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ABSTRACT: Antibody-based molecular recognition plays a dominant role in the life sciences ranging from applications in diagnostics and molecular imaging to targeted drug delivery and therapy. Here we report a generic approach to introduce protease sensitivity into antibody-based targeting by taking advantage of the intrinsic ability of antibodies to engage in multivalent interactions. Bivalent peptide ligands with dsDNA as a rigid linker were shown to effectively bridge the relatively large distance between the two antigen binding sites within the same antibody, yielding exclusively the cyclic 1:1 antibody-ligand complex. Size exclusion chromatography and small angle X-scattering were used to study the types of complexes formed between a model antibody and peptide-dsDNA conjugates displaying 1 or 2 peptide ligands and different linker lengths. Competitive binding assays using fluorescence anisotropy revealed that the interaction between bivalent peptide-dsDNA conjugate and antibody is 500-fold stronger than that of the monovalent peptide, allowing effective blocking of the antigen binding sites in a non-covalent manner. Cleavage of the linker between the peptide epitope and the DNA by matrix metalloprotease 2 disables this strong bivalent interaction and was shown to effectively restore the binding activity of the antibody in an in vitro binding assay. The approach presented here is broadly applicable, because it takes advantage of the Y-shaped multivalent presentation of antigen binding sites common to all antibodies and could be extended to control antibody activity by other input signals.
Chemical Science. 01/2013;
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ABSTRACT: The self-assembly of two enantiomerically pure hexa(oligo (p-phenylene vinylene))-substituted benzenes having 24 stereocenters was studied in pure methylcyclohexane (MCH) and in a mixture of MCH/toluene (4:1). Irrespective of the solvent a cooperative supramolecular polymerization mechanism was determined for these star-shaped molecules by using temperature-dependent CD and UV/Vis spectroscopy. Quite remarkably, a transition from one helical supramolecular state (A) to a second more thermodynamically stable supramolecular helical assembly (B) was observed. The rate of the A→B transition was strongly dependent on the nature of the solvent; being faster in the solvent mixture than in pure MCH. By using size exclusion chromatography we could relate the increased rate to a decreased stability of the supramolecular A state in the solvent mixture. Next, we mixed the two enantiomerically pure hexa-substituted benzene derivatives in a so-called majority-rules experiment, which lead to the anitcipated chiral amplification in the A state. More importantly it appeared that the A→B transition was significantly hampered in these mixed systems. Furthermore, the absence of chiral amplification in the B state revealed the formation of separated enantiomerically pure assemblies. Therefore, by using a wide variety of spectroscopic and chromatographic techniques we determined the influence of solvent and enantiomeric purity on the transition between different supramolecular states.
Chemistry 10/2012; · 5.93 Impact Factor
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ABSTRACT: In our laboratory, chloroform is increasingly required to be used as the mobile phase for the size exclusion chromatography (SEC) characterization of polyethylene glycol (PEG) derivatives, because some of the derivatives show poor solubility in many other solvents. Four types of SEC columns, all based on highly cross-linked polystyrene-polydivinylbenzene (PS/PDB) and compatible with chloroform, have been tried. However, a problem of using chloroform with all the columns tested is that retention might not be rationalized simply based on the SEC-mechanism even for the PEG standards. It was found that for the PEG standards raising the column temperature can significantly improve the SEC separation. In order to take full advantage of the temperature effect on separation, a system was developed which enables the SEC to be performed at superheated temperatures, i.e., temperatures well above the normal boiling point of the mobile phase. The improved SEC separation at elevated temperatures is most likely due to the combination of reduced adsorption of PEGs by the stationary phase and increased solubility of the solutes in the mobile phase. In this work, the SEC separation operated at temperatures above the normal boiling point of the mobile phase was called "superheated high temperature SEC".
Journal of chromatography. A 03/2012; 1237:72-9. · 4.19 Impact Factor
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ABSTRACT: 2-[(2E)-3-(4-tert-Butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB) has been considered as an excellent matrix for matrix-assisted laser desorption/ionization (MALDI) of many types of synthetic compounds. However, it might provide troublesome results for compounds containing aliphatic primary or secondary amino groups. For these compounds, strong extra ion peaks with a mass difference of 184.1 Da were usually observed, which might falsely indicate the presence of some unknown impurities that were not detected by other matrices. On the basis of the possible mechanisms proposed, these extra ions are the products of nucleophilic reactions between analyte amino groups and DCTB molecules or radical cations. In these reactions, an amino group replaces the dicyanomethylene group of DCTB forming a matrix adduct via a -C=N-bond. An aliphatic primary amine could react easily with DCTB and the reaction could start once they are mixed in a MALDI solution. For an aliphatic secondary amine, on the other hand, the reaction most likely occurs in the gas phase. Protonation of amino groups by adding acid seems to be a useful way to stop DCTB adduction for compounds with one single amino group, but not for compounds with multiple amino groups. Unlike aliphatic primary or secondary amines, aliphatic tertiary amines and aromatic amines do not yield DCTB adducts. This is because tertiary amines do not have the required transferrable H-(N) atom to form an extra -C=N-bond, while aromatic amines are not sufficiently nucleophilic to attack DCTB. In view of the possible matrix adduction, care should be taken in MALDI time-of-flight mass spectrometry (TOF MS) when DCTB is used as the matrix for compounds containing amino group(s).
Biological Mass Spectrometry 10/2010; 45(10):1195-202. · 3.41 Impact Factor
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ABSTRACT: A simple method was developed for the generation of cesium iodide (CsI) cluster ions up to m/z over 20,000 in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Calibration ions in both positive and negative ion modes can readily be generated from a single MALDI spot of CsI(3) with 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene] malononitrile (DCTB) matrix. The major cluster ion series observed in the positive ion mode is [(CsI)(n)Cs](+), and in the negative ion mode is [(CsI)(n)I](-). In both cluster series, ions spread evenly every 259.81 units. The easy method described here for the production of CsI cluster ions should be useful for MALDI MS calibrations.
Journal of the American Society for Mass Spectrometry 03/2010; 21(7):1223-6. · 4.00 Impact Factor
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ABSTRACT: In the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis of some quaternary ammonium salts (QASs), very clean spectra of the quaternary ammonium ions were recorded with a strong matrix suppression effect (MSE). The QASs also showed a considerable analyte suppression effect (ASE). It was demonstrated that the MSE and ASE of the QASs can be explained well by the cluster ionization model. According to this model, MALDI ions are formed from charged matrix/analyte clusters. Various analyte ions and matrix ions might coexist in the cluster, and they will compete for the limited number of net charges available. If enough quaternary ammonium ions are present in the cluster, they will take away the net charges, thus resulting in the MSE and ASE. Our results also suggest that 'the cluster ionization model' is not in conflict with 'the theory of ionization via secondary gas-phase reactions'. The initial MALDI ions produced from charged matrix/analyte clusters will collide with other molecules or ions in the MALDI plume. Depending on the properties of the initial ions and the composition of the MALDI plume, secondary gas-phase reactions might result from these collisions. The final ions observed are the combined results of 'cluster ionization' and 'ionization via secondary gas-phase reactions'.
Rapid Communications in Mass Spectrometry 09/2009; 23(19):3077-82. · 2.79 Impact Factor
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ABSTRACT: The covalent synthesis of complex biomolecular systems such as multivalent protein dendrimers often proceeds with low efficiency, thereby making alternative strategies based on noncovalent chemistry of high interest. Here, the synthesis of protein dendrimers using a strong but noncovalent interaction between a peptide and complementary protein is proposed as an efficient strategy to arrive at dendrimers fully functionalized with protein domains. The association of S-peptide to S-protein results in the formation of an active enzyme (ribonuclease S) and therefore serves as an ideal system to explore this synthetic approach. Native chemical ligation was used to couple four S-peptides by means of their C-terminal thioester to a cysteine-functionalized dendritic scaffold, thus yielding a tetravalent S-peptide wedge. A fully functional ribonuclease S tetramer was prepared by addition of four equivalents of S-protein. Biophysical techniques (isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and mass spectrometry) and an enzymatic activity assay were used to verify the formation of the multivalent complex. The noncovalent synthetic strategy presented here provides access to well-defined, dynamic, semisynthetic protein assemblies in high yield and is therefore of interest to the field of nanomedicine as well as biomaterials.
Chemistry 08/2009; 15(35):8760-7. · 5.93 Impact Factor
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Chemistry 07/2009; 15(35):8760 - 8767. · 5.93 Impact Factor
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ABSTRACT: Self-assembly offers the possibility to organize molecules in a given architecture through a subtle interplay between different noncovalent interactions. Although the kind of molecular association can often be predicted from information present in the individual molecules, the synthesis of supramolecular assemblies having a perfectly defined size and shape remains challenging. Here, we introduce the use of Coulombic interactions to control the supramolecular synthesis of finite, well-defined nanostructures. In particular, we demonstrate that the energy associated with the separation of ion pairs can regulate very precisely guanosine self-assembly into discrete G-quadruplexes. Assemblies comprising 8, 12, 16 or 24 guanosine molecules can be selectively and quantitatively obtained simply by tuning the stabilization of the dissociated anions in the solvent environment. Thus, factors such as solvent polarity, the nature of the anion and the cation-anion distance are shown to have a decisive role in the growth of G-quadruplexes.
Nature Chemistry 05/2009; 1(2):151-5. · 20.52 Impact Factor
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ABSTRACT: An oligo(p-phenylene vinylene) (OPV) amphiphile has been synthesized with a positively charged head group on one end and a hydrophilic ethyleneglycol wedge on the other. In water, this bolaamphiphile forms vesicles consisting of a monolayer in which the OPV surfactants are organized in a helical head-to-tail fashion. Mass spectrometry analysis and reversed-phase high performance liquid chromatography (RP-HPLC) reveals that because of the presence of two double bonds in the OPV units, photo-induced [2+2] cycloaddition takes place resulting in polymerization of the surfactants. Performing RP-HPLC allowed the OPV dimer to be isolated in a sufficient amount to perform a variety of one- and two-dimensional NMR experiments. These experiments show a highly stereoselective photochemical cycloaddition process resulting in a regioselective head-to-tail dimerization with a stereoselective syn arrangement.
Chemistry - An Asian Journal 05/2009; 4(6):910-7. · 4.50 Impact Factor
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Advanced Materials 02/2009; 21(5):597-602. · 13.88 Impact Factor
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ABSTRACT: Supramolecular complexes consisting of a single-stranded oligothymine (dTn) as the host template and an array of guest molecules equipped with a complementary diaminotriazine hydrogen-bonding unit have been studied with electrospray-ionization mass spectrometry (ESI-MS). In this hybrid construct, a supramolecular stack of guest molecules is hydrogen bonded to dTn. By changing the hydrogen-bonding motif of the DNA host template or the guest molecules, selective hydrogen bonding was proven. We were able to detect single-stranded-DNA (ssDNA)-guest complexes for strands with lengths of up to 20 bases, in which the highest complex mass detected was 15 kDa; these complexes constitute 20-component self-assembled objects. Gas-phase breakdown experiments on single- and multiple-guest-DNA assemblies gave qualitative information on the fragmentation pathways and the relative complex stabilities. We found that the guest molecules are removed from the template one by one in a highly controlled way. The stabilities of the complexes depend mainly on the molecular weight of the guest molecules, a fact suggesting that the complexes collapse in the gas phase. By mixing two different guests with the ssDNA template, a multicomponent dynamic library can be created. Our results demonstrate that ESI-MS is a powerful tool to analyze supramolecular ssDNA complexes in great detail.
Chemistry 12/2008; 15(2):352-60. · 5.93 Impact Factor
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ABSTRACT: HPLC-MS analysis of tryptic protein digests in combination with fluorescence detection is presented as a convenient and quantitative method to gain insight into the relative reactivity of lysine side chains. In this scheme (tandem) mass spectrometry was used for identification of the modified residue, whereas fluorescence detection allowed determination of their relative abundance. Our method identified 'labeling hot-spots' at two flexible parts of the collagen-binding protein CNA35, positions that were consistent with all available structural and biochemical data on the collagen-binding properties of CNA35.
Journal of Chromatography B 04/2008; 863(2):293-7. · 2.89 Impact Factor
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ABSTRACT: In the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis of various compounds synthesized in our laboratory, strong [M - H]+ ion peaks were often observed for the molecules with tertiary amino groups. In this work, the MALDI TOF MS behavior of two groups of compounds that incorporate tertiary amino moieties was investigated. One group is bisurea dimethylanilines (BUDMAs) prepared for the study of molecular recognition in thermoplastic elastomers, and the other group is the poly(propylene imine) diaminobutane dendrimers. The results clearly demonstrate the appearance of the [M - H]+ ions. In order to understand the possible mechanisms for the generation of these ions, a series of model compounds, ranging from primary to tertiary amines, were investigated. Unlike the tertiary amines, no [M - H]+ ion peaks were recorded for the primary amines, and only barely detectable ones, if any, for some secondary amines. It appears that the tertiary amino groups play an important role in the formation of these ions. In addition to MALDI TOF MS analysis, these samples were also applied to electrospray ionization (ESI) MS where no [M - H]+ ions were observed. The results indicate that the generation of [M - H]+ ion is due to the unique MALDI conditions and is likely to be formed via dehydrogenation of a protonated tertiary amine resulting in an N=C double bond. The absence of [M - H]+ ion peaks for the primary and secondary amines is probably because upon their formation these ions could easily transfer one proton to the corresponding amines in the MALDI gas-phase plume, yielding neutral imines that cannot be detected by MS.
Journal of Mass Spectrometry 04/2008; 43(8):1110-22. · 3.27 Impact Factor
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ABSTRACT: Cytochrome c' from Allochromatium vinosum is an attractive model protein to study ligand-induced conformational changes. This homodimeric protein dissociates into monomers upon binding of NO, CO or CN(-) to the iron of its covalently attached heme group. While ligand binding to the heme has been well characterized using a variety of spectroscopic techniques, direct monitoring of the subsequent monomerization has not been reported previously. Here we have explored two biophysical techniques to simultaneously monitor ligand binding and monomerization. Native mass spectrometry allowed the detection of the dimeric and monomeric forms of cytochrome c' and even showed the presence of a CO-bound monomer. The kinetics of the ligand-induced monomerization were found to be significantly enhanced in the gas phase compared with the kinetics in solution, however. Ligand binding to the heme and the dissociation of the dimer in solution were also studied using energy transfer from a fluorescent probe to both heme groups of the protein. Comparison of ligand binding kinetics as observed with UV-vis spectroscopy with changes in fluorescence suggested that binding of one CO molecule per dimer could be sufficient for monomerization.
JBIC Journal of Biological Inorganic Chemistry 09/2007; 12(6):919-28. · 3.29 Impact Factor
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Journal of the American Chemical Society 06/2007; 129(19):6078-9. · 9.91 Impact Factor
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ABSTRACT: Six anthraquinone derivatives were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Clear (pseudo) molecular ions were observed for all the compounds. Interestingly, for some derivatives, strong ions with double cation adduction were also recorded in the positive mode. It is remarkable that all these ions are singly charged. In this work, possible mechanisms for the double cation adduction were investigated and discussed. It appears that the double cation adduction was due to the electron deficient nature of the derivatives, and formed by taking up two singly charged cations and one electron. Substituents on the anthraquinone ring were found to have a significant effect on the double cation adduction. In contrast, no considerable influence of the acidity of MALDI matrix/solution was observed, even on the double proton adduction. Furthermore, it was demonstrated that double cation adduction might occur in the MALDI gas-phase plume. In addition to the anthraquinones, three more electron deficient compounds of different types, i.e. a perylene bisimide derivative (PB), 3,7-decanoylamino-4,8-dihydrobenzo[1,2-b:4,5-b']dithiophene-4,8-dione (TQ) and 6,6-phenyl C61-butyric acid methyl ester (PCBM), were also analyzed with MALDI TOF MS. The results indicate that the 'abnormal' double cation adduction might be a 'normal' phenomenon in the MALDI TOF MS analysis of many electron deficient compounds.
Journal of Mass Spectrometry 04/2007; 42(3):293-303. · 3.27 Impact Factor
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ABSTRACT: Four C3-symmetrical tris(dipeptide) disks and their precursors were characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The C3-symmetrical disks were based on a benzene-1,3,5-triscarboxamide core extended by oligopeptides with trialkoxyanilide tails. The results indicate that MALDI TOF MS is a powerful and straightforward analytical technique for characterizing C3-symmetrical disks and their precursors. Clear (pseudo)-molecular ion peaks could readily be identified. It is remarkable that strong radical ion signals were observed for all the compounds, including the anilines that were expected to be protonated prior to laser irradiation using acidic MALDI matrixes. Possible mechanisms for radical ion formation were investigated with the employment of radical scavengers, with various matrixes and with direct laser desorption/ionization (LDI). Most likely the radicals are formed by losing one electron from the aniline nitrogen and stabilized by conjugation through the phenyl ring. It appears that direct photo/thermal ionization of analytes is an important route for the radical ion formation of the compounds with trialkoxy aniline/anilide groups.
Journal of Mass Spectrometry 06/2006; 41(5):659-69. · 3.27 Impact Factor
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ABSTRACT: Host-guest interactions between the periphery of adamantylurea-functionalized dendrimers (host) and ureido acetic acid derivatives (guest) were shown to be specific, strong and spatially well-defined. The binding becomes stronger when using phosphonic or sulfonic acid derivatives. In the present work we have quantified the binding constants for the host-guest interactions between two different host motifs and six different guest molecules. The host molecules, which resemble the periphery of a poly(propylene imine) dendrimer, have been fitted with an anthracene-based fluorescent probe. The two host motifs differ in terms of the length of the spacer between a tertiary amine and two ureido functionalities. The guest molecules all contain an acidic moiety (either a carboxylic acid, a phosphonic acid, or a sulfonic acid) and three of them also contain an ureido moiety capable of forming multiple hydrogen bonds to the hosts. The binding constants for all 12 host-guest complexes have been determined by using fluorescence titrations by monitoring the increase in fluorescence of the host upon protonation by the addition of the guest. The binding constants could be tuned by changing the design of the acidic part of the guest. The formation of hydrogen bonds gives, in all cases, higher association constants, demonstrating that the host is more than a proton sensor. The host with the longer spacer (propyl) shows higher association constants than the host with the shorter spacer (ethyl). The gain in association constants are higher when the urea function is added to the guests for the host with the longer spacer, indicating a better fit. Collision-induced dissociation mass spectrometry (CID-MS) is used to study the stability of the six motifs using the corresponding third generation dendrimer. A similar trend is found when the six different guests are compared.
Chemistry 09/2005; 11(17):5126-35. · 5.93 Impact Factor
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Angewandte Chemie International Edition 09/2005; 44(32):5052-7. · 13.45 Impact Factor
