A COMPOUND FOR THE DETERMINATION OF THE PROTEIN FKBP12 AND A SENSOR UNIT COMPRISING IT
20230033380 · 2023-02-02
Assignee
Inventors
- Gabriella CAMINATI (Sesto Fiorentino (FI), IT)
- Piero PROCACCI (Sesto Fiorentino (FI), IT)
- Stefano MENICHETTI (Sesto Fiorentino (FI), IT)
- Maria Raffaella MARTINA (Sesto Fiorentino (FI), IT)
- Laura MARSILI (Sesto Fiorentino (FI), IT)
Cpc classification
B01L3/5027
PERFORMING OPERATIONS; TRANSPORTING
C07D211/06
CHEMISTRY; METALLURGY
C07D211/60
CHEMISTRY; METALLURGY
G01N33/54373
PHYSICS
International classification
C07D211/06
CHEMISTRY; METALLURGY
B01L3/00
PERFORMING OPERATIONS; TRANSPORTING
Abstract
The present invention relates to novel compounds useful as sensors for the rapid and specific determination of the FKBP12 protein, a peptidyl-prolyl cis-trans isomerase (PPlase), the levels of which in the biological fluids of a subject change if the subject is affected by pathological conditions, in particular neurodegenerative diseases, such as the Parkinson's disease and the Alzheimer's syndrome, tumour pathologies, autoimmune diseases, or if that subject is in a phase of acute rejection after organ transplantation.
Claims
1. A compound of formula (I) ##STR00009## wherein L is selected from the group consisting of thiol, organo-sulfur groups, Y is selected from —(CH.sub.2).sub.n— and —(OCH.sub.2CH.sub.2).sub.m—, where n is between 7 and 13 and m is between 2 and 6.
2. The compound of claim 1, wherein n is 8.
3. The compound of claim 1, wherein L is a thiol group.
4. A process for preparing a compound of formula (I) of claim 1, comprising the steps of: a) preparing an (S)-carbamoyl piperidine of formula (II) starting from 3-phenylpropyl-1-amine ##STR00010## b) preparing an (S)-piperidin-2-carboxy-amide of formula (IV) ##STR00011## by reacting said (S)-carbamoyl piperidine of formula (II) prepared in step a) with 4-hydroxyphenylglyoxalic acid; and c) synthesizing a spacing agent for the covalent bonding of a Y—CH.sub.2-L group to (S)-piperidin-2-carboxy-amide of formula (IV) obtained above in step b).
5. A sensor unit for detecting devices of the FKBP12 protein in biological samples, comprising a substrate having a measurement surface on at least a part of which there is a monolayer coating of a compound of formula (I) of claim 1.
6. The sensor unit of claim 19, wherein said phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylserine, dipalmitoyl phosphatidylethanolamine, dipalmitoylphosphatidic acid, dioleylphosphatidylcholine, and palmitoyloleylphosphatidylcholine.
7. The sensor unit of claim 5, wherein said coating has nanometric thickness.
8. The sensor unit of claim 19, wherein Y and L are equal to each other in said compound of formula (I) and in said spacer, when present.
9. The sensor unit of claim 5, wherein said measurement surface is a metal surface or a surface coated with a metallic coating.
10. A process for preparing the sensor unit of claim 5, comprising immersing at least part of the measurement surface of the unit in a solution of a compound of formula (I) wherein ##STR00012## L is selected from the group consisting of thiol and organo-sulfur groups, Y is selected from —(CH.sub.2).sub.n— and —(OCH.sub.2CH.sub.2).sub.m—, where n is between 7 and 13 and m is between 2 and 6 and of a spacer molecule of formula CH.sub.3—Z—CH.sub.2-L, wherein Z is equal to Y or is a group —[OCH.sub.2CH.sub.2].sub.n−1—CH.sub.2CH.sub.2-L with n ranging between 4 and 100 and L is a thiol or organo-sulfur group, in a solvent selected from water, an organic solvent, and mixtures thereof, until the surface to be coated of said sensor unit is saturated, followed by rinsing in the same above said solvent.
11. A device for detecting the FKBP12 protein in biological samples, comprising as a sensor element the sensor unit as defined in claim 5 and a detection system of said FKBP12 protein.
12. The device of claim 11, for the detection of the FKBP12 protein down to subnanomolar concentrations.
13. The device of claim 11, wherein said FKBP12 protein detection system is selected from quartz crystal microbalance (QCM), Surface Plasmon Resonance device (SPR), instrumentation for electrochemical measurements, Surface Enhanced Raman Spectroscopy (SERS), instrumentation for fluorescence measurements, and combinations thereof.
14. The device of claim 11, wherein said FKBP12 protein detection system is miniaturized and/or combined with a microfluidic system for Lab-on-chip applications and multiple analyses.
15. (canceled)
16. (canceled)
17. (canceled)
18. The compound of claim 1, wherein the organo-sulfur groups are selected from the group consisting of sulfide (—SR), disulfide (—SSR), thiolesters (—SC(O)R), and dithiocarbammates (—SC(S)NRR′) wherein R and R′, equal or different between each other, are alkyl or aryl residues, carboxylic acid, primary amine, trichlorosilane, trimethoxysilane, triethoxysilane, cholesterol, benzene, naphthalene, anthracene, phenanthrene, and pyrene.
19. The sensor unit of claim 5, wherein the monolayer coating is immersed in a matrix of a spacer molecule of formula CH.sub.3—Z—CH.sub.2-L, wherein Z is equal to Y or is a group —[OCH.sub.2CH.sub.2].sub.n−1—CH.sub.2CH.sub.2-L with n ranging between 4 and 100, or in a matrix of a phospholipid spacer molecule.
20. The sensor unit of claim 9, wherein the metal surface or a surface coated with a metallic coating is covered with graphene or graphene oxide.
21. A method for detecting the FKBP12 protein in biological samples, comprising contacting a biological sample taken from a subject with a device of claim 11.
22. The method of claim 21, further comprising comparing the concentration of FKBP12 protein in the sample to the concentration of FKBP12 protein in a healthy subject and diagnosing a condition of the subject associated with the difference in the concentration of FKBP12 protein in the sample compared to the concentration of FKBP12 in the healthy subject.
23. The method of claim 22, wherein the condition is selected from the group consisting of neurodegenerative diseases, a tumor pathology, autoimmune diseases or disorders and subjects who have undergone organ and/or tissue transplantation.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0034]
[0035]
[0036]
[0037]
[0038]
[0039]
[0040]
DETAILED DESCRIPTION OF THE INVENTION
[0041] Unless otherwise specified, in the context of the present invention the term “biological samples” means biological fluid samples such as cerebrospinal fluid, blood and blood derivatives, urine, sweat, mucus and saliva.
[0042] Subject of this invention are the novel compounds of formula (I)
##STR00001##
wherein L is a group selected from thiol (—SH), organo-sulfur groups, such as sulfide (—SR), disulfide (—SSR), thiolesters (—SC(O)R), and dithiocarbammates (—SC(S)NRR′) wherein R and R′, equal or different to each other, are selected from alkyl or aryl residues, carboxylic acid, primary amine, trichlorosilane, triethoxysilane, trimethoxysilane, cholesterol, benzene, naphthalene, anthracene, phenanthrene, and pyrene, and Y is selected from linear chains (CH.sub.2).sub.n— and —(OCH.sub.2CH.sub.2).sub.m— wherein n ranges between 7 and 13 and m ranges between 2 and 6.
[0043] In an aspect of this invention, in the compounds of formula (I), L is a group selected from thiol, sulfide, disulfide, carboxylic acid, primary amine, trichlorosilane, triethoxysilane, trimethoxysilane, cholesterol, benzene, naphthalene, anthracene, phenanthrene, and pyrene, and Y is selected from linear chains (CH.sub.2).sub.n— and —(OCH.sub.2CH.sub.2).sub.m— wherein n ranges between 7 and 13 and m ranges between 2 and 6.
[0044] Such compounds comprise, as mentioned above, a ligand group for the univocal recognition of the FKBP12 protein consisting of the aromatic pendant, an anchoring group L for the covalent attack on the surface of a support of the measurement device which can vary according to the support to which it is supposed to be bound, and a spacing agent Y having length and chemical characteristics such as to ensure correct density and molecular orientation of the receptor on the substrate surface.
[0045] Any technician skilled in the art can easily select from the L groups as defined above, the most suitable ones for the application of the specific sensor unit to be used on the substrate. For example, in the case of protein analytical measurement methods such as Surface Plasmon Resonance (SPR), Quartz Crystal Microbalance (QCM), instrumentation for electrochemical measurements, or Surface Enhanced Raman Spectroscopy, the substrate on which the sensor unit is built has a gold or silver surface. For example, in the case of the QCM method it consists of piezoelectric quartz crystals, which can be coated with gold, silver, ITO or silicon oxides or nitrides. On the other hand, in the case of different measurement methods, such as cantilevered stress measurements, the substrate forming the sensor element is a silicon oxide or nitride; in the case of spectrofluorimetric methods, the substrate is made of quartz or glass.
[0046] In the case of substrates coated with gold, the sensor unit of the invention comprises a receptor compound of general formula (I) wherein L is a group containing one or more sulfur atoms, in particular thiol, sulfide, disulphide, thiolesters or dithiocarbammates, for the attack to the surface of gold. For the chemical attack on quartz or silicon-based compounds, used in the other methods indicated above, the sensor unit of the invention typically comprises a receptor compound of general formula (I) wherein L is a silicon-containing group of formula SiX.sub.3, wherein X is Cl, OCH.sub.2CH.sub.3, and OCH.sub.3.
[0047] In the case of the analytical determination of the protein with methods such as the Surface Enhanced Raman Spectroscopy with substrates coated with metal nanoparticles, such as silver or gold, as such or veiled with graphene oxide or other graphene derivatives, the sensor element of the invention typically comprises a compound (I) wherein L is selected from primary amino groups (—NH.sub.2) or carboxylic groups (—COOH) in the case of veiling with graphene derivatives.
[0048] As regards the spacer Y in the compounds of formula (I), the definition thereof takes into account the length and the characteristics that this part of the receptor molecule must have according to the invention to allow the binding of the protein even in traces on one side and on the other the complete and stable covering of the substrate by means of the group L. Optimal results in the experimentation conducted with the compounds of the invention were obtained for the compounds of general formula (I) wherein n is 8. Replica exchange molecular dynamics calculations for spacers with chains longer than n=13, showed a folding of the compound of formula (I), which prevents both exposure of the receptor group to the solution to be analysed and a compact coating of the substrate.
[0049] The compounds of formula (I) can be prepared starting from tert-butyl (S)-2-((3-phenylpropyl) carbamoyl)piperidine-1-carboxylate of formula (II) reported below by hydrolysis of the ester group followed by functionalisation with the aromatic residue bearing the appropriate spacer Y and ligand L.
[0050] The compounds of general formula (I) are used to create a sensor unit for the devices detecting the FKBP12 protein in biological samples, by coating at least a part of the measurement surface in the form of a monolayer of a compound of formula (I) as defined above, optionally immersed in a matrix of molecules of inert spacer towards proteins. This coating typically has a nanometric thickness, and consists of a homogeneous monolayer, in which the pipecolic unit for the recognition of the FKBP12 protein are extended towards the solvent. When the spacer is present, the coating comprises compound of formula (I) and spacer, having a thickness lower than the thickness created in the monolayer by the compound (I), as schematically illustrated in
[0051] The element to be connected to the detection measurement system (transducer) can be used directly as a solid substrate; it can, for example, have a circular or rectangular shape and dimensions of a few centimetres. The modular design of this sensor unit mounted on the substrate will allow the adaptation thereof to a wide range of detection systems, such as QCM, SERS, SPR mechanical stress analysis, electrochemical orfluorescent systems, etc., as exemplified in the following Scheme 1:
TABLE-US-00001 Scheme 1 Instrumen- tation Substrate Description QCM In the case of microgravimetric detection systems the substrate can be a piezoelectric crystal cut at 5 or 10 MHz covered with an evaporated layer of gold or silver or silicon or silicon oxides or ITO. SPR In the case of detection systems based on Surface Plasmon Resonance, the gold surface can be coupled to a quartz prism by means of an elastomer. SERS In the case of investigation systems based on Surface Enhanced Raman Scattering, the surface to be covered can be in gold or quartz. The performance of the device can be increased by using nanostructured systems based on metallic nanoparticles also coupled to nanolayers of graphene derivatives Stress For stress measurement sensors the substrate can be an array analysis of cantilevers covered with gold or silver or silicon or silicon oxides/nitrides Others For other detection systems (fluorescence, electrochemical ones), glass, quartz, glassy carbon or ITO supports are preferably used. Such systems can also be used in conjunction with QCM determination.
[0052] In other words, the present sensor unit for detection devices of the FKBP12 protein, consists of a compact monolayer of receptor molecules (compound of formula (I)), optionally immersed in a matrix of spacer molecules, with control of the surface density and of the thickness of the monolayer in the nanoscale. In the following, the receptor or receptor/spacer monolayer is also referred to as a Self-Assembled Monolayer (SAM). This monolayer, in which substrate and receptor are bound by the group L of the receptor compound (I), leaves the opposite part of the molecule with the aromatic and pipecolic pendants free for binding to the FKBP12 protein. Therefore the protein, if present in a sample of biological fluid such as cerebrospinal fluid, blood or saliva for example, put in contact with the monolayer, will bind quantitatively to the receptor of formula (I) forming the monolayer, which will allow not only qualitative but also quantitative measurements being able to obtain the concentration of protein in the sample.
[0053] The sensor unit of the invention for use as a sensor in detection devices of the FKBP12 protein in biological samples comprises, on at least a part of its measurement surface, a monolayer coating of a compound of formula (I) as described above, optionally immersed in a matrix of a spacer molecule of formula CH.sub.3—Z—CH.sub.2-L, wherein Z is equal to Y or is a group —[OCH.sub.2CH.sub.2].sub.n−1—CH.sub.2CH.sub.2-L with n ranging between 4 and 100, and Y and L are defined as above, or in a matrix of a phospholipid spacer molecule, selected for example from the group consisting of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylserine, dipalmitoyl phosphatidylethanolamine, dipalmitoylphosphatidic acid, dioleylphosphatidylcholine, and palmitoyloleylphosphatidylcholine.
[0054] In an embodiment of the invention, when L is a group selected from thiol, organo-sulfur groups, carboxylic acid, primary amine, trichlorosilane, triethoxysilane, and trimethoxysilane in the compounds of formula (I), the spacer has formula CH.sub.3—Z—CH.sub.2-L, as defined above.
[0055] In an embodiment of the invention, when L is a group selected from thiol, sulfide, disulfide, carboxylic acid, primary amine, trichlorosilane, triethoxysilane, and trimethoxysilane in the compounds of formula (I), the spacer has formula CH.sub.3—Z—CH.sub.2-L, as defined above.
[0056] In a further embodiment of the invention, when L is selected from cholesterol, benzene, naphthalene, anthracene, phenanthrene, and pyrene in the compounds of formula (I), the spacer is of the phospholipid type.
[0057] The process for the preparation of the receptor monolayer on the substrate which constitutes the sensor element of the invention comprises immersing at least part of the measurement surface of the element in a solution of the compound of formula (I) in a solvent selected from water and/or ethanol until the surface to be coated is saturated, followed by rinsing in the same solvent to remove any unreacted material from the measurement surface.
[0058] In a preferred embodiment of the present invention, the sensor element comprises a monolayer comprising a compound of general formula (I) as a receptor for the FKBP12 protein, immersed in a matrix of a spacer as defined above. Also the spacer, like the receptor (I), comprises a group L intended to bind to the substrate, therefore selectable according to the same criteria indicated above for the receptor. The spacer further comprises a group CH.sub.3—[CH.sub.2].sub.n—CH.sub.2— or CH.sub.3—O[CH.sub.2CH.sub.2O].sub.mCH.sub.2CH.sub.2— wherein n and m are defined as above, inert towards the specific adsorption of the FKBP12 protein and of the other components of the biological fluids under examination. When present, the spacer is used in a molar ratio with the receptor (I) ranging between about 4:1 and about 8:1, and preferably in a molar ratio of about 6:1 in the case of a linear alkyl chain matrix, which value ensures the maximum substrate coverage of the sensor element and the best response for the interaction with the protein to be detected FKBP12 by the receptor. As shown in the following experimental part, the spacer does not bind the FKBP12 protein, but promotes the selective binding thereof to the receptor.
[0059] Preferred spacers according to the invention are compounds of formula CH.sub.3—(CH.sub.2).sub.n-L, wherein n and L are defined as above, with linear chain or hydrophilic polymers with anti-fouling properties that hinder the non-specific adhesion of proteins or other components of the biological fluids on the surface of the nanosensor by increasing the selectivity thereof towards the FKBP12 protein.
[0060] With the sensor device of the invention it is possible to conduct a diagnostic method for the diagnosis of conditions to which a change is associated in the concentration of FKBP12 protein in biological samples previously taken from a subject under examination with respect to the detectable concentration for the same protein in a healthy subject.
[0061] With the present sensor device, a method for monitoring the post-operative course in subjects who have undergone organ and/or tissue transplantations can also be realised, comprising the detection of the FKBP12 protein in a biological sample previously taken from the subject under examination with the sensor device of the invention, and a step of assessing the data obtained so that in the absence of changes in the protein concentration the subject is having a regular post-operative course, while in the presence of changes in the concentration of the FKBP12 protein in the sample examined, the subject is having transplant rejection.
[0062] Changes in the concentration of the FKBP protein that are significant for diagnostic purposes of neurodegenerative conditions, tumour pathologies and autoimmune diseases or disorders, as well as for the purpose of monitoring the post-operative course in transplanted subjects, can also be very small, of the order of picomoles/L or nanomoles/L.
[0063] If the main advantage of the present invention is that of being able to effectively detect the presence of traces of the FKBP protein, at subnanomolar concentrations, several other advantages can be granted to the invention, including the flexibility of the receptor compounds of general formula (I), adaptable to different types of analytical techniques for protein detection having different types of measurement substrates: depending on the type of substrate, it will be sufficient to select a different group L in order to be able to obtain the best results of bond of the receptor to the substrate.
[0064] Furthermore, thanks also to the presence of the matrix of spacer that are inert to the components of the biological samples together with the receptor on the substrate, a particularly selective sensor element is obtained for the detection of the FKBP protein and inert to all the other components of the biological fluids under examination; this allows, among other things, to be able to carry out the measurements also directly on the biological samples previously taken from patients, possibly with minimal pre-treatment interventions.
[0065] A further advantage of the present invention consists in the fact that the process for the preparation of the compounds (I) is a simple, inexpensive process, starting from commercially available reagents. Likewise, the process for the preparation of the self-assembled monolayer which forms the sensor element according to the invention is an extremely simple and fast process, which involves the use of only water and/or ethanol as solvents.
[0066] A still further advantage of the present invention is that of being able to carry out the detection of the FKBP12 protein with miniaturized detection systems and/or combined with microfluidic circuits, which allow to carry out the detection of the protein in a very short time and also having available only small amounts of sample to examine, as is the case with cerebrospinal fluid samples.
[0067] Last but not least, it should be noted that the experiments carried out by the inventors and described below have highlighted, as an advantage, a fair stability of the sensor element prepared with the compounds (I) over time, provided that it is kept in a container closed and saturated with N.sub.2. For shorter times, the sensor element of the invention can also be stored in aqueous or buffered solution. Furthermore, it is not a disposable sensor, but it is a sensor element that can be “regenerated” after the first use, and used to perform multiple measurements, after reconditioning with imidazole or ethanol, while maintaining the accuracy of the detection of the protein unaltered.
[0068] Illustrative, but not limiting, examples of the present invention are reported below.
EXPERIMENTAL PART
Materials and Methods
[0069] FKBP12 expressed in Escherichia coli, molecular weight 11900, purity >95% was supplied by the Interuniversity Magnetic Resonance Consortium of Paramagnetic Metalloproteins, CIRMMP (Florence, Italy). The purity of FKBP12 was determined by SDS electrophoresis. K.sub.2HPO.sub.4 and KH.sub.2PO.sub.4 for the preparation of the phosphate buffer was obtained from Sigma-Aldrich (Italy). All measurements were made using ultrapure water (resistivity=18 MΩ cm, pH=5.6 at 20° C.) obtained with a Milli-RO equipment coupled with a Milli-Q set up (Millipore, Italy).
[0070] The concentration of the stock solutions of FKBP12 in PBS buffer (pH 7.4, 0.15 M NaCl) was checked by UV/Vis measurements using ε.sub.280=9970 M.sup.−1 cm.sup.−1. The stock solution of FKBP12 was diluted to reach the concentrations used for the calibration straight line.
[0071] The formulae of the spacers used in mixture with the GPS-SH1 receptor to prepare the sensor unit of the present invention are reported below. These compounds were obtained from Sigma-Aldrich and used as they were received.
##STR00002##
[0072] 1 mM solutions of receptor in ethanol were used for the formation of the monolayer, in the case of SAM films of receptor and spacer mixed solutions were prepared using various receptor-spacer molar ratios in the interval between 1:3 and 1:10.
[0073] Both the gold-coated and the silicon-coated crystals were cleaned with piranha solution, kept in contact with the crystals for about 10 seconds, then rinsed thoroughly with water and ethanol, and dried with compressed air or N.sub.2.
[0074] The microgravimetric analyses were carried out with a Quartz-Crystal microbalance (QCM-Z 500 from KSV-Finland). This instrumentation is equipped with a Peltier element for controlling the temperature inside the measurement chamber. All experiments were performed at a temperature of T=20.0±0.1° C.
[0075] The cleaning of the instrument, of the measurement chamber, of the internal ducts, of the O-rings and of the syringe for injection of the substance was also carried out by means of repeated rinses with ultrapure water, followed by washing with ethanol. The chamber and the injection tubes of the solutions were then dried in a nitrogen flow.
[0076] The frequency and dissipation data for all recorded harmonics were processed with the QCMBrowse software which directly provides adsorbed mass in nanograms/cm.sup.2, thickness of the monolayer in nanometers and viscoelastic parameters of the system.
[0077] The minimum free energy molecular configurations of the functionalised receptor were analysed under standard conditions by simulations of replicate exchange molecular dynamics for amplified sampling of the conformational states (using the orac5.3 program together with the amber99sb force field for molecular mechanics), verifying the maintenance of the optimal exposure in aqueous solution of the carbonyl substituents on the piperidine unit for FKBP12 recognition as a function of the length and chemical characteristics of the spacing agent.
Example 1
[0078] Synthesis of the Functionalised Receptor of Formula (I) Wherein Y is (CH.sub.2).sub.8 and L is SH
##STR00003##
Step 1: Preparation of tert-butyl (S)-2-((3-phenylpropyl)carbamoyl)piperidine-1-carboxylate of formula (II)
[0079] ##STR00004##
[0080] N-Boc-L-pipecolinic acid (425 mg, 1.88 mmol), DIPEA (775 mg, 6 mmol) and HATU (684 mg, 1.8 mmol) were added to a solution of 3-phenylpropyl-1-amine (162 mg, 1.2 mmol) in 10 ml of anhydrous DMF. The additions are carried out at a constant temperature of 0° C., keeping the reaction flask under constant stirring in N.sub.2 atmosphere. When all the reagents had dissolved and the solution in the reaction flask was homogeneous, the temperature was allowed to rise to room temperature and maintained for 15 hours.
[0081] The reaction solution was then mixed with 30 mL of ethyl acetate, and the phases were allowed to separate; the organic phase was washed several times with a saturated solution of NH.sub.4Cl and brine and dried on anhydrous Na.sub.2SO.sub.4. The aqueous solution was then filtered and evaporated by rotary evaporator until a yellow oil was obtained which was purified by flash chromatography column on silica gel using a mixture of DCM:EtOAc/8:1 as eluent. The expected derivative of the title was isolated as a colourless oil (330 mg, 80% yield) and characterized as previously described.
Step 2: Preparation of 4-hydroxyphenylglyoxalic acid of formula (III)
[0082] ##STR00005##
[0083] Freshly distilled thionyl chloride (1.67 mL) was added dropwise at room temperature to a suspension of D-4-hydroxyphenylglycine (1,050 mg, 6.2 mmol) in methanol (25 mL). The reaction solution was left under stirring for 15 hours, then the solvent was removed by means of a rotary evaporator to obtain the methyl ester of the D-4-hydroxyphenylglycine in the form of a white solid, in quantitative yield. It was used for the subsequent steps without further purification.
[0084] The methyl ester of D-4-hydroxyphenylglycine (650 mg, 3.00 mmol) prepared as described above, then glyoxylic acid (2200 mg, 30 mmol) and copper (II) sulfate pentahydrate (740 mg, 3.00 mmol) were added in a freshly prepared buffer solution with 2.5 moles of pyridine and 0.5 moles of acetic acid in 38 mL of water. The reaction mixture was kept under stirring for 10 hours at room temperature, then extracted three times with portions of dichloromethane (20 mL). The organic phases were then combined, washed three times with 1 N HCl and dried over anhydrous Na.sub.2SO.sub.4. The organic phase was then concentrated in the rotary evaporator until a raw product was obtained, purified on a chromatographic column with eluent (CHCl.sub.3). Methyl-4-hydroxyphenylglyoxylate (300 mg, 40% yield) was thus obtained in the form of a yellow solid, used without further purification as follows.
[0085] The methyl-4-hydroxyglyoxylate (410 mg, 2.3 mmol) was dissolved in a 2.5 M NaOH solution (20 mL) and the reaction mixture was kept under stirring for 2 hours at room temperature. The reaction mixture was then washed with diethyl ether (2×10 mL) and acidified with concentrated HCl up to pH=2. The aqueous solution was then extracted with ethyl acetate (5×10 mL) and the combined organic phases, washed with brine (2×10 mL) and dried over anhydrous Na.sub.2SO.sub.4. After filtration, the removal of the solvent with the rotary evaporator delivered the 4-hydroxyphenylglyoxalic acid of formula (III) of the title in the form of a yellow solid (310 mg, 83% yield), characterized by comparison with an authentic sample.
Step 3: Preparation of (S)-1-(2-(4-hydroxyphenyl)-2-oxoacetyl)-N-(3-phenylpropyl)piperidine-2-carboxamide of formula (IV)
[0086] ##STR00006##
[0087] 4-hydroxyphenylglyoxalic acid (148 mg, 0.88 mmoles) prepared as above, DIPEA (460 mg, 3.06 mmol) and HATU (410 mg, 1.08 mmol) were added to a solution of (2S)-N-(3-phenylpropyl)piperidine-2-carboxamide (177 mg, 0.72 mmol) in anhydrous DMF (30 mL), keeping the reaction mixture at a constant temperature of 0° C. in N.sub.2 atmosphere. The reaction mixture has assumed a yellow colour and is kept under stirring at room temperature for 48 hours, maintaining the N.sub.2 atmosphere. The reaction mixture was then diluted with ethyl acetate (in 30 mL) and the phases were allowed to separate; the organic phase was washed with a saturated NH.sub.4Cl solution and brine (3×25 mL), and dried over anhydrous Na.sub.2SO.sub.4. The solution was filtered and evaporated with a rotary evaporator until a yellow oil was obtained. The raw thus obtained was purified on chromatographic column with eluent DCM:EtOAc/8:2 so as to obtain (S)-1-(2-(4-hydroxyphenyl)-2-oxoacetyl)-N-(3-phenylpropyl)piperidine-2-carboxamide (190 mg, yield 67%) of the title of formula (IV), in the form of a white solid. NMR, IR and mass spectroscopic analyses provided the following data:
[0088] .sup.1H NMR, 400 MHz, CDCl.sub.3, δ:1.25-1.88 (m, 8H, CH.sub.2-16+CH.sub.2-8+CH.sub.2-18+CH.sub.2— 18*); 2.22-2.30 (m, 2H, CH.sub.2-17); 2.62-2.66 (m, 2H, CH.sub.2-7); 2.68-2.76 (m, 2H, CH2-7*); 3.25-3.36 (m, 4H, CH.sub.2-9, H-15, H-9*); 3.47-3.50 (m, 1H, H-15); 4.13-4.14 (m, 1H, H-13*); 4.59-4.63 (m, 1H, H-15*); 5.18 (m, 1H, H-13); 6.33 (bs, 1H, NH-10); 6.83-6.85 (d, J=8 Hz, 1H.sub.arom); 6.87-6.90 (d, J=12 Hz, 1H.sub.arom); 6.92-6.94 (m, 1H.sub.arom); 7.15 (s, 1H, OH.sub.para); 7.17-7.19 (m, J=8 Hz, 2H.sub.meta); 7.24-7.29 (m, 1H.sub.arom); 7.80-7.82 (d, J=8 Hz, 2H H.sub.orto). The spectrum .sup.1H-NMR showed the presence of a 1:0.7 mixture of two rotamers. For the signals where the attribution to the different rotamers has been possible, the symbol (*) refers to the minority rotamer.
[0089] .sup.13C NMR, 100 MHz, CDCl.sub.3, δ:20.21; 20.22; 24.8; 26.29; 26.31; 30.90; 30.01; 30.06; 39.56; 44.61; 51.96; 57.06 (C-13); 116.28; 116.34; 125.95; 125.98; 128.12; 128.60; 128.25; 128.29; 128.38; 128.42; 132.41; 132.66; 140.99; 141.07; 168.37 (2C, amide C═O-19, C-26); 170.46 (amide C═O-11); 190.58 (ketone C═O).
[0090] ESI-MS: m/z 417.25 [M+Na].sup.+, 811 [2M+Na].sup.+ (positive mode); 787 [2M-H].sup.− (negative mode).
[0091] IR (CDCl.sub.3), cm.sup.−1:3578 (w, NH stretch.), 3315 (w, NH stretch.), 3028 (w, CH stretch.), 2947 (CH stretch.), 1664 (N—C═O stretch.), 1601 (Ph-C═O stretch).
[0092] [α].sup.25.sub.D=−7.68°, (c=40 mg in 2 mL DCM).
Step 4: Preparation of (S)-4-(2-oxo-2-(2-((3-phenylpropyl)carbamoyl)piperidin-1-yl)acetyl)phenyl 11-(tritylthio)undecanoate
[0093] ##STR00007##
[0094] 11-(tritylthio)undecanoic acid (56 mg, 0.12 mmol) is added at room temperature to a solution of (S)-4-(2-oxo-2-(2-(3-phenylpropylcarbamoyl)piperidin-1-yl)acetyl)phenyl acetate (100 mg, 0.25 mmol) in anhydrous DMF (20 mL). The reaction mixture was then brought to 0° C. and DIC (45 μL, 0.30 mmol) and DMAP (14 mg, 0.12 mmol) were added. The reaction mixture was kept under constant stirring at room temperature for 24 hours. The separated organic phase was then washed with saturated solutions of NH.sub.4Cl and NaHCO.sub.3, (3×30 mL) and dried over anhydrous Na.sub.2SO.sub.4. The solution was filtered and the solvent removed by rotary evaporator until a yellow oil was obtained, purified on chromatographic column with the eluent Et.Pet:EtOAc/7:3 to obtain the product of the title of formula (V) (41 mg, yield 41%) in the form of a light yellow oil. The product was characterized as follows by NMR and IR spectroscopy.
[0095] .sup.1H NMR, 400 MHz, CDCl.sub.3, δ: 1.19-1.42 (m, 16H, 6CH.sub.2alkyl+CH.sub.2-8+CH.sub.2-18+CH.sub.2-18*); 1.62-1.76 (m, 4H, 2CH.sub.2akyl); 1.88-1.94 (m, 2H, CH.sub.2-16); 2.12-2.15 (t, 2H, J=5.4, CH.sub.2, C-41); 2.28-2.32 (m, 2H, CH.sub.2-17); 2.55-2.59 (t, 2H, J=5.4, CH.sub.2, C-32); 2.65-2.81 (m 3H, CH.sub.2-7+CH.sub.2-7*); 3.20-3.47 (m, 5H, CH.sub.2-9+CH.sub.2-15, H-9*); 4.07-4.09 (m, 1H, H-13*); 4.61-4.64 (m, 1H, H-15*); 5.16-5.19 (m, 1H, H-13); 6.05 (bs, 1H, NH-10); 6.70 (bs, 1H, NH*); 7.19-7.21 (m, δH, H.sub.arom); 7.25, 7.29 (m, 10H, H.sub.arom); 7.40-7.42 (m, δH, H.sub.arom); 7.90-8.02 (m, 2H, CH-24+CH-28). The spectrum .sup.1H-NMR shows the presence of a rotamer mixture 1:0.72. For the signals where the attribution to the different rotamers has been possible, the symbol (*) refers to the minority rotamer.
[0096] .sup.13C NMR, 100 MHz, CDCl.sub.3, δ: 20.41; 20.68; 24.76; 25.25; 25.74; 26.00; 28.50; 20.90; 29.02; 20.12; 29.30; 31.27; 33.0133.19; 34.37; 39.42; 44.57; 51.83; 57.02; 122.40; 127.47:127.76; 128.40; 129.50; 131.33; 145.07; 155.95; 156.25; 167.31; 168.20; 169.36 (amide C═O); 171.40; 190.22; 191.10 (ketone C═O).
[0097] IR (CDCl.sub.3):2931 (CH stretch.), 1711 (C═O stretch.), 1601 (Ph-C═O stretch.).
[0098] [α].sup.25.sub.D=−3.45 (c=44 mg in 2 mL DCM)
Step 5: Preparation of (S)-4-(2-oxo-2-(2-((3-phenylpropyl)carbamoyl)piperidin-1-yl)acetyl)phenyl 11-mercaptoundecanoate of formula (VI)—GPS-SH1
[0099] ##STR00008##
[0100] TFA (10 μL, 0.14 mmoles) and Et.sub.3SiH (14 μL, 0.089 mmoles) were added dropwise to a solution of (S)-4-(2-oxo-2-(2-((3-phenylpropyl)carbamoyl)piperidin-1-yl)acetyl)phenyl-11-(tritylthio)undecanoate (15 mg, 0.018 mmol) in anhydrous DMF (10 mL). The reaction mixture was then left under constant stirring for 45 minutes at room temperature. Then the solvent was removed by rotary evaporator and the reaction raw purified by column chromatography with eluent Et.Pet.:EtOAc/6:4) to obtain the product GPS-SH1 of the title of formula (VI) (7.0 mg, yield 70%) in the form of a light yellow oil. This product was characterized by NMR, IR and mass spectroscopy, obtaining the following data:
[0101] .sup.1H NMR, 400 MHz, CDCl3, δ: 1.25-1.42 (m, 16H, .sup.6CH.sub.2akyl+CH.sub.2-8+CH.sub.2-18+CH.sub.2-18*); 1.57-1.76 (m, 4H, 2CH.sub.2akyl); 1.90-1.94 (m, 2H, CH.sub.2-16); 2.25-2.35 (m, 2H, CH.sub.2-17); 2.40-2.55 (q, J=6, J=6.4, 2H, CH.sub.2, CH-41); 2.56-2.58 (t, J=8, 2H, CH.sub.2, CH.sub.2— 32); 2.65-2.75 (m 3H, CH.sub.2-7+CH.sub.2-7*); 3.20-3.49 (m, 5H, CH.sub.2-9+CH.sub.2-15, H-9*); 4.07-4.09 (m, 1H, H-13*); 4.60-4.64 (m, 1H, H-15*); 5.16-5.19 (m, 1H, H-13); 6.05 (bs, 1H, NH-10); 6.70 (bs, 1H, NH*); 7.19-7.23 (m, 3H, H.sub.arom); 7.25-7.30 (m, 4H, H.sub.arom); 7.90-8.02 (m, 2H, CH-24+CH-28). The spectrum .sup.1H-NMR shows the presence of a mixture of two rotamers 1:0.70. For the signals where the attribution to the different rotamers has been possible, the symbol (*) refers to the minority rotamer.
[0102] .sup.13C NMR, 100 MHz, CDCl.sub.3, δ: 20.41; 20.68; 24.76; 25.25; 25.74; 26.00; 28.50; 20.90; 29.02; 20.12; 29.30; 31.27; 33.0133.19; 34.37; 39.42; 44.57; 51.83; 57.02; 122.40; 127.76; 145.07; 155.95; 156.25; 167.31; 168.20; 169.36 (amide C═O); 171.40; 190.22; 191.10 (ketone C═O).
[0103] IR (CDCl.sub.3):2922 (CH stretch.), 1729 (C═O stretch.), 1601 (Ph-C═O stretch.), 1522 (C═C stretch).
[0104] Exact mass for C.sub.34H.sub.46N.sub.2O.sub.5S. Calculated 595.3161; Found 594.3164.
[0105] [α].sup.25D=−3.18 (c=6 mg in 2 mL DCM).
Example 2
[0106] Preparation of the Self-Assembled Monolayer (Here Also Referred to as SAM, from Self-Assembled Monolayer)
[0107] The functionalised GPS-SH1 receptor prepared as described above in Example 1 was solubilized in ethanol forming a 1 mM solution, which was used to coat a gold-coated piezoelectric quartz crystal.
[0108] The C12-SH, C18-SH and PEG-SH spacers, having the above reported formulas, were used together with the functionalised GPS-SH1 receptor, to prepare mixed solutions in ethanol with various receptor-spacer molar ratios in the interval between 1:3 and 1:10. These mixed solutions have also been used to coat gold substrates of the type indicated above.
[0109] After contact by immersion for about 4 hours of time, the substrates were rinsed with abundant water and ethanol, then dried with compressed air or N.sub.2.
[0110] A compact monolayer of receptor or receptor/spacer molecules was formed on the gold substrate by adding increasing concentrations of receptor or receptor/spacer respectively on the substrate. The formation of the monolayer over time was monitored with microgravimetric measurements by means of a quartz crystal microbalance (QCM) with dissipation analysis instrumentation. With this instrumentation, the frequency variation of the crystal covered with gold was measured, which is proportional to the quantity of mass adsorbed on the surface, and conveys the thickness thereof.
[0111] Once reached the saturation of the surface, completely covered by the molecules of the receptor or of the receptor/spacer mixture, i.e. when no change in frequency is observed, the rinsing and drying process was carried out as described above to eliminate any residual unbound material.
[0112] The following Table 1 shows the structural parameters for the monolayer chemisorbed on the gold surface when formed by the GPS-SH1 receptor alone, or by the GPS-SH1 receptor inserted in a matrix of the two C12-SH and PEG-SH spacers in ratio receptor:spacer 1:6.
TABLE-US-00002 TABLE 1 Structural characteristics of the nanosensor Average area Adsorbed mass available per Monolayer SAM composition density, ng/cm.sup.2 molecule, Å.sup.2 thickness, Å GPS-SH1 264 39 20 GPS-SH1/C12-SH 140 24.sup.a 18 1:6 GPS-SH1/PEG-SH 223 117.sup.a 6 1:6 .sup.acalculated using the average molecular weight for the 1:6 mixture.
[0113] The optimal theoretical structure of a GPS-SH1 molecule in extended conformation was obtained from theoretical calculations providing a value of 26 Å for the length of the molecule at maximum extension, from the same calculations the cross section for the molecule of 29.8 Å.sup.2 was estimated in the conformation shown in
[0114] For the monolayer consisting of GPS-SH1 only on gold, the comparison between experimental measurements and theoretical calculations indicates that the receptor molecule assumes an extended conformation with the thiol group (—SH) anchored to the surface and a tilt angle of 13° with respect to the normal to the surface; this indicates that the molecules are compacted in an almost vertical conformation in the nanostructure with an average experimental area of 40 Å.sup.2.
[0115] As reported above in Table 1, in the case of mixed SAM GPS-SH1 and C12SH, the average thickness of the monolayer remains almost unchanged while the average area available for each molecule decreases to 24 Å.sup.2. In the case of mixed GPS-SH1 and PEG-SH platforms the measurements resulted in an average thickness of 6 Å, much lower than that of GPS-SH1 alone, indicating that the group available for protein binding extends beyond out of the surface of the SAM towards the solution, while in the case of the C18-SH spacer, on the other hand, stable SAMs with compact covering, suitable for the construction of the sensor platform of the invention, were not obtained.
Example 3
[0116] Detection of FKBP12 with the Self-Assembled Monolayer of Example 2
[0117] The gold-coated piezoelectric quartz support covered with the SAMs as described above in Example 2, was brought into contact with solutions of increasing concentration of the FKBP12 protein and the concentration of the protein bound to the self-assembled monolayer SAM at saturation was recorded with gravimetric measurements.
[0118] Two different miniaturized versions of an open chamber QCM quartz crystal microbalance (from QCM KSV, Finland and an OpenQCM, Italy modified by the inventors) were also used as a device, allowing the use of small measurement volumes (less than 100 microliters) of the solution to analyse. In this case, in addition to being able to use samples with low volume availability (biological fluids, cerobrospinal fluid, CSF, etc.), it was also possible to significantly shorten the response times that might be significantly high in standard instrumentation for very low concentrations of FKBP12 due to of the diffusion process of the protein from the solution to the surface.
[0119]
[0120] Again with reference to
TABLE-US-00003 TABLE 2 Structural characterization of nanosensor + FKPP12 Langmuir model Langmuir FIT SAM composition Kd.sup.a, pM (M/Area).sub.max, ng/cm.sup.2 GPS-SH1 750 123 GPS-SH1/C12-SH 1:6 + 25 250 FKBP12 GPS-SH1/PEG-SH 1:6 + 112 223 FKBP12
[0121] The trend shown in
Δm/A=(Δm/A).sub.sat[C]/[C]+k.sub.d
[0122]
[0123] The application of this model to the data obtained from the microgravimetric measurements allowed to obtain a detailed picture of the interaction mechanism and the energetics involved, in particular we obtained the maximum surface density of FKBP12 (to saturation), the dissociation constant Kd for FKBP12/receptor in the nanosensor and free energy of physi-sorption. The values obtained are shown in Table 3 below, for all the types of SAMs examined.
TABLE-US-00004 TABLE 3 Structural characterization of nanosensor + FKPP12 Experimental data Area available Ratio Area Langmuir FIT SAM for FKBP12 to FKBP12/Area Kd.sup.a, (Maa/Area).sub.max, composition saturation, Å.sup.2 GPS-SH1.sup.b pM ng/cm.sup.2 GPS-SH1 1910 56:1 750 123 GPS- 881 8:1 25 250 SH1/C12-SH 1:6 + FKBP12 GPS- 730 6:1 112 223 SH1/PEG-SH 1:6 + FKBP12 .sup.aIn the case of mixed pseudo-Kd systems. .sup.bin the case of mixed systems the area for each receptor molecule was considered equal to the average area.
Example 4
[0124] Construction of the Calibration Straight Line for FKBP12
[0125] The data reported in
[0126] From the analysis of the calibration straight lines obtained for the systems examined, the linearity intervals and the experimental limit of detection (LOD) were obtained (wherein LOD means the smallest amount of measurable analyte with acceptable accuracy and precision) for each sensor tested, considering that the maximum sensitivity to the mass in the cell for liquids of the instrumentation used is 0.5 ng/cm.sup.2. This limit may vary depending on the analytical instrumentation adopted for the same SAM used. The results obtained are summarized in the following Table 4.
TABLE-US-00005 TABLE 4 Characteristics of the response of the GPS- SH1 nanosensor/spacer to the FKBP12 protein Limit of Detection Linear (LOD).sup.a Nano-sensor interval/pM pM R.sup.2 GPS-SH1 10-800 8.0 0.99983 GPS-SH1/C12-SH 1:6 10-50 8.7 0.98842 GPS-SH1/PEG-SH 1:6 10-100 8.2 0.99043 .sup.acalculated as LOD = 3x (y-intercept standard deviation)/slope of the calibration curve.
Example 5
[0127] Duration of Operation of the SAM and the Reconditioning Thereof
[0128] The microgravimetric measurements described above were repeated on freshly prepared SAMs or aged up to 4 months, finding the same results, at least for SAMs stored in closed containers saturated with N.sub.2.
[0129] Measurements were also carried out on SAMs already used for a measurement, after reconditioning with imidazole or with ethanol. In both cases, the frequency measurement of the GPS-SH1-covered QCM support prior to the addition of FKBP12 and after rinsing with the reconditioning solvent turned out to be identical, demonstrating the detachment and/or the denaturation of the protein by adding the solvent. The same result was achieved by leaving in incubation the SAM in solution of the reconditioning solvent.
[0130] Therefore, the SAM of the invention is not a disposable device, but it can be reused for subsequent uses if reconditioned after the first measurement, with a solvent suitable for detaching any protein bound in the first measurement.
[0131] The present invention has been described herein with reference to a preferred embodiment. It is to be understood that there may be other embodiments that relate to the same inventive nucleus, all falling within the scope of protection of the claims provided below.
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