Access to bifunctionalized biomolecular platforms using oxime ligation

Accepted Manuscript Access to bifunctionalized biomolecular platforms using oxime ligation Karel Křenek, Radek Gaž ák, Gour Chand Daskhan, Julian Garcia, Michele Fiore, Pascal Dumy, Miroslav Šulc, Vladimír Křen, Olivier Renaudet PII: DOI: Reference:

S0008-6215(14)00181-5 http://dx.doi.org/10.1016/j.carres.2014.04.020 CAR 6737

To appear in:

Carbohydrate Research

Received Date: Revised Date: Accepted Date:

28 March 2014 28 April 2014 29 April 2014

Please cite this article as: Křenek, K., Gaž ák, R., Daskhan, G.C., Garcia, J., Fiore, M., Dumy, P., Šulc, M., Křen, V., Renaudet, O., Access to bifunctionalized biomolecular platforms using oxime ligation, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/j.carres.2014.04.020

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Access to bifunctionalized biomolecular platforms using oxime ligation

Karel Křenek,a Radek Gažák,a Gour Chand Daskhan,b Julian Garcia,b Michele Fiore,b Pascal Dumy,b Miroslav Šulc,a Vladimír Křen*,a Olivier Renaudet*,b,c

a

Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská

1083, CZ-14220 Praha 4, Czech Republic, b

Département de Chimie Moléculaire, UMR CNRS 5250 & ICMG FR 2607,

Université Joseph Fourier, BP53, 38041 Grenoble Cedex 9, France, c

Institut Universitaire de France, 103 boulevard Saint-Michel, 75005 Paris, France c

*E-mail: [email protected], [email protected]

Keywords Chemoselective ligation; glycocluster; cyclopeptide; multivalency; oxime

Note We previously reported the synthesis of original neoglycopeptide conjugates and the evaluation of their immunological properties towards NK cells. Due to the inability to reproduce the immunological data (Int. J. Mol. Sci. 2014, 15, 1271-1283), we have recently decided to fully retract this paper (J. Am. Chem. Soc. 2014, 136, 11561156). The present manuscript describes this synthetic strategy which was fully validated, updated and enhanced with additional experiments.

Abstract This paper describes an efficient oxime ligation strategy to prepare multivalent conjugates wherein peptides alone or in combination with carbohydrate or oxime group were coupled to a cyclopeptide scaffold. To demonstrate the versatility of this approach, two classes of conjugates have been prepared. In one class, we attached two or four peptide sequences to the cyclopeptide core together with free oxime groups, while the second class contains an additional substitution with four or two monosaccharides. The well-defined structure of these conjugates was confirmed by high-resolution mass spectrometry.

Introduction Carbohydrates and proteins are key structural motifs that exist in all living systems where they play central roles in a wide range of biological events [1-3]. Synthetic carbohydrates alone or combined with peptides and proteins represent promising tools to decipher these complex biological processes as well as to develop potential therapeutic and diagnostic agents [4-15]. However, chemical construction of such structures is complicated due to difficulties associated with the low compatibility of peptide/protein and carbohydrate chemistries, the fragility of glycosidic linkage and the presence of multiple functionalities. Chemoselective and bioorthogonal strategies have been developed to circumvent these difficulties and provide access to complex bioconjugates [16-20]. Among these methods, we and other groups have focused on oxime ligation that consists in using two functionalities namely, aldehyde and aminooxy groups that are highly reactive in aqueous buffers [21]. This strategy has been used successfully to synthesize numbers of original glycosylated constructs for applications going from cell targeting to surface modifications [22-25]. As a part of a long term program, we have demonstrated the utility of cyclopeptide scaffolds, commonly known as Regioselectively Addressable Functionalized Templates (RAFT) [26], for the multivalent presentation of carbohydrate or peptide ligands in well-defined spatial orientation [27-28]. The utilization of this scaffold is indeed interesting to us due to its conformational stability and the presence of two independent domains that can be functionalized regioselectively. In particular, we demonstrated recently the robustness of oxime ligation to prepare well-defined cyclopeptide-based glycodendrimers displaying clusters of carbohydrates on the upper domain of the scaffold [29-33]. In this paper, we show that both addressable domains of the cyclopeptide can be functionalized successively with peptides and

carbohydrates using an oxime-based ligation process. To demonstrate the versatility of our strategy we have prepared two classes of compounds (Figure 1). In the first one, we have combined two or four peptides together with free oxime groups, i.e. compounds 1 and 3 respectively. The second classes display an additional substitution with four or two GalNAc attached through an oxime ether linkage, i.e. compounds 2 and 4, respectively.

Figure 1. Structure of oxime-peptide (1 and 3) and carbohydrate-peptide (2 and 4) conjugates.

Results and Discussion

We have previously showed that the synthesis of glycodendrimers [31] using successive oxime conjugations is efficient but should be performed following a strict sequence to avoid side reactions. In particular, we have observed that the simultaneous presence of both aminooxy and oxime functionalities within a single molecule can induce trans-oximation reactions [31]. To avoid this problem, two different cyclodecapeptide cores with orthogonal protecting groups have been prepared (Scheme 1) as key intermediates for the nonglycosylated and glycosylated peptide conjugates 1-4.

Scheme 1. Synthesis of oxime-peptide (1) and carbohydrate-peptide (2) conjugates.

The first cyclopeptide 5 display four lysines pre-functionalized with serines [34] on the upper

domain

and

two

lysines

protected

with

N-(1-(4,4-dimethyl-2,6-

dioxocyclohexylidene)ethyl) (Dde) [35] on the lower domain. Dde protecting groups were first removed using a solution of

3%

of hydrazine

in DMF. N-

Hydroxysuccinimidyl ester of ethoxyethylidene aminooxy acetic acid linker (Eei-AoaOSu) [36] was next coupled to the free lysines to afford compound 6. Final

deprotection of the four serine and two aminooxy moieties was performed by acidolysis with a solution of trifluoroacetic acid/triisopropylsilane/water (TFA/TIS/H2O, 95:2.5:2.5) to provide the fully deprotected scaffold 7. The molecular assembly was performed using successive oxime coupling and aldehyde formation. For this purpose, we have synthesized a pentapeptide that contains diverse functionalities (i.e. carboxylic acids and alcohol). This model peptide was terminated with a serine which can be easily converted into glyoxylic aldehyde (COCHO) by oxidative cleavage with sodium periodate [37-38]. The oxime coupling was performed with an excess of the peptide-COCHO and the aminooxy-containing scaffold 7 in a mixture of CH3CN/H2O containing 0.1% of TFA. The conversion was complete and we obtained quantitatively a peptide conjugate displaying two peptide and four serine moieties. An additional treatment with sodium periodate afforded four COCHO functions that were used to conjugate either hydroxylamine or aminooxy αGalNAc [39] to the second addressable domain of the scaffold. The oxime coupling was performed under similar conditions and the desired compounds 1-2 were obtained quantitatively as confirmed by analytical HPLC of the crude mixtures (Figure 2). (a)

(b)

Figure 2. HPLC profile (linear gradient: 5 to 90% CH3CN in 15 min, λ = 250 nm) of the crude reaction mixture of (a) compound 1; (b) compound 2.

We followed the same procedure to prepare the conjugates 3 and 4 from the scaffold 8, which contains reverse functionalities, i.e. two pre-functionalized and four protected lysines (Scheme 2). No difference of reactivity was observed and both compounds 3 and 4 were obtained as pure products after HPLC purification in similar yields.

Scheme 2. Synthesis of oxime-peptide (3) and carbohydrate-peptide (4) conjugates.

The fragmentation of oxime ether linkages during ionization process of mass spectroscopy spectrometry analysis are well documented in the literature [31, 33, 40]. To avoid this problem and received high resolution MS data we have chosen matrixassisted laser desorption/ionization ion source combined with Fourier transform ion cyclotron resonance as detector, time-of-flight mass spectroscopy (MALDI-TOFFTICR-MS) for mass spectrometry experiments. The average monoisotopic molecular weight of the synthesized conjugates 1-4 was measured by MALDI-TOF FT-ICR MS mass spectroscopy analysis using α-cyano-4-hydroxycinnamic acid (CCA) or 2,5dihydrobenzoic acid (DHB) as MALDI matrices for compounds 1-3 and 4, respectively (Table 1).

Table 1 MALDI-TOF FT-ICR MS analysis of conjugates 1-4. Found mass (m/z)

Error (ppm)

Cpnd

Formula

Calcd. mass

1

C118H188N32O46

2812.324768 [M+Na]+ 2812.31664

2.9

2

C150H240N36O66

3624.642258 [M+Na]+ 3624.64305

0.3

3

C174H278N42O70

4076.955767 [M+H]+

4076.95899

0.8

4

C190H304N44O80

4483.114512 [M+H]+

4483.19100

17.0

We next performed molecular modelling study with peptide-glycoconjugates 2 and 4. Energy minimizations were calculated in vacuo using Insight II/Discover. We first observed that minimized structures are conformationally stable and the cyclopeptide remains rigid in both cases. In addition, no steric clashes were observed between the peptides fragments and the carbohydrates attached onto both addressable domains of the cyclopeptide. This suggests that such scaffolds can be functionalized with

diverse recognition or structural elements in well-defined spatial orientation without interfering with their biological function.

(a)

(b)

Figure 3. Molecular modelling of (a) compound 2; (b) compound 4.

Conclusion

In summary, we have described a straightforward strategy for the synthesis of bifunctionalized cyclopeptide scaffolds bearing peptide and carbohydrates on two separated addressable domains. By employing successive and reproducible oxime ligations, new classes of peptide-conjugates 1-3 and 2-4 were prepared using attachment of either peptide-aldehyde, hydroxylamine or aminooxy αGalNAc onto RAFT scaffolds displaying aminooxy and masked aldehyde groups. The well-defined structure of these conjugates was determined by high-resolution mass spectrometry. Molecular modelling study also confirmed the spatial separation of both addressable domains, thus providing an attractive biomolecular platform for diverse biomedical applications.

Experimental General methods. All chemical reagents were purchased from Aldrich (SaintQuentin Fallavier, France) or Acros (Noisy-Le-Grand, France) and were used without further purification. Protected amino acids and Fmoc-Gly-Sasrin resin were obtained from Advanced ChemTech Europe (Brussels, Belgium), Bachem Biochimie SARL (Voisins-Les-Bretonneux, France) and France Biochem S.A. (Meudon, France). PyBOP was purchased from France Biochem. Reaction progress was monitored by reverse-phase HPLC on Waters equipment using C18 columns. Analytical HPLC (Nucleosil 120 Å 3 µm C18 particles, 30 × 4.6 mm2) was performed at 1.3 mL/min and preparative HPLC (Delta-Pak 300 Å 15 µm C18 particles, 200 × 25 mm2) at 22 mL/min with UV monitoring (214 nm and 250 nm) using a linear A–B gradient (buffer A: 0.09% TFA in H2O; buffer B: 0.09% TFA in 90% CH3CN). Routine mass spectra were recorded on a VG Platform II by electron spray ionization in the positive mode. Accurate mass spectra were obtained for compounds 1-4 on a MALDI-APEX Qe-FT-

ICR mass spectrometer equipped with a 9.4 T superconducting magnet and a ion Combi Source (Bruker-Daltonics, Bremen, Germany). Positive mass spectra were obtained by accumulating ions in the collision hexapole and running the quadrupole mass filter in non-mass-selective RF-only mode so that ions of a broad m/z range (850–5000) were allowed to enter the analyzer cell. All spectra were calibrated externally using the monoisotopic [M+H]+ ions of Pepmix2 calibrant (BrukerDaltonics, Germany). A 5 mg/mL solution of CCA or 10 mg/mL solution DHB in 50% CH3CN and 0.3% CH3CO2H was used as matrix. A 1 µL of sample dissolved in CH3OH was mixed with a 1.0 µL of the matrix solution. A 0.3 µL of mixture was loaded on the target and allowed to dry at ambient temperature.

Synthesis of compound 5. The linear precursor peptide (0.25 mmol; analytical RPHPLC: Rt = 12.2 min (5 to 100% B in 15 min, 214 nm); ESI-MS: calcd. for C118H203N20O31 2397.5 [M+H]+; found: m/z 2397.9) was cyclized in CH2Cl2 (500 mL) with PyBOP (156 mg; 0.3 mmol) and DIPEA (82 µL; 0.5 mmol; pH 8). After stirring for 1 h at room temperature, the solution was evaporated and peptide 5 was recovered by precipitation in Et2O. Analytical RP-HPLC: Rt = 14.9 min (5 to 100% B in 15 min, 214 nm); ESI-MS: calcd. for C118H201N20O 30 2378.5 [M+H]+; found: m/z 2378.1.

Synthesis of compound 6. Protected cyclodecapeptide 5 (0.25 mmol) was treated for 1 h at room temperature with a solution of 3% hydrazine in DMF (50 mL). After evaporation and precipitation in Et2O, Eei-Aoa-OSu (129 mg; 0.5 mmol) and DIPEA (83 µL; 0.5 mmol) were added and the solution was stirred in DMF (25 mL). The coupling reaction was monitored by analytical HPLC and reached completion after 2 h. The solvent was then evaporated and the excess of the activated ester removed by precipitation in Et2O. Yield: 52% (300 mg) from the corresponding linear peptide

sequence (3 steps); analytical RP-HPLC: Rt = 14.1 min (5 to 100% B in 15 min, 214 nm); ESI-MS: calcd. for C110H195N22O 32 2336.4 [M+H]+; found: m/z 2336.2.

Synthesis of compound 7. Crude compound 6 (196 mg; 0.084 mmol) was treated with a cocktail of TFA/TIS/H2O (50 mL; 95:2.5:2.5). After 2 h of stirring at room temperature, the solution was evaporated and fully deprotected cyclopeptide 7 was recovered by precipitation in Et2O. Analytical RP-HPLC: Rt = 5.7 min (5 to 60% B in 15 min, 214 nm); ESI-MS: calcd. for C66H119N22O22 1571.9 [M+H]+; found: m/z 1571.9.

Synthesis of compound 8. Cyclodecapeptide 8 was synthesized for the linear peptide precursor (0.25 mmol; analytical RP-HPLC: Rt = 10.5 min (5 to 100% B in 15 min, 214 nm); ESI-MS: calcd. for C114H185N18O27 2239.3 [M+H]+; found: m/z 2238.6) by following the procedure described for 5. Analytical RP-HPLC: Rt = 12.9 min (5 to 100% B in 15 min, 214 nm); ESI-MS: calcd. for C114H183N18O26 2220.4 [M+H]+; found: m/z 2220.1.

Synthesis of compound 9. Compound 9 was synthesized by following the procedure described for 7. Analytical RP-HPLC: Rt = 5.7 min (5 to 60% B in 15 min, 214 nm); ESI-MS: calcd. for C64H115N22O22 1543.8 [M+H]+; found: m/z 1543.6.

Synthesis of compound 1. Peptide 7 (14 mg; 0.0069 mmol) was dissolved in a mixture of CH3CN/H2O/TFA (2 mL; 1:1:0.1) and peptide-CHO (27 mg; 0.041 mmol) was added to the solution. The mixture was stirred at 37°C overnight then acetone (1 mL) was added. The resulting peptide conjugate (was used without further treatment. Analytical RP-HPLC: Rt = 12.6 min (5 to 40% B in 15 min, 214 nm); ESI-MS: calcd for

C122H205N32O46 2854.5 [M+H]+; found: m/z 2854.3. Sodium periodate (59 mg; 0.28 mmol) was then added to the crude solution and the mixture purified by RP-HPLC after 1 h to isolate the corresponding oxidized conjugate in 95% yield (18 mg) from 7 (2 steps). Analytical RP-HPLC: Rt = 12.9 min (5 to 60% B in 15 min, 214 nm). This compound (9 mg; 0.0033 mmol) was finally stirred at 37°C with hydroxylamine hydrochloride (1.8 mg; 0.026 mmol) in CH3CN/H2O/TFA (2 mL; 1:1:0.1). After RPHPLC purification, compound 1 was obtained with a 70% yield (6.5 mg). Analytical RP-HPLC: Rt = 7.6 min (5 to 100% B in 15 min, 214 nm); MALDI-FT-ICRTOF HRMS: calcd for C118H188N32O46 2812.324768 [M+Na]+; found: 2812.31664.

Synthesis of compound 2. Compound 2 was obtained from the previous oxidized peptide (9 mg; 0.0033 mmol) and aminooxy αGalNAc (8 mg; 0.033 mmol) in CH3CN/H2O/TFA (2 mL; 1:1:0.1) following the procedure described for 1. Yield: 80% (9.5 mg); analytical RP-HPLC: Rt = 7.1 min (5 to 100% B in 15 min, 214 nm); MALDIFT-ICRTOF HRMS: calcd for C150H240N36O66 3624.642258 [M+Na]+; found: 3624.64305.

Synthesis of Compound 3. Compound 3 was prepared following the 3-step procedure described for 1. Compound 9 (12 mg; 0.0068 mmol) was first treated with peptide-CHO (28 mg; 0.041 mmol). The resulting conjugate was then oxidized with sodium periodate (29 mg; 0.14 mmol) in water (7 mL) and purified by RP-HPLC after 1 hour. Yield: 91% (25 mg) from 9 (2 steps); analytical RP-HPLC: Rt = 8.3 min (5 to 100% B in 15 min, 214 nm). This peptide (11 mg; 0.0027 mmol) was reacted with hydroxylamine hydrochloride (0.8 mg; 0.011 mmol) in CH3CN/H2O/TFA (2 mL; 1:1:0.1) to obtain 3 after purification. Yield: 68% (7.5 mg); analytical RP-HPLC: Rt =

8.4 min (5 to 100% B in 15 min, 214 nm); MALDI-FT-ICRTOF HRMS: calcd for C174H278N42O70 4076.955767 [M+H]+; found: 4076.95899.

Synthesis of compound 4. Compound 4 was obtained from the previous oxidized peptide (14 mg; 0.0034 mmol) and aminooxy αGalNAc (3 mg; 0.014 mmol) in CH3CN/H2O/TFA (2 mL; 1:1:0.1) following the procedure described for 3. Yield: 66% (10 mg); analytical RP-HPLC: Rt = 8.1 min (5 to 100% B in 15 min, 214 nm); MALDIFT-ICRTOF

HRMS:

calcd

for

C190H304N44O80 4483.114512

[M+H]+;

found:

4483.19100.

Molecular modeling. Structure calculations were performed in vacuo using InsightII / Discover (Version 2005, Accelrys, SanDiego,CA,USA) software, and the energy of the system was calculated by the consistent CVFF force field (version 2.3). To shorten the range of Coulomb interaction, a distance-dependent relative dielectric constant, εr, was used (εr = 4r). The resulting molecule was subjected to 2000 iterations of steepest descent minimization, followed by 3500 iterations of conjugate gradient minimization and the convergence of minimization was followed until the RMS derivative was less than 0.01 kcal.mol-1.

Acknowledgements This work was supported by the Université Joseph Fourier (UJF), the Centre National de la Recherche Scientifique (CNRS), the “Communauté d’agglomération GrenobleAlpes Métropole” (Nanobio Program), the Ligue contre le cancer (MF) and the ANR12-JS07-0001-01 “VacSyn” (GCD). O.R. acknowledges support from the Labex

Arcane (ANR-11-LABX-003), and O.R.&V.K. acknowledge European project “MultiGlycoNano” ESF COST chemistry action CM1102 (MSMT LD13042).

References 1. Varki, A. Glycobiology 1993, 3, 97-130. 2. Collins, B. E.; Paulson, J. C. Curr. Opin. Chem. Biol. 2004, 8, 617-625. 3. Imberty, A.; Varrot, A. Curr. Opin. Struct. Biol. 2008, 18, 567-576. 4. Sears, P.; Wong, C.-H. Science 2001, 291, 2344-2350. 5. Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357-2363. 6. Pratta, M. R.; Bertozzi, C. R. Chem. Soc. Rev. 2005, 34, 58-68. 7. J. D. Warren, Geng, X.; Danishefsky, S. J. Top. Curr. Chem. 2007, 267, 109-141. 8. Seeberger, P. H.; Werz, D. B. Nature, 2007, 446, 1046-1051. 9. Horlacher, T.; Seeberger, P. H. Chem. Soc. Rev. 2008, 37, 1414-1422. 10. Gamblin, D. P.; Scanlan, E. M.; Davis, B. G. Chem. Rev. 2009, 109, 131-163. 11. Gaidzik, N.; Westerlind, U.; Kunz H. Chem. Soc. Rev. 2013, 42, 4421-4442. 12. Rouhanifard, S. H.; Nordstrøm, L. U.; Zheng, T.; Wu, P. Chem. Soc. Rev. 2013, 42, 4284-4296. 13. Park, S.; Gildersleeve, J. C.; Blixt, O.; Shin, I. Chem. Soc. Rev. 2013, 42, 43104326. 14. Johnson, M. A.; Bundle, D. R. Chem. Soc. Rev. 2013, 42, 4327-4344. 15. Unverzagt, C.; Kajihara, Y. Chem. Soc. Rev. 2013, 42, 4408-4420. 16. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 20042021. 17. Meldal, M.; Tornøe, C. W. Chem. Rev. 2008, 108, 2952-3015. 18. Dirksen, A.; Dawson, P. E. Curr. Opin. Chem. Biol. 2008, 12, 760-766.

19. Sletten, E. M.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6974-6998. 20. Dondoni, A.; Marra, A. Chem. Soc. Rev. 2012, 41, 573-586. 21. Rose, K. J. Am. Chem. Soc. 1994, 116, 30-33. 22. Lemieux, G. A.; Bertozzi, C. R. Trends Biotechnol. 1998, 16, 506-513. 23. Peri, F.; Nicotra, F. Chem. Commun. 2004, 623-627. 24. Hudak, J. E.; Yu, H. H.; Bertozzi, C. R. J. Am. Chem. Soc. 2011, 133, 1612716135. 25. Ulrich, S.; Boturyn, D.; Marra, A.; Renaudet, O.; Dumy, P. Chem. Eur. J. 2014, 20, 34-41. 26. Dumy, P.; Eggleston, M.; Cervigni, S.; Sila, U.; Sun, X.; Mutter, M. Tetrahedron Lett. 1995, 36, 1255-1258. 27. Boturyn, D.; Defrancq, E.; Dolphin, G. T.; Garcia, J.; Labbé, P.; Renaudet, O.; Dumy, P. J. Pept. Sci., 2008, 14, 224-240. 28. Galan, M. C.; Dumy, P.; Renaudet, O. Chem. Soc. Rev. 2013, 42, 4599-4612. 29. Renaudet, O.; Dumy, P. Org. Lett. 2003, 5, 243-246. 30. André, S.; Renaudet, O.; Bossu, I.; Dumy, P.; Gabius, H.-J. J. Pept. Sci. 2011, 17, 427-437. 31. Bossu, I.; Šulc, M.; Křenek, K.; Dufour, E.; Garcia, J.; Berthet, N.; Dumy, P.; Křen, V.; Renaudet, O. Org. Biomol. Chem. 2011, 9, 1948-1959. 32. Berthet, N.; Thomas, B.; Bossu, I.; Dufour, E.; Gillon, E.; Garcia, J.; Spinelli, N.; Imberty, A.; Dumy, P.; Renaudet, O. Bioconjugate Chem. 2013, 24, 1598-1611.

33. Thomas, B.; Berthet, N.; Garcia, J.; Dumy, P.; Renaudet, O. Chem. Commun. 2013, 49, 10796-10798. 34. Foillard, S.; Ohsten Rasmusssen, M.; Razkin, J.; Boturyn, D.; Dumy, P. J. Org. Chem. 2008, 73, 983-991. 35. Bycroft, B. W.; Chan, W. C.; Chabra, S. R.; Hone, N. D. J. Chem. Soc. Chem. Commun. 1993, 778-779. 36. Duléry, V.; Renaudet, O.; Dumy, P. Tetrahedron 2007, 63, 11952-11958. 37. Geoghegan, K. F.; Stoh, J. G. Bioconjugate Chem. 1992, 3, 138-146. 38. El-Mahdi, O.; Melnyk, O. Bioconjugate Chem. 2013, 24, 735-765. 39. Renaudet, O.; Dumy, P. Org. Biomol. Chem. 2006, 4, 2628-2636. 40. Nazarpack-Kandlousy, N.; Chernushevich, I. V.; Meng, L. J.; Yang, Y.; Eliseev, A. V. J. Am. Chem. Soc. 2000, 122, 3358-3366.