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Peptide-Appended Permethylated β-Cyclodextrins with Hydrophilic and Hydrophobic Spacers

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† Institute for Molecules and Materials, Radboud University, Heijendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
‡ Okklo Life Sciences BV, Pivot Park, Molenstraat 110, 5349 TD Oss, The Netherlands
§ QareFree, Blauwoogvlinder 2, 4814 ST Breda, The Netherlands
*E-mail: [email protected]. Tel: +31 (0)24 36 52016. Fax: +31 (0)24 36 53393.
Cite this: Bioconjugate Chem. 2017, 28, 8, 2160–2166
Publication Date (Web):July 11, 2017
https://doi.org/10.1021/acs.bioconjchem.7b00319
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Abstract

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A novel synthetic methodology, employing a combination of the strain-promoted azide–alkyne cycloaddition and maleimide–thiol reactions, for the preparation of permethylated β-cyclodextrin-linker-peptidyl conjugates is reported. Two different bifunctional maleimide cross-linking probes, the polyethylene glycol containing hydrophilic linker bicyclo[6.1.0] nonyne-maleimide and the hydrophobic 5′-dibenzoazacyclooctyne-maleimide, were attached to azide-appended permethylated β-cyclodextrin. The successfully introduced maleimide function was exploited to covalently graft a cysteine-containing peptide (Ac-Tyr-Arg-Cys-Amide) to produce the target conjugates. The final target compounds were isolated in high purity after purification by isocratic preparative reverse-phase high-performance liquid chromatography. This novel synthetic approach is expected to give access to many different cyclodextrin–linker peptides.

Introduction

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Cyclodextrins (CDs) are cyclic oligosaccharides containing six, seven, or eight (α-1,4)-linked d-glucopyranoside units, named α-, β-, and γ- cyclodextrin, respectively. (1) These macromolecules have a hydrophilic exterior which makes them water-soluble and a hydrophobic interior that can accommodate small lipophilic molecules. As such, CDs are used as pharmaceutical excipients that can solubilize various poorly soluble drugs through the formation of water-soluble drug–CD complexes. Hence, CDs are applied in the pharmaceutical industry (2-5) to promote the bioavailability, safety, stability, and solubility of drug molecules. (6) Moreover, CDs (mainly β-CDs as depicted in Scheme 1) have been used for many years in biochemical studies to extract cholesterol from plasma membranes. (7-10)

Scheme 1

Scheme 1. Synthetic Routes to Obtain Maleimide-Functionalized PMβCD Derivatives and the Target Conjugates: (A) via Hydrophilic Linker BCN-Mal; (B) via Hydrophobic Linker DIBAC-Mal
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The functionality and applications of the cyclodextrins can be further expanded by the addition of functional groups that allow for cell and tissue specific targeting. The conjugation of small peptides is especially interesting in this respect. However, the synthesis of cyclodextrin–peptide conjugates with different spacers/linkers presents a nontrivial scientific challenge, due to the presence of multiple various reactive groups. Despite several previous reports on the attachment of small peptides directly to the CD core, (11, 12) there is as yet no flexible synthetic route that allows for insertion of linkers of choice between the peptide and CD. In this study, we have exploited the −SH of a cysteine-based model peptide to successfully accomplish its covalent grafting to an azide-appended permethylated β-cyclodextrin (PMβCD) via hydrophilic and hydrophobic linkers, utilizing the strain-promoted azide–alkyne cycloaddition (SPAAC) reaction followed by the maleimide–thiol coupling.
As proof of concept, we applied a multistep strategy (Scheme 1, cartoon representation of β-CD reproduced from ref 13) to use the polyethylene glycol (PEG)-containing hydrophilic linker bicyclo[6.1.0]-nonyne-maleimide (BCN-Mal, derived from BCN-PEG-maleimide 7) and the hydrophobic linker 5′-dibenzoazacyclooctyne-maleimide (DIBAC-Mal, derived from DIBAC-maleimide 11), with a model cysteine containing peptide (Ac-Tyr-Arg-Cys-Amide) to provide the desired conjugates by the final maleimide-cysteine coupling reaction step. This model peptide is of interest because it contains the phenolic hydroxyl and guanidine groups in addition to the reactive thiol of cysteine. The reason for selecting PMβCD-N34 instead of βCD-N33 as the starting point for the conjugation to access the target conjugates 10 and 13 (Scheme 1) is that both the latter and model peptide 9 contain multiple reactive (nucleophilic) groups which could give rise to side products. (14)
Previous studies by Shi et al. (15, 16) have shown that permethylated CDs are soluble both in water and in organic solvents and do not undergo side reactions during coupling. (17) Thus, we envisaged that the use of permethylated azide-appended β-CD instead of its hydroxyl analogue will simplify the synthetic approach. The mono-6-azido-permethylated β-CD 4 was obtained from precursor β-CD 1 by a selective tosylation of the primary hydroxyl group on the outer face to give mono 6-OTs-β-CD 2, (18) followed by azide displacement resulting in hydroxyl azide-appended β-CD 3, (19) and permethylation of the remaining hydroxyl groups to afford the key intermediate 4, (14, 20, 21) which is the starting point for access to a wide range of compounds of the β-CD family. As method for the conjugation of the permethylated azide-appended β-CD 4 with the cross-linking reagent [either the hydrophilic BCN-Mal 7 (path A in Scheme 1) or the hydrophobic DIBAC-Mal 11 (path B in Scheme 1)] we chose a click reaction, viz., the SPAAC reaction between permethylated azide-appended β-CD and alkyne-appended BCN (or DIBAC), because it is metal-free and the resultant product is therefore suitable for application in living systems. (22) The coupling of 4 with the commercially available bifunctional hydrophilic cross-linking reagent (BCN-Mal) 7 resulted in maleimide-functionalized PMβCD 8 (path A). The model peptide 9 (Ac-Tyr-Arg-Cys-Amide) was synthesized by standard Fmoc solid-phase peptide synthesis (see Supporting Information for details) and reacted with its terminal cysteine thiol to the maleimide in 8 to give the target conjugate with hydrophilic linker PMβCD-Mal-BCN-Peptide 10. Similarly, the introduction of the hydrophobic linker DIBAC-Mal 11 was carried out using the same synthetic approach (SPAAC reaction followed by maleimide–thiol coupling to peptide 9) to afford the final target conjugate 13 (path B).

Results and Discussion

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Synthesis of mono-6-azido PMβCD 4

The synthesis of key intermediate 4 has been reported (22) but was modified to improve the yield of the desired product. Regioselective tosylation of a single hydroxyl group at the 6-position was achieved according to a reported method (18) by reacting β-CD 1 with 1.2 equiv of tosyl chloride in the presence of 8 N NaOH and water/acetonitrile as a solvent to afford the compound 2 in a yield of 26%. 1H and 13C NMR corroborated the identity of 6-β-CD-OTs 2 (Figures S2 and S3). ESI-MS analysis confirmed the formation of target compound 2, but also revealed the presence of traces of impurities (unreacted and ditosylated β-CD, Figure S1). It is not straightforward to remove these impurities due to the high polarity of these compounds. Since TLC, 1H NMR, ESI-MS, and LC-MS had proven the structure and acceptable purity of the product, it was used in the next step without further purification. The azidation step was performed with excess sodium azide in water. After precipitation in acetone followed by centrifugation, the mono-6-azido β-CD (N3-βCD) 3 was obtained in a yield of 85%. ESI-MS showed the mass of the compound 3 (Figure S4), and 1H and 13C NMR identified its chemical structure (Figures S5 and S6). As in the case of 2, the presence of some β-CD, remaining from the previous step, was apparent from the 1H NMR spectrum of the target azide-appended CD, as confirmed by LC-MS. After recrystallization from acetone, the azido-β-CD intermediate was used subsequently without further purification. The next step, the capping of all remaining hydroxyl groups, was performed with excess methyl iodide (25.0 equiv) in the presence of the strong base NaH (25.0 equiv) and anhydrous DMF as a solvent compatible with this reaction. The purity and the chemical structure of 4 were determined by LC-MS, ESI-MS, 1H NMR, and 13C NMR (Figures S7, S8, S9, and S10, respectively). Since the crude product showed a single spot in TLC, it could only be purified by a prep-HPLC system provided with two UV detectors at 254 and 215 nm. It eluted at 17.0 min (Figure S11) and the yield after this step was 34%. Analytical HPLC (Figure S12) showed that compound 4 was pure, and the impurities remaining from previous steps had been effectively removed.

Synthesis of Maleimide-Functionalized Hydrophilic Linker-Appended PMβCD 8

The linker BCN-Mal 7 possesses two functional ends, dibenzocyclooctyne moiety for click reaction with azide functionalized compounds and maleimide end for cysteine-maleimide conjugation reaction. The starting materials for the preparation of 7, BCN-(PEO)3-NH25 and Mal-NHS 6, were obtained from SynAffix B.V. (Oss, The Netherlands). The bifunctional reagent was obtained using PBS (pH 7.2) with DMSO or DMF as solvent (1:1 v/v). The LC-MS and ESI-MS data showed the formation of the product as a main peak (Figures S13 and S14), with only traces of a side product in LC-MS. Without the buffer, only a small amount of the target BCN-Mal is formed, and the unwanted BCN-Mal-BCN (Figure S15) represents the dominant product; this is formed due to a high pH value which causes the amine group from BCN-(PEO)3-NH2 to attack both electrophilic sites, the NHS and the maleimide moieties, in Mal-NHS. The product 8 was obtained by SPAAC reaction in 42% yield after purification by flash column chromatography on a Biotage system. TLC analysis clearly indicated the yellow spot for the double bond maleimide moiety after dipping in KMnO4 stain. The formation of 8 was indicated by LC-MS and MALDI-TOF MS analyses (Figures S16 and S17) and confirmed by 1H NMR (Figure S18), showing a singlet around 6.7 ppm for the triazole. IR analysis showed that the azide peak of 4 at 2100–2200 cm–1 completely disappeared after the reaction with BCN-Mal 7.

Synthesis, prep-HPLC Purification, and Characterization of the Peptide 9

The model peptide Ac-YRC-NH2 (9) was synthesized with a semiautomated peptide synthesizer (Labortec SP 640, Bubendorf, Switzerland) with standard Fmoc α-protection (23) [see experimental protocol S6 and Scheme 1 in the Supporting Information] from the protected amino acids Fmoc-Cys(Trt)–OH, Fmoc-Arg(Pbf)–OH, and Fmoc-Tyr(tBu)–OH. Coupling reactions of Fmoc amino acids were achieved in DMF applying amino acid/HOBt/DIPCDI/Breipohl resin in the molar ratio 3.0:3.6:3.3:1.0. Acetylation of the N-terminus was achieved with Ac2O/DIPEA in DMF using a molar ratio 1:1 (equiv). The coupling reactions and Fmoc deprotections were monitored at intervals with the color Kaiser test (24, 25) until completed. Upon prep-HPLC purification, the product 9 gave a chromatogram with a major peak in addition to a small peak representing its associated dimer form in MS as determined by LC-MS and MALDI-TOF MS (Figures S19 and S20), {calcd. [M + H]+ 482.2; obsd. 482.3}. The model peptide was characterized and checked for purity by various analytical techniques including 1H NMR, 13C NMR, prep-HPLC, and analytical HPLC (Figures S21, S22, S23, S24, respectively).

Preparation, prep-HPLC Purification and Characterization of the PMβCD-Hydrophilic Linker-Peptide Conjugate 10

One of the most widespread specific covalent coupling reactions is the conjugation of a protein or peptide bearing a thiol moiety which can react with maleimide reagents. The cysteine thiol can be efficiently and site-specifically labeled with maleimides, disulfides, or haloacetyl compounds. (26) Maleimide can be coupled to sulfhydryl groups when the pH of the reaction is between 6.5 and 7.5. The formed thioether linkage is stable but can be cleaved with reducing agents, such as TCEP, dithiothreitol (DTT), and β-mercaptoethanol (βME).
Based on the previously published procedures from our group, (27, 28) it was our goal to access the target conjugates by reacting PMβCD-BCN-Mal 8 with model peptide 9 at suitable conditions (Scheme 1A). The conjugation of maleimide attached to hydrophilic linker appended-PMβCD 8 with thiol cysteine-based model peptide 9 was accomplished in PBS buffer at pH 7.2 for 4 h at room temperature employing TCEP.HCl as reducing agent and a small amount of DMF. It is essential to degas the buffer for 1 h by allowing a flow of nitrogen, since a cysteine-containing peptide is prone to oxidation. Also, a freshly prepared stock solution of the odorless tris(2-carboxyethyl)phosphine reducing agent (TCEP-HCl) at pH 7 was used for the reaction. This circumvents the problems of adding TCEP.HCl in solid form which potentially results in a sharp drop of pH.
However, as confirmed by LC-MS and ESI-MS analyses, the conjugate 10 was not produced efficiently due to an impurity which appeared at a retention time very close to that of the main conjugate. It is probably from the starting material peptide 9 which is added in excess relative to the maleimide-attached PMβCD 8 in the final reaction. It was decided to systematically optimize the reaction conditions by variation of the buffer and its pH values, reaction time, temperature, and the stoichiometry of the reactants. Varying the pH in the range of 6–7.8 did not result in reduction of the amount of impurity in the reaction product, nor did raising the temperature to 37 °C at pH 7.2, or adding the maleimide-attached PMβCD 8 in steps.
When we explored the effect of the molar ratio of 9 and 8 on the reaction, we found that besides the stoichiometry other factors were also important, viz., the PBS buffer (100 μM, pH 7.2), using a fresh solution (100 μM) of TCEP-HCl, and incubation for 4 h at room temperature. The best results were obtained when using 1.3 equiv of the model peptide relative to the Mal-appended PMβCD. The presence of the target conjugate 10 in the product was supported by the observation of a significant doubly charged peak [M+2H]2+ at 1206.3 in ESI-MS (Figure S25) and the observation of the [M + H]+ and [M + Na]+ adducts in MALDI-TOF analysis (Figure S26), and further confirmed by its 1H NMR spectrum, in which the absence of maleimide protons at 6.7 ppm indicates that the addition of thiol peptide has taken place (Figure S27). LC-MS for the crude product of the conjugation reaction gave a strong peak at 1206.2 corresponding to the calculated mass of the target compound 10, but also an unidentified impurity at 1090.6 (20% of strongest peak, Figure 1a). Attempted separation by gradient prep-HPLC was not successful, but the final conjugate could be purified by prep-RP-HPLC in isocratic mode. Notably, the compound was successfully separated from the impurity by this method as confirmed by LC-MS analysis before and after purification (Figure 1b, Figure S28).

Figure 1

Figure 1. (a) LC-MS of the conjugate 10 before prep-RP-HPLC isocratic purification. The main peak at 7.91 min (top trace) represents the target conjugate as testified by the double- and triple-charged molecular ions [M+2H]2+ 1206.2 and [M+3H]3+ 803.9, analyzed at retention time 7.74–8.41 min (middle trace). The shoulder due to the impurity at 8.34 min corresponds to m/z 1090.6, analyzed at retention time 8.22–8.41 min (bottom trace). The relative intensities of the peaks of target and impurity do not necessarily reflect the amounts formed because of likely differences in the response factors. (b) LC-MS of the conjugate 10 after prep-HPLC isocratic purification. The main peak represents the target conjugate at 7.96 min as testified by the double- and triple-charged molecular ions [M+2H]2+ 1206.2 and [M+3H]3+ 803.9, analyzed at retention time 7.78–8.41 min.

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Even after separation, it was not possible to establish the identity of the impurity, as there was not enough material for NMR analysis and the LC-MS and ESI-MS results were insufficient to hypothesize a relevant structure. It could not be correlated to any known structure originating from the reactants, or to possible side products.

Preparation, RP-HPLC Purification, and Characterization of the PMβCD-Hydrophobic Linker-Peptide Conjugate 13

In a parallel approach we explored cyclodextrin–peptide conjugates with hydrophobic spacers. The synthesis of maleimide attached to hydrophobic linker appended PMβCD 12 was carried out identically to that of 8, except for using the hydrophobic linker DIBAC-Mal 11, which was used directly as obtained commercially. Intermediate 12 was synthesized and purified by a Biotage chromatographic system. The mass of 12 was confirmed by MALDI-TOF MS (Figure S30) and the formation of the triazole ring by 1H NMR which showed a singlet at approximately 6.7 ppm (Figure S31). Analysis by LC-MS revealed two very close chromatographic peaks in a 1:1 ratio which obviously result from regioisomerism (Figure S29). These regioisomers were not separated and used as a mixture for the rest of the synthesis, since they exhibit identical reactivity toward azides. The triazole isomers were combined for the conjugation with peptide 9 to PMβCD-hydrophobic linker-peptide 13. An excess (1.3 equiv) of the model peptide 9 relative to the Mal-appended hydrophobic linker PMβCD was used under the same conditions that afforded 10 from 8 and 9. LC-MS analysis of the product of the reaction of 12 and 9 showed the mass of the target conjugate 13 at 1174.8 [M+2H]+ (Figure 2b) as well as an unidentified impurity at 1059.2 (Figure 2a), which is comparable to that obtained for the side product of the reaction of 8 and 9 to give 10, although there were slight differences in the retention times and the final yields. Likewise, the final target conjugate 13 with the hydrophobic linker could be obtained in pure form after isocratic prep-HPLC purification (Figure S32) in a yield of 14%. The final product was further confirmed by the disappearance of the maleimide protons at 6.7 ppm in the 1H NMR spectrum (Figure S33). The LC-MS analysis of the final conjugate before and after prep-HPLC isocratic purification is shown in Figure 2a and b, respectively.

Figure 2

Figure 2. (a) LC-MS of the conjugate 13 before prep-HPLC isocratic purification. The main peak represents the target conjugate at 8.13 min (top trace) with its corresponding formation of double-charged molecular ion [M+2H]2+ 1174.8, analyzed at retention time 7.75–8.17 min (middle trace), and the minor peak the overlapping impurity at 8.55 min with its corresponding mass formation 1059.2, analyzed at retention time 8.45–8.71 min (bottom trace). The relative intensities of the peaks of target and impurity do not necessarily reflect the amounts formed because of likely differences in the response factors. (b) LC-MS of the conjugate 13 after prep-HPLC isocratic purification. The main peak represents the target conjugate at 8.01 min with its corresponding formation of double-charged molecular ion [M+2H]2+ 1174.8, analyzed at retention time 7.86–8.40 min.

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Conclusions

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The synthesis and characterization of the conjugates PMβCD-BCN-Mal-peptide 10 and PMβCD-DIBAC-Mal-peptide 13 with hydrophilic and hydrophobic linkers have been successfully accomplished. We developed a robust and reliable synthetic route toward modified cyclodextrins that can be conjugated to peptides. These novel peptide-β-CD conjugates can give rise to new developments in the field of cyclodextrin derivatives, for example, in drug delivery. Remarkably, this is, to the best of our knowledge, the only synthetic route toward a combination of an azide-appended cyclodextrin and a peptide via suitable hydrophilic and hydrophobic linkers. We consider this novel methodology for the preparation of PMβCD-linker-peptide derivatives to be a promising valuable research tool for the development of interesting therapeutics.

Experimental Procedures

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General Methods

1H NMR spectra were recorded on a Varian Inova-400 (400 MHz) spectrometer at 300 K and chemical shifts are given in parts per million (δ) relative to tetramethylsilane as an internal reference (δ = 0.00 ppm). Coupling constants are reported as J-values in Hz. The following abbreviations are used to designate the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. 13C NMR spectra were recorded on a Bruker Avance III 500 MHz NMR and the chemical shifts are internally referenced to the residuals selected solvent. Mass spectra were confirmed by ESI-MS (Thermo Finnigan LCQ Advantage Max). Liquid chromatography electrospray ionization mass spectrometry was measured on a Shimadzu LCMS-QP8000 (Duisburg, Germany) single quadrupole benchtop mass spectrometer operating in a positive ionization mode. Infrared (IR) spectra were recorded on a IR-ATR Bruker TENSOR 27 spectrometer; only the absorption frequencies (cm–1) of major peaks are reported. Optical rotations of samples were measured on a Perkin & Elmer Polarimeter 241 using a 10 cm, 1 mL cell. All reactions were magnetically stirred and monitored by TLC on Kieselgel 60 F254 (Merck, Darmstadt, Germany); Spots were visualized under UV light (254 nm) and were stained with ninhydrin, 2,4-dinitrophenylhydrazine (DNP), or aqueous KMnO4 (depending on the reaction), followed by heating on a hot plate. Rf values were obtained with solvent mixtures indicated. Unreacted TCEP reagent and degassed PBS buffer (pH 7.2) were removed by size exclusion chromatography using a Sephadex G10 column. The model peptide was synthesized on a semiautomated peptide synthesizer (Labortec SP 640, Labortec AG) by applying the Fmoc-protocol. Accurate molecular weights of the model peptide and compounds were confirmed by ESI-MS technique using a Micromass Platform II (Altrincham, United Kingdom) single quadrupole benchtop mass spectrometer operating in a positive ionization mode. The mass of each compound was measured and the observed monoisotopic [M + H] + values were correlated with the calculated [M + H]+ values by use of (ChemBioDraw Ultra 14) Cambridge software program. Other adducts [M + Na]+ and [M+K]+ have also been detected and identified. The room temperature in the reactions is in the range 20–25 °C.
Compounds 8 and 11 were purified by flash column chromatography on a Biotage Isolera One chromatography system (silicycle 4g cartridge, size 230–400 mesh 40–63 μm, Quebec, Canada) using a 15% methanol/dichloromethane gradient. Analytical HPLC was performed on a Shimadzu LC-20A Prominence system (Shimadzu, ‘s Hertogenbosch, The Netherlands) equipped with a C18 ReproSil column, 150 × 3 mm, particle size 3 μm (Screening Devices, Amersfoort, The Netherlands). Lyophilization was achieved using an ilShin Freeze-Dryer (ilShin, Ede, The Netherlands).

Compound Characterization

The identity of the compounds was confirmed and further characterized by TLC, 1H NMR, 13C NMR, LC-MS, Maldi-Tof MS, ESI-MS, IR, and optical rotation [α]D, see Supporting Information for details.

Reverse-Phase HPLC Characterization of the Final Conjugates 10 and 13

Both final conjugates were purified on a Phenomenex Gemini-NX 3u C18 110A reversed-phase column (150 × 21.2 mm) with HPLC system using an isocratic elution at a constant flow rate of 10 mL/min at 30 °C. The analytical procedure for both conjugates was as follows: in the isocratic phase, the acetonitrile concentration was kept at 40% for 25 min, followed by a gradient from 40% to 100% over 3 min. Conjugates 10 and 13 elute at 15.5 and 20.4 min, respectively. Buffer A: 0.1% (v/v) TFA in water; Buffer B: CH3CN containing 0.1% (v/v) TFA. All the samples were filtered through a 0.22-μm syringe filter before injection.

LC-MS Characterization and Analytical HPLC Analysis

LS-MS analysis for all the compounds was performed on a Thermo Finnigan LCQ-Fleet ESI-ion trap (Thermofischer, Breda, The Netherlands) equipped with a Phenomenex Gemini-NX C18 column, 50 × 2.0 mm, particle size 3 μM (Phenomenex, Utrecht, The Netherlands). An acetonitrile/water gradient containing 0.1% formic acid was used for elution (5–100%, 1–20 min, flow 0.2 mL min–1).

Analysis of Purified Compounds and the Model Peptide by MALDI-TOF Mass Spectroscopy

The purified compounds and lyophilized fractions of model peptide were analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometer (Bruker Biflex III Maldi-Tof MS, Germany). 10 μL of the required compound was mixed with 10 μL of the supernatant (1:1) Maldi matrix solution (α-cyano-4-hydroxycinnamic acid; 5.0 mg weighed in an Eppendorf vial and dissolved with acetonitrile/water 125.0 μL:125.0 μL (v/v); α-CHCA). From this mixture, 5 μL was spotted onto a stainless steel Maldi target plate (MTP target frame III of Bruker, Germany). Sample spots were left to dry for 30 min at ambient temperature prior to analysis by the technique. The samples were analyzed in positive-ion mode using the reflection method in the molecular weight range 400–4000. All the pure targeted compounds and the model peptide revealed the corresponding peaks.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00319.

  • Details of synthetic procedures and characterization (LC-MS, MALDI-TOF MS, ESI-MS, 1H NMR, 13C NMR, HPLC) for compounds 2, 3, 4, 7, 8, 9, 10, 11, 12, and 13 (PDF)

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Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Author
  • Authors
    • Abbas H. K. Al Temimi - Institute for Molecules and Materials, Radboud University, Heijendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
    • Thomas J. Boltje - Institute for Molecules and Materials, Radboud University, Heijendaalseweg 135, 6525 AJ Nijmegen, The Netherlands;  Orcidhttp://orcid.org/0000-0001-9141-8784
    • Daniel Zollinger - Okklo Life Sciences BV, Pivot Park, Molenstraat 110, 5349 TD Oss, The Netherlands;  QareFree, Blauwoogvlinder 2, 4814 ST Breda, The Netherlands
    • Floris P. J. T. Rutjes - Institute for Molecules and Materials, Radboud University, Heijendaalseweg 135, 6525 AJ Nijmegen, The Netherlands;  Orcidhttp://orcid.org/0000-0003-1538-3852
  • Notes
    The authors declare no competing financial interest.

Acknowledgment

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This work was supported by the Dutch Organization for Scientific Research (NWO) within the framework of FONDS NCI-KIEM project 731.014.104. Dr. Stephan Peters, Dr. Alex Zwiers (Okklo Life Sciences), Dr. Marco Felici, Jan Dommerholt, Dr. Roel Hammink, Dr. Vijayendar Reddy Yedulla, Dr. Dennis W. P. M. Löwik, and Dr. Rajat K. Das (Radboud University) are gratefully acknowledged for their insightful discussions.

Abbreviations List

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Ac2O

acetic anhydride

β-CD

beta-cyclodextrin

BCN-Mal

bicyclo[6.1.0] nonyne-maleimide

βME

beta-mercaptoethanol

CD

cyclodextrins

DIBAC-Mal

5′-dibenzoazacyclooctyne-maleimide

DIPCDI

N,N′-disopropylcarbodiimide

DIPEA

N,N-diisopropylethylamine

DMF

dimethylformamide

DTT

dithiothreitol

ESI-MS

electrospray ionization-mass spectrometry

HOBt

1-hydroxybenzotriazole

HPLC

high-performance liquid chromatography

LC-MS

liquid chromatography mass spectrometry

MALDI-TOF

matrix-assisted laser-induced desorption-time-of-flight

NHS

N-hydroxysuccinimide

PBS

phosphate-buffered saline

PEG

polyethylene glycol

PEO

poly(ethylene oxide)

PMβCD

permethylated β-cyclodextrin

SPAAC

strain-promoted azide–alkyne cycloaddition

TCEP.HCl

tris(2-carboxyethyl)phosphine hydrochloride

TLC

thin layer chromatography

References

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This article references 28 other publications.

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    Loftsson, T. and Brewster, M. E. (1997) Cyclodextrins as pharmaceutical excipients Pharm. Technol. 5, 26– 34
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    Tsai, Y., Tsai, H., Wu, C., and Tsai, F. (2010) Preparation, characterisation and activity of the inclusion complex of paeonol with β-cyclodextrin Food Chem. 120, 837– 841 DOI: 10.1016/j.foodchem.2009.11.024
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    Yannakopoulou, K. (2012) Cationic cyclodextrins: cell penetrating agents and other diverse applications J. Drug Delivery Sci. Technol. 22, 243– 249 DOI: 10.1016/S1773-2247(12)50035-3
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    Arun, R. (2008) Cyclodextrins as Drug Carrier Molecule: A Review Sci. Pharm. 76, 567– 598 DOI: 10.3797/scipharm.0808-05
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    Zidovetzki, R. and Levitan, I. (2007) Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies Biochim. Biophys. Acta, Biomembr. 1768 (6) 1311– 1324 DOI: 10.1016/j.bbamem.2007.03.026
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    Use of cyclodextrins to manipulate plasma membrane cholesterol content: Evidence, misconceptions and control strategies
    Zidovetzki, Raphael; Levitan, Irena
    Biochimica et Biophysica Acta, Biomembranes (2007), 1768 (6), 1311-1324CODEN: BBBMBS; ISSN:0005-2736. (Elsevier Ltd.)
    A review. The physiol. importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, esp. as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compds., which, due to the presence of relatively hydrophobic cavity, can be used to ext. cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extn., cyclodextrins may remove cholesterol from both raft and nonraft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addn., other hydrophobic mols. such as phospholipids may also be extd. from the membranes by cyclodextrins. The authors review the evidence for the specific and nonspecific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extn. Finally, the authors discuss useful control strategies that may help to verify that the obsd. effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.
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    Irie, T., Fukunaga, K., and Pitha, J. (1992) Hydroxypropylcyclodextrins in parenteral use. I: Lipid dissolution and effects on lipid transfers in vitro J. Pharm. Sci. 81, 521– 3 DOI: 10.1002/jps.2600810609
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    Ohtani, Y., Irie, T., Uekama, K., Fukunaga, K., and Pitha, J. (1989) Differential effects of alpha-, beta- and gammacyclodextrins on human erythrocytes Eur. J. Biochem. 186, 17– 22 DOI: 10.1111/j.1432-1033.1989.tb15171.x
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    Differential effects of α-, β- and γ-cyclodextrins on human erythrocytes
    Ohtani, Yoshiro; Irie, Tetsumi; Uekama, Kaneto; Fukunaga, Kazuhiro; Pitha, Josef
    European Journal of Biochemistry (1989), 186 (1-2), 17-22CODEN: EJBCAI; ISSN:0014-2956.
    The α-, β-, and γ-cyclodextrins are cyclic hexamers, heptamers, and octamers of glucose, resp., and thus are hydrophilic; nevertheless, they have the ability to solubilize lipids through the formation of mol. inclusion complexes. The vol. of lipophilic space involved in the solubilization process increases with the no. of glucose units in the cyclodextrin mol. and, consequently, cyclodextrins were found to have different effects on human erythrocytes: (a) in the induction of shape change from discocyte to spherocyte the potency was α > γ, but with β-cyclodextrin hemolysis occurred before the change was complete; (b) in the increase of fluorescence intensity of 1-anilinonaphthalene-8-sulfonate in cyclodextrin-pretreated membranes, the obsd. potency was β » γ > α; (c) in the release of K and Hb, the potency was β > α > γ. The potencies of cyclodextrin for solubilizing various components of erythrocytes were α > β » γ for phospholipids, β » γ > α for cholesterol, and β » γ > α for proteins. The solubilization potencies were derived from concn./final-effect curves. The above processes occurred without entry of solubilizer into the membrane, since (a) β-[14C]cyclodextrin did not bind to erythrocytes and (b) cyclodextrins did not enter the cholesterol monolayer. A study of the [3H]cholesterol in erythrocytes indicated that β-cyclodextrin extd. this lipid from membrane into a new compartment located in the aq. phase that could equilibrate rapidly with addnl. erythrocytes. Therefore, the effects of cyclodextrins differ from those of detergents that first incorporate themselves into membranes then ext. membrane components into supramol. micelles.
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    Ohvo, H. and Slotte, J. P. (1996) Cyclodextrin-mediated removal of sterols from monolayers: effects of sterol structure and phospholipids on desorption rate Biochemistry 35, 8018– 24 DOI: 10.1021/bi9528816
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    Cyclodextrin-Mediated Removal of Sterols from Monolayers: Effects of Sterol Structure and Phospholipids on Desorption Rate
    Ohvo, Henna; Slotte, J. Peter
    Biochemistry (1996), 35 (24), 8018-8024CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    In this study, we have examd. a no. of parameters which affect the rate of sterol desorption from a model membrane surface (a monolayer at the air/water interface) to cyclodextrins (CD) in the aq. subphase. The desorption expts. were carried out at a const. lateral surface pressure with a zero-order trough, which allowed for a detn. of desorption rates which were unaffected by monolayer substrate concn. At a surface pressure of 20 mN/m (30 °C), 0.9 mM β-CD caused a desorption of about 13 pmol of cholesterol per min and square centimeter of monolayer area. The desorption of cholesterol proceeded linearly as a time function and was sensitive to the concn. of β-CD in the subphase. The rate of cholesterol desorption increased as the monolayer surface pressure increased (3 → 35 mN/m) but decreased slightly with increasing temp. (15 → 30 °C). The rate of sterol desorption appeared to be influenced by the relative polarity of the sterols. Oxidized sterols desorbed significantly faster than cholesterol (e.g., 4-cholesten-3-one desorbed 8.4-fold faster than cholesterol), whereas less polar sterols desorbed at slower rates [e.g., 20(R)-isoheptyl-5-pregnen-3β-ol, a cholesterol analog with a ten-carbon branched side chain, desorbed at 1/10 of the rate of cholesterol]. Cholesterol desorption from a monolayer membrane contg. both cholesterol and a phospholipid was much slower than from a pure cholesterol monolayer. When the effect of dipalmitoylphosphatidylcholine and N-palmitoylsphingomyelin on cholesterol desorption rate was compared, it was found that cholesterol desorption was much more retarded from sphingomyelin monolayers as compared to that from phosphatidylcholine monolayers. Taken together, the results of this study show that the β-CD-enhanced desorption of cholesterol (and other sterols) from monolayer membranes is influenced by the polarity of the desorbing mols., as well as by lipid/lipid interactions in the membranes. Since β-CD has no surface activity of its own, it appears to be a useful, nonintrusive catalyzer of cholesterol desorption and is expected to become a valuable probe in membrane and cell research.
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    White, R. J., Plieger, P. G., and Harding, D. R. K. (2010) Synthesis of bi-functionalised peptide derivatives based on a β-cyclodextrin core with drug delivery potential Tetrahedron Lett. 51 (5) 800– 803 DOI: 10.1016/j.tetlet.2009.11.118
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    Ruiz Garcia, Y., Zelenka, J., Pabon, V., Iyer, A., Budìšinský, M., Kraus, T., Smith, C. I. E., and Madder, A. (2015) Cyclodextrin-peptide conjugates for sequence specific DNA binding Org. Biomol. Chem. 13, 5273– 5278 DOI: 10.1039/C5OB00609K
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    Loukou, C., Changenet-Barret, P., Rager, M. N., Plaza, P., Martin, M. M., and Mallet, J. M. (2011) The design, synthesis and photochemical study of a biomimetic cyclodextrin model of Photoactive Yellow Protein (PYP) Org. Biomol. Chem. 9, 2209 DOI: 10.1039/c0ob00646g
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    The design, synthesis and photochemical study of a biomimetic cyclodextrin model of Photoactive Yellow Protein (PYP)
    Loukou, Christina; Changenet-Barret, Pascale; Rager, Marie-Noelle; Plaza, Pascal; Martin, Monique M.; Mallet, Jean-Maurice
    Organic & Biomolecular Chemistry (2011), 9 (7), 2209-2218CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    The design, synthesis and study of the photophys. and photochem. properties of the first biomimetic cyclodextrin (CD) model of photoactive yellow protein (PYP) are described. This model bears a deprotonated trans-p-coumaric acid chromophore, covalently linked via a cysteine moiety to a permethylated 6-monoamino β-CD. NMR and UV/Visible spectroscopy studies showed the formation of strong self-inclusion complexes in water at basic pH. Steady-state photolysis demonstrated that, unlike the free chromophore in soln., excitation of the model mol. leads to the formation of a photoproduct identified as the cis isomer by NMR spectroscopy. These observations provide evidence that the restricted CD cavity offers a promising framework for the design of biomimetic models of the PYP hydrophobic pocket.
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    Shi, J., Guo, D.-S., Ding, F., and Liu, Y. (2009) Unique Regioselective Binding of Permethylated â-Cyclodextrin with Azobenzene Derivatives Eur. J. Org. Chem. 2009, 923– 931 DOI: 10.1002/ejoc.200800829
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    Liu, Y., Shi, J., and Guo, D.-S. (2007) Novel Permethylated â-Cyclodextrin Derivatives Appended with Chromophores as Efficient Fluorescent Sensors for the Molecular Recognition of Bile Salts J. Org. Chem. 72, 8227– 8234 DOI: 10.1021/jo071131m
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    Diaz, E., Jankowski, C. K., Hocquelet, C., del Rio, F., and Barrios, H. (2008) The unambiguous assignment of NMR spectra of per-O-methylated 6-mono and 6,6-diamino-Beta-cyclodextrines Can. J. Chem. 86, 726– 736 DOI: 10.1139/v08-063
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    Miyake, K., Irie, T., Hirayama, F., and Uekama, K. Improved solubility and oral bioavailability of cyclosporin A by hydrophilic cyclodextrin complexation. In Proceedings of the Ninth International Symposium on Cyclodextrins. Torres Labandeira, J. J. and Vila-Jato, J. L., eds. Kluwer Academic Publishers: Dordrecht, 1999, 293− 296.
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    Lovrinovic, M. and Niemeyer, C. M. (2007) Microtiter plate-based screening for the optimization of DNA-protein conjugate synthesis by means of expressed protein ligation ChemBioChem 8 (1) 61– 67 DOI: 10.1002/cbic.200600303
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    Molina, A. G., Kungurtsev, V., Virta, P., and Lönnberg, H. (2012) Acetylated and Methylated â-Cyclodextrins as Viable Soluble Supports for the Synthesis of Short 2′ Oligodeoxyribonucleotides in Solution Molecules 17, 12102– 12120 DOI: 10.3390/molecules171012102
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    Felici, M., Marzá-Pérez, M., Hatzakis, N. S., Nolte, R. J. M., and Feiters, M. C. (2008) â-Cyclodextrin-Appended Giant Amphiphile: Ag-gregation to Vesicle Polymersomes and Immo-bilisation of Enzymes Chem. - Eur. J. 14, 9914– 9920 DOI: 10.1002/chem.200801429
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    21
    β-Cyclodextrin-appended giant amphiphile: aggregation to vesicle polymersomes and immobilisation of enzymes
    Felici, Marco; Marza-Perez, Maria; Hatzakis, Nikos S.; Nolte, Roeland J. M.; Feiters, Martin C.
    Chemistry - A European Journal (2008), 14 (32), 9914-9920CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A giant amphiphile consisting of polystyrene end-capped with permethylated β-cyclodextrin was synthesized and found to form vesicular structures when injected as a soln. in THF into water. The ability of the cyclodextrins on the surface of the polymersomes to form inclusion complexes with hydrophobic compds. was tested by carrying out a competition expt. with a fluorescent probe sensitive to the polarity of the surrounding medium. 1-Adamantol can displace the fluorescent probe from the cavities of the cyclodextrin moieties of the polymersomes. The recognition of mols. by cell membranes in nature is often based on interactions with specific membrane receptors. To mimic this behavior, the enzyme horseradish peroxidase was modified with adamantane groups through a poly(ethylene glycol) spacer and its interaction with the polymersomes was investigated. It was established that the presence of adamantane moieties on each enzyme allowed a host-guest interaction with the multifunctional surface of the polymersomes.
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    Van Hest, J. C. M. and van Delft, F. L. (2011) Protein modification by strain-promoted alkyne-azide cycloaddition ChemBioChem 12, 1309– 1312 DOI: 10.1002/cbic.201100206
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    22
    Protein Modification by Strain-Promoted Alkyne-Azide Cycloaddition
    van Hest, Jan C. M.; van Delft, Floris L.
    ChemBioChem (2011), 12 (9), 1309-1312CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Strain-promoted reactions between cyclooctynes and 1,3-dipoles have emerged as a versatile technol. for the modification of proteins. A broad range of methods has been developed for the introduction of azides (and other dipoles) into proteins, and a range of cyclooctynes has now become synthetically readily accessible or com. available. The emergence of these techniques will greatly expand the ability to prep. homogeneous protein conjugates (PEGylation, dimerization, spin-labeling, etc.) without the need for metals or reagents; this could be of great value for, for example, the manuf. of biopharmaceuticals. The recent demonstration that cyclooctyne can be genetically encoded into a protein will facilitate the direct read-out of protein tracking in living cells and potentially whole organisms, and is therefore a substantial extension of the bioconjugation toolbox. Cycloaddns. with other, more reactive dipoles than azide (e.g., nitrones or nitrile oxides) is also feasible now. Although the reactivity of cyclooctyn-3-ol is relatively low, the genetic encoding of more reactive cyclic alkynes is a logical next step.
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  23. 23
    Merrifield, R. B. (1963) Solid Phase Peptide Synthesis. The Synthesis of a Tetrapeptide J. Am. Chem. Soc. 85, 2149– 2154 DOI: 10.1021/ja00897a025
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    Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides Anal. Biochem. 34 (2) 595– 598 DOI: 10.1016/0003-2697(70)90146-6
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    24
    Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides
    Kaiser, Emil; Colescott, R. L.; Bossinger, C. D.; Cook, P. I.
    Analytical Biochemistry (1970), 34 (2), 595-8CODEN: ANBCA2; ISSN:0003-2697.
    A rapid color test is described for detecting ≥5 micromoles free terminal amino group/g in the reaction mixt. from solid-phase peptide synthesis according to R. B. Merrifield (1968). After washing the solid phase with CH2Cl2, a 10-20 mg sample of the resin peptide is treated with 2-3 drops each of the following reagents: 500 mg ninhydrin/10 ml EtOH; 80 g PhOH/ 20 ml EtOH; and 20 ml 0.001M KCN dild. to 100 ml with pyridine. The mixt. is heated 2-5 min at 100°. The development of an intense blue color on the beads and in the soln. (except that terminal proline or terminal β-benzyl aspartate gave a brown or red-brown color) is a pos. result. In the absence of untreated terminal amino groups, the beads remained white and the soln. yellow (neg. result). The formation of pale blue beads and a light green soln. was a slightly pos. result.
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  25. 25
    Sarin, V. K., Kent, S. B., Tam, J. P., and Merrifield, R. B. (1981) Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction Anal. Biochem. 117, 147– 157 DOI: 10.1016/0003-2697(81)90704-1
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    25
    Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction
    Sarin, Virender K.; Kent, Stephen B. H.; Tam, James P.; Merrifield, R. B.
    Analytical Biochemistry (1981), 117 (1), 147-57CODEN: ANBCA2; ISSN:0003-2697.
    The title technique involves the reaction of the free amine with ninhydrin under carefully controlled conditions and the detn. of the resulting chromophore in soln. at 570 nm. All of the blue chromophore of the ninhydrin reaction with primary amines is found in soln. and the beads of the polystyrene support are colorless. An ion-exchange mechanism for the formation of blue beads by previous methods is proposed. The technique is useful for measuring the total no. of peptide chains on the resin and, esp., for monitoring the the progress of the coupling reaction. It is possible to det. when the formation of a peptide bond is >99.9% complete.
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  26. 26
    Kim, Y., Ho, S. O., Gassman, N. R., Korlann, Y., Landorf, E. V., Collart, F. R., and Weiss, S. (2008) Efficient site-specific labeling of proteins via cysteines Bioconjugate Chem. 19 (3) 786– 791 DOI: 10.1021/bc7002499
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    26
    Efficient Site-Specific Labeling of Proteins via Cysteines
    Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon
    Bioconjugate Chemistry (2008), 19 (3), 786-791CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    Methods for chem. modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome anal. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidn. before the reaction, and the effective removal of unreacted mols. prior to fluorescence studies. Here, the authors describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estd. using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than ∼95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addn. to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivs. This facile and rapid procedure is well suited for high throughput proteome anal.
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  27. 27
    Georgieva, J. V., Brinkhuis, R. P., Stojanov, K., Weijers, C. A. G. M., Zuilhof, H., Rutjes, F. P. J. T., Hoekstra, D., van Hest, J. C. M., and Zuhorn, I. S. (2012) Peptide-mediated blood-brain barrier transport of polymersomes Angew. Chem., Int. Ed. 51, 8339– 8342 DOI: 10.1002/anie.201202001
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    27
    Peptide-Mediated Blood-Brain Barrier Transport of Polymersomes
    Georgieva, Julia V.; Brinkhuis, Rene P.; Stojanov, Katica; Weijers, Carel A. G. M.; Zuilhof, Han; Rutjes, Floris P. J. T.; Hoekstra, Dick; van Hest, Jan C. M.; Zuhorn, Inge S.
    Angewandte Chemie, International Edition (2012), 51 (33), 8339-8342, S8339/1-S8339/16CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Polymersomes tagged with a dodecamer peptide that recognizes gangliosides GM1 and GT1b are shown to cross the blood-brain barrier, both in an in vitro model and in vivo. The combination of polymeric vesicles with a small GM1-binding peptide and GM1/GT1b ganglioside as targeting sites for blood-brain barrier transport is unprecedented.
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  28. 28
    Stojanov, K., Georgieva, J. V., Brinkhuis, R. P., van Hest, J. C. M., Rutjes, F. P. J. T., Dierckx, R. A. J. O., de Vries, E. F. J., and Zuhorn, I. S. (2012) In vivo biodistribution of prion- and GM1-targeted polymersomes following intravenous administration in mice Mol. Pharmaceutics 9, 1620– 1627 DOI: 10.1021/mp200621v
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    28
    In Vivo Biodistribution of Prion- and GM1-Targeted Polymersomes following Intravenous Administration in Mice
    Stojanov, Katica; Georgieva, Julia V.; Brinkhuis, Rene P.; van Hest, Jan C.; Rutjes, Floris P.; Dierckx, Rudi A. J. O.; de Vries, Erik F. J.; Zuhorn, Inge S.
    Molecular Pharmaceutics (2012), 9 (6), 1620-1627CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
    Due to the aging of the population, the incidence of neurodegenerative diseases, such as Parkinson's and Alzheimer's, is expected to grow and, hence, the demand for adequate treatment modalities. However, the delivery of medicines into the brain for the treatment of brain-related diseases is hampered by the presence of a tight layer of endothelial cells that forms the blood-brain barrier (BBB). Furthermore, most conventional drugs lack stability and/or bioavailability. These obstacles can be overcome by the application of nanocarriers, in which the therapeutic entity has been incorporated, provided that they are effectively targeted to the brain endothelial cell layer. Drug nanocarriers decorated with targeting ligands that bind BBB receptors may accumulate efficiently at/in brain microvascular endothelium and hence represent a promising tool for brain drug delivery. Following the accumulation of drug nanocarriers at the brain vasculature, the drug needs to be transported across the brain endothelial cells into the brain. Transport across brain endothelial cells can occur via passive diffusion, transport proteins, and the vesicular transport pathways of receptor-mediated and adsorptive-mediated transcytosis. When a small lipophilic drug is released from its carrier at the brain vasculature, it may enter the brain via passive diffusion. On the other hand, the passage of intact nanocarriers, which is necessary for the delivery of larger and more hydrophilic drugs into brain, may occur via active transport by means of transcytosis. In previous work we identified GM1 ganglioside and prion protein as potential transcytotic receptors at the BBB. GM1 is a glycosphingolipid that is ubiquitously present on the endothelial surface and capable of acting as the transcytotic receptor for cholera toxin B. Likewise, prion protein has been shown to have transcytotic capacity at brain endothelial cells. Here we det. the transcytotic potential of polymersome nanocarriers functionalized with GM1- and prion-targeting peptides (G23, P50 and P9), that were identified by phage display, in an in vitro BBB model. In addn., the biodistribution of polymersomes functionalized with either the prion-targeting peptide P50 or the GM1-targeting peptide G23 is detd. following i.v. injection in mice. We show that the prion-targeting peptides do not induce efficient transcytosis of polymersomes across the BBB in vitro nor induce accumulation of polymersomes in the brain in vivo. In contrast, the G23 peptide is shown to have transcytotic capacity in brain endothelial cells in vitro, as well as a brain-targeting potential in vivo, as reflected by the accumulation of G23-polymersomes in the brain in vivo at a level comparable to that of RI7217-polymersomes, which are targeted toward the transferrin receptor. Thus the G23 peptide seems to serve both of the requirements that are needed for efficient brain drug delivery of nanocarriers. An unexpected finding was the efficient accumulation of G23-polymersomes in lung. In conclusion, because of its combined brain-targeting and transcytotic capacity, the G23 peptide could be useful in the development of targeted nanocarriers for drug delivery into the brain, but appears esp. attractive for specific drug delivery to the lung.
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  • Abstract

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    Scheme 1

    Scheme 1. Synthetic Routes to Obtain Maleimide-Functionalized PMβCD Derivatives and the Target Conjugates: (A) via Hydrophilic Linker BCN-Mal; (B) via Hydrophobic Linker DIBAC-Mal
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    Figure 1

    Figure 1. (a) LC-MS of the conjugate 10 before prep-RP-HPLC isocratic purification. The main peak at 7.91 min (top trace) represents the target conjugate as testified by the double- and triple-charged molecular ions [M+2H]2+ 1206.2 and [M+3H]3+ 803.9, analyzed at retention time 7.74–8.41 min (middle trace). The shoulder due to the impurity at 8.34 min corresponds to m/z 1090.6, analyzed at retention time 8.22–8.41 min (bottom trace). The relative intensities of the peaks of target and impurity do not necessarily reflect the amounts formed because of likely differences in the response factors. (b) LC-MS of the conjugate 10 after prep-HPLC isocratic purification. The main peak represents the target conjugate at 7.96 min as testified by the double- and triple-charged molecular ions [M+2H]2+ 1206.2 and [M+3H]3+ 803.9, analyzed at retention time 7.78–8.41 min.

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    Figure 2

    Figure 2. (a) LC-MS of the conjugate 13 before prep-HPLC isocratic purification. The main peak represents the target conjugate at 8.13 min (top trace) with its corresponding formation of double-charged molecular ion [M+2H]2+ 1174.8, analyzed at retention time 7.75–8.17 min (middle trace), and the minor peak the overlapping impurity at 8.55 min with its corresponding mass formation 1059.2, analyzed at retention time 8.45–8.71 min (bottom trace). The relative intensities of the peaks of target and impurity do not necessarily reflect the amounts formed because of likely differences in the response factors. (b) LC-MS of the conjugate 13 after prep-HPLC isocratic purification. The main peak represents the target conjugate at 8.01 min with its corresponding formation of double-charged molecular ion [M+2H]2+ 1174.8, analyzed at retention time 7.86–8.40 min.

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  • References

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      Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides Anal. Biochem. 34 (2) 595– 598 DOI: 10.1016/0003-2697(70)90146-6
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      24
      Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides
      Kaiser, Emil; Colescott, R. L.; Bossinger, C. D.; Cook, P. I.
      Analytical Biochemistry (1970), 34 (2), 595-8CODEN: ANBCA2; ISSN:0003-2697.
      A rapid color test is described for detecting ≥5 micromoles free terminal amino group/g in the reaction mixt. from solid-phase peptide synthesis according to R. B. Merrifield (1968). After washing the solid phase with CH2Cl2, a 10-20 mg sample of the resin peptide is treated with 2-3 drops each of the following reagents: 500 mg ninhydrin/10 ml EtOH; 80 g PhOH/ 20 ml EtOH; and 20 ml 0.001M KCN dild. to 100 ml with pyridine. The mixt. is heated 2-5 min at 100°. The development of an intense blue color on the beads and in the soln. (except that terminal proline or terminal β-benzyl aspartate gave a brown or red-brown color) is a pos. result. In the absence of untreated terminal amino groups, the beads remained white and the soln. yellow (neg. result). The formation of pale blue beads and a light green soln. was a slightly pos. result.
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    25. 25
      Sarin, V. K., Kent, S. B., Tam, J. P., and Merrifield, R. B. (1981) Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction Anal. Biochem. 117, 147– 157 DOI: 10.1016/0003-2697(81)90704-1
      [Crossref], [PubMed], [CAS], Google Scholar
      25
      Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction
      Sarin, Virender K.; Kent, Stephen B. H.; Tam, James P.; Merrifield, R. B.
      Analytical Biochemistry (1981), 117 (1), 147-57CODEN: ANBCA2; ISSN:0003-2697.
      The title technique involves the reaction of the free amine with ninhydrin under carefully controlled conditions and the detn. of the resulting chromophore in soln. at 570 nm. All of the blue chromophore of the ninhydrin reaction with primary amines is found in soln. and the beads of the polystyrene support are colorless. An ion-exchange mechanism for the formation of blue beads by previous methods is proposed. The technique is useful for measuring the total no. of peptide chains on the resin and, esp., for monitoring the the progress of the coupling reaction. It is possible to det. when the formation of a peptide bond is >99.9% complete.
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    26. 26
      Kim, Y., Ho, S. O., Gassman, N. R., Korlann, Y., Landorf, E. V., Collart, F. R., and Weiss, S. (2008) Efficient site-specific labeling of proteins via cysteines Bioconjugate Chem. 19 (3) 786– 791 DOI: 10.1021/bc7002499
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      26
      Efficient Site-Specific Labeling of Proteins via Cysteines
      Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon
      Bioconjugate Chemistry (2008), 19 (3), 786-791CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      Methods for chem. modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome anal. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidn. before the reaction, and the effective removal of unreacted mols. prior to fluorescence studies. Here, the authors describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estd. using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than ∼95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addn. to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivs. This facile and rapid procedure is well suited for high throughput proteome anal.
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    27. 27
      Georgieva, J. V., Brinkhuis, R. P., Stojanov, K., Weijers, C. A. G. M., Zuilhof, H., Rutjes, F. P. J. T., Hoekstra, D., van Hest, J. C. M., and Zuhorn, I. S. (2012) Peptide-mediated blood-brain barrier transport of polymersomes Angew. Chem., Int. Ed. 51, 8339– 8342 DOI: 10.1002/anie.201202001
      [Crossref], [CAS], Google Scholar
      27
      Peptide-Mediated Blood-Brain Barrier Transport of Polymersomes
      Georgieva, Julia V.; Brinkhuis, Rene P.; Stojanov, Katica; Weijers, Carel A. G. M.; Zuilhof, Han; Rutjes, Floris P. J. T.; Hoekstra, Dick; van Hest, Jan C. M.; Zuhorn, Inge S.
      Angewandte Chemie, International Edition (2012), 51 (33), 8339-8342, S8339/1-S8339/16CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Polymersomes tagged with a dodecamer peptide that recognizes gangliosides GM1 and GT1b are shown to cross the blood-brain barrier, both in an in vitro model and in vivo. The combination of polymeric vesicles with a small GM1-binding peptide and GM1/GT1b ganglioside as targeting sites for blood-brain barrier transport is unprecedented.
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    28. 28
      Stojanov, K., Georgieva, J. V., Brinkhuis, R. P., van Hest, J. C. M., Rutjes, F. P. J. T., Dierckx, R. A. J. O., de Vries, E. F. J., and Zuhorn, I. S. (2012) In vivo biodistribution of prion- and GM1-targeted polymersomes following intravenous administration in mice Mol. Pharmaceutics 9, 1620– 1627 DOI: 10.1021/mp200621v
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      28
      In Vivo Biodistribution of Prion- and GM1-Targeted Polymersomes following Intravenous Administration in Mice
      Stojanov, Katica; Georgieva, Julia V.; Brinkhuis, Rene P.; van Hest, Jan C.; Rutjes, Floris P.; Dierckx, Rudi A. J. O.; de Vries, Erik F. J.; Zuhorn, Inge S.
      Molecular Pharmaceutics (2012), 9 (6), 1620-1627CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
      Due to the aging of the population, the incidence of neurodegenerative diseases, such as Parkinson's and Alzheimer's, is expected to grow and, hence, the demand for adequate treatment modalities. However, the delivery of medicines into the brain for the treatment of brain-related diseases is hampered by the presence of a tight layer of endothelial cells that forms the blood-brain barrier (BBB). Furthermore, most conventional drugs lack stability and/or bioavailability. These obstacles can be overcome by the application of nanocarriers, in which the therapeutic entity has been incorporated, provided that they are effectively targeted to the brain endothelial cell layer. Drug nanocarriers decorated with targeting ligands that bind BBB receptors may accumulate efficiently at/in brain microvascular endothelium and hence represent a promising tool for brain drug delivery. Following the accumulation of drug nanocarriers at the brain vasculature, the drug needs to be transported across the brain endothelial cells into the brain. Transport across brain endothelial cells can occur via passive diffusion, transport proteins, and the vesicular transport pathways of receptor-mediated and adsorptive-mediated transcytosis. When a small lipophilic drug is released from its carrier at the brain vasculature, it may enter the brain via passive diffusion. On the other hand, the passage of intact nanocarriers, which is necessary for the delivery of larger and more hydrophilic drugs into brain, may occur via active transport by means of transcytosis. In previous work we identified GM1 ganglioside and prion protein as potential transcytotic receptors at the BBB. GM1 is a glycosphingolipid that is ubiquitously present on the endothelial surface and capable of acting as the transcytotic receptor for cholera toxin B. Likewise, prion protein has been shown to have transcytotic capacity at brain endothelial cells. Here we det. the transcytotic potential of polymersome nanocarriers functionalized with GM1- and prion-targeting peptides (G23, P50 and P9), that were identified by phage display, in an in vitro BBB model. In addn., the biodistribution of polymersomes functionalized with either the prion-targeting peptide P50 or the GM1-targeting peptide G23 is detd. following i.v. injection in mice. We show that the prion-targeting peptides do not induce efficient transcytosis of polymersomes across the BBB in vitro nor induce accumulation of polymersomes in the brain in vivo. In contrast, the G23 peptide is shown to have transcytotic capacity in brain endothelial cells in vitro, as well as a brain-targeting potential in vivo, as reflected by the accumulation of G23-polymersomes in the brain in vivo at a level comparable to that of RI7217-polymersomes, which are targeted toward the transferrin receptor. Thus the G23 peptide seems to serve both of the requirements that are needed for efficient brain drug delivery of nanocarriers. An unexpected finding was the efficient accumulation of G23-polymersomes in lung. In conclusion, because of its combined brain-targeting and transcytotic capacity, the G23 peptide could be useful in the development of targeted nanocarriers for drug delivery into the brain, but appears esp. attractive for specific drug delivery to the lung.
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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00319.

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