INTERROGATION OF A SONOGASHIRA CROSS ...

Nucleosides, Nucleotides and Nucleic Acids, 30:168–184, 2011 C Taylor and Francis Group, LLC Copyright ! ISSN: 1525-7770 print / 1532-2335 online DOI: 10.1080/15257770.2010.548844

INTERROGATION OF A SONOGASHIRA CROSS-COUPLING OF 8-BROMOGUANOSINE WITH PHENYLACETYLENE ON AMBERLITE: EVIDENCE FOR Pd/Cu ION BINDING AND PROPAGATION OF Pd/Cu NANOPARTICLES

Andrew G. Firth,1,2 Karen Wilson,1 Christoph G. Baumann,2 and Ian J. S. Fairlamb1 1 Department of Chemistry, University of York, Heslington, York, United Kingdom 2 Department of Biology, Area 10, University of York, Heslington, York, United Kingdom The reactivity of Amberlite (IRA-67) base “heterogeneous” resin in Sonogashira cross-coupling of 8-bromoguanosine 1 with phenylacetylene 3 to give 2 has been examined. Both 1 and 2 coordinate to Pd and Cu ions, which explains why at equivalent catalyst loadings, the homogeneous reaction employing triethylamine base is poor yielding. X-ray photo-electron spectroscopy (XPS) has been used to probe and quantify the active nitrogen base sites of the Amberlite resin, and post-reaction Pd and Cu species. The PdCl2 (PPh3 )2 precatalyst and CuI cocatalyst degrade to give Amberlite-supported metal nanoparticles (average size ∼2.7 nm). The guanosine product 2 formed using the Amberlite Pd/Cu catalyst system is of higher purity than reactions using a homogeneous Pd precatalyst, a prerequisite for use in biological applications. !

Keywords

Nucleoside; alkyne; palladium; cross-coupling; inhibition; catalyst recycling

INTRODUCTION Palladium-mediated cross-coupling reactions[1] are among the most powerful and widely used synthetic transformations for the formation of carboncarbon and carbon-heteroatom bonds.[2] The Sonogashira cross-coupling reaction[3]—the union of a Csp atom with a Csp2 atom, mediated by a Pd0 Received 7 November 2010; accepted 14 December 2010. Current affiliation for K. W: School of Chemistry, Cardiff University, Park Place, Cardiff, CF10 3AT, UK. The authors are grateful to the BBSRC (CASE studentship award to A. G. F.), the University of York, and Replizyme Ltd. (Dr. K. Darley) for funding these studies. The authors thank the Royal Society for a generous equipment grant and funding for a research fellowship (I. J. S. F.), and Meg Stark for technical assistance with the electron microscopy. Address correspondence to Ian J. S. Fairlamb, Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK. E-mail: [email protected]; Christoph G. Baumann, Department of Biology, Area 10, University of York, Heslington, York, YO10 5YW, UK. E-mail: [email protected]; Karen Wilson, School of Chemistry, Cardiff University, Park Place, Cardiff, CF10 3AT, UK (current address). E-mail: [email protected]

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catalyst and CuI cocatalyst, to give vinyl and aryl substituted acetylenes—is an extremely versatile transformation.[4] It has been employed successfully to prepare natural products,[5] pharmaceutical agents,[6] and advanced materials and bioprobes.[7] Given these extensive applications, new reaction conditions continue to emerge, particularly reactions that can be conducted on solid-support allowing the metal catalysts/cocatalysts, for example, Pd0 and CuI, and byproducts to be removed easily from the desired product. On a general note, improvements to these methodologies facilitates broader use in both academic and industrial arenas.[8] An effective Sonogashira cross-coupling protocol using the solid-supported base, Amberlite IRA-67 (a weakly basic gel-type resin with tertiary amine functionality), rather than NEt3 or NHEt2 , was reported in 2005.[9] Under these apparent heterogenized conditions,[10] it was shown that an acetate-protected propargyl amine could be effectively coupled with 8-bromo-2# -deoxyguanosine in high yields; such products are very difficult to attain in pure form using traditional reaction conditions. We have reported in preliminary form the Sonogashira cross-coupling of unprotected[11] multifaceted organohalide substrate[12] 8-bromoguanosine 1 with phenylacetylene 3 affords 2, which requires that a low palladium loading and an optimized PdII/CuI ratio is used (Scheme 1).[13]

SCHEME 1 Sonogashira cross-coupling.

A problem for the cross-coupling of haloguanosines is the inherent binding affinity of Pd and Cu ions to guanine moieties vide infra (through N 7 and O6, or N 1 and O6; Figure 1).[14] These coordination modes are known to play a significant inhibitory role in the Suzuki cross-coupling (arylation) of haloguanosines in aqueous media, as demonstrated comprehensively by Shaughnessy and Western,[15] which is an issue for other cross-couplings of these substrates, especially unprotected derivatives. Therefore, it was hypothesized that the use of an amine derivatised solid “heterogeneous” base could generally improve the couplings of 1 with terminal acetylenes, simplifying the work-up procedure by eliminating the need for addition of a “homogeneous” base and sequestering the metal catalyst/cocatalyst from the product, whose purity ought to be consequently improved.

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FIGURE 1 Coordination of purine bases to Pd and Cu.

In this article, we have examined the coordinating ability of compounds 1 and 2 to Pd and Cu ions, the findings of which highlight the importance of the ratio of Pd and Cu for successful Sonogashira cross-couplings of 1.[13] A detailed investigation concerning the application of Amberlite IRA-67 in the synthesis of C-modified guanosine nucleosides is then reported. The solid-base resins, from pre- and post-reaction, and the reaction products, have been analyzed by x-ray photoelectron spectroscopy (XPS)[16] and elemental analysis to assess product purity and efficiency of the solid base in trapping halide by-products and sequestering metals from the reaction. The potential for subsequent recycling of the basic resin, and the recovered nanoparticulate palladium species, from these reactions has been further examined.

RESULTS AND DISCUSSION The application of Amberlite IRA-67[17] (hereafter, Amberlite) in Sonogashira cross-coupling was first compared with a conventional homogeneous reaction using NEt3 . A slightly modified procedure to that reported[9] was employed using 10 mol% of PdCl2 (PPh3 )2 (precatalyst) instead of 10 mol% Pd(PPh3 )4 or 10% Pd/C. Efficient cross-coupling of 1 with 3 was observed to give 2 in 83% yield (Equation (1), see Experimental section for full details) with negligible metal contamination detected by elemental analysis. By contrast, the equivalent homogeneous reaction (Equation (2)), using NEt3 as the base, gave 2 in only 20% yield.[13] The product 2 was found to be very impure (from elemental analysis: 1.63 wt% Pd; 1.28 wt% Cu), due to the


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uptake of Pd and Cu. This outcome led us to evaluate the coordination of Pd and Cu ions to both 1 and 2.

1

Pd(PPh3 )Cl2 (10 mol%), Cul(20 mol%), ≡ −Ph (2 equiv.) 3 −−−−−−−−−−−−−→ Amberlite IRA-67, DMF, [1] = 0.12 M, 110◦ C, 18 h

1

Pd(PPh3 )Cl2 (10 mol%), Cul(20 mol%), ≡ −Ph 2 equiv. 3 −−−−−−−−−−−−−→ Et3 N (3 equiv.), DMF, [1] = 0.12 M, 110◦ C, 18 h

2 (83%)

2 (20%)

(1)

(2)

Guanosine Coordination to Pd0 and PdII: Implications for Sonogashira Cross-Couplings Given our findings with the homogeneous reaction run at higher catalyst loadings we examined the coordination ability of both 1 and 2 toward Pd0, as this or a related species is likely the active species in the catalytic cycle of the Sonogashira reaction.[18] A stable and convenient Pd0 complex possessing PPh3 ligands is Pd(η2-dba)(PPh3 )2 (dba = E,E-dibenzylideneacetone), formed by reaction of Pd2 dba3 and PPh3 (1:2, Pd:PPh3 ) in a variety of solvents.[19] Reaction of Pd(η2-dba)(PPh3 )2 (1.0 equiv.) with 1 in DMF at 25◦ C (1.0 equiv.) was monitored by 31P NMR spectroscopy (202 MHz; Figure 2, spectrum A). Several phosphorus signals are observed in spectrum A (Figure 2). Compound 1 does not completely displace dba from the coordination sphere of Pd(η2-dba)(PPh3 )2 —the broad signals at δ 26.2 and 24.2 ppm represent the two nonequivalent phosphorus signals from Pd(η2-dba)(PPh3 )2 . Two new singlets at δ 17.2 and 17.5 ppm are attributed to the geometric cis- and trans-Pd0(PPh3 )(N -7,O-6)S complexes (ratio = 0.8:1). A broad signal at δ −2.75 ppm is observed and attributed to exchanging PPh3 which is slow on the NMR timescale. Three other phosphorus signals were detected at δ 21.7, 20.2, and 25.9 ppm. The latter is OPPh3 [20]; the other two phosphorus signals are not characterized. An identical reaction was run in DMSO-d 6 , and a similar spectrum obtained (spectrum not shown). The two signals at about δ 17 ppm were found to be in a ratio of 1:1, indicating that a coordinating solvent molecule (in this case DMSO), in addition to one PPh3 (absence of 31P spin-spin coupling), is involved in stabilizing this species. The addition of three equivalents of 1 with respect to Pd(η2-dba)(PPh3 )2 (in DMSO-d 6 ), under identical conditions, increased

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FIGURE 2 Pd0 coordination of 1 and 2: A) 31P NMR spectrum of Pd(η2-dba) (PPh3 )2 and 8bromoguanosine (1:1) in DMF at 25◦ C (35 mM); B) as for A with an additional three equivalents of 1 using DMSO instead of DMF; C) 31P NMR spectrum of Pd(η2-dba)(PPh3 )2 and 2 (1:1) in DMF at 25◦ C (35 mM).

the intensity of the signals at δ 17.2 and 17.5 ppm, without affecting their ratio, at the expense of Pd(η2-dba)(PPh3 )2 (spectrum B, Figure 2). The reaction of 2 with Pd(η2-dba)(PPh3 )2 in DMF is shown in spectrum C (Figure 2), which again shows the formation of two new phosphorus signals at ca. δ 17 ppm. These experiments confirm that N ,O-coordination from 1 or 2 can effectively compete with dba-coordination for Pd0. This is a remarkable finding, particularly given the fact that over 90 equivalents of additional PPh3 are required to completely displace dba from the coordination sphere of Pd(η2-dba)(PPh3 )2 to give Pd0(PPh3 )3 .[19] In the reaction shown in Scheme 1 and Equations (1) and (2), the absence of dba would cause Pd0 complexes containing 1 and 2 to predominate in the reaction mixture. We have also determined that 1 competes with phosphine coordination in PdCl2 (PPh3 )2 in DMF in the absence of NEt3 and CuI (note: avoiding PdII to Pd0 reduction). The four resonances observed at δ 21.6, 21.5, 21.0, and 20.5 ppm, which do not exhibit 31P spin-spin coupling, are likely PdCl2 (PPh3 )(N-7-1) or PdCl2 (PPh3 )(N -7,O-6)S type complexes. In addition, we observe PdCl2 (PPh3 )2 at δ 25.5 ppm. Infrared (IR) spectroscopic studies provide further evidence for coordination of the exocyclic O-6 atom from the guanine moiety—relative to 1 ν(C O) was reduced by 70 cm−1, appearing at 1630 cm−1, which is similar to that reported for other PdII guanosine complexes.[21] Finally, it is interesting to note that on addition of CuI (1.0 equiv.) all the phosphorus signals, with the exception of δ 25.5 ppm, disappear. This key experiment indicates that CuI is effectively competing with PdII coordination to guanosine.

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Examination of Sonogashira Cross-Couplings on Amberlite There are several advantages associated with the use of Amberlite in Sonogashira cross-couplings. The Amberlite, possessing dimethylamino groups on the resin surface, will sequester Pd and Cu species, also effectively competing with guanosine coordination. Use of Amberlite facilitates removal of these metal residues by filtration on reaction completion. In addition, the dimethylamino groups can act as base sites, meaning that other bases, for example, NEt3 or Et2 NH, need not be used. Overall, the use of Amberlite allows metal recovery and simple removable of the protonated base resin from post-reaction samples. The modified physical form of the resin (from Equation (1)) was subsequently probed in detail by XPS and electron microscopy, to provide a detailed insight into the degree of Pd and/or Cu uptake by the resin, and changes in the surface properties. In addition, in order to assess the recycling potential of the Amberlite, XPS was also used to determine whether all the free base sites in the resin were protonated. The surface composition of the pre- and post-reaction Amberlite resins was first evaluated (Table 1) (a survey of the complete XP spectra are shown in Figure 3). The C, O, and N regions for the parent material were consistent with that expected for Amberlite, which is an acrylic-divinylbenzene resin functionalized with tertiary amino substituents. Post-reaction, adsorbed Cl, Pd, I, Br, and Cu can be detected on the surface of the resin; however, there is a negligible change in the C, O, and N content of the material. This suggests efficient recovery of the organic product 2 from the resin, and that there is no leaching of amine functionality. These results are consistent with the bulk elemental analysis data which indicate that the Pd and Cu content of the resin is 0.4 and 0.5 wt%, respectively. It is interesting to note that no P was detected on the resin suggesting that “ligand-free” Pd has bound to the Amberlite surface. High resolution XPS was employed to probe the nature of the Amberlite bound Pd. Pd 3d spectra (shown in Figure 4a) reveal two peaks at 334.9 and 340.2 eV, which are consistent with the spin-orbit split 3d5/2 and 3d3/2 binding energy for Pd0, respectively. A small high binding energy shoulder present on both

TABLE 1 Surface composition of Amberlite pre and post-reaction Sonogashira cross-couplings of 1 with phenylacetylene 3 to give 2 (by XPS) Surface composition/atomic% Amberlite Pre-reaction Amberlite Post-reaction 10 mol% Pd, 20 mol% Cu

C

N

O

Cl

I

Br

Pd

Cu

72.6 69.1

11.2 11.7

15.9 16.2

0.3 0.7

— 0.3

— 1.2

— 0.5

— 0.3

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FIGURE 3 Survey XP spectra of Amberlite pre and post-Sonogashira reaction.

peaks at 337 and 342 eV also suggests the presence of some higher oxidation state Pd species, possibly PdII. The N 1s spectrum reveals a single N environment at 399.3 eV for the parent Amberlite, which is consistent with the expected amine environment (Figure 4b). Post-reaction, a new state evolves at 401.8 eV, indicative of tertiary amine protonation to form a quaternary ammonium salt. This new high binding energy component comprises ∼19% of the total N 1s signal, suggesting the surface contains only ∼2.2 atomic % reactive surface tertiary amines, which form the ammonium salt. This is also consistent with the total amount

FIGURE 4 a) Pd 3d XP spectra and b) N 1s XP spectra of Amberlite pre and post-reaction, and postreaction after reuse in a recycling experiment.

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of halide detected on the surface (∼1.4 atomic%), suggesting a large proportion of the amine groups are either inaccessible or unreactive towards HX formed during synthesis of 2. The presence of some free base sites within the resin, and high concentrations of adsorbed Pd0 and CuI, led us to evaluate recycling (initially without basification of the resin). In the recycling experiment, the black post-reaction Amberlite solid resin was added to a DMF solution containing 1 and 3 and heated to 110◦ C for 18 hours. However, this gave 2 in very poor yield (7%), which suggests the remaining nitrogen sites in the Amberlite resin are inaccessible. This hypothesis was confirmed by addition of three equivalents of NEt3 to the post-reaction Amberlite which increased the yield of 2 to 43%. Therefore, Pd0 and CuI species bound to the resin can be activated to promote a modest cross-coupling, provided that additional base is added. XPS analysis of the reused material (Table 2) revealed the Pd and Cu content was virtually unchanged, suggesting the supported Pd/Cu moieties are stable, while the bromide content rose from 1.15 to 4.82 atomic %. It is also interesting to note that the proportion of N present as NR2 H+ increases slightly (Figure 4b), which would be consistent with additional binding of bromide anion. To assess whether the presence of residual surface NR2 groups is due to their inaccessibility, Amberlite was reacted with aqueous HBr to determine the limiting number of amine groups that could be protonated. Similar observations were made, with only 50% of the amino (NR2 ) groups converted to NR2 H+ groups (Figure 5). A previous study[22] established that the loading of dimethylamino groups on the surface of Amberlite was 3.8 mmol/g (by titration with HCl). A value of 2.48 (±0.07) mmol/g was found in this study for the Amberlite. Based on the XPS results there should be 4.4 mmol/g of NR2 groups titratable by HBr, which is largely consistent with these bulk titrations. The morphology of the Amberlite resins were subsequently investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM revealed that the pre-reaction resin morphology is quite different to that observed post-reaction (Figures 6 and 7). The surface of the fresh resin has deep invaginations (Figure 6), which are clogged with material in the post-reaction resin (Figure 7). The XPS data shows the postreaction resin has accumulated Pd, Cu, Br, and I. Based on this XPS data, we TABLE 2 Comparison of resin composition after first use and reuse Surface composition/ atomic % After first use After reuse

C

N

O

Cl

I

Br

Pd

Cu

69.1 67.1

11.7 11.3

16.2 15.9

0.7 —

0.3 0.2

1.2 4.8

0.5 0.4

0.3 0.2

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406

404

402

400

398

Binding Energy / eV FIGURE 5 N 1s XP spectrum of Amberlite treated with HBr(aq) .

FIGURE 6 SEM image of Amberlite pre-Sonogashira reaction.

FIGURE 7 SEM image of Amberlite post-Sonogashira reaction.

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FIGURE 8 A) TEM image of Amberlite post-reaction showing the metal containing nanoparticles; scale bar = 50 nm. B) Particle size distribution in nanometers (the histogram was generated using 14 bins; range of 0 to 8 nm, in KaleidaGraph; N = the total number of particles counted).

postulate that the material clogging these surface invaginations is composed of these metals and halides. The formation of metal-containing nanoparticles[23] on the post-reaction Amberlite was revealed by TEM (Figure 8a). The nanoparticles are very well dispersed, with diameters in the range of 1 to 8 nm (Figure 8b). This observation supports the hypothesis that metal-containing particles are taken up by the resin, and suggests the reactions are being catalysed by leached metal species (most likely homogeneously, given the high reaction temperatures used here and highly polar solvent, DMF). The latter point is also supported by the modest catalyst/cocatalyst recycling that can occur upon re-use of the Amberlite resin in a cross-coupling reaction. It is worth noting that palladium nanoparticles of similar size[24] are often prepared for immobilized systems,[25] and are able to catalyze these types of reactions.[26] The purity of product 2 was further examined by XPS, mainly to assess whether the use of Amberlite reduced the presence of contaminants (Table 3). TABLE 3 Surface composition of 2 produced using 10 mol% Pd, 20 mol% Cu using Amberlite or NEt3 as the base Surface composition/atomic%a Expected for 2 (C18 N5 O5 ) NEt3 routeb Amberlite routec a Atomic%

C

N

O

I

Pd

Cu

64.2 65.4 66.2

17.9 12.9 16.7

17.9 20.7 16.8

— 0.2 —

— 0.4 0.1

— 0.3 0.1

determinations within ± 0.1 atomic%.

b The Cu content (1.23 wt%) is similar to that observed by elemental analysis (1.28 wt%); the Pd content

is slightly higher by XPS (2.84 wt%), as compared to elemental analysis (1.63 wt%). c The Pd and Cu content was determined to be < 0.3 wt% by elemental analysis (limit of detection).

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These results indicate that following a standard reaction and workup protocol, there is significant metal and halide contamination of the product. However, the use of Amberlite, reduces the Pd, Cu, and iodide content in the final product. Hence, the use of the solid-supported base on Amberlite improves the separation of both the metals and residual halide from this type of product. CONCLUSION We have shown that compounds 1 and 2 effectively coordinate Pd and Cu ions in DMF, which we believe explains the poorly performing homogeneous reaction at high Pd loadings of about 10 mol% (Equation (2)). The Amberlite-mediated reaction, however, under otherwise identical conditions (at equivalent Pd loading), performs much better. Effectively, the Amberlite appears to compete for Pd and Cu ion binding, which is beneficial to the global efficacy of the cross-coupling process (Equation (1)). Our comprehensive physical measurements have demonstrated that excess Amberlite is required for efficient Sonogashira cross-coupling of 1 with phenylacetylene 3 to give 2 on this resin. The reason for this is that only one fifth of the resin contains active amino-base sites. Crucially, we have shown that the palladium precatalyst, PdCl2 (PPh3 )2 , and CuI cocatalyst, degrade to give metal-containing nanoparticles supported by the Amberlite IRA-67 resin. The Pd, Cu and I uptake (and retention) by Amberlite is generally excellent, and it has been established that Pd/Cu recycling is possible by treating the post-reaction Amberlite with three equivalents of NEt3 , which allows for modest recycling. Two key questions emerge from our study: 1) Why does Pd-coordination to the Amberlite resin produce a better catalyst system than when the Pd competitively binds to the substrate 1? and 2) Why coordination to 1 appears less important when working at the low Pd concentrations? Our recent published study[24c] shows that surface-catalyzed processes are operative with Pd-PVP stabilized nanoparticles in related cross-coupling processes. In that case a different reaction mechanism is operative in comparison to the anticipated homogeneous process. Therefore, we believe that 1 is interacting differently with the Amberlite-resin Pd catalyst system. This catalyst system operates more effectively than the homogeneous Pd catalyzed conditions[13] at equivalent Pd loadings, thus our observations allow us to conclude that Amberlite coordination to Pd0 is competitive with guanosine-type substrates. Finally, it is not clear why guanosine coordination is less important when working at lower Pd concentrations. It is important to note that there is a beneficial Pd/Cu ratio at the lower loadings which could ultimately alter the structure of the catalytically active species. Our findings indicate that further comprehensive mechanistic studies are required to address these questions.


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Prior to this study, the uptake of all these elements by guanosine Sonogashira cross-coupling products had not been probed comprehensively. Indeed, a survey of the literature reveals that very little elemental analysis data is available for products derived from direct cross-couplings of unprotected halogenated guanosines. The uptake of metals/halides could impede the accurate determination of fluorescent properties and limit the bio-compatibility of the cross-coupled products prepared using traditional routes. This finding is likely to be important for the derivatisation of oligonucleotides containing purine ring systems, where significant metal uptake can be seen using cross-coupling and related reactions.[27] EXPERIMENTAL General Remarks Solvents were dried where necessary using standard procedures prior to use and stored under an argon atmosphere. Nitrogen gas was oxygen-free and was dried immediately prior to use by passage through an 80 cm column containing sodium hydroxide pellets and silica. Argon gas was used directly via balloon transfer or on a Schlenk line. TLC analysis was performed routinely using Merck 5554 aluminum backed silica plates or Macherey-Nagel polygram ALOX N/UV254 aluminum oxide coated plastic sheets. Compounds were visualized using UV light (254 nm) and a basic aqueous solution of potassium permanganate or acidic DNP (dinitrophenol hydrazine). Mass spectrometry was carried out using a Fisons Analytical (VG) Autospec instrument. 1H NMR spectra were recorded at 400 MHz using a JEOL ECX 400 spectrometer or at 500 MHz on a Bruker AV 500 spectrometer; 31P NMR spectra at 202 MHz (1H decoupled) and referenced to H3 PO4 . Chemical shifts are reported in parts per million (δ) downfield from an internal tetramethylsilane reference. Coupling constants (J values) are reported in Hertz (Hz), and spin multiplicities are indicated by the following symbols: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad). 8-Bromoguanosine has been previously synthesized.[28] Stoichiometric Reactions of (1) and (2) with Pd and Cu Ions: 31 P NMR Spectroscopic Studies In a typical Pd0 experiment, in a 2 mL sample vial was added a dry DMF (1 mL) solution of Pd(η2-dba)(PPh3 )2 prepared in a glove-box by the reaction of Pd2 dba3 (10 mg, 1.09 × 10−5 mol) and PPh3 (11.4 mg, 4.37 × 10−5 mol, 2 equiv. with respect to Pd). After 10 minutes at 25◦ C, the mixture was filtered (through glass wool tightly packed in a glass pipette) into a Young’s NMR tube. This complex was run unlocked by 31P NMR spectroscopy {in a separate experiment Pd(η2-dba)(PPh3 )2 was referenced to a locked

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DMF sample containing (CD3 )2 CO}. After about 1 hour, 8-bromoguanosine 1 (7.9 mg, 2.18 × 10−5 mol, 1 equiv.) was added to the sample in a glove-box. No filtration was necessary. The sample was left to stand for 0.5 hours and then a 31P NMR spectrum was recorded. Spectroscopic data is given in the main text of this article. Similar NMR spectroscopic experiments were conducted for 8(phenylethynyl)guanosine 2 (8.4 mg, 2.18 × 10−5 mol). In a typical PdII experiment, in a Young’s NMR tube, 8-bromoguanosine 1 (8.4 mg, 2.18 × 10−5 mol) and PdCl2 (PPh3 )2 (15.3 mg, 2.18 × 10−5 mol) were dissolved in dry DMF (0.7 mL) and then left to equilibrate for 1 hour at 25◦ C). The sample was then run unlocked (to mirror the reaction conditions as closely as possible) by 31P NMR spectroscopy {in a separate experiment PdCl2 (PPh3 )2 was referenced to a locked DMF sample containing (CD3 )2 CO). CuI (4.2 mg, 2.18 × 10−5 mol, 1 equiv.) was added to the sample (under N2 ), and then a further 31P NMR spectrum recorded. Spectroscopic data is given in the main text of this article. Sonogashira Alkynylation of 8-Bromoguanosine (1) on Amberlite Base Resin A reaction tube was charged with 8-bromoguanosine 1 (0.54 g, 1.5 mmol), PdCl2 (PPh3 )2 (105 mg, 10 mol%), CuI (57 mg, 20 mol%) and Amberlite IRA-67 (1.34 g). The reaction vessel was evacuated and backfilled with N2 (3x), then dry DMF (12 mL) and phenylacetylene (306 mg, 3.0 mmol) were added. The reaction tube was sealed and placed in an oil bath at 110◦ C. After 18 hours, the reaction mixture was cooled, and filtered. After removal of solvents under reduced pressure with gentle heating (approximately 35◦ C), the crude product was purified by washing with boiling water (4 × 25 mL), ethyl acetate (3 × 25 mL), and finally diethyl ether (2 × 25 mL) to give the 8-(phenylethynyl)guanosine (2) as a light grey solid (0.47 g, 83%); mp 234–236◦ C (decomp.), ν max (DMSO)/cm−1 3421 (OH), 3328 (NH), 1695 (CO), 1639 and 1602 (NH); δH (400MHz; DMSO) 3.55 (1 H, m, C(5# ’)H) 3.65 (1 H, m, C(5# )H), 3.88 (1 H, m, C(4# )H), 4.16 (1 H, m, C(3# )H), 4.96–4.99 (2 H, m, C(5# )OH, C(3# )OH), 5.16 (1 H, d, J 5.2, C(2# )OH), 5.51 (1 H, d, J 6.4, C(2# )H), 5.89 (1 H, d, J 6.4, C(1# )H), 6.63 (2 H, s, NH2 ), 7.51 (3 H, m, Ph), 7.65 (2 H, d, J 2.0, Ph) and 10.91 (1 H, s, NH); δC (128MHz; DMSO) 61.9, 70.5, 70.9, 79.5, 85.6, 88.2, 92.6, 117.5, 120.4, 128.9, 129.4, 129.9, 131.5, 151.2, 153.9 and 156.0; m/z (FAB) 384.1299 (MH+ C18 H18 N5 O5 requires 384.1307), 384 (2.5%, MH+), 277 (14), 185 (100). Standard Sonogashira alkynylation of 8-bromoguanosine (1) with NEt3 as base A reaction tube was charged with 8-bromoguanosine 1 (0.54g, 1.5 mmol), PdCl2 (PPh3 )2 (105 mg, 10 mol%), and CuI (57 mg, 20 mol%). The reaction


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vessel was evacuated and backfilled with N2 (3x), then dry DMF (12 mL), dry NEt3 (459 mg, 4.5 mmol) and phenylacetylene (306 mg, 3.0 mmol) were added. The reaction tube was sealed and placed in an oil bath at 110◦ C. After 18 hours, the reaction mixture was allowed to cool, and then filtered. After removal of solvents under reduced pressure with gentle heating (approximately 35◦ C), the crude product was purified by washing with boiling water (4 × 25 mL), ethyl acetate (3 × 25 mL), and finally diethyl ether (2 × 25 mL) to give the 8-(phenylethynyl)guanosine (2) as a grey solid (0.11 g, 20%); mp 234–236◦ C (decomp.). The characterization data for 2 was identical to the reaction detailed above. Details of the titration study The dimethylamino group loading on Amberlite IRA-67 was measured using a HCl back-titration method.[22] Amberlite (100 mg) was added to HCl (20 mL, 0.05 M) and stirred for 24 hours. An aliquot (10 mL) of this suspension was removed and titrated against NaOH (0.05 M). Phenolphthalein was used as an end-point indicator in the titration. A blank titration was done using 10 mL of HCl that had not been treated with Amberlite. The procedure was repeated in triplicate and yielded an average loading of 2.48 (+/− 0.07) mmol/g. X-ray photoelectron spectrometry XPS measurements were performed using a Kratos AXIS HSi instrument equipped with a charge neutralizer and Mg Kα X-ray source. Spectra were recorded at normal emission using an analyzer pass energy of 20 eV and an X-ray power of 144 W, and were energy referenced to the valence band and adventitious carbon. Quantification was performed using appropriate elemental relative sensitivity factors. Scanning and transmission electron microscopy SEM analysis was performed using a JEOL 6490LV and an acceleration voltage of 5 kV. Amberlite IRA-67 (pre- and post-reaction) was mounted on a SEM stub and coated with gold/palladium using a sputter coater. The very thin (∼7 nm) heavy metal coating was applied from above. TEM analysis was performed using a FEI Tecnai G2 (equipped with a CCD camera) and an acceleration voltage of 120 kV. Amberlite IRA-67 (pre- and post-reaction) was ground up into a fine powder using a pestle and mortar. The powder was suspended in ethanol (2 mL) and left to settle (10 minutes). A small amount of solution (10 µl) was then pipetted on to TEM copper discs for viewing. The particle diameter was measured along the longest axis first, and then along the orthogonal axis. A mean value was computed from these two measurements. The histogram was generated using 14 bins (range = 0–8 nm) in Kaleidagraph (v4.1, Synergy Software, Reading, PA, USA).

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Elemental Analysis Analysis was performed using an inductively coupled plasma-optical emission spectrometer (ICP-OES, Fisons Instruments Ltd.). Amberlite IRA-67 and reaction products were decomposed using concentrated acid prior to analysis. REFERENCES 1. Negishi, E. Handbook of Organopalladium Chemistry for Organic Synthesis, Wiley InterScience, New York, 2002, vol. 1. 2. a) Heck, R.F. Palladium Reagents in Organic Syntheses, Academic Press, New York, 1985; b) Tsuji, J. Palladium Reagents and Catalysts, Wiley, Chichester, 1995. 3. Sonogashira, K. Development of Pd-Cu catalyzed cross-coupling of terminal acetylenes with sp2carbon halides. J. Organomet. Chem. 2002, 653, 46–49. 4. Chinchilla, R.; Nájera, C. The Sonogashira reaction: a booming methodology in synthetic organic chemistry. Chem. Rev. 2007, 107, 874–922. 5. Nicolaou, K.C.; Bulger, P.G.; Sarlah, D. Palladium-catalyzed cross-coupling reactions in total synthesis. Angew. Chem., Int. Ed. 2005, 44, 4442–4489. 6. Itami, K.; Yoshida, J. Multisubstituted olefins: platform synthesis and applications to materials science and pharmaceutical chemistry. Bull. Chem. Soc. Jap. 2006, 79, 811–824. 7. Selected referenecs: a) Collings, J.C.; Parsons, A.C.; Porrès, L.; Beeby, A.; Batsanov, A.S.; Howard, J.A.K.; Lydon, D.P.; Low, P.J.; Fairlamb, I.J.S.; Marder, T.B. Optical properties of donor-acceptor phenylene-ethynylene systems containing the 6-methylpyran-2-one group as an acceptor. Chem. Commun. 2005, 2666–2668; b) Zhang, X.; Bernet, B.; Vasella, A. Oligonucleotide analogues with integrated bases and backbone Part 14: synthesis and association of ethynylene-linked selfcomplementary tetramers. Helv. Chim. Acta 2007, 90, 792–819; c) Seela, F.; Zulauf, M. Synthesis of 7-alkynylated 8-aza-7-deaza-2# -deoxyadenosines via the Pd-catalysed cross-coupling reaction. J. Chem. Soc., Perkin Trans. 1 1998, 3233–3239; d) Seela, F.; Zulauf, M. Palladium-catalyzed cross-coupling of 7-iodo-2# -deoxytubercidin with terminal alkynes. Synthesis1996, 726–730. 8. Carey, J.S.; Laffan, D.; Thomson, C.; Williams, M.T. Analysis of the reactions used for the preparation of drug candidate molecules. Org. Biomol. Chem. 2006, 4, 2337–2347. 9. Garg, N.K.; Woodroofe, C.C.; Lacenere, C.J.; Quake, S.R.; Stoltz, B.M. A ligand-free solid-supported system for Sonogashira couplings: applications in nucleoside chemistry. Chem. Commun. 2005, 4551–4553. 10. Yin, L.; Liebscher, J. Carbon-carbon coupling reactions catalyzed by heterogeneous palladium catalysts. Chem. Rev. 2007, 107, 133–173. 11. The use of unprotected nucleosides in cross-coupling reactions remains relatively rare, see: Crisp, G.T.; Gore, J. Palladium-catalysed attachment of labels with acetylenic linker arms to biological molecules. Tetrahedron 1997, 53, 1523–1544, and reference 12. For a general review on the Pdcatalyzed reactions of nucleosides, see: Agrofoglio, L. A.; Gillaizeau, I.; Saito, Y. Palladium-assisted routes to nucleosides. Chem. Rev. 2003, 103, 1875–1916; see also Thoresen, L.H.; Jiao, G.-S.; Haaland, W.C.; Metzker, M.L.; Burgess, K. Rigid, conjugated, fluoresceinated thymidine triphosphates. Chem. Eur. J. 2003, 9, 4603–4610. 12. Halogenated guanosines contain a labile ribose motif possessing secondary and primary hydroxyl moieties capable of binding metal ions. In addition, the purine moiety can participate in metal binding via several coordination modes. 13. Firth, A.G.; Fairlamb, I.J.S.; Darley, K.; Baumann, C.G. Sonogashira alkynylation of unprotected 8brominated adenosines and guanosines: Fluorescence properties of compact conjugated acetylenes containing a purine ring. Tetrahedron Lett. 2006, 47, 3529–3533. Very recently, an elegant method for the 8-alkynylation of 8-bromo-2# -deoxyguanosine was reported by the Shaughnessy group, see: Cho, J.H.; Prickett, C.D.; Shaughnessy, K.H. Efficient Sonogashira coupling of unprotected halonucleosides in aq ueous solvents using water-soluble palladium catalysts. Eur. J. Org. Chem. 2010, 3678– 3683.


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14. a) Pneumatikakis, G. 1:1 Complexes of palladium(II) and platinum(II) with caffeine and their interaction with nucleosides. Inorg. Chim. Acta 1984, 93, 5–11; b) Dehand, J.; Jordanov, J. Interaction of cis-diaminotolueneplatinum(II) with nucleosides: evidence for guanosine O(6).N(7) chelation by platinum. J. Chem. Soc. Chem. Commun. 1976, 598–599. 15. Western, E.C.; Shaughnessy, K.H. Inhibitory effects of the guanine moiety on the Suzuki couplings of unprotected halonucleosides in aqueous media. J. Org. Chem. 2005, 70, 6378–6388. Studies by Wagner have demonstrated that it is possible to conduct Suzuki-Miyaura cross-couplings on phosphorylated nucleosides in aqueous media using a solubilising phosphine ligand, see: Collier, A.; Wagner, G. A facile two-step synthesis of 8-arylated guanosine mono- and triphosphates (8-aryl GXPs). Org. Biomol. Chem. 2006, 4, 4526–4532. 16. XPS has been used extensively to study palladium nanoparticles (heterogenized species) in various applications (including cross-coupling processes). For selected references, see: Lengke, M.F.; Fleet, M.E.; Southam, G. Synthesis of palladium nanoparticles by reaction of filamentous cyanobacterial biomass with a palladium(II) chloride complex. Langmuir 2007, 23, 8982–8987, and references cited therein. 17. Amberlite IRA-67 (CAS no. 80747–90-6) was purchased from Sigma-Aldrich (Acrylaic (gel) matrix; 16–50 mesh; 1.6 meq(mL exchange capacity; ∼60% moisture). 18. Cai, C.; Vasella, A. Oligosaccharide analogues of polysaccharides. Part 5. Studies on the crosscoupling of alkynes and haloalkynes. Helv. Chim. Acta 1995, 78, 2053–2064, and references cited therein. 19. Amatore, C.; Jutand, A. Role of dba in the reactivity of palladium(0) complexes generated in situ from mixtures of Pd(dba)2 and phosphines. Coord. Chem. Rev. 1998, 180, 511–528. 20. Berg, J.M.; Holm, R.H. Model for the active sites of oxo-transfer molybdoenzymes: reactivity, kinetics, and catalysis. J. Am. Chem. Soc. 1985, 107, 925–932. 21. Canty, A.J.; Tobias, R.S. Heavy metal nucleoside interactions. Inorg. Chem. 1979, 18, 413–417. 22. Lee, T.-J.; Chun, B.C.; Chung, Y.-C. Detoxification of reactive compounds by a cyclic electrolytic system with surface-modified ion-exchange resin. React. Funct. Poly. 2003, 56, 37–44. 23. Reetz, M.T.; de Vries, J.G. Ligand-free Heck reactions using low Pd-loading. Chem. Commun. 2004, 1559–1563. 24. The application of recoverable nanosized palladium(0) catalysts (∼7 nm) on polyvinylpyrrolidinone (PVP) has been reported for the Sonogashira alkynylation reaction, see: a)Li, P.; Wang, L.; Lia, H. Application of recoverable nanosized palladium(0) catalyst in Sonogashira reaction. Tetrahedron 2005, 61, 8633–8640; b) For an original report on the use of Pd(PVP nanoparticles in cross-coupling processes, see: Bradley, J.S.; Millar, J.M.; Hill, E.W. Surface chemistry on colloidal metals: A highresolution nuclear magnetic resonance study of carbon monoxide adsorbed on metallic palladium crystallites in colloidal suspension. J. Am. Chem. Soc. 1991, 113, 4016–4017. For another important application, see: c) Ellis, P.E.; Fairlamb, I.J.S.; Hackett, S.F.J.; Wilson, K.; Lee, A.F. Evidence for the surface catalysed Suzuki-Miyaura reaction over Pd nanoparticles: an operando XAS study. Angew. Chem. Int. Ed. 2010, 49, 1820–1824. 25. We refer to “immobilized” as palladium coordinating to a polymer support, or similar support. It is important to note that palladium can be leached out of such a support (∼5–10 nm nanoparticles), see: a) Broadwater, S.J.; McQuade, D.T. Investigating PdEnCat catalysis. J. Org. Chem. 2006, 71, 2131–2134; b) Choudary, B.M.; Madhi, S.; Chowdari, N.S.; Kantam, M.L.; Sreedhar, B. Layered double hydroxide supported nanopalladium catalyst for Heck-, Suzuki-, Sonogashira-, and Stille-Type coupling reactions of chloroarenes. J. Am. Chem. Soc. 2002, 124, 14127– 14136. 26. a) Gaikwad, A.V.; Holuigue, A.; Thathagar, M.B.; ten Elshof, J.E.; Rothenberg, G. Ion-leaching and atom-leaching mechanisms from palladium nanoparticles in cross-coupling reactions. Chem. Eur. J. 2007, 13, 6908–6913. It is acknowledged that the initial oxidative addition reaction with the organohalide could occur on the metal surface, for example, defect site, of the palladium nanoparticle (cluster), before being released into solution to participate in a “homogenous” reaction. For an excellent comprehensive discussion of this important aspect in the Heck reaction, see: b) Köhler, K.; Kleist, W.; Pröckl, S.S. Genesis of coordinatively unsaturated palladium complexes dissolved from solid precursors during Heck coupling reactions and their role as catalytically active species. Inorg. Chem. 2007, 46, 1876–1883.

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A. G. Firth et al.

27. Ropartz, L.; Meeuwenoord, N.J.; Van Der Marel, G.A.; van Leeuwen, P.W.N.M.; Slawina, A.M.Z.; Kamer, P.C.J. Phosphine containing oligonucleotides for the development of metallodeoxyribozymes. Chem. Commun. 2007, 1556–1558. 28. Sheu, C.; Foote, C.S. Reactivity toward singlet oxygen of a 7,8-dihydro-8-oxoguanosine(“8hydroxyguanosine”) formed by photooxidation of a guanosine derivative. J. Am. Chem. Soc. 1995, 117, 6439–6442.