Introduction

Molecular container compounds are hollow spherical hosts with cavities that allow accommodation of one or multiple guest molecules1,2,3. They are of great interest as nanoreactors4,5, in which fleeting intermediates are stabilized6,7,8,9, reactions accelerated10,11 and regio- and stereochemistry altered12,13,14, as well as for solar energy conversion15, nanodevice fabrication16, drug delivery17, storage and separation technology18. The discovery of self-assembly processes involving hydrogen bonding or metal coordination, in which multiple building blocks spontaneously assemble to form spherically and cylindrically shaped molecular capsules held together by hydrogen bonding or metal–ligand interactions, has tremendously increased the diversity of capsules with respect to shape and size19,20,21,22,23. The efficiency of such self-assembly approaches is best demonstrated in the quantitative multicomponent synthesis of structurally well-defined molecular spheres with cavity diameters that reach 5 nm (see refs. 24,25). The synthesis of similar-sized nanocapsules, in which building blocks are covalently linked, is especially desirable for biomedical applications. We recently reported a nearly quantitative one-pot 18-component synthesis of an octahedral nanocontainer that is built up from six bowl-shaped cavitands and 12 linker units held together by 24 newly formed imine bonds (Fig. 1) (ref. 26). Our approach strongly surpasses earlier multistep covalent synthesis of related nanocontainers in its simplicity and efficiency, which should facilitate applications in medicinal, analytical, chemical and material sciences27,28. Important was the choice of imine bonds to connect building blocks during the synthesis. Imine bond formation is reversible, which provides an error correction mechanism such that ultimately the thermodynamically most stable product—in this case 1—is obtained29,30. A subsequent reduction of all 24 imine bonds fixes the structure and produces amino groups that allow further functionalization of the nanocapsule. From molecular mechanics calculations, a cavity volume of approximately 1,700 Å3 was estimated for 1 and 4, which is sufficient for encapsulation of multiple small organic molecules or a small biomacromolecule. We see potential use of suitably functionalized or immobilized nanocapsules in drug delivery, wastewater detoxification, separation technology or as building blocks for new sensors.

Figure 1: 18-component synthesis of nanocontainer 1 and imine bond reduction.
figure 1

Conditions: (a) CF3COOH catalytic, CHCl3, room temperature; (b) 1. NaBH4, CHCl3/CH3OH; 2. HCl conc. in CH3OH; 3. NaOH; 4. RP-HPLC CH3OH/H2O/CF3COOH.

The synthesis of 1 involves condensation of six tetraformylcavitands 2 with twelve 1,2-ethylene-diamines 3 in chloroform in the presence of catalytic amounts of trifluoroacetic acid (TFA). Cavitand 2 can be prepared in gram quantities in four steps from resorcinol and hexanal according to the literature procedures (Fig. 2) (see refs. 3134 and Boxes 1,2,3,4). It is stable in the solid state but the formyl groups slowly oxidize in aerated solution, which substantially lowers the yield of its condensation reaction with 3. Upon mixing 2 and 3, hexamer 1 forms slowly at room temperature (22 °C). Equilibrium is reached after 2–3 days. The reaction is best monitored by 1H NMR spectroscopy (Fig. 3).

Figure 2: Synthesis of cavitand 2 (refs. 3134).
figure 2

Conditions: (a) EtOH-H2O-conc. HCl, 60 °C; (b) N-bromosuccinimide, 2-butanone, room temperature; (c) BrCH2Cl, K2CO3, DMF, 65 °C; (d) 1. BuLi, THF, −78 °C; 2. DMF, −78 °C → room temperature; 3. 5% NH4Cl·(aq).

Figure 3: 1H-NMR spectra (25 °C, CDCl3, a and c, 400 MHz, b, 300 MHz) of products formed upon mixing 2 with two equivalents of 3 in CHCl3 containing catalytic amounts of CF3CO2H after 100 min (a), 24 h (b) and 69 h (c).
figure 3

Imine proton resonances of hexamer 1 and tetramer 8 are marked with arrows.

Spectra taken at an early stage of the reaction show substantial amounts of tetramer 8, which subsequently converts into 1 (Figs. 3b and 4). After full equilibration, the hexamer to tetramer ratio is approximately 15:1 (Fig. 3c). We have carried out this reaction on a half-gram scale and do not find a decrease in the yield of 1 as long as the reactants are of high purity. On the other hand, the condensation reaction is remarkably solvent dependent35. In solvents other than chloroform, the yield of 1 is considerably lower and other nanocages form preferentially. For example, in tetrahydrofuran (THF), tetramer 8 is the major condensation product and octamer 9 in dichloromethane (Fig. 4). For a discussion of the solvent effect on the outcome of the condensation reaction between 2 and 3, the reader is referred to ref. 35.

Figure 4: Tetrahydrofuran.
figure 4

Tetrameric (8) and octameric (9) nanocapsules.

The reduction of all imine bonds of 1 with NaBH4 is quantitative and leads initially to boramines -CH2N(BH2)-, which have to be hydrolyzed under acidic conditions. Hydrolysis is slow and should be carried out at room temperature. We observe substantial acetal cleavage if hydrolysis is carried out at elevated temperature. An intramolecular acid catalysis by the ammonium groups might contribute to this side reaction (Fig. 5) (ref. 35).

Figure 5: Proposed intramolecular acid-catalyzed acetal hydrolysis of 4.
figure 5

Reproduced with permission from J. Am. Chem. Soc. 128, 14120–14127 (2006). Copyright 2006 American Chemical Society.

Support for this cleavage mechanism comes from the observation of substantial acetal cleavage, if solid 4·24CF3CO2H is heated to 80 °C under vacuum for 24 h. At room temperature, side reactions are minimized and 4·24CF3CO2H is obtained in 50–65% yield after purification by reversed-phase high-pressure liquid chromatography (HPLC). Nanocontainer 4·24HCl is soluble in methanol:water 9:1, but precipitates if the water content is increased.

Materials

Reagents

  • Resorcinol (Fisher Scientific, cat. no. R254-500)

  • Hexanal (Sigma-Aldrich, cat. no. 115606)

  • N-bromosuccinimide (Fisher Scientific, cat. no. B81255)

  • Bromochloromethane (Sigma-Aldrich, cat. no. 135267)

  • Anhydrous K2CO3 (Fisher Scientific, cat. no. P208-500)

  • 1,2-Ethylenediamine (3) (Sigma-Aldrich, cat. no. 391085)

  • Trifluoroacetic acid (Acros Organics, cat. no. 293812500)

  • Chloroform-d (Sigma-Aldrich, cat. no. 151823)

  • Sodium borohydride (NaBH4; Sigma-Aldrich, cat. no. 452882)

  • 4 Å molecular sieves, 8–12 meshes (Acros Organics, cat. no. 197250050)

  • NH4Cl

  • Hydrochloric acid (minimum 37% w/w)

  • Ethanol (95%)

  • Methyl ethyl ketone (Sigma-Aldrich, cat. no. 360473)

  • Acetone

  • Methanol

  • Dichloromethane

  • Ethylacetate

  • Sodium hydroxide

  • Anhydrous MgSO4

  • Sea sand

  • Flash silica gel (Sorbent Technology, cat. no. 10930-25)

  • Aluminum backing silica gel thin-layer chromatography (TLC) plates with fluorescence indicator (Sorbent Technology, cat. no. 1634126)

  • HPLC solvent A: 0.1 % TFA in methanol

  • HPLC solvent B: 0.1 % TFA in distilled water

Equipment

  • Three-necked round-bottomed flasks, 250, 1,000 and 2,000 ml

  • Round-bottomed flask, 250 ml

  • Reflux condenser

  • Addition funnel, 250 ml

  • Mechanical stirrer assembly

  • Thermometer, −10 to 110 °C

  • Low-temperature thermometer, −90 to 40 °C

  • Teflon-coated magnetic stir bars

  • Heating mantle for 2,000 ml round-bottomed flasks

  • Rotavaporator (Buchi, Model R-3000); vacuum pump (Buchi, Model V-500); vacuum controller (Buchi, Model V-800)

  • Vacuum pump (Fisher Scientific, Maximal C plus, model M2C)

  • HPLC with UV detector: dual pump solvent delivery, Varian prostar 210; UV-visible detector, Varian prostar 345

  • HPLC column: Vydac, protein and peptide C18, 5 μm, 300 Å, 4.6 × 250 mm, cat. no. 218TP54; Vydac, protein and peptide C18, 10 μm, 300 Å, 22 × 250 mm, cat. no. 218TP1022

  • HPLC syringe: Hamilton Microliter 750 syringe, 500 μl, 710 syringe, 100 μl

  • Glass-threaded vials (Fisher Scientific, 1.8 ml, cat. no. 03-339-21A; 11.1 ml, cat. no. 03-339-21E)

  • Three-port Schlenck line

  • Chromatography column with a 250 ml reservoir, 2.56 cm i.d. × 30.72 cm length

Reagent Setup

  • Chloroform Chloroform (Sigma-Aldrich, cat. no. 319988) may contain substantial amounts of HCl, which catalyzes the slow decomposition of nanocage 1. It should be freshly purified by filtration through a pad of Na2CO3 before use.

  • THF Dry and deoxygenate THF (Fisher Scientific, cat. no. T425-4) either with a solvent purification system (e.g., SolvTek) by passing it under nitrogen through an activated alumina column and a column containing a copper oxygen scrubber or by distilling it under nitrogen from sodium with sodium benzophenone ketyl as an indicator.

  • N,N -dimethylformamide Evacuate N,N-dimethylformamide (DMF, Sigma-Aldrich, cat. no. 494488) for 5 min at 0.1–1 mm Hg, in order to remove trace amounts of amines. Vent with nitrogen or argon and store over activated molecular sieves (4Å) under argon or nitrogen.

  • n -Butyl lithium n-Butyl lithium (BuLi, 2.5 M solution in hexanes; Sigma-Aldrich, cat. no. 230707) slowly decomposes at room temperature. Even though an excess of BuLi is used, the concentration of BuLi should be determined as described in detail in refs. 36,37, if the BuLi solution has been stored for a longer time at room temperature.

Equipment Setup

  • HPLC Set up a preparative HPLC method with a flow rate of 15 ml min−1 and UV-visible detection at λ = 280 nm.

    Table 1 Table 2
  • Flash column chromatography Pack a chromatography column with 150 ml of suspended silica gel in dichloromethane (CH2Cl2). Cover the top of the silica gel with a 0.5 cm layer of sand.

  • TLC Fill TLC tank with 95:5 (v/v) CH2Cl2/EtOAc.

Procedure

  1. 1

    Dry a 250 ml three-necked round-bottomed flask containing a Teflon-coated magnetic stir bar in an oven (at least 1 h). Remove the flask from the oven and connect it to a Schlenck line. Evacuate hot flask and allow it to cool to room temperature under vacuum. Vent flask with argon and keep it under argon during the following steps (Steps 2–10).

  2. 2

    Weigh out 65.3 mg (72.6 μl; 1.09 mmol) ethylenediamine (3) into a 1.8 ml vial. Add 1.0 ml chloroform into the vial and close the cap.

    Critical Step

    Ethylenediamine is added in a 2.8 mol% excess relative to the tetraformyl cavitand (2), as the condensation reaction is accelerated by the presence of trace amounts of ethylenediamine. It should be of high purity in order to obtain a high yield. Ethylenediamine can react with carbon dioxide and be oxidized in air. Therefore, exposure to air should be minimized.

  3. 3

    Place 490 mg (0.528 mmol) tetraformyl cavitand (2) into an 11.1 ml vial. Add 2.0 ml chloroform and sonicate for 1 min.

  4. 4

    Transfer the solution into the reaction flask using a 500 μl glass syringe. Rinse the vial four times with 0.5 ml chloroform each. Add additional 30.0 ml chloroform into the reaction flask.

    Critical Step

    In solution, the formyl groups of 2 are easily oxidized by dissolved oxygen. As the yield of the condensation step also depends on the purity of the tetraformyl cavitand, exposure to air should be minimized and the reaction flask should be kept under positive argon pressure.

  5. 5

    Transfer the ethylenediamine solution into the reaction flask using a 500 μl glass syringe. Rinse the vial six times with 0.5 ml each of chloroform. Add additional 3.0 ml chloroform into the reaction flask so that the total volume of chloroform is 41.0 ml.

  6. 6

    While under argon, sonicate the solution for 2 min by immersing it in an ultrasound bath. This removes any dissolved oxygen, which will be exchanged for argon.

  7. 7

    Add 3.9 μl (0.053 mmol) TFA into the reaction flask. This initiates the condensation reaction. The solution may turn pale yellow as the reaction proceeds. The color is due to the formation of trace amounts of side products. However, this does not affect the reaction.

    Caution

    TFA is highly corrosive. If a Hamilton microliter syringe or an Eppendorf pipetter is used for the addition of the TFA, the syringe should be rinsed immediately with plenty of water and any TFA vapor inside the Eppendorf pipetter should be removed by passing a stream of air through the pipetter body.

  8. 8

    Check the stoichiometry 30 min after TFA addition. Take out a 0.2 ml aliquot of the reaction solution and place it into a 10 ml round-bottomed flask. Remove solvent at rotavaporator at room temperature and remove residual solvent using a high vacuum line (room temperature, 10 min).

  9. 9

    Dissolve the residue in 0.7 ml chloroform-d and record 1H NMR spectrum. Add more ethylenediamine if residual aldehyde signal is observed (δ = 10.31 p.p.m.). Add more tetraformyl cavitand if residual ethylenediamine signal is more than 3% (δ = 3.0–2.7 p.p.m.) (see Fig. 6). Check the stoichiometry again 30 min later, if more reagent was added.

    Figure 6: Partial 1H NMR spectra (25 °C, CDCl3, (a), 400 MHz, (b), 300 MHz) of products formed upon mixing 2 with two equivalents of 3 in CHCl3, containing catalytic amounts of CF3CO2H, after 100 min (a) and 24 h (b).
    figure 6

    The correct stoichiometry (0–2.8% excess amino groups) can be verified and if necessary adjusted as follows: ([I(3) − I(1)2]/[I(1)2 + I(2)2]100%), if positive, is equal to the excess of amino groups in the reaction mixture (in %) based on the initial amount of 2 added, or to the excess of formyl groups, if negative (I(n) and I(n′) are the integrals of the signals (n) and (n′) in spectra a and b, respectively). In this example (spectrum a), [14.40 − 1.00 × 2]/[1.00 × 2 + 66.42 × 2]100% = 9.2% excess of amino groups are present. Therefore, an additional amount of 2 (6.4–9.2% based on the initially added 2) should be added, in order to reduce the excess of amine to below 2.8%. After adjustment of the reaction stoichiometry in this way (spectrum b), the excess amount of amino groups is [I(3′)]/[I(2′)2]100% = [1.00]/[76.65 × 2]100% = 0.65% based on the total amount of 2.

    Critical Step

    The yield of the condensation step is highest if the stoichiometry between ethylenediamine and tetraformyl cavitand is 2:1.

  10. 10

    Continue stirring for an additional 41 h at room temperature under argon.

    Pause point

    Can be left for 41 h at room temperature.

    Critical Step

    We usually check the progress of the reaction by 1H NMR spectroscopy as described in Steps 8 and 9 before proceeding to the reduction step (Step 11). If the integral of the nanocage imine signal (see Fig. 3c) is smaller than 60% of the total imine integral, the reaction time should be increased by 24 h, after which another 1H NMR spectrum should be recorded.

    Troubleshooting

  11. 11

    Stir the reaction mixture vigorously (500–600 r.p.m.). Add 3.8 g NaBH4 and stir for 3 min.

    Caution

    NaBH4 is toxic and highly flammable.

    Troubleshooting

  12. 12

    Add 0.4 ml dry methanol (dried with 3 Å molecular sieves for at least 24 h) into the reaction flask and stir for 5 min. Add an additional 3.7 ml dry methanol. Continue stirring overnight at room temperature.

    Critical Step

    The equilibrium of the condensation reaction can be affected by water present in methanol and methanol itself. Water shifts the equilibrium toward the starting materials. Too much methanol also lowers the yield.

    Pause point

    Can be left overnight at room temperature.

    Troubleshooting

  13. 13

    Remove the solvent at the rotavaporator. Add 150 ml water into the flask and sonicate for 10 min.

  14. 14

    Filter off the crude boramine mixture through a medium-porosity (10–16 μm) sintered glass filter funnel. Wash the white solid three times with 10 ml water each and air-dry it for 10 min.

    Critical Step

    Before proceeding to Step 15, the completion of the reduction should be checked by 1H NMR spectroscopy in CDCl3. The absence of imine protons at δ = 8.4 ± 0.2 indicates complete reduction. Otherwise, add 40 ml chloroform and repeat Steps 11 and 12.

  15. 15

    Prepare a solution composed of 150 ml methanol and 15 ml concentrated hydrochloric acid.

  16. 16

    Transfer the crude boramine into a 250 ml round-bottomed flask containing a stirring bar. Rinse the funnel with 10 ml MeOH/HCl solution and transfer the rinse solution into the flask. Pour the leftover MeOH/HCl solution into the flask.

  17. 17

    Continue stirring for an additional 3.5 days at room temperature. Check the progress of the hydrolysis using ESI MS or MALDI-TOF MS. This is carried out by neutralizing a small sample (0.2–0.5 ml) with 1 M NaOH (aq) and extracting it into dichloromethane before sample preparation for MS in order to remove chloride ions.

    Critical Step

    The product yield depends critically on the reaction time and is usually highest after about 3.5 days. If the reaction time is too short, the hydrolysis of B-N bond is incomplete. On the other hand, if the reaction time is too long, a noticeable number of the acetal groups (-OCH2O-) are cleaved.

  18. 18

    Dissolve 7.5 g (187.5 mmol) NaOH in 10 ml water. Cool the solution to room temperature. Slowly add the solution into the reaction flask until the reaction mixture is slightly basic. Check pH of the solution with pH paper.

  19. 19

    Remove methanol at the rotavaporator at room temperature.

    Critical Step

    Connect a high-vacuum pump to the rotavaporator in order to remove methanol at room temperature. Amine is sensitive to oxidation. Minimize contact with air to reduce oxidation.

  20. 20

    Filter the mixture through a fine-porosity (4.0–5.5 μm) sintered glass filter funnel. Wash the white residue with enough water to remove all inorganic salts. Air-dry the residue for 30 min.

  21. 21

    Transfer the residue into a 25 ml round-bottomed flask and dry it at high vacuum line at room temperature overnight. The crude product is a fine powder and may have a slight yellow color.

    Pause point

    The product can be stored in freezer at −20 °C.

  22. 22

    Dissolve 100 mg crude product in 500 μl methanol containing 30 μl TFA. Suck the solution into a 1 ml plastic syringe and pass it through a plastic syringe filter (0.20–0.45 μm).

  23. 23

    Inject the solution onto a preparative reversed-phased HPLC column at a flow rate of 15 ml min−1. Monitor fractions by UV-visible at λ = 280 nm.

  24. 24

    Collect fractions containing the major product (retention time tR 13.6 min). Evaporate the solvent to obtain a white crystalline powder. It might be necessary to repurify overlapping fractions containing 4 24TFA and the by-product M-C (Figs. 7 and 8).

    Figure 7: ESI MS spectra of fractions collected from HPLC purification (CH3OH/H2O/TFA 98:2:0.1).
    figure 7

    (a) Pure hexamer 4; (b) hexamer 4 plus nanocontainer lacking one acetal group.

    Figure 8: Typical HPLC profile of the crude reaction mixture after imine reduction and boramine hydrolysis.
    figure 8

    The elution order is reduced tetrameric nanocontainer 8, hexameric nanocontainer 4 (M) and nanocontainer 4 lacking one acetal group (M-C). (Conditions: column, Vydac RP-18, 5 μm, 300 Å, 4.6 × 250 mm; mobile phase, CH3OH/H2O/TFA gradient 85:15:0.1 to 98:2:0.1 over 15 min, then isocratic; flow, 1 ml min−1; detection λ = 280 nm.)

  25. 25

    Purify the remaining crude product in 100 mg portions by repeating Steps 22–24.

Troubleshooting

Troubleshooting advice can be found in Table 1.

Table 1 Troubleshooting table.

Timing

Step 1: 1.5 h

Steps 2–7: 1 h

Steps 8 and 9: 1 h

Step 10: 41 h

Steps 11 and 12: 8 h

Steps 13 and 14: 1 h

Steps 15 and 16: 0.5 h

Step 17: 3.5 days

Steps 18–21: 1.5 h

Steps 22–25: 8 h

Anticipated results

Typical yields

The typical yield of the condensation step is approximately 75% as determined by integration of the imine proton signal of 1 at δ = 8.34 relative to the total integration of the imine proton signals in the 1H NMR spectrum of crude 1 (Fig. 3c). The yield also depends on the reaction stoichiometry and is highest if 2 and 3 are added in an exact 1:2 ratio (82%). However, under these conditions, the reaction rate is slowest, as excess amino groups catalyze the transimination steps needed to reach equilibrium. An excellent compromise between yield and rate is the use of a small excess of amine (maximal 3%), which always gives greater than 70% yield. The subsequent reduction of 1 is essentially quantitative. The hydrolysis of the resulting boramine requires careful attention. The correct length of the hydrolysis step is very critical. If it is too short or too long, the presence of unhydrolyzed B-N groups or partially cleaved OCH2O acetal groups will strongly reduce the yield of 4 and make its purification more difficult. The exact time of the completion of this step is best determined by MALDI-TOF or ESI mass spectrometry. The ESI mass spectrum of 4 is shown in Figure 7. Each unhydrolyzed B-N bond increases the mass of the hexameric nanocage by approximately 12 mass units and each cleaved acetal reduces the mass by 12 (see Fig. 7b). After complete hydrolysis, a small fraction of the acetal groups (typically less than 1%) will be cleaved. Fortunately, these by-products can be separated from 4 by HPLC. A representative HPLC profile of the crude product 4 24TFA is shown in Figure 8. The product peak partially overlaps with that of a nanocontainer lacking one acetal (M-C). ESI-mass spectra of the purified product 4 and that of an overlapping fraction containing 4 and the nanocontainer lacking one acetal (M-C) are shown in Figure 7a,b. After HPLC, the typical isolated yield of the final product 4 is 60% based on the starting material tetraformyl cavitand 2.

Analytical data

Condensation product 1: 1H NMR (400 MHz, CDCl3, 22 °C) δ 8.34 (s, 24H, CHN); 7.12 (s, 24H, Haryl); 5.70 (d, J = 7.5 Hz, 24H, OCHoutHO); 4.83 (t, J = 8 Hz, 24H, CH(CH2)4CH3); 4.46 (d, J = 7.5 Hz, 24H, OCHinHO); 3.75 (sb, 48H, NCH2); 2.25–2.15 (m, 48H, CHCH2(CH2)3CH3); 1.5–1.3 (m, 144H, CHCH2(CH2)3CH3); 0.91 (t, J = 7.1 Hz, 72H; CHCH2(CH2)3CH3).

13C NMR (100 MHz, CDCl3, 22 °C;) δ 157.7, 153.6, 138.8, 124.5, 121.7, 100.5, 63.2, 36.7, 32.3, 30.1, 27.9, 23.0, 14.4.

FT-IR (CHCl3) v 2,956.8 (s), 2,929.6 (s), 2,872.1 (sh), 2,855.6 (s) 1,641.5 (s), 1,602.6 (m), 1,587 (m), 1,361.3 (m), 1,112.3 (w), 1,088.9 (m), 980 (s).

ESI-MS (CH2Cl2/CH3CN (1:5) (m/z) 1,954.9 (100%, [M+3H]3+); 1,466.9 (13%, [M+4H]4+); 1,173.7 (3%, [M+5H]5+).

Final product 4: 1H NMR (CD3OD; 0.4 vol% CF3COOD; 7 °C; 300 MHz) δ 7.55 (s, 24H, Haryl); 6.16 (d, J = 6.9 Hz, 24H, OCHoutHO); 4.85 (t, J = 7.6 Hz, 24H, CH(CH2)4CH3); 4.43 (d, J = 6.9 Hz, 24H, OCHinHO); 4.16 (sb, 48H, NCH2Ar); 3.59 (sb, 48H, N(CH2)2N); 2.38 (sb, 48H, CHCH2(CH2)3CH3); 1.6–1.2 (m, 144H, CHCH2(CH2)3CH3); 0.92 (t, J = 7.1 Hz, 72H; CHCH2(CH2)3CH3).

13C NMR (CD3OD; 0.4 vol% CF3COOD; 22 °C; 75 MHz) δ 160.5 (q; J = 37.8 Hz), 155.1, 139.9, 124.6, 119.9, 101.2, 44.4, 42.7, 38.5, 33.1, 30.9, 29.1, 24.0, 14.6.

ESI-MS (CH3OH/H2O/TFA (98/2/0.1) (m/z) 1,478.9 ([M+4H]4+); 1,507.3 ([M+4H+TFA]4+); 1,535.5 ([M+4H+2TFA]4+); 1,564.1 ([M+4H+3TFA]4+); 1,592.5 ([M+4H+4TFA]4+); 1,620.7 ([M+4H+5TFA]4+); 1,649.1 ([M+4H+6TFA]4+); 1,677.4 ([M+4H+7TFA]4+); 1,706.0 ([M+4H+8TFA]4+). MALDI-TOF MS (m/z) 5912.28 ([M+H]+, 100%).