 150bar, the critical point for the binary mixture. The maximum in theproduct ratio coincided with the maximum cage effect seen in ourearlier pressure studies. This would imply that under conditionswhere the influence of the co-solvent cage on reactants was thegreatest (that is, where diffusional processes were most impeded),single methylation was most favored. However, both product yieldsappeared to benefit the most from reactions carried out in thesubcritical region where a cage effect was observable, but notoptimized. As pointed out earlier, drastic changes in fluid compress-ibility around the critical region can impose drastic changes in themacroscopic thermodynamic properties of the system, which in turn

FIG. 13. Possible mechanisms for the for-mation of N,a,a-trimethyl benzylethyl-amine (TMBEA). Channel A depicts ni-trogen-carbon bond scission allowingsecondary methylation. Channel B depictscarbon-carbon bond scission without sec-ondary methylation.

TABLE 1. Effect of Solvent Composition on Product Distributiona

Solvent composition Relative yieldb Single-to-doublemethylation

product ratiocCarbon dioxide Acetonitrile Deprenyl TMBEA

0.90 0.10 0.32 0.05 6.40.79 0.21 0.28 6 0.05 0.09 6 0.02 3.11 6 0.120.28 0.72 0.46 6 0.08 0.30–0.06 1.53 6 0.10

TMBEA 5 N,N, a-trimethyl benzylethyl amine.a Reactions were carried out for 10 min at 95°C, 275 bar pressure and with the UV lamp on using 0.38 mmol methyl iodide and 0.90 mmol nordeprenyl.b Relative yields based on GC integrated peak areas where each product area was divided by the nordeprenyl peak. While these yields were not absolute,they did provide information on systematic trends since the amount of nordeprenyl used was a constant factor.c The single-to-double methylation product ratio was based on the peak area ratio of deprenyl-to-TMBEA.

Solvent Cages in PET Radiotracer Synthesis 451

can affect reaction rate constants. These effects may or may not bedirectly related to changes in the diffusional processes.

Effect of the Amount of Methyl Iodide

When considering application of this strategy to labeling organiccompounds with carbon-11, we should not have to be concernedabout controlling the degree of multi-methylation, because theamount of mass associated with [11C]methyl iodide in a no-carrier-added tracer synthesis is somewhat smaller than the amounts ofcarrier methyl iodide used in these bench studies. Thus one wouldassume the opportunity for secondary methylation would be greatlydiminished. We tested this premise in a final study where theamount of methyl iodide was varied relative to all other reactionconditions remaining constant. Results in Figure 12 show the ratioof deprenyl product to TMBEA product, or single-to-double meth-ylation, as a function of the amount of methyl iodide. This amountwas varied from 0.15 to 9.6 mmol. As expected, single-to-doublemethylation ratios increased nearly 16-fold as the amount of methyliodide decreased.

Application to the Synthesis of [11C]-L-Deprenyl

The utility of this strategy hinges on whether one can demonstratean improvement in radiochemical yield using no-carrier-added[11C]methyl iodide, and less starting materials. Using the apparatusdescribed in Figure 1, we tested the method using 5 mCi of[11C]methyl iodide. Figure 14 illustrates a comparison of tworadio-HPLC traces obtained from both the caged reaction (traceA), and a conventional PET synthesis methodology (trace B).Reaction conditions for the caged experiment were selected basedon what knowledge was gathered on the conditions that optimizedthe deprenyl product yield, while providing some degree of a cageeffect. Obviously, we wanted to operate in the subcritical regionwhere the methylation reaction appeared to proceed with greatestefficiency. However, too low a pressure might promote secondarymethylation, although the results in Figure 12 suggest that thisshould not be a concern. We arbitrarily selected an operatingpressure of 83 bar only because the pressure curves indicatedreasonable yields of deprenyl in this range, as well as provided a highsingle-to-double methylation ratio. The other conditions includedreacting 5 mCi of no-carrier-added [11C]methyl iodide with 100 mgnordeprenyl for 10 min at 95°C with the UV light on. In addition,the acetonitrile concentration was 20% relative to CO2. Theconditions for the conventional synthesis were described previously.Results showed that greater than 90% radiochemical yield of[11C]-L-deprenyl (based on a balance of carbon-11 radioactivitymade before and after the reaction) could be obtained through thismethod using 40 times less starting substrate than in the conven-tional PET tracer synthesis. One should note that Trace A wasexpanded considerably relative to Trace B in order to show thesmall radioactive peak eluting earlier in the chromatogram that wasidentified as [11C]TMBEA. It would appear that significantlycleaner chemistry, as well as a higher radiochemical yield could beachieved using the high-pressure reaction methodology rather thanthe conventional alkylation method. More importantly, thesestandards can be attained using far less starting material. Thisfeature alone should provide significant improvement in the “qual-ity” of radiotracer made for PET by minimizing the amount ofcontaminant labeling substrate present in the final formulation.

It should be noted that final product specific activity was not anissue in this study. Although [11C]deprenyl specific activity was less

than 1 Ci/mmol at EOB, this activity was low only because theprecursor specific activity was not optimal due to the short targetirradiation used to make [11C]carbon dioxide.

CONCLUSIONS

The present work has demonstrated that molecular cages formed byacetonitrile co-solvent cluster formation in supercritical carbondioxide fluid and in subcritical liquid carbon dioxide can havesufficient structural integrity, as well as a long enough lifetime toaffect diffusional processes, and impact on chemistry occurringbetween potential reactants trapped within them. For 15% concen-trations of acetonitrile in carbon dioxide at 95°C, we observed amaximum cage effect at around 150 bar pressure, which coincidedwith the estimated critical loci for this binary mixture. We alsoobserved that a cage effect could be sustained throughout thesupercritical regime, but at a somewhat lower level. This behaviorappears to be similar to that of other co-solvent systems reported inthe literature, although this cage effect is the first reported for abinary mixture of acetonitrile and carbon dioxide. The cagebehavior we observed in the subcritical region for this mixture isinteresting, but impossible to comment on at this time owing to thelack of parallel studies below the critical point.

This work has also demonstrated an important application of thisphenomenon in the preparation of [11C]-labeled radiotracers. It waspossible to label [11C]-L-deprenyl in a high-pressure environment

FIG. 14. Comparison of radio-high performance liquid chro-matography (HPLC) traces. Trace A represents the high-pressure reaction of no-carrier-added [11C]methyl iodide at95&C, 83 bar in 20:80 acetonitrile:carbon dioxide with 100 mgof nordeprenyl. Trace B represents the conventional syn-thetic procedure using 4 mg of nordeprenyl reacted withno-carrier-added 11CH3I at 130&C in 200 mL of 3:1 dimethylformamide:dimethyl sulfoxide and 300 mL of acetonitrile.

452 R. A. Ferrieri et al.

where solute caging caused by co-solvent clustering was observed.We also demonstrated that it is possible to carry out an alkylationwith [11C]methyl iodide at a significantly higher radiochemicalyield using this strategy and with far less starting material than withconventional synthetic practices.

This research was carried out at Brookhaven National Laboratory undercontract DE-AC02-98CH10886 with the U.S. Department of Energyand supported by its Office of Biological and Environmental Research,and also supported by the National Institutes of Health, GrantNS-15380.

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