BIOSENSORS

Abstract

Provided are molecular entities capable of transforming, aggregating or assembling into a three dimensional biosensors.

Claims

1-43. (canceled)

44. A biosensor comprising: a peptide moiety; and a moiety capable of altering fluorescence emission, the moiety comprising at least one metal ion, the biosensor being associated with at least one probe molecule having a fluorescent label, the probe molecule being selected to partially or fully interact with at least one target molecule.

45. The biosensor according to claim 44, being in a form selected from tubular structure; fibrilar structures; spheres; joint spherical structures; spherical aggregates; distorted spherical aggregates; linear, two-dimensional or three-dimensional arrays; and single molecule forms.

46. The biosensor according to claim 44, self-assembled into a three dimensional form in solution.

47. The biosensor according to claim 44, wherein the peptide comprises an amino acid is selected amongst alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine valine, pyrrolysine and selnocysteine, or an amino acid analog selected from homo-amino acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids and ,-disubstituted amino acids.

48. The biosensor according to claim 44, wherein the peptide comprises an aromatic amino acid.

49. The biosensor according to claim 44, wherein the peptide moiety comprises an amino acid selected from phenylalanine (Phe) and glycine (Gly).

50. The biosensor according to claim 49, wherein the peptide moiety comprises the amino acid motif Phe-Gly or Gly-Phe.

51. The biosensor according to claim 44, wherein the moiety capable of altering fluorescence emission is selected to modulate fluorescence emission from the florescence labeled probe molecule.

52. The biosensor according to claim 51, wherein modulation is selected from attenuation, quenching, enhancement, shift in wavelength, shift in polarity and changing fluorescence lifetime, or wherein modulation is by quenching, and wherein the moiety capable of altering fluorescence emission is a fluorescence quenching moiety.

53. The biosensor according to claim 52, wherein the fluorescence quenching moiety is selected from aliphatic amines, aromatic amines, halogenated moieties, and electron scavengers.

54. The biosensor according to claim 44, wherein the at least one metal ion is an electron scavenger.

55. The biosensor according to claim 54, wherein the metal is Cu or Cd or Pd or Mn or Eu or As or Cs or Zn or Hg or Ni or Co.

56. The biosensor according to claim 54, wherein the at least one metal ion is directly associated with an amino acid of the peptide moiety or with a ligand group associated with an amino acid of the peptide moiety.

57. The biosensor according to claim 44, wherein one or both of said probe molecule and target molecule is a nucleic acid.

58. The biosensor accordion to claim 57, wherein the probe molecule is a probe nucleic acid and the target molecule is a target nucleic acid.

59. The biosensor according to claim 58, wherein the probe nucleic acid is selected to hybridize to the target nucleic acid through a portion or portions of the probe sequence that are substantially complementary to a target nucleic acid sequence.

60. The biosensor according to claim 51, wherein the labelled probe molecule is at least one nucleic acid associated with a fluorescence label.

61. The biosensor according to claim 57, wherein the probe nucleic acid and the target nucleic acid are each, independently, selected from DNA, single stranded DNA (ssDNA), double-stranded DNA (dsDNA), cDNA; and RNA.

62. The biosensor according to claim 61, wherein the probe nucleic acid and the target nucleic acid are each, independently, selected from aptamers, ribonucleotides, deoxyribonucleotides, ribonucleotide polyphosphate molecules, deoxyribonucleotide polyphosphate molecules, peptide nucleotides, nucleoside polyphosphate molecules, metallonucleosides, phosphonate nucleosides and modified phosphate-sugar backbone nucleotides.

63. The biosensor according to claim 58, wherein the probe nucleic acid is dsDNA and the target nucleic acid is a ssDNA.

64. A method of determining the presence of at least one target nucleic acid in a sample, the method comprising: contacting said sample with at least one biosensor according to claim 44, permitting association between a probe molecule and the target nucleic acid in the sample; and measuring emission from the sample to determine the presence or absence of the at least one target nucleic acid in the sample; whereby an emission of fluoresce indicates the presence of at least one nucleic acid in the sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0076] FIG. 1 depicts an exemplary synthetic methodology adopted for the synthesis of biosensors of the invention referred to herein as L and LM.

[0077] FIGS. 2A-R provide microscopic analysis of the self-assembled structures formed by L and LM. Left-hand panels: representative TEM micrographs; middle panels: HR-SEM micrographs; and right-hand panel: AFM micrographs (two-dimensional representation) of the self-assembled structures formed by L and LM under different solvents (see for example Table 1 for details): FIGS. 2A, B, Ccondition 1, FIGS. 2D, E, Fcondition 2, FIGS. 2G, H, Icondition 3, FIGS. 2J, K, Lcondition 4, FIGS. 2M, N, Ocondition 5 and FIGS. 2P, Q, Rcondition 6.

[0078] FIGS. 3A-E depicts self-assembly of LM in different solvents. FIG. 3Aschematic representation of the various self-assembled structures formed by LM in different solvents. FIG. 3BUV-vis absorbance spectra of LM (3 mg/mL) in methanol (line 1), 50% ethanol (line 2), and water (line 3). Deconvoluted FT-IR spectra of self-assembled LM in (FIG. 3c) methanol, (FIG. 3d) 50% ethanol and (FIG. 3e) water. The dashed line indicates the original FTIR spectra and the solid line represents the deconvoluted curves with a Gaussian function.

[0079] FIG. 4 depicts by way of an illustration DNA detection by LM. This is a schematic representation of an embodiment of the invention directed at detecting target HIV DNA (T1) using LM as a sensing platform. In the first step, the fluorescein-labelled probe DNA (P1) interacts and adsorbs on the self-assembled aggregated spherical structures of LM; this triggers the florescence quenching. Then, the presence of the target DNA (T1) induces fluorescence recovery owing to the conformational change of P1 (dye-labelled SS DNA) into a duplex form.

[0080] FIGS. 5A-D provide (FIG. 5A) fluorescence spectra of the probe DNA P1 (50 nM) under different conditions (excitation at 494 nm): (1) P1 in tris-HCl buffer (pH =7.4); (2) P1+400 nM HIV DNA (T1); (3) P1+0.016 mM LM+400 nM HIV DNA (T1); (4) Probe DNA+0.016 mM LM. (FIG. 5B) Fluorescence intensity ratio of P1 (red markers) and P1-LM (blue markers) with (F/F.sub.0-1) plotted against the logarithm of the concentration (Log C) of HIV1 DNA (T1). Excitation: 494 nm, emission: 521 nm. (FIG. 5C) The effect of LM dosage (0.539 mM) on the emission intensity of the probe DNA (P1) (50 nmol L.sup.1). (FIG. 5D) Fluorescence quenching of P1 (50 nm) in Tris-HCl buffer by LM as a function of time (green markers), and fluorescence restoration of P1-LM in Tris-HCl buffer (pH=7.4) by T1 (HIV1) (200 nM) as a function of time (grey markers). Excitation: 494 nm, and emission: 521 nm. SD was calculated based on 3 experiments.

[0081] FIGS. 6A-D depict optimisation of the detection system. FIG. 6AFluorescence spectra of the fluorescein-labelled probe DNA-LM conjugate (P1-LM) after incubation with increasing concentrations of target HIV DNA (T1) ranging from 0 to 500 nM, at room temperature; excitation wavelength=494 nm; incubation time=180 min. FIG. 6BThe graph plots (F.sub.T-F.sub.M)/F.sub.M (emission recovery) versus target-DNA (T1) concentrations. [probe DNA]=50 nmol, [LM]=0.016 mM, incubation time=180 min; emission intensities are measured at 521 nm. FIG. 6CFluorescence intensity changes (F.sub.T/F.sub.M-1) of the fluorescein-labelled probe DNA (P1)+LM towards the target DNA (T1A, 100 nM), (T1B, 200 nM) and single-base mismatched DNA (MT1, 1 M). F.sub.M and F.sub.T are the emission intensities of P1-LM at 521 nm in the absence and presence of different targets. FIG. 6DFluorescence response (F/F.sub.0) of P1-LM incubated in the presence of 500 nM T1 with or without (control) several interfering proteins at a concentration of 2 M; F and F.sub.0 are the emission intensities of P1-LM/T1 and P1-LM at 521 nm, respectively.

[0082] FIGS. 7A-B depict size distribution obtained from the DLS measurement for the spherical particles formed by (FIG. 7A) LM in methanol and (FIG. 7B) L in 50% ethanol.

[0083] FIGS. 8A-C show (FIG. 8A) TEM micrograph of self-assembled structures formed by LM in 50% ethanol. HR-SEM micrographs of (FIG. 8B) self-assembled structures formed by LM in water and (FIG. 8C) L in water.

DETAILED DESCRIPTION OF EMBODIMENTS

Results and Discussion

Synthesis and Characterization

[0084] As depicted in FIG. 1, for exemplification purposes, to generate a complex of the invention, molecule (5) that comprises two repeating units of Phe-Gly-Phe (Phe=phenylalanine and Gly=glycine) linked by an ethylene diamine linker (see Experimental below). This exemplary peptide unit was chosen because it was assumed that the aromatic Phe will interact with DNA fragments through aromatic interactions. In addition, the free NH group will promote intermolecular hydrogen bonding and together with aromatic interaction, it will lead to the peptide's self-assembly. It was further assumed that the ethylene diamine bridge will act as a flexible linker of the two peptides, which will provide structural freedom in the self-assembly process. The de-protected N-terminus of 5 was then coupled to the metal chelator 4-(bis(pyridin-2-ylmethyl)amino)-benzaldehyde to produce the ligand (L). Considering the metal binding affinity of the 2,2-dipicolylamine (DPA)-based receptor, the metal-peptide conjugate (LM, ligand-metal conjugate) was synthesized by reacting an aqueous solution of Cu(ClO.sub.4).sub.2 with a methanol solution of L. All synthesized products were isolated and characterised by standard analytical techniques as well as spectroscopic and biophysical methods.

[0085] In addition, all UV-Vis absorption spectra of L and LM were recorded in order to detect the change in the optical spectral pattern of the peptide (L) in the presence and absence of the metal ion.

[0086] To trigger the self-assembly of L and LM, the two compounds were dissolved in different solvents having different polarities. First, the compound (either L or LM) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) to a concentration of 100 mg/mL. Then, each solution was diluted with methanol, 50% ethanol or water (Table 1). The polarized solvents (methanol<<50% ethanol<<water) allowed the peptides to self-assemble.

TABLE-US-00001 TABLE 1 The different self-assembly conditions for L and LM and the resulting assemblies Condition Compound Solvent Concentration Assemblies 1 L Methanol 2 or 3 mg/mL No distinct structures 2 L 50% 2 or 3 mg/mL Spheres Ethanol 3 L Water 2 or 3 mg/mL Distorted spherical aggregates 4 LM Methanol 2 or 3 mg/mL Spheres 5 LM 50% 2 or 3 mg/mL Joint Ethanol spherical structures 6 LM Water 2 or 3 mg/mL Spherical aggregates

[0087] High-resolution scanning electron microscopy (HR-SEM) analysis revealed that in methanol L did not form any distinct structures (FIG. 2B) but LM can form spherical assemblies with nanomeric dimensions (FIG. 2K). Dynamic light scattering (DLS) analysis revealed that the average diameter of the particles was 71427 nm (FIG. 7A). In 50% ethanol, L self-assembled into spherical nanostructures (average diameter 52613 nm; FIG. 7B and FIG. 2E), whereas LM formed nanospheres that were joined together (FIG. 2N). It had been suggested that for such a structure, the nanospheres are first arranged in an ordered array and then commutate with each other through neck formation.

[0088] For further insights into the morphology of the self-assembled nanomeric spherical structures formed by L and LM under different conditions of solvents, transmission electron microscopy (TEM) analysis was performed. This analysis revealed a difference between the nanospheres formed under conditions 2 and 4; Table 1 (FIG. 2D and J): The spheres formed under condition 4; Table 1 by LM had a comparable lighter contrast with a white center and look like a hollow sphere, which suggests a lower electron density. The TEM analysis of the self-assembled structures formed under condition 5; Table 1, further confirmed the morphology of joined nanospheres through neck formation (FIG. 2M, FIG. 8A).

[0089] AFM analysis further supported the results obtained by HR-SEM and TEM as different nanostructures formed by L and LM in different solvents. Under condition 2 (Table 1), the nanospheres formed by L in 50% ethanol had a height ranging from 5 nm to 32 nm. (FIG. 2F, FIG. 9A-C). The height of the spheres formed by LM in 100% methanol (condition 4, Table 1) ranged from 11 nm to 79 nm (FIG. 2, FIGS. 9D-F). These findings are in good agreement with the results obtained by the DLS measurements. Under condition 5, where the nanospheres communicated with each other through neck formation, the height of the spheres varied from 92 nm to 434 nm (FIG. 2O, FIGS. 9G-I). From the height analysis, it is clear that the spheres formed by LM in 50% ethanol (condition 5) are much higher than the spheres formed by LM under condition 4 and by L under condition 2. When the self-assembly of L and LM was examined in water, it was found that both L and LM self-assembled into a 3D network of aggregated spheres. The spherical structures in the network formed by L and LM were different in size and morphology. L formed 3D aggregates of distorted spherical assemblies (FIGS. 2G-I) when compared with LM (FIG. 2P-R).

[0090] Overall, both L and LM formed spherical structures with diverse morphologies in solvents having different polarities. The self-assembly process is controlled by the molecular parameters of the peptide-based monomer, which depends on peptide-peptide and metal-peptide interaction energies that govern the stabilities and average size of the self-assembled structures.

[0091] The structural transformation of peptide assemblies is dependent on the solvent's polarity. This may have a potential implication on solvent-induced controlled molecular self-assembly. Water, which has the highest polarity, may lead to the formation of a stronger hydrogen binding network, which prompted transition of LM from individual nanospheres into fused spherical aggregates (FIG. 3A).

[0092] UV-vis absorption spectra of LM were also recorded in the different polar protic solvents, used for the self-assembly process. As the solvent's polarity increased, the absorption maximum of LM, corresponding to the intercomponent charge transfer transition, shifted towards longer wavelengths (327.fwdarw.347.fwdarw.364 nm) (FIG. 3B). These solvent-dependent spectral shifts arise from either non-specific interactions (dielectric enrichment) or specific bonds such as hydrogen bonding. This effect can be determined by using a solvent polarity scale or solvatochromic parameters. The excited states for most of the electronic transitions are more polar than their ground states because a greater charge separation is observed in the excited state. In a protic polar solvent the dipole-dipole interactions and hydrogen bonds stabilised the electronic excited state relative to the ground state; hence, the absorption in a more polar solvent will be shifted towards longer wavelengths (lower energy) in comparison with a less polar solvent.

[0093] To obtain an insight into the secondary structure of the different self-assembled structures of LM formed in the different solvents, Fourier transform infrared (FT-IR) analysis was generated and each spectrum was deconvoluted. The FT-IR spectra of the spherical structures formed in methanol exhibited two major peaks at 1633 cm.sup.1 and 1685 cm.sup.1, indicating an anti-parallel -sheet structure (FIG. 3C). The spectra of the self-assembled structures (connecting spheres) formed in 50% ethanol exhibited one minor peak at 1615 cm.sup.1, which may relate either to extended hydrated structures or to a -sheet and another major peak at 1667 cm.sup.1, which corresponds to -turn configuration (FIG. 3D). The spherical aggregates formed in water exhibited one major peak at 1668 cm.sup.1 ascribed to -turn and another minor peak at 1605 cm.sup.1, indicating a disordered and random structure (FIG. 3E).

[0094] The nature of the nanostructures formed by LM in water was further studied by X-ray powder diffraction (XRD) and compared to the assemblies in water formed by L. The XRD pattern indicated that both L and LM had amorphous and monoclinic crystalline structures. The XRD spectrum of LM exhibited sharp peaks along with the characteristic crystalline reflections in a wide range of 2 (2-40); however, the intensities of these characteristic peaks were comparatively lower for L. Furthermore, the crystalline area and degree of crystallinity (%) were lower for L (200.2, 13.07%) compared with LM (305.6, 22.89%). In addition, the crystallite size for L and LM was 109.6 nm and 119.3 nm, respectively. Taken together, these structural analyses suggest that these spherical structures are not well ordered. However, it is noted that the spherical aggregates formed by LM in water are stable for at least one month at room temperature and atmospheric pressure.

Optical Detection of DNA

[0095] Structures having aromatic moieties with electron systems can interact with biomolecules such as DNA and proteins. In addition, some metal ions such as Zn.sup.2+ and Cu.sup.2+ are usually used as coordination centers, which have intrinsic fluorescence quenching properties. The detection or sensing of biological samples under physiological conditions (such as aqueous medium) is of major importance. Furthermore, the quenching efficiency of LM in water (spherical aggregates)>LM in ethanol (connecting spheres)>LM in methanol (individual spheres). This result suggests that the spherical aggregates formed by LM in water have a larger surface area for the adsorption of the probe DNA. This leads to a higher quenching efficiency with better optical response. Previous reports also showed that the association of ssDNA/GO is much faster than SWCNT and CNPs due to the presence of a larger adsorption area. It is therefore assumed that the surface area of the self-assembled nanostructures and the charge properties of the ssDNA are the main contributors to the adsorption rate. Hence, it is considered the metal-peptide complex, LM, with its self-assembled network as a smart sensing platform comparable to several MOFs.

[0096] The self-assembled LM was utilized as a biosensor, where Cu.sup.2+ acted as a coordination center, the electron system mediated non-covalent bonding and fluorescein (FLC)-labeled ssDNA was used as a probe. The HIV-1U5 long terminal repeat sequence was used as the target analyte since early detection of HIV is highly important. Emission quenching was used as the basis for optical sensing.

[0097] FIG. 4 provides a schematic representation of this optical detection platform. - interactions were assumed between the nucleobases of the DNA molecule and LM, along with the d.sup.9 metal (Cu.sup.2+) coordination of LM with the chelating site of the probe DNA, would bind the dye-labeled ssDNA and almost completely quench the fluorescence of the dye. The d.sup.9 metal center of Cu.sup.2+ can strongly quench the fluorescence of the dye owing to a photo-induced electron transfer process. The presence of a target molecule was assumed to alter the conformation of the dye-labeled ssDNA and that this conformational change will result in the release of the dye-labeled DNA in the duplex form, reversing the emission quenching effect. This result could give a turn-on emission response that is sensitive and selective to the target molecule. To validate our design, used following oligonucleotides were used: A Fluorescein (FLC)-labeled ssDNA sequence (P1) 5-AGTCAGTGTGGAAAATCTCTAGC-FLC-3, a synthetic oligonucleotide (T1) (5-GCTAGAGATTTTCCACACTGACT-3) from the HIV1-U5 long terminal repeat sequence as complementary target DNA, and the single-base mismatch sequence (M1) 5-GCTAGAGATTGTCCACACTGACT-3 (mismatch underlined). Four consecutive experiments were carried out to validate the feasibility and reproducibility of the experimental results.

[0098] Since LM, the target and the probe DNA are water-soluble, the detection system is homogeneous. This type of homogeneous detection of nucleic acid with fluorescent probes has several natural advantages, such as ease of operation, rapid hybridization kinetics and potential compatibility with real-time monitoring and in situ cellular imaging. FIG. 5A shows the fluorescence emission spectra of P1 under different conditions. The probe DNA P1 (in Tris-HCl buffer pH=7.4) in the absence of LM had strong fluorescence emission owing to the presence of fluorescein (FIG. 5A, curve 1). In the presence of LM, up to 93% fluorescence quenching occurred (FIG. 5A, curve 4). This observation indicates a strong interaction between P1 and LM and high fluorescence quenching efficiency of LM. The P1-LM complex had a marked fluorescence enhancement upon the addition of the target DNA (T1, the complementary sequence of P1) (FIG. 5A, curve 3). The fluorescence of the free P1 was, however, barely influenced by the addition of only T1 (FIG. 5A, curve 2).

[0099] FIG. 5B illustrates the fluorescence intensity changes (F/F.sub.0-1) for P1 and P1-LM upon the addition of different concentrations of T1, where F.sub.0 and F are the fluorescence intensities at 521 nm in the absence and the presence of T1, respectively. With P1, no significant variation in the fluorescence intensity was found in the target concentration range. However, for the P1-LM conjugate, a dramatic increase in the fluorescence intensity was observed. In a reverse experiment, when we added LM to the mixture of P1/T1, the emission was barely affected and the quenching efficiency was significantly weaker than that in the presence of only P1. Thus, it may be concludes that LM weakly interacts with dsDNA. Due to this markedly weaker emission quenching, the ratio of the signal-to-background can be increased when LM is introduced. This result suggests that the sensing mechanism is based on the fluorescence quenching of the dye-labelled ssDNA (P1) upon binding to LM. More specifically, when P1 was hybridized with its complementary target T1 to form a duplex, it became rigid/stable, and conformational changes released the duplex from the LM network, reversing the emission quenching effect. Because the interactions between the double-stranded DNA (dsDNA) and LM were rather weak, it led to observable fluorescence that is the basis of a signal-on DNA sensor.

[0100] The ssDNA adsorption on the GO surface is very fast and reaches equilibrium within one minute. The release of the duplex from the GO surface with a maximum emission recovery occurs within 30 minutes with a lower detection limit (LOD) of 10 nM. The emission quenching of the dye-labelled ssDNA by SWCNT exhibits a rapid reduction in the first hour followed by a slow decrease over the subsequent 2-3 hr. The release of the duplex from the wall of CNT in the presence of the complementary target reaches a plateau after 3 hr with an LOD of 4 nM. The CNPs reaches about 82% fluorescence quenching within 30 minutes and DNA hybridization occurs at a rate comparable to ssDNA adsorption and yields the best fluorescence response after 40 minutes (LOD>10 nM). Cu(H.sub.2dtoa)-based MOF sensing of ssDNA has a quenching efficiency of 84.5% and the incubation time considered for maximum emission recovery is more than 4 hr with an LOD of 3 nM.

[0101] For better quantification of the target DNA, several factors were optimized, such as the incubation time and the dosage of LM. The effect of the dosage of LM was studied (FIG. 4C). The fluorescence intensity of 50 nmol probe DNA (P1) decreased with increasing LM dosage from 0-27 L (the concentration of the LM stock solution was 1 mg mL.sup.1, 0.539 mM) and then reached a plateau. When 27 L of LM was added, about 90% of the fluorescence emission quenching was observed. Hence, 27 L (stock solution 0.539 mM) of LM was chosen as the standard dosage for the following experiments. The kinetic behaviour of P1 and LM as well as P1-LM conjugate in the presence of T1 was studied by monitoring the emission intensity as a function of time (FIG. 4D). The fluorescence of P1 was quenched completely by LM within 30 min; therefore, 30 min was chosen as the quenching time for subsequent experiments. The interaction of ssDNA with LM was rapid at room temperature and reached equilibrium within 30 minutes. However, the formation and release of dsDNA from LM was relatively slow. The dynamic behaviour of this sensing technique was studied by monitoring the fluorescence recovery of P1 as a function of incubation time in the presence of T1 (FIG. 4D). This analysis revealed that an increase in the incubation time results in a gradual increase in the fluorescence intensity and that it achieved a plateau after three hours. Hence, three hours was chosen as the optimum incubation time of the target DNA (T1).

[0102] FIG. 5A describes the fluorescence spectra of the FLC-labelled P1-LM complex conjugate upon the addition of increasing concentrations of the target DNA (T1). Increasing the concentration target DNA induced a gradual increase in the fluorescence intensity, implying that more P1 was released from the LM network. FIG. 5B shows that a linear relationship exists between the change in the fluorescence intensity (F.sub.T-F.sub.M)/F.sub.M and the concentration of target ss-DNA (T1) in the range of 4 nM to 100 nM, where F.sub.M and F.sub.T were the fluorescence intensities at 521 nm in the absence and presence of T1 calculated by:

(F.sub.TF.sub.M)/F.sub.M=0.0022C+0.0607, R=0.993,

[0103] where C is the concentration of the target DNA and R is the regression coefficient of the equation. Based on this method, the detection limit of the target ss-DNA was estimated to be 1.4 nmol L.sup.1 (defined as S/N=3). This detection limit is lower than those obtained by systems that rely on carbon nanostructures including SWNT, GO, CNPs platform and electrochemical-DNA sensors. The major limit of carbon-based nanomaterials such as SWCNT, GO, and CNPs is their lack of (or low) solubility in water. Therefore, sensing platforms that are based on these materials cannot support homogeneous detection of biomolecules. Homogeneous detection technique has potential compatibility in detecting lower concentrations of targeted molecules with better sensitivity and in-situ cellular imaging. Moreover, this detection system, which we propose, exhibits excellent reproducibility because the relative standard deviation (RSD) for parallel experiments of four newly prepared ss-DNA solutions (100 nmol L.sup.1) was only 6.1%.

[0104] Usually, DNA-intercalating dyes can interact in a non-specific manner with DNA fragments and proteins. This greatly limits the application of the intercalating dyes in DNA detection. To evaluate the specificity of the system, an attempt was made to detect a one-base mismatched DNA fragment (MT1). In the presence of MT1, the fluorescent recovery was low. This indicates lower (F.sub.T-F.sub.M)/F.sub.M owing to the existence of an unstable duplex conformation (FIG. 5C). The introduction of the complementary DNA (T1) resulted in a marked fluorescence recovery and a larger (F.sub.T-F.sub.M)/F.sub.M. This result shows that a one-base mismatch in the target DNA sequence can be discriminated by the system. Furthermore, in order to confirm the specificity of this detection system, the emission spectra of P1 was recorded in the presence of MT1. The emission of the labelled dye was barely affected. However, the introduction of MT1 to the LM-P1 conjugate failed to produce a significant emission recovery. This is because MT1 cannot hybridize and release the probe ssDNA from LM by forming a stable duplex conformation or because LM possibly competitively destabilizes the mismatched duplex, which leads to insignificant emission recovery. This DNA-sequence signal specificity is higher than that of linear DNA probes, which cannot discriminate single-base mismatch targets. A further investigation was carried out in the presence of several possible interfering proteins such as bovine serum albumin (BSA), alkaline phosphatase (ALP), pepsin and lysosome (FIG. 5D). The interaction of ss-DNA with proteins mainly occurs through non-covalent binding; however, owing to the stability of the dsDNA structure formed between P1 and the perfectly complementary T1, its emission response (F/F.sub.0) was not affected by the coexisting proteins, showing the good specificity of the present strategy.

[0105] Moreover, when L was used instead of LM in this sensing system, an insignificant emission response of P1 was observed. This experimental observation suggests that the low optical response of P1 in the presence of L is attributed to the absence of Cu(II) from the network. Cu(II) in LM has a d.sup.9 electronic configuration that facilitates its binding to the probe DNA (P1), along with - stacking, and it quenches the fluorescence of FLC owing to the photoinduced electron transfer (PET) process. It is well known that Cu(II) complexes with free coordination sites efficiently quench the emission of fluorophores with the binding properties in molecular beacon oligonucleotide probes; this triggers PET. The advantage of these types of optical quenchers is their ability to interact reversibly with probe DNA through space or weak contact interactions. This makes the detection of targeted DNA by an optical sensing stand feasible. The results from these studies suggest that LM can differentiate between ssDNA and ds DNA. This does not only offer selective detection of DNAit may be possible to extend this optical sensing application to a wide spectrum of analytes by complementing LM with functional nucleic structures (e.g., aptamers). More importantly the emission quenching response of P1 after introducing LM in the presence of polyanions such as polyacrylic acid is comparable to that without polyacrylic acid. Further addition of increasing concentrations of T1 leads a steady increase in the emission recovery. This result suggests that this newly developed receptor sensing stand is stable and functions in the presence of polyanions such as polyacrylic acid.

Experimental Section

Materials

[0106] All chemicals and solvents are commercially available and were used as supplied unless otherwise stated Amino acids such as Phenylalanine and Glycine were purchased from Novabiochem (Darmstadt, Germany) Cu(ClO4).sub.2 was purchased from Sigma Aldrich (St Louis, Mo., USA). 2-(chloromethyl) pyridine hydrochloride was purchased from Sigma Aldrich (St Louis, Mo., USA), aniline, ethylenediamine and phosphoryl chloride were purchased from Merck (Darmstadt, Germany) (1-Hexadecyl) trimethyl ammonium chloride was purchased from Alfa Aser (Ward Hill, China), N,N-dicyclohexyl-carbodiimide, Di-tert-butyl dicaronate and 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) were purchased from Alfa Aser (Heysham, England); hydroxybenzotriazole was purchased from Chem-Implex International (Wood Dale, Ill., USA). The following DNA sequences were synthesized by Sigma(Rehovot, Israel):

[0107] Probe DNA: 5-AGTCAGTGTGGAAAATCTCTAGC-FLC-3 (FLC=Fluorescein) Target DNA: 5-GCTAGAGATTTTCCACACTGACT-3 Single-base mismatch DNA: 5-GCTAGAGATTGTCCACACTGACT-3. The probe DNA (P1) is the complementary sequence of HIV-1 U5. The target DNA (T1) is the HIV-1 U5 sequence. Single-base mismatched DNA has a single-base mismatch in the underlined position.

Self-Assembly of L and LM

[0108] A fresh stock solution of L and LM was prepared by dissolving the lyophilised forms of L and LM in HFP to a concentration of 100 mg/mL. Then, we blended these peptides in several different proportions and diluted them in the desired solvent as indicated in Table 1. The polarized solvent allowed the molecules to self-assemble.

Preparation of the Complex Probe DNA-LM and Assay of the Target DNA

[0109] First, 1.0 mg of LM (which was previously self-assembled in water and dried) was dispersed in 1 mL triple distilled water (TDW) by sonication. Next, 27 L of this suspension were mixed with 50 nM probe DNA with oscillation at RT in order to obtain a symmetrical and limpid solution. The fluorescence intensity of the solution was detected; then different concentrations of the target DNA were added to the solution containing P1-LM complex with oscillation at 35 C. for 3 h (except for the time-course study). The complex obtained was used immediately to measure fluorescence. DNA fragments in buffer solutions were prepared by dissolving the DNA in 0.5 mM Tris-HCl buffer (pH 7.4, containing 100 mM NaCl and 5 mM MgCl.sub.2).

High-Resolution Scanning Electron Microscopy (HR-SEM)

[0110] A 10 L drop of the solution of either L or LM in the different solvents was placed on a glass cover slip and allowed to dry at RT. The substrates were then coated with gold using a Polaron SC7640 Sputter Coater. SEM analysis was performed using a high-resolution scanning electron microscope (HR-SEM, Serion equipped with X-MAX20 SDD Inca 450 EDS LN.sup.2 free detector) operating at 1 kV.

Transmission Electron Microscopy (TEM)

[0111] A 10 L drop of the solution of either L or LM in the different solvents was placed on a 200-mesh copper grid, covered by carbon-stabilized Formvar film (Electron Microscopy Science, PA, USA). After 1 min, excess fluid was removed from the grid. The samples were analysed using a Tecnai T12 G.sup.2 Spirit (Cryo-TEM) operating at 120 kV.

Atomic Force Microscopy Analysis

[0112] Topography images of the structures on glass cover slips were taken using JPK NanoWizard3 (JPK instruments, Germany) working in AC mode. Si.sub.3N.sub.4 cantilever probes with a spring constant of 3 Nm.sup.1 and a resonance frequency of 75 kHz were used.

Fourier Transform Infrared Spectroscopy (FT-IR)

[0113] Fourier transform infrared spectra were recorded using a Nicolet 6700 FT-IR spectrometer with a deuterated triglycine sulfate (DTGS) detector (Thermo Fisher Scientific, Mass., USA). The metal-peptide complex solutions were deposited on a CaF.sub.2 window and dried under vacuum. The peptide deposits were resuspended with D.sub.2O and subsequently dried to form thin films. The re-suspension procedure was repeated twice to ensure a maximal hydrogen-to-deuterium exchange. The measurements were taken using 4 cm.sup.-1 resolution and averaging 2000 scans. The transmittance minimal values were determined by the OMNIC analysis program (Nicolet).

UV-Vis Spectroscopy

[0114] UV-Vis absorption spectra of the monomeric form of L, LM in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) and the UV-Vis absorption spectra of self-assembled metallo peptides (LM) in the different solvents (methanol, 50% ethanol and water) were recorded using a UV/Vis spectrophotometer (SHIMADZU, UV-1650PC).

Fluorescence Spectroscopy

[0115] For the quantitative homogeneous detection of the target DNA, the fluorescence measurements were performed at RT using a fluorescent spectrometer (Perkin Elmer LS 55). The emission spectra were collected from 500 to 650 nm with an excitation wavelength of 494 nm. Both the excitation and emission slit widths were set to 5.0 nm. The fluorescence intensity at 521 nm was used for quantitative analysis. The quenching efficiency (Q.sub.E,%) was calculated by the formula: Q.sub.E=(1F.sub.M/F.sub.0)100%, where F.sub.M and F.sub.0 are fluorescence intensities at 521 nm in the presence and the absence of LM. The fluorescence recovery was calculated by the formula: RE=(F.sub.T/F.sub.M1)100%, where F.sub.T and F.sub.M are fluorescence intensities at 521 nm in the presence and the absence of the target DNA after introducing LM.

X-Ray Diffraction (XRD) Analysis

[0116] The phase of the product was identified by X-ray powder diffraction (X-Ray DiffractometerD8 Advance), using Cu K (=0.15406 nm) and a solid state NaI dynamic scintillation detector. The full Diffrac.sup.Plus package software was used for data acquisition, phase analysis, crystallography and thin film characterisation.

Dynamic Light Scattering (DLS) Analysis

[0117] Dynamic light scattering measurements of the assemblies were performed using a Nano-zeta sizer (Malvern instruments), model ZEN3600).

Peptides Synthesis:

[0118] Peptides were synthesized by conventional solution-phase methods. Peptide coupling was mediated by dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBt). The products were purified by column chromatography using silica gel (100-200 mesh) as the stationary phase and an n-hexane-ethyl acetate mixture as an eluent. The final compounds were fully characterized by Bruker 500 MHz 1H-NMR spectroscopy, and mass spectroscopy (Applied Biosystems Voyager-DE Pro MALDI-TOF and Accela Autosampler, Thermo Scientific (CCQ Fleet)).

[0119] Synthesis of BOC Phe-OH: A solution of L-phenylalanine (3.30 g, 20 mmol) in a mixture of dioxane (40 mL), water (20 mL) and 1 M NaOH (20 mL) was stirred and cooled in an ice-water bath. Di-tertbutylpyrocarbonate (4.583 g, 21 mmol) was added and stirring continued at room temperature (RT) for 6 h. Then the solution was concentrated in vacuum to about 10-15 mL, cooled in an ice-water bath, covered with a layer of ethyl acetate (about 50 mL) and acidified with a dilute solution of KHSO4 to pH 2-3 (determined by Congo red). The aqueous phase was extracted with ethyl acetate and this was done repeatedly. The ethyl acetate extracts were pooled, washed with water, dried over anhydrous Na.sub.2SO.sub.4 and evaporated in a vacuum. The pure material was obtained as a waxy solid. Yield 4.45 g (16.8 mmol, 84.0%).

##STR00002##

[0120] Synthesis of NH.sub.2-Gly-OMe Hydrochloride: 4.5 g (60 mmol) of L-glycine was dissolved in 90 mL of MeOH and cooled in an ice bath. Then, 12 ml of SOCl2 was added dropwise and stirred for 8 h. The excess solvent was evaporated under rotary vacuum. The dried crystalline solid product obtained was L-glycine methyl ester hydrochloride. Yield 6.30 g (50.4 mmol, 85.0%).

[0121] Synthesis of BOC-Phe-Gly-OMe (1): 4.0 g (15 mmol) of Boc-Phe-OH was dissolved in 40 ml dry DCM in an ice-water bath. H-gly-OMe. HCl 2.507 g (20.0 mmol) and Et.sub.3N 4 ml, 30 mmol) were then added to the reaction mixture, followed immediately by the addition of 3.30 g (16.0 mmol) dicyclohexylcarbodiimide (DCC) and 2.16 g (16.0 mmol) of HOBt. The reaction mixture was allowed to warm-up to RT and was stirred for 48 h. DCM was evaporated and the residue was dissolved in ethyl acetate (60 mL). The dicyclohexylurea (DCU) was filtered off. The organic layer was washed with 2M HCl (350 mL), brine (250 mL), 1 M sodium carbonate (350 mL) and brine (250 mL) and finally dried over anhydrous sodium sulfate. It was then evaporated under vacuum to yield Boc-Phe-Gly-OMe as a white solid. The product was purified by silica gel (100-200 mesh) using n-hexane-ethyl acetate (3:1) as eluent. Yield: 3.65 g (10.85 mmol, 72.35%). .sup.1H NMR (CDCl.sub.3, 400 MHz, ppm): 7.31-7.28 (m, 2H, ArH of Phe), 7.23-7.20 (m, 3H, ArH of Phe), 6.52 (b, 1H, NH Phe), 5.05 (b, 1H, NH Gly), 4.42-4.41 (m, 1H, CH, Phe) 4.07-3.91 (dd, 2H, CH2-Gly), 3.73 (s, 3H, OMe), 3.14-3.05 (m, 2H, CH, Phe) 1.39 (s, 9H, Boc). ESI-MS (m/z): [M]=336.38 (calculated); 336.59 (observed), [M+Na+H]+=360.38 (calculated); 360.11 (observed); [M+K+H]+=376.38 (calculated); 376.29 (observed), [M+2Na]+=382. 38 (calculated); 381.21 (observed).

[0122] Synthesis of BOC-Phe-Gly-OH (2): To 3.0 g (8.91 mmol) of Boc-Phe-Gly-OMe, 30 mL MeOH and 2M 15 mL NaOH were added and the progress of saponification was monitored by thin layer chromatography (TLC). The reaction mixture was stirred. After 10 h, the methanol was removed under vacuum; the residue was dissolved in 50 mL of water and washed with diethyl ether (250 mL). Then, the pH of the aqueous layer was adjusted to 2 using 1M HCl and extracted with ethyl acetate (350 mL). The extracts were pooled, dried over anhydrous sodium sulfate and evaporated under vacuum to obtain the compound as a waxy solid. Yield: 2.72 g (8.46 mmol, 95%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz, ppm): 12.56 (s, 1H, COOH), 8.23 (t, 1H, NH Gly), 7.27, 7.16 (m, 5H, ArH of Phe), 6.89 (d, 1H, NH Phe), 4.23-4.17 (m, 1H, CH, Phe) 3.86-3.72 (m, 2H, CH2 Gly), 3.03-2.69 (m, CH, Phe) 1.28 (s, 9H, Boc). ESI-MS (m/z): [M+Na+H]+=346.35 (calculated); 346.42 (observed); [M+K+H]+=362.35 (calculated); 362.37 (observed).

[0123] Synthesis of BOC-Phe-NHCH.sub.2CH.sub.2NH-Phe-Boc (3) and NH2-Phe-NHCH.sub.2CH.sub.2NH-Phe-NH.sub.2 (4): 3.0 g (11.27 mmol) of Boc-Phe-OH were dissolved in 40 ml dry DCM in an ice-water bath. Ethylenediamine (340 mg) (5.63 mmol) was then added to the reaction mixture, followed immediately by the addition of 2.79 g (13.5 mmol) dicyclohexylcarbodiimide (DCC) and 1.82 g (13.5 mmol) of HOBt. The reaction mixture was allowed to warm-up to RT and was stirred for 48 h. DCM was evaporated and the residue was dissolved in ethyl acetate (60 mL) and the dicyclohexylurea (DCU) was filtered off. The organic layer was washed with 2M HCl (350 mL), brine (250 mL), 1 M sodium carbonate (350 mL) brine (250 mL) and dried over anhydrous sodium sulfate; finally it was evaporated under vacuum to yield BOC-Phe-NHCH2CH2NH-Phe-Boc (3) as a white solid. The product was purified by silica gel (100-200 mesh) using n-hexane-ethyl acetate (4:1) as eluent. Yield: 2.04 g (3.61 mmol, 64.18%). .sup.1H NMR (CDCl.sub.13, 400 MHz, ppm): 7.34-7.30 (m, 3H, ArH of Phe), 7.19-7.17 (m, 2H, ArH of Phe), 5.77 (b, 1H, NH Phe), 5.10 (b, 1H, NH Ethelynediamine), 4.77-4.11 (m, 1H, CH, Phe) 3.18-3.16 (m, 2H, CH.sub.2-Ethylenediamine), 3.02-2.93 (m, 2H, CH Phe) 1.41 (s, 9H, Boc). ESI-MS (m/z): [M+Na+2H]+=579.67 (calculated); 579.74 (observed); [M+K+H]+=595.67 (calculated); 596.10 (observed).

[0124] Next, 2 g (3.60 mmol) of compound 3 were dissolved in 25 mL of DCM in an ice bath. Then, 6 mL of TFA were added and stirred for 2h. The progress of the reaction was monitored by TLC. After the reaction was completed, all solvents were evaporated in a rotary evaporator. The product was then dissolved in water, neutralized with NaHCO.sub.3 solution, extracted with ethyl acetate, dried over anhydrous sodium sulphate and evaporated by rotary evaporator to obtain an oily product 4, which was immediately used for the next reaction. Yield: 1.194 g (3.36 mmol, 93.6%). ESI-MS (m/z): [M+Na+2H]+=379.44 (calculated); 379.93 (observed); [M+2Na]+=400.44 (calculated); 400.25 (observed).

##STR00003##

[0125] Synthesis of BOC-Phe-Gly-Phe-NHCH.sub.2CH.sub.2NH-Phe-Gly-Phe-Boc (5) and NH.sub.2-Phe-Gly-Phe-NHCH.sub.2CH.sub.2NH-Phe-Gly-Phe-NH2 (6): 2.554 g (7.90 mmol) of Boc-Phe-Gly-OH were dissolved in 30 ml dry DCM in an ice-water bath. Compound 4 (1.274 g 3.6 mmol) was then added to the reaction mixture, followed immediately by the addition of 1.96 g (9.48 mmol) dicyclohexylcarbodiimide (DCC) and 1.28 g (9.48 mmol) of HOBt. The reaction mixture was allowed to warm-up to RT and stirred for 48 h. DCM was evaporated and the residue was dissolved in ethyl acetate (50 mL) and the dicyclohexylurea (DCU) was filtered off. The organic layer was washed with 2M HCl (350 mL), brine (250 mL), 1 M sodium carbonate (350 mL), brine (250 mL), dried over anhydrous sodium sulfate and evaporated in a vacuum to yield BOC-Phe-Gly-Phe-NHCH.sub.2CH.sub.2NH-Phe-Gly-Phe-Boc (5) as a white solid. The product was purified by silica gel (100-200 mesh) using n-hexane-ethyl acetate (4:1) as eluent. Yield: 2.09 g (2.20 mmol, 62.21%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz, ppm): 8.11-8.05 (m, 2H, NH Phe and Gly), 7.28-7.23 (m, 8H, ArH Phe), 7.22-7.18 (m, 4H, ArH Phe), 6.97 (d, J=8.3 Hz, 1H, NH Phe), 4.46-4.42 (m, 1H, CH, Phe) 4.22-4.17 (m, 1H, CH Phe), 3.83-3.78 (dd, 1H, CH2 Gly), 3.69-3.63 (dd, 1H, CH2 Gly) 3.09-2.96 (m, 4H, CH Phe), 2.87-2.36 (m, 2H, CH.sub.2 EDA), 1.30 (s, 9H, Boc). ESI-MS (m/z): [M+2H]+=965.13 (calculated); 965.61 (observed); [M+H3O]+=982.13 (calculated); 982.60 (observed); [M+Na+H]+=987.14 (calculated); 987.44 (observed).

##STR00004##

[0126] Next, 1.5 g (1.58 mmol) of compound 5 was dissolved in 20 mL of DCM in an ice bath. Then, 4 mL of TFA were added and stirred for 2 h. The progress of the reaction was monitored by TLC. After the reaction was completed, all solvents were evaporated in a rotary evaporator. The product was then dissolved in water, neutralised with NaHCO3 solution, extracted with ethyl acetate, dried over anhydrous sodium sulphate, and evaporated by a rotary evaporator to obtain an oily product 6, which was immediately used for the next reaction. Yield: 1.103 g (1.47 mmol, 93.3%). ESI-MS (m/z): [M]=762.89 (calculated); 762.75 (observed); [M+Na]+=785.89 (calculated); 785.80 (observed); [M+K]+=801.89 (calculated); 801.62 (observed).

[0127] Synthesis of Phenyl-bis-pyridin-2ylmethyl-amine (1/): To a solution of 2-chloromethylprydine hydrochloride (2 g, 12 mmol) in H.sub.2O (0.5 ml), aniline (0.558 g, 6 mmol), 5 N NaOH (6 ml) and hexadecytrimethylammonium chloride (20 mg) were added under N.sub.2 protection. The mixture was stirred vigorously for 24 h at RT. It was then extracted with CH.sub.2Cl.sub.2, and the extract was washed with H.sub.2O and dried with MgSO.sub.4. After the solvent was evaporated, the desired product was obtained as a beige solid via column chromatography (silica, CH.sub.2Cl.sub.2/AcOEt, 4/1, v/v). Yield: 850.6 g (3.08 mmol, 51.2%). .sup.1H NMR (CDCl.sub.3, 400 MHz, ppm): 8.60 (d, J=6.8 Hz, 2H ArH), 7.63 (t, J=7.6 Hz, 2H ArH), 7.28 (d, J=8 Hz, 2H ArH), 7.19-7.15 (m, 4H ArH), 6.74-6.70 (m, 3H ArH), 4.84 (s, 4H CH2). ESI-MS (m/z): [M+H]+=276.14 (calculated); 276.17 (observed).

##STR00005##

[0128] Synthesis of 4-(Bis-Pyridin-2-ylmethyl-amino-benzaldehyde (2): POCl.sub.3 (1 ml, 17 mmol) was added to the solution of DMF (2 ml, 26 mmol) in 2 portions within 30 min, and cooled in an ice bath. Then, the solution was stirred for 30 min. Compound 1 (0.800 g, 2.89 mmol) in DMF (1.25 ml) was added in portions within 20 min. The mixture was heated for 3 h at 90 C., poured into H.sub.2O (5 ml), and then neutralized to pH 6-8 with K.sub.2CO.sub.3 along with stirring. The mixture was extracted with CH2Cl2, and dried with Na.sub.2SO.sub.4. Via column chromatography (silica, petroleum:acetone, 5:3, v/v), the desired product was obtained as a yellow sticky oil. Yield: 294.3 mg (0.97 mmol, 33.5%). .sup.1H NMR (CDCl.sub.3, 400 MHz, ppm): 9.75 (s, 1H, CHO), 8.60 (d, J=5.8 Hz, 2H ArH), 7.68-7.61 (m, 4H ArH), 7.20 (d, J=7.8 Hz, 4H ArH), 6.78 (d, J=8.4 Hz, 2H ArH), 4.89 (s, 4H CH2). ESI-MS (m/z): [M]=303.13 (calculated); 303.33 (observed).

[0129] Synthesis of L: 2 (200 mg, 0.65 mmol) was added to a solution of 6 (490.2 mg, 0.655 mmol) dissolved in 20 ml of methanol. The resulting mixture was stirred for 8 h. After the reaction was completed (confirmed by TLC), the reaction mixture was filtered off and a light brown oily residue was obtained upon removal of the solvent from the filtrate under vacuum. This oily residue upon treatment with n-hexane yielded a light brownish solid precipitate, which was collected by decantation as well as proper washing and drying to afford L as a pure product. Yield: 518.6 mg, 60.5%. 9.04 (d, J=6.0 Hz, 2H ArH of DPA based receptor), 8.59 (s, 1H NCH), 8.13-8.11 (m, 2H, NH Phe and Gly), 7.71-7.66 (m, 14H ArH), 7.22 (d, J=7.8 Hz, 4H ArH), 6.73 (d, J=8.2 Hz, 2H ArH), 5.32 (s, 4H, CH2 of DPA), 4.12-4.07 (m, 1H, CH Phe), 3.89-3.84 (m, 1H, CH Phe), 3.54-3.16 (m, 2H, CH.sub.2 Gly), 2.38-2.35 (m, 4H, CH Phe), 2.12-2.09 ((m, 2H, CH2 EDA), ESI-MS (m/z): [M/2]=666.32 (calculated); 666.37 (observed); [M+2H]+=1335.58 (calculated); 1335.64 (observed).

##STR00006##

[0130] Synthesis of LM: L (200 mg, 0.15 mmol) was dissolved in 20 mL of methanol and then a solution of Cu(ClO.sub.4).sub.2.6H.sub.2O (138 mg, 0.372 mmol) in 5 mL HPLC water was added in a dropwise manner into it. The resulting solution was stirred for 10 h at RT. The desired compound was then precipitated in a pure form by slow evaporation of the solvent at RT. The precipitate was filtered, washed with cold water and dried. Yield: 160 mg, 57.5%. ESI-MS (m/z): [M+2H]+=1854.86 (calculated); 1855.31 (observed).

##STR00007##