OPTIMIZED ANALYTE DERIVATIZATION FOR SYNERGISTIC APPLICATION WITH CRYSTAL SPONGE METHOD

Abstract

The invention provides a sample preparation method (100) comprising: providing a sample (10) comprising an organic molecule (20), wherein the organic molecule (20) comprises a target group (21), wherein the target group (21) is a nucleophilic group and/or an acidic group; a derivatization stage (110) comprising: derivatizing the target group (21) of the organic molecule (20) with a moiety (31) comprising one or more of (i) a hydrocarbon comprising group and (ii) a 3rd period atom comprising group, wherein the 3rd period atom is selected from the group consisting of Si, P, and S, thereby providing a derivatized organic molecule (30); a separation stage (120) comprising: subjecting the sample (10) to a separation process to provide a fraction (35) comprising the derivatized organic molecule (30); and a preparation stage (130) comprising: introducing the derivatized organic molecule (30) into a porous single crystal (40), to provide a derivatized organic molecule doped porous single crystal (50).

Claims

1-15. (canceled)

16. A sample preparation method comprising: providing a sample comprising an organic molecule, wherein the organic molecule comprises a target group, wherein the target group is a nucleophilic group, and/or an acidic group; a derivatization stage comprising: derivatizing the target group of the organic molecule with a moiety comprising one or more of (i) a hydrocarbon comprising group and (ii) a 3.sup.rd period atom comprising group, wherein the 3.sup.rd period atom is selected from the group consisting of Si, P, and S, thereby providing a derivatized organic molecule; a separation stage comprising: subjecting the sample to a separation process to provide a fraction comprising the derivatized organic molecule; and a preparation stage comprising: introducing the derivatized organic molecule into a porous single crystal, to provide a derivatized organic molecule doped porous single crystal.

17. The sample preparation method according to claim 16, wherein the sample comprises a protic solvent, wherein the separation stage further comprises executing a solvent exchange by replacing at least part of the protic solvent by an aprotic solvent, and wherein, the separation stage comprises subjecting the sample to process.

18. The sample preparation method according to claim 16, wherein the porous single crystal comprises a metal-organic framework material, wherein the metal-organic framework material is tpt-ZnX.sub.2 based where X═Cl or Br or I.

19. The sample preparation method according to claim 16, wherein the organic molecule is an organic biomolecule, and wherein the target group is selected from the group consisting of —OH, —COOH, —NH.sub.2, —NRH, and —SH.

20. The sample preparation method according to claim 16, wherein the moiety comprises a hydrocarbon comprising group, the hydrocarbon group comprising an aliphatic group and/or an alkyl group and/or a methyl group, and/or an aromatic group.

21. The sample preparation method according to claim 20, wherein the aromatic group comprises a phenyl group or a benzyl group.

22. The sample preparation method according to claim 16, wherein the moiety comprises the 3.sup.rd period atom comprising group, wherein the 3.sup.rd period atom is selected from the group consisting of Si, P, and S.

23. The sample preparation method according to claim 22, wherein the 3rd period atom comprises Si, and wherein the moiety comprises a group selected from the group consisting of —SiR.sub.3, —SiArR.sub.2, —SiAr.sub.2R, and —SiAr.sub.3, wherein R is selected from the group consisting of methyl, ethyl, propyl, and isopropyl, and wherein Ar is —C.sub.6H.sub.5.

24. The sample preparation method according to claim 16, wherein the separation stage comprises providing N fractions, wherein N≥2 and wherein the preparation stage comprises contacting the N fractions with N porous single crystals, respectively, to provide N organic molecule doped porous single crystals.

25. The sample preparation method according to claim 16, wherein the sample preparation method further comprises a pre-analysis stage after the separation stage, the pre-analysis stage comprising subjecting at least part of the fraction to a mass spectrometry process to attempt to identify the derivatized organic molecule, wherein the pre-analysis stage comprises providing the fraction to the preparation stage if the identification of the derivatized organic molecule with the mass spectrometry process is unsuccessful, and wherein the pre-analysis stage comprises terminating the sample preparation method if the identification of the derivatized organic molecule with the mass spectrometry process is successful.

26. An X-ray analysis method of an organic molecule, the method comprising a sample providing stage and an analysis stage, wherein the sample providing stage comprises providing the derivatized organic molecule doped porous single crystal obtained by the method of claim 16, and wherein the analysis stage comprises subjecting the derivatized organic molecule doped porous single crystal to single crystal X-ray analysis.

27. The X-ray analysis method according to claim 26, comprising subjecting each of N derivatized organic molecule doped porous single crystals, wherein N≥2, to a single crystal X-ray analysis, respectively.

28. A system comprising: a derivatization unit, configured to derivatize a target group of an organic molecule with a moiety comprising one or more of (i) a hydrocarbon comprising group and (ii) a 3.sup.rd period atom comprising group, wherein the 3.sup.rd period atom is selected from the group consisting of Si, P, and S, thereby providing a derivatized organic molecule, wherein the target group is a nucleophilic group, and/or an acidic group; a separation unit, functionally coupled to the derivatization unit, configured to subject a sample comprising the derivatized organic molecule to a separation process to provide a fraction comprising the derivatized organic molecule; a preparation unit, functionally coupled to the separation unit, configured to introduce the derivatized organic molecule into a porous single crystal, to provide a derivatized organic molecule doped porous single crystal; an analysis unit, functionally coupled to the preparation unit, configured to subject the organic molecule doped porous single crystal to single crystal X-ray analysis; and a control system, configured to control the derivatization unit, the separation unit, the preparation unit and the analysis unit.

29. The system according to claim 28, wherein the separation unit comprises one or more of a LC system, a GC system, a LCMS system, or a GCMS system.

30. The system according to claim 28, further comprising a solvent exchange unit, functionally coupled to the separation unit and to the preparation unit, configured to solvent exchange the fraction comprising the derivatized organic molecule from the separation unit and to provide a solvent-exchange fraction comprising the derivatized organic molecule to the preparation unit, and wherein the separation unit is configured to provide N fractions, wherein N≥2, and wherein the preparation unit is configured to introduce the derivatized organic molecule of each of the N fractions into a respective porous single crystal, to provide respective derivatized organic molecule doped porous single crystals.

31. The system according to claim 28, wherein the system is configured to execute: a) a sample preparation method comprising: providing a sample comprising an organic molecule, wherein the organic molecule comprises a target group, wherein the target group is a nucleophilic group, and/or an acidic group; a derivatization stage comprising: derivatizing the target group of the organic molecule with a moiety comprising one or more of (i) a hydrocarbon comprising group and (ii) a 3.sup.rd period atom comprising group, wherein the 3.sup.rd period atom is selected from the group consisting of Si, P, and S, thereby providing a derivatized organic molecule; a separation stage comprising: subjecting the sample to a separation process to provide a fraction comprising the derivatized organic molecule; a preparation stage comprising: introducing the derivatized organic molecule into a porous single crystal, to provide a derivatized organic molecule doped porous single crystal; and/or b) an X-ray analysis method of an organic molecule, the method comprising a sample providing stage and an analysis stage, wherein the sample providing stage comprises providing the derivatized organic molecule doped porous single crystal obtained by the method of step a), and wherein the analysis stage comprises subjecting the derivatized organic molecule doped porous single crystal to single crystal X-ray analysis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0130] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which:

[0131] FIG. 1A-B schematically depict embodiments of the methods and the system according to the invention;

[0132] FIG. 2A-B schematically depict embodiments of the derivatization stage; and

[0133] FIG. 3 schematically depicts an embodiment of the single porous crystal (or: the preparation stage). The schematic drawings are not necessarily to scale.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0134] FIG. 1A schematically depicts the sample preparation method 100. The sample preparation method 100 may comprise providing a sample 10 comprising an organic molecule 20, wherein the organic molecule 20 comprises a target group 21, wherein the target group 21 is a nucleophilic group and/or an acidic group. The sample preparation method may further comprise a derivatization stage 110, a separation stage 120, and a preparation stage 130. The derivatization stage 110 may comprise derivatizing the target group 21 of the organic molecule 20 with a moiety 31, especially a moiety 31 comprising one or more of (i) a hydrocarbon comprising group and (ii) a 3rd period atom comprising group, especially wherein the 3rd period atom is selected from the group consisting of Si, P, and S. The derivatization stage may (thereby) provide a derivatized organic molecule 30, especially the sample 10 comprising the derivatized organic molecule. In the depicted embodiment, the derivatization stage 110 comprises contacting the organic molecule with a reactant 25 such that the target group 21 of the organic molecule 20 is derivatized with the moiety 31. The separation stage 120 may comprise subjecting the sample 10 to a separation process to provide a fraction 35 comprising the derivatized organic molecule 30. The preparation stage 130 may comprise introducing the derivatized organic molecule 30 into a porous single crystal 40, to provide a derivatized organic molecule doped porous single crystal 50.

[0135] In embodiments, the organic molecule 20 may be selected from the group consisting of an organic biomolecule, especially an organic biological molecule, or especially a biologically active organic molecule.

[0136] In embodiments, the separation stage 120 may comprise providing N fractions 35, wherein N≥2, and wherein the preparation stage 130 comprises contacting the N fractions with N porous single crystals 40, respectively, to provide N organic molecule doped porous single crystals 50.

[0137] FIG. 1A further depicts an embodiment of the X-ray analysis method 200 of an organic molecule 20 as described herein. The X-ray analysis method may comprise a sample providing stage and an analysis stage 240, wherein the sample providing stage may comprise providing the derivatized organic molecule doped porous single crystal 50 obtainable according to the sample preparation method 100, and wherein the analysis stage 240 may comprise subjecting the organic molecule doped porous single crystal 50 to single crystal X-ray analysis.

[0138] In embodiments wherein the sample providing stage comprises providing N organic molecule doped porous single crystals 50, the X-ray analysis method 200, especially the analysis stage 240, may comprise subjecting each of the N organic molecule doped porous single crystals 50 to a single crystal X-ray analysis, respectively.

[0139] FIG. 1A further depicts an embodiment of the system 300 according to the invention. The system may comprise a derivatization unit 310, a separation unit 320, a preparation unit 330, an analysis unit 340 and a control system 350. The derivatization unit 310 may be configured to derivatize a target group 21 of an organic molecule 20 with a moiety 31, especially a moiety 31 comprising one or more of (i) a hydrocarbon comprising group and (ii) a 3rd period atom comprising group, especially wherein the 3rd period atom is selected from the group consisting of Si, P, and S. The derivatization unit 310 may be configured to provide a derivatized organic molecule 30. The separation unit 320 may be functionally coupled to the derivatization unit 310. The separation unit 320 may be configured to subject a sample 10 comprising the derivatized organic molecule 30 to a separation process to provide a fraction 35 comprising the derivatized organic molecule 30. The preparation unit 330 may be functionally coupled to the separation unit 320. The preparation unit may be configured to introduce the derivatized organic molecule 30 into a porous single crystal 40, especially to provide a derivatized organic molecule doped porous single crystal 50. The analysis unit 340 may be functionally coupled to the preparation unit 330. The analysis unit may be configured to subject the organic molecule doped porous single crystal 50 to single crystal X-ray analysis. The control system 350 may be configured to control one or more of the derivatization unit 310, the separation unit 320, the preparation unit 330 and the analysis unit 340.

[0140] In the depicted embodiment, the sample preparation method 100 is carried out using the system 300 as described herein. Hence, in embodiments, the system 300 may be configured to execute the sample preparation method 100 as described herein and/or the X-ray analysis method 200 as described herein. It will be clear to the person skilled in the art, however, that the sample preparation method 100 and/or the X-ray analysis method 200 may also be carried out without using the system 300 according to the invention.

[0141] FIG. 1B schematically depicts another embodiment of the sample preparation method (100). For visualization purposes only, the process steps are indicated with solid arrows, whereas the flow of analyte is indicated with hyphenated arrows. In the depicted embodiment, the separation stage 120 comprises subjecting the sample 10 to a chromatography process 122, especially an LC process 122, 122a or a GC process 122, 122b. In the depicted embodiment, the separation stage 120 further comprises subjecting the sample 10 to a mass spectrometry process 124. Hence, the separation stage may comprise subjecting the sample to an LCMS process 125, 125a or a GCMS process 125, 125b. in further embodiments, at least part of the fraction 35 comprising the derivatized organic molecule may be subjected to a mass spectrometry process 124. The remainder of the fraction 35 may be provided to the separation stage.

[0142] In further embodiments, the separation stage 120 may comprise executing a solvent exchange by replacing at least part of a first solvent, especially a protic solvent, by a second solvent, especially an aprotic solvent. Especially, the separation stage may comprise executing a solvent exchange by replacing at least part of a first solvent in the sample 10 by a second solvent. Especially, the separation stage 120 may comprise first executing a solvent exchange and then subjecting the sample 10 to an LC process 122, 122a or a GC process 122, 122b. Further, the separation stage 120 may comprise subjecting the sample 10 to an LC process 122, 122a or a GC process 122, 122b and then executing a solvent exchange. Hence, after the GC and/or LC process, the fraction comprising the derivatized organic molecule may be dissolved in a first solvent, and the sample preparation stage may comprise executing the solvent exchange by replacing at least part of the first solvent by a second solvent.

[0143] In the depicted embodiment, the sample preparation method 100 further comprises an optional pre-analysis stage 145, the pre-analysis stage 145 comprising assessing whether the (derivatized) organic molecule 20 is identifiable based on fragmentation patterns obtained from the mass spectrometry process 124. If the (derivatized) organic molecule is (uniquely) identified, the sample preparation process may be terminated. If the (derivatized) organic molecule has not yet been identified, the fraction 35 comprising the derivatized organic molecule 30 may be subjected to the preparation stage 130. Hence, in the depicted embodiment, the process may continue from the pre-analysis stage 145 to the preparation stage 130 or may be terminated after the pre-analysis stage 145.

[0144] Specifically, in the depicted embodiment, the sample preparation method 100 further comprises a pre-analysis stage 145 after the separation stage 120, the pre-analysis stage 145 comprising subjecting at least part of the fraction 35 to a mass spectrometry process 124 to attempt to identify the derivatized organic molecule 30, wherein the pre-analysis stage 145 comprises providing the fraction 35 to the preparation stage 130 if the identification of the derivatized organic molecule 30 with the mass spectrometry process 124 is unsuccessful, and wherein the pre-analysis stage 145 comprises terminating the sample preparation method 100 if the identification of the derivatized organic molecule 30 with the mass spectrometry process 124 is successful.

[0145] FIG. 1B further schematically depicts another embodiment of the system 300. In the depicted embodiment, the system 300, especially the separation unit 320, comprises a chromatography unit 322, especially an LC unit 322, 322a (or: “LC system”) or a GC unit 322, 322b (or “GC system”). In the depicted embodiment, the separation unit 320 may comprise a mass spectrometry unit 324 (also: “mass spectrometry system”) configured to subject the sample to a mass spectrometry process 124. Hence, in embodiments, the separation unit may comprise one or more of an LCMS unit 325a (or “LCMS system”) and a GCMS unit 325b (or: “GCMS system”).

[0146] In further embodiments, the system may comprise a solvent exchange unit. The solvent exchange unit may be functionally coupled to the separation unit 320 and to the preparation unit 330. In further embodiments the separation unit may comprise the solvent exchange unit. In further embodiments, the preparation unit may comprise the solvent exchange unit. The solvent exchange unit may be configured to solvent exchange the fraction 35 comprising the derivatized organic molecule 30 and to provide a solvent-exchange fraction.

[0147] Especially, the solvent exchange unit may be configured to solvent exchange the fraction 35 comprising the derivatized organic molecule 30 from the separation unit 320 and to provide a solvent-exchange fraction comprising the derivatized organic molecule 30 to the preparation unit 330.

[0148] In the depicted embodiment, the system 300 further comprises an optional pre-analysis unit 345, wherein the pre-analysis unit 345 may be configured to assess whether the organic molecule 20 is identifiable based on fragmentation patterns obtained from the mass spectrometry unit 324. If the (derivatized) organic molecule is (uniquely) identified, a sample preparation process may be terminated. If the (derivatized) organic molecule has not yet been identified, the fraction 35 comprising the derivatized organic molecule 30 may be provided to the preparation unit 330.

[0149] FIG. 2A-B schematically depict embodiments of the derivatization stage 110. The derivatization stage mat comprise derivatizing the target group 21 of the organic molecule 20 with a moiety 31 comprising one or more of (i) a hydrocarbon comprising group and (ii) a 3.sup.rd period atom comprising group, wherein the 3.sup.rd period atom is selected from the group consisting of Si, P, and S, thereby providing a derivatized organic molecule 30.

[0150] In particular, FIG. 2A schematically depicts derivatizing two target groups 21 of the organic molecule 20 daidzein having targets groups 21 comprising —OH with a moiety 31 comprising methyl to provide the derivatized organic molecule 30 dimethyldaidzein. In alternative embodiments, the organic molecule daidzein may be derivatized with a moiety 31 comprising trimethylsilyl to provide the trimethylsilyl derivative of daidzein as described in C. S. Creaser, M. R. Koupai-Abyazani and G. R. Stephenson, Journal of Chromatography, 1989, 478, 415-21, which is hereby herein incorporated by reference.

[0151] FIG. 2B schematically depicts an embodiment comprising derivatizing the organic molecule 20 benzylamine having a target group 21 comprising —NH.sub.2 with a moiety 31 comprising trimethylsilyl to provide its trimethylsilyl derivative. The derivatization may be performed using the procedure described in C. Bellini, T. Roisnel, J. -F. Carpentier, S. Tobisch and Y. Sarazin, Chem. Eur. J. 2016, 22,15733-15743; A. V. Lebedev, A. B. Lebedeva, V. D. Sheludyakov, S. N. Ovcharuk, E. A. Kovaleva and 0. L. Ustinova, Russian Journal of General Chemistry, 2006, 76, 469-477, which is hereby herein incorporated by reference. In further embodiments, the organic molecule 20 phenethylamine may be derivatized with a moiety 31 comprising trimethylsilyl, especially using the same procedure.

[0152] FIG. 3 schematically depicts an embodiment of the derivatized organic molecule doped porous single crystal 50, i.e. the derivatized organic molecule 30 has been introduced into the porous single crsystal 40. In the depicted embodiment, the derivatized organic molecule 30 comprises dimethyldaidzein.

[0153] In the depicted embodiment, the porous single crystal 40 comprises a metal-organic framework material. Specifically, in the depicted embodiment the porous single crystal 40 comprises tpt-ZnX.sub.2, wherein X═Cl or Br or I.

[0154] Experimental methods.

Example 1: Derivatizations of 4′,7-dihydroxy isoflavone (daidzein)

[0155] Procedure 1—absorption of an organic molecule into a single porous crystal. Specifically, procedure 1 describes absorption of an organic molecule into a crystalline sponge comprising [(ZnCl.sub.2).sub.3(tpt).sub.2.(cyclohexane).sub.x] (tpt=1,3,5-tris(4-pyridyl)triazine. The organic molecule is first dissolved in trichloro methane (chloroform), i.e., the sample comprises the organic molecule in trichloro methane. The procedure comprises the steps: [0156] i—1 mg of organic molecule is dissolved in 1 ml of chloroform at room temperature. (Proportionally lower quantities can be used, depending on the available amount of analyte.) [0157] ii—A single crystal of [(ZnCl.sub.2).sub.3(tpt).sub.2.(cyclohexane).sub.x] (tpt=1,3,5-tris(4-pyridyl)triazine) crystal sponge of about 100 μm diameter, which has been visually inspected under a microscope and found to be without twinning or visible cracks, is placed in a septum screw-top glass vial with conically pointed bottom, submerged in 50 μl of cyclohexane. [0158] iii—4.5 μl of the solution obtained in step (i) (containing 4.5 μg of analyte) is added to the crystalline sponge in cyclohexane as described in step (ii). [0159] iv—The screw-top is closed, and the septum is pierced with a medical-type syringe needle, which may be left in that position to enable slow solvent evaporation. This assembly is incubated at 50° C. for 24 or more hours. Most of the solvent may evaporate in the process. [0160] v—After 24 or more hours the process is complete, and the crystal can be used for single-crystal X-ray diffraction for determination of the analyte's chemical structure.

[0161] The experiments A-C described herein were performed using above-described procedure 1.

Experiment A

[0162] Procedure 1 was applied to 4′,7-dihydroxy isoflavone (daidzein) as model organic molecule. It was observed that at 20° C. about 0.005 mg analyte could be dissolved in 1 ml chloroform. This means that the solution of 1 mg analyte in 1 ml solvent required by Step 1 of the above Standard Procedure cannot be prepared, due to limited solubility.

Experiment B

[0163] 4′,7-Dihydroxy isoflavone was derivatized into 4′,7-dimethoxy isoflavone. Methylation can be performed e.g. with dimethyl carbonate or with methyl iodide and potassium carbonate.

Experiment C

[0164] The derivatized organic molecule (4′,7-dimethoxy isoflavone obtained from Experiment B) was dissolved in chloroform. A solution of 1 mg analyte in 1 ml chloroform could be prepared without difficulty, owing to the reduced polarity of 4′,7-dimethoxy isoflavone as compared with the underivatized analyte 4′,7-dihydroxy isoflavone. Addition of 4.5 μlstandard solution to [(ZnCl.sub.2).sub.3(tpt).sub.2.(cyclohexane).sub.x] (tpt =1,3,5-tris(4-pyridyl)triazine) crystal sponge in 50 μl of cyclohexane and incubation at 50° C. for 24 h or more (see Procedure 1) resulted in analyte absorption and subsequent successful determination of the analyte structure with X-ray analysis.

[0165] This X-ray analysis was carried according to the procedure (Procedure 2) as follows:

[0166] Single crystal X-ray diffraction measurement was conducted on a Rigaku Oxford Diffraction XtaLAB Synergy-R diffractometer using Cu-Kα X-ray radiation (λ=1.54184 Å), equipped with a HyPix-ARC 150° Hybrid Photon Counting (HPC) detector (Rigaku, Tokyo, Japan) at a temperature of 100 K using a Cryostream 800 nitrogen stream (Oxford Cryostreams, UK). The software CrysAlisPro ver. 171.41.68) was used for calculation of measurement strategy and data reduction (data integration, empirical and numerical absorption corrections and scaling).

[0167] All crystal structures were modeled using OLEX2 [Dolomanov O V, Bourhis L J, Gildea R J, Howard J A K, and Puschmann H (2009) OLEX2: a complete structure solution, refinement and analysis program. J. Appl. Crystallogr. 42: 339-341.], solved with SHELXT ver. 2014/5 and refined using SHELXL ver. 2018/1 [Sheldrick G M (2015) Crystal structure refinement with SHELXL. Acta Crystallogr. C Struct. Chem. 71: 3-8.]. Non-hydrogen atoms were refined anisotropically. Hydrogen atoms were fixed using the riding model. Populations of the guests in the crystal were modelled by least-square refinement of a guest/solvent disorder model under the constraint that the sum of them should equal to 100%.

[0168] The framework is refined without using restraints. Two 4′,7-dimethoxy isoflavone molecules could be found in the asymmetric unit translationally disordered and disordered with cyclohexane and refined using the disorder model. Some bonds and angles were fixed using DFIX and DANG commands. Results of the refinement can be taken from Table 1.

TABLE-US-00001 TABLE 1 Crystal data and structure refinement for sponge soaked with 4′,7-dimethoxy isoflavone. Empirical formula C.sub.17H.sub.74Cl.sub.6N.sub.12O.sub.4Zn.sub.3 Formula weight 1568.23 Temperature/K 100.01(10) Crystal system monoclinic Space group C2/c a/Å 33.2791(5) b/Å 14.5035(2) c/Å 31.6896(4) α/° 90 β/° 102.087(2) γ/° 90 Volume/Å.sup.3 14956.3(4) Z 8 ρ.sub.calc g/cm.sup.3 1.393 μ/mm.sup.−1 3.532 F(000) 6464.0 Crystal size/mm.sup.3 0.238 × 0.107 × 0.085 Radiation Cu Kα (λ = 1.54184) 2Θ range for data collection/.sup.° 5.432 to 134.156 Index ranges −39 ≤ h ≤ 39, −17 ≤ k ≤ 10, −37 ≤ 1 ≤37 Reflections collected 48777 Independent reflections 13311 [Rint = 0.0183, Rsigma = 0.0150] Data/restraints/parameters 13311/559/1217 Goodness-of-fit on F.sup.2 1.117 Final R indexes [I >= 2σ (I)] R.sub.1 = 0.0644, wR.sub.2 = 0.1604 Final R indexes [all data] R.sub.1 = 0.0691, wR.sub.2 = 0.1627 Largest diff. peak/hole/e Å.sup.−3 0.89/−0.52

[0169] In conclusion, the solubility problem observed in Experiment A was resolved through analyte derivatization according to Experiment B. By means of Experiment C, analyte derivatization was confirmed to extend the scope of applicability of the CS method.

[0170] Experiments D-M can be performed.

Experiment D

[0171] 4′,7-Dihydroxy isoflavone is converted into its trimethylsillyl derivative using the procedure described in C. S. Creaser, M. R. Koupai-Abyazani and G. R. Stephenson, Journal of Chromatography, 1989, 478, 415-21, which is hereby herein incorporated by reference.

Experiment E

[0172] The trimethylsillyl derivative (obtained from Experiment D) is dissolved in dichloromethane (1 mg/1 mL). Addition of 4.0 μl standard solution to [(ZnCl.sub.2).sub.3(tpt).sub.2.(cyclo-hexane).sub.x] (tpt=1,3,5-tris(4-pyridyl)triazine) crystal sponge in 50 μl of cyclohexane and incubation at 50° C. for 24 h (see Procedure 1) results in analyte absorption and subsequent determination of the analyte structure in XRD (see Procedure 2).

Example 2: Derivatization of Benzylamine or Phenethylamine

Experiment F

[0173] Primary amines are nucleophilic, and they tend to destroy the crystal sponge during analyte soaking procedure. For example, addition of 4.0 μl standard solution of benzylamine or Phenethylamine (dissolved 1 mg/1 mL in dichloromethane) to [(ZnCl.sub.2).sub.3(tpt).sub.2.(cyclohexane).sub.x] (tpt=1,3,5-tris(4-pyridyl)triazine) crystal sponge in 50 μl of cyclohexane and incubation at 50° C. for 24 h (see Procedure 1 as in Example 1) results in completely cracked sponge crystals and subsequent determination of the analyte structure using XRD is not possible.

Experiment G

[0174] Benzylamine or Phenethylamine is converted into its trimethylsillyl derivative using the procedures described in C. Bellini, T. Roisnel, J. -F. Carpentier, S. Tobisch and Y. Sarazin, Chem. Eur. J. 2016, 22,15733-15743; and A. V. Lebedev, A. B. Lebedeva, V. D. Sheludyakov, S. N. Ovcharuk, E. A. Kovaleva and O. L. Ustinova, Russian Journal of General Chemistry, 2006, 76, 469-477, which are hereby herein incorporated by reference.

Experiment H

[0175] The trimethylsillyl derivative (obtained from Experiment G) is dissolved in dichloromethane (1 mg/1 mL). Addition of 4.0 μl standard solution to [(ZnCl.sub.2).sub.3(tpt).sub.2.(cyclohexane).sub.x] (tpt=1,3,5-tris(4-pyridyl)triazine) crystal sponge in 50 μl of cyclohexane and incubation at 50° C. for 24 h (see Procedure 1 as in Example 1) results in analyte absorption and subsequent determination of the analyte structure in XRD (See Procedure 2 as in example 1).

Experiment I

[0176] Benzyl trimethylsilyl ether is dissolved in dichloromethane (1 mg/1 mL). Addition of 4.0 μl standard solution to [(ZnCl.sub.2).sub.3(tpt).sub.2.(cyclohexane).sub.x] (tpt=1,3,5-tris(4-pyridyl)triazine) crystal sponge in 50 μl of cyclohexane and incubation at 50° C. for 24 h (see Procedure 1 as in Example 1) results in analyte absorption and subsequent determination of the analyte structure in XRD (see Procedure 2 as in example 1). Benzyl trimethylsilyl ether may be a commercially available silylated derivative of benzyl alcohol.

[0177] The framework is refined without using restraints. One Benzyl trimethylsilyl ether molecule could be found in the asymmetric. Some bonds and angles were fixed using DFIX and DANG commands. Results of the refinement can be taken from Table 2.

TABLE-US-00002 TABLE 2 Crystal data and structure refinement for sponge soaked with Benzyl trimethyl silyl ether. Empirical formula C.sub.42.44H.sub.34.02Cl.sub.6N.sub.12O.sub.0.71Si.sub.0.71Zn.sub.3 Formula weight 1151.64 Temperature/K 100.00(10) Crystal system monoclinic Space group C2/c a/Å 32.8428(12) b/Å 14.4175(3) c/Å 31.0244(15) α/° 90 β/° 99.428(4) γ/° 90 Volume/Å.sup.3 14492.0(9) Z 8 ρ.sub.calc g/cm.sup.3 1.056 μ/mm.sup.−1 3.561 F(000) 4647.0 Crystal size/mm.sup.3 0.173 × 0.055 × 0.025 Radiation Cu Kα (λ = 1.54184) 2Θ range for data collection/° 5.776 to 134.15 Index ranges −39 ≤ h ≤ 38, −8 ≤ k ≤ 17, −37 ≤ 1 ≤ 37 Reflections collected 40240 Independent reflections 12853 [Rint = 0.0581, Rsigma = 0.0479] Data/restraints/parameters 12853/66/630 Goodness-of-fit on F.sup.2 1.015 Final R indexes [I >= 2σ (I)] R.sub.1 = 0.1926, wR.sub.2 = 0.5242 Final R indexes [all data] R.sub.1 = 0.2073, wR.sub.2 = 0.5440 Largest diff. peak/hole/e Å.sup.−3 1.38/−2.73

Experiment J

[0178] N-Benzyl-1,1,1-trimethylsilanamine is dissolved in dichloromethane (1 mg/1 mL). Addition of 4.0 μl standard solution to [(ZnCl.sub.2).sub.3(tpt).sub.2.(cyclohexane).sub.x] (tpt=1,3,5-tris(4-pyridyl)triazine) crystal sponge in 50 μl of cyclohexane and incubation at 50° C. for 24 h (see Procedure 1 as in Example 1) results in analyte absorption and subsequent determination of the analyte structure in XRD (see Procedure 2 as in example 1). N-Benzyl-1,1,1-trimethylsilanamine may be a commercially available silylated derivative of benzyl amine.

Example 3

[0179] Despite several trials, the structure of Oseltamivir (ethyl(3R,4R,5S)-4-acetamido-5-amino-3-pentan-3-yloxycyclohexene-1-carboxylate) could not successfully be elucidated by the crystalline sponge method. Therefore, the primary amine function was derivatized by acylation. After derivatization the crystalline sponge method could successfully be applied.

Experiment K

[0180] Oseltamivir was derivatized with acetic anhydride as is shown in reaction scheme 1 below:

##STR00001##

Derivatization was carried out as follows: Oseltamivir phosphate (199.6 mg) and dimethylaminopyridin (104.6 mg) were mixed in dichloromethane (2 ml). Triethylamine (200 μl) was added to the suspension and acetic anhydride (130 μl) was added dropwise over 30 s. After 2.5 h the reaction progress was checked by thin layer chromatography (silicagel 60 F254; DCM/MeOH 95:5). After completion of the reaction the solution was washed with HCl (6 mol/l), saturated NaHCO.sub.3 solution, water, saturated NaCl solution and dried over Na.sub.2SO.sub.4. The solvent was removed under reduced pressure and dissolved in water/methanol (1:1) and the solvent was slowly evaporated to yield a colorless powder. The product was purified by column chromatography (silicagel, DCM/MeOH 95:5).

Experiment L

[0181] The derivatized Oseltamivir (obtained from Experiment K) was dissolved in dichloromethane (1 mg/1 mL). 2.0 μl of a standard solution of the derivatized Oseltamivir was added to a [(ZnCl.sub.2).sub.3(tpt).sub.2] (tpt=1,3,5-tris(4-pyridyl)triazine) crystal sponge in 40 μl of cyclohexane and incubated at 50° C. for 21 h (see Procedure 1 as in Example 1). This resulted in analyte (Oseltamivir derivative) absorption for subsequent determination of the analyte structure in XRD.

Experiment M

[0182] Single crystal X-ray diffraction measurement was conducted according to Procedure 2 (see Procedure 2 as in example 1) including measurement on a Rigaku Oxford Diffraction XtaLAB Synergy-R diffractometer using Cu-Kα X-ray radiation (λ=1.54184 Å), equipped with a HyPix-ARC 150° Hybrid Photon Counting (HPC) detector (Rigaku, Tokyo, Japan) at a temperature of 100 K using a Cryostream 800 nitrogen stream (Oxford Cryostreams, UK). The software CrysAlisPro ver. 171.41.68) was used for calculation of measurement strategy and data reduction (data integration, empirical and numerical absorption corrections and scaling).

[0183] All crystal structures were modeled using OLEX2 [Dolomanov O V, Bourhis L J, Gildea R J, Howard J A K, and Puschmann H (2009) OLEX2: a complete structure solution, refinement and analysis program. J. Appl. Crystallogr. 42: 339-341.], solved with SHELXT ver. 2014/5 and refined using SHELXL ver. 2018/1 [Sheldrick G M (2015) Crystal structure refinement with SHELXL. Acta Crystallogr. C Struct. Chem. 71: 3-8.]. Non-hydrogen atoms were refined anisotropically. Hydrogen atoms were fixed using the riding model. Populations of the guests in the crystal were modelled by least-square refinement of a guest/solvent disorder model under the constraint that the sum of them should equal to 100%.

[0184] The framework is refined without using restraints. One ZnCl.sub.2 moiety is disordered and refined using disorder model. One Oseltamivir molecule could be found in the asymmetric unit. Some bonds and angles were fixed using DFIX and DANG commands. Results of the refinement can be taken from Table 3.

TABLE-US-00003 TABLE 3 Crystal data and structure refinement for sponge soaked with Oseltamivir. Empirical formula C.sub.90H.sub.75C1.sub.12N.sub.26O.sub.5Zn.sub.6 Formula weight 2418.38 Temperature/K 99.9(4) Crystal system monoclinic Space group C2 a/Å 32.7057(5) b/Å 14.37810(10) c/Å 31.2441(6) α/° 90 β/° 101.413(2) γ/° 90 Volume/Å.sup.3 14401.9(4 Z 4 ρ.sub.calc g/cm.sup.3 1.115 μ/mm.sup.−1 3.521 F(000) 4884.0 Crystal size/mm.sup.3 0.155 × 0.077 × 0.037 Radiation Cu Kα (λ = 1.54184) 2Θ range for data collection/.sup.° 5.514 to 149.288 Index ranges −39 < h < 40, −10 < k < 17, −38 < 1 < 39 Reflections collected 129250 Independent reflections 23539 [R.sub.int = 0.0314, R.sub.sigma = 0.0329] Data/restraints/parameters 23539/192/1328 Goodness-of-fit on F.sup.2 1.034 Final R indexes [I >= 2σ (I)] R.sub.1 = 0.0502, wR.sub.2 = 0.1427 Final R indexes [all data] R.sub.1 = 0.0776, wR.sub.2 = 0.1573 Largest diff. peak/hole/e Å.sup.−3 0.47/-0.51 Flack Parameter 0.129(11)

[0185] From the above data the crystal structure for Oseltamivir was successfully obtained. In conclusion, the derivatization of Oseltamivir allowed structure elucidation using the crystalline sponge (CS) method for XRD cystallography where with the underivatized Oseltamivir the crystal structure could not be successfully elucidated using the the crystal sponge method.

[0186] The term “plurality” refers to two or more. Furthermore, the terms “a plurality of” and “a number of” may be used interchangeably.

[0187] The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%.

[0188] Moreover, the terms “about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90%-110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

[0189] The term “comprise” includes also embodiments wherein the term “comprises” means “consists of”.

[0190] The term “and/or” especially relates to one or more of the items mentioned before and after “and/or”. For instance, a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term “comprising” may in an embodiment refer to “consisting of” but may in another embodiment also refer to “containing at least the defined species and optionally one or more other species”.

[0191] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0192] The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

[0193] The term “further embodiment”, and similar terms, may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

[0194] It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

[0195] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

[0196] Use of the verb “to comprise” and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0197] The article “a” or “an” preceding an element does not exclude the presence of a plurality of such elements.

[0198] The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0199] The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

[0200] The term “controlling” and similar terms herein especially refer at least to determining the behavior or supervising the running of an element, such as a unit. Hence, herein “controlling” and similar terms may e.g. refer to imposing behavior to the element (determining the behavior or supervising the running of an element), etc., such as e.g. measuring, displaying, actuating, opening, shifting, changing temperature, etc. Beyond that, the term “controlling” and similar terms may additionally include monitoring. Hence, the term “controlling” and similar terms may include imposing behavior on an element and also imposing behavior on an element and monitoring the element. The controlling of the element can be done with a control system (also: “controller”). The control system and the element may thus at least temporarily, or permanently, functionally be coupled. The element may comprise the control system. In embodiments, the control system and element may not be physically coupled. Control can be done via wired and/or wireless control. The term “control system” may also refer to a plurality of different control systems, which especially are functionally coupled, and of which e.g. one control system may be a master control system and one or more others may be slave control systems.

[0201] The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method respectively.

[0202] The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.