Neighboring Effect in Fragmentation Pathways of Cage

Article pubs.acs.org/JPCA

Neighboring Effect in Fragmentation Pathways of Cage Guanylhydrazones in the Gas Phase Marina Šekutor, Zoran Glasovac,* and Kata Mlinarić-Majerski* Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička c. 54, P.O. Box 180, 10 002 Zagreb, Croatia S Supporting Information *

ABSTRACT: ESI−MS/MS investigation of the mono- and bis(guanylhydrazone) derivatives 1−5 based on adamantane and pentacycloundecane (PCU) skeleton was described. Elimination of neutral guanidine is the most abundant reaction channel in the case of 2,4-adamantyl and PCU derivatives 4 and 5, while the elimination of CH2N2 fragment is preferred for other compounds. This was attributed to the cage opening of adamantane or PCU skeletons in the former case leading to the formation of the cyclohexyl- or cyclopropylcarbinyl carbocation stabilized by the conjugation with the guanylhydrazone subunit. The main fragmentation pathways observed experimentally were analyzed by using DFT calculations. All investigated bis(guanylhydrazone)s formed dications and their abundances were found to be proportional to the interguanidine distance in the considered ions. Calculation of the first and the second proton affinities supported qualitative interpretation of the dication abundance. Close contact of two guanidine subunits is thus confirmed to be crucial in determining preferential fragmentation pathway and to suppress formation of the dication.

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INTRODUCTION Guanidinium ions are known to be a biologically active structural motif present in a number of pharmacophores.1 They are also a part of many important building blocks in cells, such as the amino acid arginine and the purine base guanine present in the nucleic acid. From the technological point of view, guanidines are interesting as strong neutral organic bases.2 Their high basicity is attributed primarily to very efficient stabilization of the protonated form by Y-conjugation3−5 and high affinity toward formation of the hydrogen bonds with surrounding molecules.6 In such systems, Coulombic interaction with the surroundings is expected to be smaller than for the systems with highly localized charge. Moreover, Jungwirth and co-workers found that two guanidinium cations can form stable stacked pairs within a small cluster with three water molecules.7 This feature of the guanidinium cation has been recently employed in the formation of relatively stable dications in the gas phase starting from bis(guanidine)s8 by using electrospray ionization (ESI) mass spectrometry.9 High basicity, suitable geometry, and the relative ease in formation of the bis(guanidine) dications make guanidine an important fragment in molecular recognition; most often expressed through hydrogen bonding to various oxo-functional groups and anions.6b,10 In this respect, guanylhydrazones are not the exception. Recently, bis(guanylhydrazone)s were found to be potent stabilizers of G-quadruplex by intercalation supported by binding of the guanidine subunit to phosphate groups.11 However, authors noticed low cell uptake of the drug and suggested its structural modification to increase lipophilicity. For this purpose, usage of polycyclic cage © 2013 American Chemical Society

compounds such as adamantane and pentacycloundecane could be beneficial.12 These compounds are versatile structural moieties that have found numerous applications, from biological chemistry13 to material sciences.14 Furthermore, their rigid skeleton allows for a design of compounds with welldefined geometry and a control of positioning of the guanidine subunit. Recently, bis(guanylhydrazone) derivatives containing the adamantane skeleton were successfully tested as butyrylcholinesterase inhibitors.15 The docking studies performed in that study showed significant difference in binding to the enzyme depending on the structure of the active compound. Until now, no systematic study of such compounds has been performed. Detailed insight in the intrinsic properties and reactivity of such guanylhydrazone derivatives would give us a powerful tool for future design of potent biologically active guanylhydrazones. It was, thus, our aim to combine the structural motifs of rigid cage compounds and two guanylhydrazone moieties in order to study intrinsic properties and the gas-phase reactivity in a series of novel guanylhydrazones with particular emphasis on the importance of the guanidine−guanidine interactions. The underlying feature of this series of compounds is a varying spatial distance between two guanidine substituents which is the largest for the adamantane substituted in 2,6-positions and somewhat smaller for 2,4-substituted adamantane, and for the Received: November 8, 2012 Revised: February 15, 2013 Published: February 17, 2013 2242

dx.doi.org/10.1021/jp311049f | J. Phys. Chem. A 2013, 117, 2242−2252

The Journal of Physical Chemistry A

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derivative 5 was prepared in a similar manner starting from the pentacyclo[5.4.0.02,6.03,10.05,9]undecane-8,11-dione.16 Preparation of 8,11-Bis(N,N′-guanidino)iminopentacyclo[5.4.0.02,6.03,10 .05,9 ]undecane 5. Pentacyclo[5.4.0.0 2,6.03,10.05,9]undecane-8,11-dione (174 mg, 1.0 mmol) was dissolved in 10 mL of absolute ethanol, and then Na2CO3 (106 mg, 1.0 mmol) and aminoguanidine hydrochloride (221 mg, 2.0 mmol) were added into the solution. The reaction mixture was refluxed for 5 h and filtered after cooling, and the solvent was evaporated, yielding the crude product. The obtained solid was washed with ethyl acetate/ethanol solvent mixture yielding PCU guanylhydrazone 5 as a mixture of 3 isomers, 226 mg (79%). 1H NMR (300 Hz, CD3OD, TMS, δ/ppm): 1.58−1.67 (m, 1H), 1.89−1.97 (m, 1H), 2.54−2.65 (m, 2H), 2.86− 2.97 (m, 3H), 3.06−3.16 (m, 1H), 3.58−3.63 (m, 1H), 3.72−3.83 (m, 1H). 13C NMR (75 Hz, CD3OD, TMS, δ/ppm): 38.0, 38.1, 38.9, 39.0, 39.2, 41.5, 41.6, 42.1, 42.2, 44.0, 44.5, 46.3, 46.6, 46.7, 47.5, 47.6, 52.1, 52.8, 160.8, 166.0, 166.3; IR (KBr, ν̃/cm−1): 3402(br), 3277(br), 3168(m), 2926(w), 1683(m), 1667(s), 1558(m), 1011(w). HRMS− MALDI m/z [M + Na]+ calcd for C13H18N8Na, 309.1547; found, 309.156. ESI−MS/MS Experiments. ESI−MS/MS experiments were performed using an Agilent Technologies 1200 series HPLC system equipped with a binary pump, a vacuum membrane degasser, an automated autosampler and injector interfaced with 6410 triple quadrupole mass spectrometer with electrospray ionization source (ESI) (Agilent Technologies Inc., Palo Alto, CA) operating in a positive ion mode. Tandem mass spectrometry (MS/MS) was used for the study of fragmentation pathways of compounds 1−5. Solvent used for the analysis was methanol and the collision gas was nitrogen. Deuteration experiments were performed using a methanol/D2O mixture. All data acquisition and processing was performed using Agilent MassHunter software. Computational Details. All calculations were performed by Gaussian03 program package.17 Geometrical optimization of the

8,11-PCU derivative, it approximates the length of one C−C bond. Herein, we describe the ESI mass spectra of guanylhydrazones 1−5 (Figure 1) and the fragmentation patterns of the

Figure 1. Structures of the cage guanylhydrazones 1−5.

generated mono- and dications. In addition to the experimental results, we also present theoretical determination of the first and the second proton affinities of the studied compounds that facilitated an elucidation of the fragmentation pathways and thereby helped us to ascertain the neighboring effects.

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EXPERIMENTAL AND THEORETICAL METHODS

Synthesis. Adamantane-substituted guanylhydrazones 1-4 were synthesized by coupling the respective ketone with aminoguanidine hydrochloride in the presence of an equimolar amount of Na2CO3 according to our previously published procedure,15 and the PCU-

Scheme 1. Main Fragmentation Pathways of Adamantyl Guanylhydrazonesa

a

The calculated activation energies and reaction enthalpies are given in kcal mol−1. 2243



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Figure 2. Transition state structures obtained for three competitive fragmentation pathways of 1H+ optimized at the B3LYP/6-311+G(d,p) level of theory.

mol−1 in spite of the different chemistry taking place within.26 Elimination of ammonia must be preceded by a [1,3]-hydrogen shift8,23b,c with activation barrier of ca. 48 kcal mol−1 (pathway A, Scheme 1). On the basis of the calculation results, this step is a rate-determining step along pathway A. Similar finding has been observed earlier as well.8 On the other hand, the fragmentation via pathway C has no need for preorganization by proton migration. Elimination of CH2N2 fragment proceeds in a two-step process. It starts with preorganization of the structure which is endothermic by 14 kcal mol−1. The barrier for the first step is estimated to be 15 kcal mol−1 and it is kinetically insignificant. The second step includes C−N bond breaking which is identified as the rate-determining step with the barrier of 46 kcal mol−1. The bond cleavage is strongly coupled with the proton transfer from the carbodiimide part toward hydrazone amino nitrogen (TSB2, Figure 2) preserving charge on the adamantylhydrazone. Previous investigations showed absence of pathway B for the permethylated derivatives8 except if the fragmentation processes were initiated by the electron impact ionization procedure.24 This confirms the role of the proton migration in activation of the specific reaction channels. Thus, accessing the intermediate structure suitable for the elimination of ammonia appears to be the critical step that determines the low population of the corresponding reaction channel while this is not the case for the extrusion of CH2N2. Qualitatively the same results were also obtained at the BMK/6-311+G(3df,2p)//B3LYP/6311+G(d,p) level of theory (Table S1 in the Supporting Information). The TS structures for all rate-determining steps are shown in Figure 2. ESI−MS/MS of 2 shows a fragmentation pattern similar to that of 1 with elimination of carbodiimide (pathway B) more favored over the other paths although upon increasing the collision energy pathway C becomes almost as important as pathway B. Elimination of ammonia is still the least favorable process. It is noteworthy to add that parallel to guanidine elimination, formation of the ion with m/z = 60 was also observed. We assigned the structure of this ion to guanidinium cation based on the deuteration experiments. When the pentadeuterated parent ion (m/z (MD+) = 212; 5D) was mass selected and fragmented, approximately equal amounts of two ions at m/z = 64 and 65 appear. The latter corresponds to the hexadeuterated guanidinium cation which implies deuteration of the guanidine with some residual D2O after the bond cleavage. Since we have used MeOH/D2O mixture as a solvent, appearance of both D5 and D6 guanidinium cations was not a surprise.

structures and verification of the minima and transition state structures were done at the B3LYP/6-311+G(d,p) level of theory.18 Electronic energies were also recalculated using BMK19/6-311+G(3df,2p) approach at the B3LYP optimized geometries. Enthalpies were obtained by correcting electronic energies for unscaled zero-point vibrational energy and work term (Ew = RT) as implemented in Gaussian03. Reaction paths for the fragmentations were also analyzed by IRC calculations20 starting from transition state structures toward minima in both directions. All energies are given in kcal mol−1 (1 kcal mol−1 = 4.184 kJ mol−1). The structures at the stationary points were generated by MOLDEN 5.0.21

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RESULTS AND DISCUSSION ESI−MS/MS spectra of guanidine 1 showed monocation MH+ (m/z = 207) as the basic and practically only peak. Fragmentation of the MH+ using CID approach with nitrogen as the collision gas resulted in elimination of neutral NH3, CH2N2, and CH5N3 as the primary processes (pathways A, B, and C, respectively, Scheme 1). These typical fragmentation pathways have also been observed by others using different ionization techniques.22−24 The main fragmentation pathway for MH+ was found to be elimination of CN2H2 fragment that can correspond either to carbodiimide or cyanamide, while the reaction channels that encompasses extrusion of ammonia or guanidine were shown to be much less abundant. The first two processes (A and B) were also present in phenylenebis(guanidine)s, described by Schröder and co-workers,8 but in opposite relative abundance. The pathway C has been identified as one of the main fragmentation pathways in the protonated arginine,23 but not in phenylenebis(guanidine)s8 which is presumably a consequence of low stability of the phenyl cation that would be formed upon extrusion of guanidine. The nitrenium cation formed by extrusion of guanidine can be stabilized by ring enlargement of the adamantane subunit resulting in a cyclic 4-azatricyclo[4.3.1.13,8]undec-3-ene structure (1ctc+).25 Tricyclic cation formed in this way can undergo [1,3]-hydrogen shift associated with ring-opening ending up in the 1cH+ as the most stable structure with m/z = 148. Reaction enthalpies and activation energies (Ea) of each fragmentation process were calculated in order to rationalize the obtained results. All three pathways (formation of the 1aH+−1cH+) were predicted to be endothermic. Pathway B is thermodynamically strongly favored over pathways A and C by 14.8 and 10.0 kcal mol−1, respectively. Additionally, elimination of guanidine is slightly less endothermic than elimination of ammonia (ΔErel = 4.8 kcal mol−1). In line with the thermochemistry of these two processes, pathway C is slightly more populated than pathway A. The calculated barriers for the rate-determining steps in pathways A and C fall within 1.5 kcal 2244



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Figure 3. Ion fragmentation products at collision energies of Elab = 15 eV for compounds 1, 3, 4, and 5 that demonstrate an almost complete absence of CH2N2 elimination (pathway B) for compounds 4 and 5.

Figure 4. Normalized abundance of precursor and product ions for different fragmentation pathways of guanylhydrazonium cations 1H+ and 3H+5H+. Pathway A for 5H+ is corrected for the abundance of the ion at m/z = 253.

the monoprotonated 2,4-derivative is observed already at Elab = 5 eV implying that a close proximity of the second guanidine subunit activates this process. The ratio of these two pathways is similar to that found for arginine at higher energies.23a Similar changes in relative abundance of product ions but lesser in extent were also observed for PCU derivative 5 (Figure 4d). In fragmentation of compound 5, pathway A is even slightly more abundant than pathway C at lower energies. It is interesting to note that an ion at m/z = 253, corresponding to the loss of two molecules of ammonia, was also observed and this pathway was used to correct the total fragment ion

Introduction of the second guanylhydrazone unit affects the fragmentation of the monocation and the relative intensities of the daughter ions depend on the position of the substituent and the structure of the hydrocarbon subunit (Figures 3 and 4). The effect is least pronounced in the case of 2,6-adamantyl derivative 3 where pathway C becomes more abundant relative to that for 1. On the other hand, in a 2,4-disubstituted adamantyl derivative 4, we have observed an almost complete absence of pathway B. Pathway C thus became the most abundant reaction channel, while the elimination of ammonia remains a low intensity process. Elimination of guanidine from 2245



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Scheme 2. Calculated Reaction Enthalpies for Elimination Processes from Bis(guanylhydrazone)s 3−5

to be similar to the ones calculated for the fragmentation of 1H+.27 Comparison of the ΔE values given in Schemes 1 and 2 shows that elimination of ammonia via pathway A becomes less endothermic for all three bis(guanylhydrazone)s while the same holds for the elimination of guanidine from 4H+ and 5H+. Observed change in thermodynamics of pathway A arises from the fragmentation of the neutral guanidine subunit rather than the protonated one. On the other hand, obtained reaction energies for pathway C clearly show neighboring effect of one

abundance for pathway A (Figure 4d). In contrast to 2,4adamantyl derivative 4, in 5 one can still notice a weak signal at m/z = 245 [M + H - CH2N2]+, although this fragmentation pathway is strongly disfavored with respect to other pathways. To rationalize these trends, we calculated reaction energies for the guanidine extrusion from bis(guanylhydrazone)s 3−5. For this purpose, extrusion of NH3 and CH2N2 was assumed to occur from the neutral guanidine subunit as suggested earlier.8 On the other hand, guanidine was eliminated from the protonated side. The barriers for these processes were assumed 2246



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Figure 5. Structures of 3ctcH+, 4cH+, and 5cH+ optimized at B3LYP/6-311+G(d,p).

its almost complete disappearance. On the basis of the calculation results (Scheme 2, path ii) we assume that the sequential loss of CH2N2 and NH3 fragments from the monocations might take place simultaneously with the onestep elimination of guanidine. It is easy to conceive that elimination of ammonia from 4bH+and 5bH+ assisted with the proton donation by the neighboring guanidinium subunit (Scheme 2, path ii) is exothermic and consequently favorable process which might be responsible for their almost complete loss from the CID spectra.30 Recent MS investigations on arginine and its fragments showed occurrence of the CH2N2 extrusion when no proton-donating functional group is present (fragmentation of the 4-guanidinobutan-1-al).31 On the other hand, no trace of this process was evidenced upon fragmentation of the unsubstituted arginine.31 Additionally, MS experiments conducted on the arginine containing oligopeptides with no free carboxylic groups also produced ions by losing the CH2N2 fragment.32 These data also support our assumption that a close contact of the reaction center with a good proton-donating functional group suppresses pathway B in our experiments. We have also analyzed whether low population of the A channel could be due to a similar secondary fragmentation process as described above. Extrusion of ammonia from 3H+− 5H+ led to structures 3aH+−5aH+ bearing carbodiimide and guanidine moieties. CID experiments conducted on these ions selected from the source indicated the importance of the neighboring guanidine group on the fragmentation mechanism. While in 3aH+ elimination of cyanamidyl radical (CHN2, m/z = 41) took place, producing thus daughter ion at m/z = 219, elimination of fragments with both m/z = 41 and 42 occurred upon dissociation of 4aH+. The same experiment that was conducted starting from 5aH+ resulted in the loss of the CH2N2 fragment, while the loss of fragment with m/z = 41 was not observed. Since no daughter ions with m/z = 219 were observed upon CID experiments on either 3H+ or 4H+, we have concluded that no secondary fragmentation processes are responsible for the low abundance of pathway A. The same experiments also show that in the case of 5, elimination of guanidine could also aggregate two stepwise eliminations of ammonia and CH2N2 fragments but to a lesser extent. Fragmentation of the Dications. MS spectra of dications derived from bis(guanylhydrazone)s 3−5 were also investigated. Analogously to previous investigations,8 all considered bis(guanidine)s tend to form dications by monoprotonation of each guanidine subunit. The dication/monocation intensity ratio in the source spectra decreases from 3 to 5 amounting to 1.50, 0.98, and 0.06 in 3, 4, and 5, respectively. Upon examining the optimized structures of three possible conformations of protonated bis(guanidine)s 4H+ and 5H+ (Figure 6), we have found that the dication signal intensity is qualitatively

guanylhydrazone subunit on the fragmentation of the other. Extrusion of the neutral guanidine from derivative 3 leads to the formation of a ring opened “cyano” product 3ctc+ (Figure 5). Thermodynamics of this process is unfavorable with respect to the other two competitive pathways but is similar to the corresponding one in 1H+. Again, low abundance of pathway A is most likely determined by the preorganization step which has a relatively high barrier and a rigid TS structure. The other results presented in Scheme 2 predict the elimination of guanidine to be thermodynamically the most favored process in the case of bis(guanylhydrazone)s 4 and 5 producing the ring opened structures (Figure 5). This finding agrees well with the experimental results at higher energies presented in Figure 4.28 Geometrical optimization of 3ctcH+, 4cH+, and 5cH+ cations led to structures presented in Figure 5 as the most stable minima. The optimized structures of other fragmentation products are given in Supporting Information (Figure S3). Formation of these structures is due to heterolytic scission of one of the bonds adjacent to hydrazone imino group initiated by the guanidine extrusion. Electron density shifts from the cleaved bond toward the “imino” part of the molecule, thus producing a cyano group. Consequently, a lack of electron density remains on the carbon atom in the α-position relative to the guanylhydrazone fragment and this state is stabilized by conjugation. Similar “imino to cyano” transformation was observed earlier in solvolytic experiments.29 Proposed mechanism for the cage opening is presented in Scheme 3. Scheme 3. Proposed Mechanism for the Formation of 4cH+ Ion

In the case of derivative 5, additional four-membered ring contraction takes place resulting in a formation of the cyclopropylcarbinyl cation where the cation center is additionally conjugatively stabilized by the imino double bond of the second guanylhydrazone moiety (5cH+, Figure 5). Absence of the ions formed by pathway B, in the case of 4H+ and 5H+, is somewhat surprising regarding the thermochemistry of this process given in Scheme 2. Applied calculations predict this process to be only 4 (4H+) and 7 (5H+) kcal mol−1 more endothermic than pathway C and practically isoenergetic to pathway A. Although one can expect significant decrease in the abundance of pathway B, calculated energies do not explain 2247



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Figure 6. Distance between central guanidine carbon atoms (d(Cgv−Cgv)/Å) in optimized structures of three conformations of protonated bis(guanidine)s 4H+ and 5H+. Analogous interguanidine distances in dications are given in parentheses.

Scheme 4. Primary Fragmentation Channels for Dications 3H22+ and 4H22+

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Table 1. First and Second Gas-Phase Basicities (GB) and Proton Affinities (PA) of Various Isomers of Guanylhydrazones Calculated at the B3LYP/6-311+G(d,p) Level of Theory molecule 1 1H+ 3 3H+ 3H22+ 4 4H+ 4H22+ 4 4H+ 4H22+ 4 4H+ 4H22+ 5 5H+ 5H22+ 5 5H+ 5H22+ 5 5H+ 5H22+

isomer

in−in in−in in−in in−out in−out in−out out−out out−out out−out up−up up−up up−up up−down up−down up−down down−down down−down down−down

H(298)/au (G(298)/au) −648.89114 −649.27363 −907.20889 −907.59304 −907.89787 −907.20751 −907.60649 −907.87841 −907.20819 −907.59398 −907.88908 −907.20841 −907.59359 −907.89311 −944.07438 −944.46269 −944.75635 −944.07459 −944.46229 −944.75936 −944.07449 −944.46272 −944.75715

(−648.94293) (−649.32835) (−907.27373) (−907.66117) (−907.96725) (−907.27223) (−907.66987) (−907.94714) (−907.27325) (−907.66200) (−907.95779) (−907.27339) (−907.66153) (−907.96183) (−944.13827) (−944.52944) (−944.82384) (−944.13869) (−944.52876) (−944.82687) (−944.13850) (−944.53004) (−944.82469)

PA(298 K)/akcal mol‑1

Hrel(conf)/bkcal mol‑1

GB(298 K)/c kcal mol‑1

Grel(conf)b/kcal mol‑1

241.5 − 242.5 192.8 − 251.8 172.1 − 243.6 186.7 − 243.2 189.4 − 245.2 185.8 − 244.8 187.6 − 245.1 186.5 −

− − − − − 0.0 0.0 0.0 −0.4 7.9 −6.7 −0.6 8.1 −9.2 0.0 0.0 0.0 −0.1 0.3 −1.9 −0.1 0.0 −0.5

235.6

− − − − − 0.0 0.0 0.0 −0.6 4.9 −6.7 −0.7 5.2 −9.2 0.0 0.0 0.0 −0.3 0.4 −1.9 −0.2 −0.4 −0.5

236.8 185.8 243.2 167.7 − 237.7 179.3 − 237.3 182.2 − 239.2 178.5 238.5 180.8 − 239.4 178.6 −

a

PA(BH+) = H°(B) - H°(BH+) + H°(H+); H°(H+) = (5/2) RT. bRelative enthalpies of different conformers for investigated bis(guanidine)s 4 and 5 and their mono and bis protonated forms with “in−in” or “up−up” conformers being the reference structures. cGB(BH+) = G°(B) - G°(BH+) + G°(H+); G°(H+) = −6.28 kcal mol−1.

monocations what is a thermodynamically favored process. Although we cannot discriminate between these two processes, two important trends were observed. Tendency toward formation of the cage opened carbocation by the elimination of guanidine, as the one described for the monocations, is evident. Second, if two guanidine subunits are close enough, the proton or hydrogen atom transfer from one guanidine subunit to another could take place leading to the elimination of a neutral CH2N2 fragment. This process cannot occur in 2,6derivative 3aH+ due to the large interguanidine distance producing thus an ion with m/z = 219 only. This ion appears during fragmentation of the dication 3H22+ but not from the monocation 3H+, most likely due to the higher energy of the intermediate 3aH+ obtained by the charge separation process. Appearance of both signals in the case of 4H22+ could be rationalized in a similar way. It has been shown experimentally that compound 4 exists as a mixture of three isomers in the solution.15 Since it is known that under ESI−MS conditions structures of the ions may closely resemble those in the solution,34−38 we expect all three isomeric structures of 4H+ (Figure 6) to contribute to the formation of the ion at m/z = 260. Regarding interguanidine distances, one can expect “out− out” conformation to dominate in the dication formation and to act similarly to 3H+ upon fragmentation. On the other hand, “in−in” conformation will be prone to a “proton assisted” type of fragmentation resulting in the elimination of neutral CH2N2 rather than cyanamidyl radical. A relatively large abundance of the ion at m/z = 218 indicates that a certain degree of conversion to “in−in” conformation possibly took place during the first step of the fragmentation process. The results obtained for 5H22+ perfectly follow this reasoning. No daughter ion that would correspond to the

proportional to the interguanidine distance in the considered bis(guanylhydrazone)s. This was attributed to the Coulombic repulsion of the two charged guanidine subunits which is expected to be the largest in 5H22+. It should be noted, however, that interguanidine distance in “in−in” isomer of 4H22+ is even shorter than in 5H22+ and most likely it does not contribute in dication formation. Fragmentation of dications 3H22+, 4H22+, and 5H22+ led to either elimination of NH4+ in a charge separation process or to the loss of neutral NH3 (Scheme 4). Additionally, formation of the dication daughter ion (m/z = 109.6) by elimination of a neutral guanidine was unequivocally confirmed only in the case of 3H22+. Depending on the starting dications, appearance of signals with m/z at 218 or 219 was also observed, indicating loss of fragments with m/z = 60 or 59, respectively. These ions can be formed by elimination of either guanidinium cation ([CH6N3]+) or guanidine radical cation ([CH5N3]•+) in one step, or by sequential loss of ammonium cation and either cyanamide or cyanamidyl radical (Scheme 4). In the case of 3H22+, only ion at m/z = 219 and no 218 was formed, while in the case of 5H22+ the opposite situation was found.33 Upon fragmentation of 2,4-derivative 4H22+ both ions (m/z = 218 and 219) were observed. CID experiments conducted on the ion at m/z = 260 (4aH+) produce both 218 and 219 daughter ions (see above), thus supporting existence of the stepwise process at least in parallel to the one-step elimination of the guanidinium ions. Abundance of the ion at m/z = 219 at low energies (Elab = 0 eV) is higher than the m/z = 260 indicating that this ion could be a primary fragmentation product. Since dications are highly energetic ions, one may expect homolytic (N−N) bond cleavage to occur upon CID experiments. Furthermore, such a process leads to the formation of two 2249



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therefore not surprising that the “in−in” conformer of 4 has the lowest second PA, while the highest value was obtained for the “out−out” conformer where interguanidine distance is more than 8 Å. The second PA of 5 is similar for all three conformers and is lower than for the bis(guanidine) “out−out” conformer of 4 for ca. 2 kcal mol−1. The second PAs of all examined bis(guanylhydrazone)s are predicted to be slightly higher than for phenylenebis(guanidine) (PA2,0K = 177 kcal mol−1).8 In that work, Schröder and co-workers generated dications starting from a MeOH solution of bishydrochloride salts. In contrast to that, we have used a MeOH solution of neutral compounds. It is therefore expected that the intensity of ions in our experiments depends on the degree of protonation by the solvent. In this case, subtle differences in the second proton affinities will have more impact on the intensity of dications. Consequently, low PA2 of 5H+ could be one of the main reasons for difficulties in dication 5H 2 2+ formation in the gas-phase under the applied experimental conditions.

elimination of cyanamidyl radical is observed whatsoever. Optimized geometries of three isomers of monocation 5H+, closely resembling structures of the dication 5H22+, show similar interguanidine distances, and it decreases upon extrusion of the ammonium cation. Consequently, hydrogen transfer can be expected irrespectively of the actual ion structure. Basicity of the Investigated Guanylhydrazones and Their Monocations. To qualitatively estimate influence of the charge−charge interaction between two guanidine subunits on the basicities of guanylhydrazones, we have calculated the first and the second gas-phase basicities (GB) and proton affinities (PA) for all bis(guanidine) derivatives (Table 1). In this respect, monoguanidine serves as the reference compound in which no charge−charge interaction is present. Both Gibbs energies and enthalpies show the same trend with respect to conformational changes of the investigated structures (Table 1) and will not be discussed separately. Comparison of the enthalpies calculated for all three conformers of 4H+ shows stabilization of the “in−in” conformer of monocation with respect to the other two conformers. This could be ascribed to the formation of two hydrogen bonds which allow efficient electron density drift from the neutral to the protonated guanidine subunit in a similar manner as in proton sponges.39 In neutral and bisprotonated structures 4 and 4H22+ no such stabilization is possible. In these cases, two electron rich or electron poor subunits tend to move away from each other to minimize unfavorable interactions. As a consequence, “in−in” conformers of 4 and 4H22+ become the least stable isomers. Inspection of the three most stable conformations of 5H+ structure reveals that the approximate distance between guanidine subunits is larger than in “in−in” conformation of 4H+. Besides that, no intramolecular hydrogen bonds are present in either of the 5H+ conformations due to practically parallel orientation of the guanidine planes. Therefore, only “through-space” interaction of cation-π-electron density between guanidine subunits in 5H+ is possible. Consequently, “up−down” monocation, with the largest interguanidine distance, is predicted to be slightly less stable than other two conformers. However, due to the similar distance between guanidine groups (ca. 6 Å) in all three isomers, the difference in enthalpies among the conformers is practically negligible. In spite of the absence of direct hydrogen bonding, two guanidine subunits are close enough that one can still expect proton/ hydrogen atom transfer between two guanidine groups during the fragmentation processes. The qualitative energetic considerations are corroborated by calculating the first and second PA and GB values for all bis(guanidine)s (Table 1). Again, trend in GBs completely follows the trend in PAs so we will limit our discussion on the calculated PA values. The lowest first PA (PA1) was obtained for 2,6-bis(guanidine), while the largest first PA was obtained for “in−in” 2,4-derivative 4. Calculated PA1(4) of 252 kcal mol−1 is by ca. 10 kcal mol−1 higher than for 3 and places it in the category of superbases. This is not surprising since in the “in−in” 2,4-derivative two intramolecular hydrogen bonds (IMHBs) can be established. Such beneficial influence of IMHB to PAs is well-known40 and has been applied previously in designing new superbases.41,42 The same reasoning could be applied in analysis of the second PAs (PA2). In this case interactions between two guanidine fragments in dications are repulsive in nature. It is

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CONCLUSIONS Conducted ESI−MS/MS investigation of several bis(guanylhydrazone)s showed importance of the guanylhydrazone−guanylhydrazone neighboring effect on the activation of the observed fragmentation pathways. Preferred reaction channel is dictated by close proximity of the guanylhydrazone subunits in derivatives 4 and 5 and their charge. In addition to the expected elimination of ammonia and carbodiimide, extrusion of guanidine was also observed. In the case of monocations, the loss of neutral guanidine was supported by the formation of either cyclohexyl or cyclopropylcarbinyl carbocation additionally stabilized by conjugation with a guanylhydrazone subunit in thermodynamically favorable processes. On the other hand, protonated guanylhydrazone fragment cannot stabilize (or even destabilizes) such intermediates which changes the preference of the dications for the loss of either charged guanidinium cation or neutral guanidine. While in 2,6-adamantyl derivative 3 elimination of the neutral guanidine is preferred, in other two bis(guanylhydrazone) derivatives elimination of guanidinium cation is favored. Calculated first and second proton affinities confirm interactions of two guanidine subunits through the establishing of intramolecular hydrogen bonds or through the “charge− charge” or “charge-dipole” electrostatic interactions along the interguanidine distance. These processes significantly affect energetics of the protonation. The highest first and the lowest second proton affinity for the “in−in” isomer of 4 indicate a different abundance of this isomer in monocation and dication signal. Furthermore, inability of 5 to achieve the conformation with the sufficiently separated guanidine fragment leads to relatively low second proton affinities for all isomers and consequently to a very weak signal for the dication. This dication is expected to be of relative high energy and consequently with high affinity toward anions.

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ASSOCIATED CONTENT

* Supporting Information S 13

C NMR spectra of the mixture of isomers for bis(guanylhydrazone)s 4 and 5, energies of all investigated structures calculated using BMK and B3LYP density functionals, structures of the products formed by fragmentation pathways A and B and optimized geometries of all investigated 2250



Article

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structures given in Cartesian coordinates. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: (Z.G.) [email protected]; (K.M.-M.) majerski@ irb.hr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Croatian Ministry of Science, Education and Sports for financial support of this study (Grant Nos. 0980982933-2911 and 098-0982933-2920). We would also like to thank the Computing Center of the University of Zagreb (SRCE) for allocation of computer time on the Isabella cluster as well as Lidija Brkljačić for recording the ESI−MS/MS spectra.

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