New fluorescent derivatives of oligopropylamines

New fluorescent-tagged amines have been obtained starting from 4-chloro-7-nitro-2,1,3-benzoxadiazole and oligopropylamines containing fragments of N -methylpropylamine close to those found in diatom frustules. Optical properties of new dyes and staining of silica structures in vivo and in vitro are described.


Introduction
Oligopropylamines (OPAs) are intensively studied nowadays for several reasons: (i) these compounds are structurally close to biogenic amines such as spermine, spermidine and polyamines from diatoms 1,2 (ii) OPAs are prospective building blocks for more complicated structures, i.e. dendrimers, graft and branched polymers; (iii) OPAs are effective catalysts or initiators of various processes including epoxy-resin synthesis and sol-gel reactions. Weakly basic fluorescent dyes are known as lysotrackers -compounds capable to penetrate into acidic cytoplasmic vesicles of eukaryotic cells. 3 Recently we reported 4 the synthesis of two representatives of fluorescence-tagged methylated OPAs by the reaction between 4-chloro-7nitro-2,1,3-benzoxadiazole (Cl-NBD) and the corresponding amines. These compounds were used for in vivo staining of diatom cells and we have found that NBD-N2 and NBD-N3 were not removed from stained diatom frustules by drastic treatment with H2SO4 and H2O2. Fluorescent silica can also be obtained by chemical precipitation from its precursors in the presence of NBD-N2 or NBD-N3. This work was aimed at the synthesis of other tagged OPAs including derivatives bearing two 7-nitro-2,1,3-benzoxadiazole (NBD) groups or having a free NH group. Fluorescence properties of new dyes and their activity during in vivo and in vitro staining of silica structures have been studied too.

Results and Discussion
We have synthesized four new NBD-tagged OPAs and also have studied UV-Vis spectral properties of them and two substances obtained earlier (Table 1). Synthetic procedures were similar to the previously published ones 4 and in the case of single-tagged derivatives of di-NH amines a 10-20 fold excess of the amine was used. Purification of the target compounds was done by re-precipitation and flash-chromatography. HPLC and HRMS tools were applied to confirm purity and structure of the tagged amines. NMR signals were assigned based on the data from Ref. [5][6][7] and from Ref. 8 for 13 C NMR of NBD ring. The NBD-tagged OPAs show basic properties and titration of NBD-N3 hydrochloride from acidic medium (an excess of HCl was added to adjust pH = 2) proceeds with three inflections which correspond to removal of the corresponding proton from the amine. A fourth inflection was expected at the beginning of the first proton removal but the low value of the corresponding pKBH+ (2.8) does not allow this inflection to be observed. The other protonation constants are 7.1 and 9.6. Titration of the NBD derivative of diethylamine gives pKBH+ = 2.6, so the value 2.8 obtained for NBD-N3 corresponds to the nitrogen attached to the heterocycle. These pK values allow estimation of protonation degrees at various pH, which are 100, 50, 35 and near 0% at pH 2.0, 5.5, 7.0 and 10.0 correspondingly. NBD-N3H shows similar basic properties with pKBH+ values of 9.5, 6.6 and 2.8.
Methylated OPA aggregates in water medium at neutral and alkali pH values 9 due to hydrophobic interactions. The introduction of NBD moieties must increase the aggregation and we have found 2NBD-N2 to give unstable emulsion at pH 7-10. 2NBD-N3 solutions are stable at high pH values but dynamic light scattering (DLS) data show a presence of 1300-1500 nm particles at pH 7 and 10. DLS of the solutions of singly substituted OPAs gives a relatively noisy autocorrelation function at pH 10 corresponding to a small amount of 400-600 nm particles.
Self-association of the dye molecules is often accompanied by a hypochromic effect in UV-Vis spectra. [10][11][12][13][14][15] Spectra of mono-NBD derivatives of OPAs are similar to the known NBDamines 5 and contain two main bands near 350 and 500 nm (Table 1, Figure 1) irrespective of pH values. A decrease of absorbance at 500 nm was observed for NBD-N2H at pH 10 only. 2NBD-N3 spectra at pH 2.0 and 5.5 ( Figure 2) are similar to mono-NBD amines but the 500 nm band is moved to 475 nm and its absorption coefficient is lower than  (Table 2). 2NBD-N2 and 2NBD-N3 show several times lower fluorescence activity compared with mono-substituted derivatives. The decrease of fluorescence is understandable at neutral and high pH values because aggregation often results in fluorescence quenching. 16 On the other hand, there is now aggregation at pH 2 where 2NBD-N2 and 2NBD-N3 have at least one or two protonated nitrogens correspondingly. As mentioned above, the absorption spectra of twice-substituted molecules have maximum near 475 nm and a shoulder near 500 nm. We supposed that this band is a superposition of two bands related to isolated NBD units (500 nm) and to NBD rings involved in some kind of intramolecular interaction. 2NBD-N3 spectrum decomposition into Lorentz curves allowed us to extract 500 nm band ( Figure 4). Recalculation of the quantum yield based on this band gives a value of 0.022 which is equal to mono-substituted OPAs. Thus, mono-NBD derivatives of OPAs show similar absorbance and fluorescence properties at pH 2-7 and the corresponding spectra are similar with spectra of diethylamino-NBD (data not shown). Di-NBD amines show anomalies which can be attributed to an intramolecular interaction between NBD rings. Recently 5 fluorescence quenching has been found for a compound containing NBD and naphthyl ring capable of donor-acceptor interactions. In the case of two NBD groups the interaction is possible due to protonation of nitrogen attached to one ring. This protonation must increase acceptor properties of the corresponding ring thus providing non-equivalence of the NBD moieties. Quantum-chemical calculations ( Figure 5) confirm this hypothesis: the most stable conformation of protonated NBD-N2 contains closely located rings. The other kind of interaction is possible for singly protonated 2NBD-N3 (at pH 5.5-7.0): hydrogen bond between NH + and nitrogen in one of the heterocycles gives rise to a stable conformation with coplanar rings. It is known 17 that absorbance near 500 nm and the corresponding fluorescence are connected with a charge transfer, the amino group acting as the electron  donor and the nitro group as the acceptor. So, any donor-acceptor interaction involving NBD ring can obstruct the charge transfer responsible for the fluorescence. We recognize that these speculations are preliminary and thorough quantum-chemical and spectroscopic investigations are necessary to understand the behavior of the OPAs bearing two NBD rings.
The new NBD-tagged OPAs were used in synthesis of fluorescent-tagged silica ( Figure 6) and for in vivo staining of diatom cells (Figure 7). The OPAs inclusion in silica obtained by in vitro precipitation proceeds through hydrogen and/or ionic bonds between amine nitrogens and silanol groups. This explains the observed more intensive fluorescence of silica samples obtained using NBD derivatives of OPAs with three nitrogen atoms. But it does not explain the higher activity of NBD-N3 comparing with NBD-N3H. Staining of diatom frustules with fluorescence-tagged OPAs is realized 4 by non-specific penetration of amines into the cell followed by its concentration in acidic vesicles, including SDV (Silica Deposition Vesicle)a special cell organelle in which parts of new silica frustules are synthesized. 19,20 The data obtained with new NBD-tagged OPAs (Figure 7) confirm our previous observation about the ability of N3 fluorescent dyes to stain not only biogenic silica but also other intracellular vesicles in contrast to N2 derivatives which stain silica frustules only. This fact had been explained 4 by the low affinity of NBD-N2 to acidic functions in comparison with NBD-N3 because NBD-N2 has only one nitrogen atom capable of durable interaction with weak acids. The low activity of 2NBD-N2 and 2NBD-N3 in diatom staining is not unexpected taking into account their reduced fluorescent activity, one or even absence of free amine nitrogens and the inclination to aggregation giving large particles which have no any possibilities to penetrate into the cell. NBD-N3 is unexpectedly more active as compared with NBD-N3H in in vivo staining similarly to the in vitro experiments. We have done also a chromatography experiment (Figure 8) which confirms the higher affinity of NBD-N3 to silica comparing with NBD-N3H. A possible reason for this difference is the lower value of the second protonation constant of NBD-N3H (6.6) comparing with NBD-N3 (7.1) which must decrease the ability of NBD-N3H to interact with low-acidic silica structures. NBD-tagged OPAs are non-toxic for the diatom cells in long-term cultivation experiments (up to one month) in 0.5-4 µM concentraition.

Conclusions
Thus, we have elaborated synthetic procedures for several new NBD-tagged oligopropylamines. Two of these substances bear terminal NH groups which allow their use in the design of more complicated fluorescent-tagged structures including polymers and dendrimers. Absorption and fluorescence properties of twice-substituted amines depend on the pH of the water solution. Aggregation at high pH values results in hypochromic effect. Protonation in the acidic area facilitates donor-acceptor interactions between rings which give a new band in the visible spectra but this transition is not active in fluorescence. Fluorescent silica materials can be obtained by in vitro precipitation and also in vivo by cultivation of diatoms in the presence of NBD-tagged oligopropylamines.

Experimental Section
General. 1 H and 13 C NMR spectra were recorded using DPX 400 Bruker spectrometer in DMSO-d6. Absorption, excitation and emission spectra were obtained with a Cintra 20 UV/VIS spectrophotometer (Sangji, Korea) and Shimadzu RF-5301 PC spectrofluorimeter with a 10 mm quartz cell. The following 10 mM buffer solutions were used: HCl, acetic acid/NaOH, 1methylimidazole/HCl and ammonia/HCl at pH values 2.0, 5.5, 7.0 and 10.0 correspondingly. Semiempirical quantum-chemical calculations (PM3 method) were performed with the HyperChem program. 22 Epifluorescence microscopy was performed using an inverted microscope Axiovert 200 with a mercury lamp HBO 50W/AC ASRAM. To obtain fluorescent images, a ×100 immersion objective was used. Excitation was performed at 450-490 nm (Ex), emission was observed at 515 nm (Em) and beam splitting at 510 nm. Zeiss LSM710 microscope was used to obtain confocal images with the following parameters: excitation 488 nm, detector slit -501-572 nm. Scanning electron microscopy (SEM) was performed using an FEI Quanta 200 instrument. An acetone suspension of silica frustules was placed on aluminium sample holders and then sputter coated with gold using an SDC 004 (BALZERS) device. DLS experiments were performed using a LAD-079 instrument built in the Institute of Thermophysics (Novosibirsk, Russia). All solutions were purified from dust using filter units with 0.45 m pore size (Sartorius 16555-Q Minisart syringe filters). The experiments were performed at 20 C ± 0.02 C. Measurements were done with 650 nm solid-state laser at 90º scattering angle. Autocorrelation functions were analyzed with a polymodal model using random-centroid optimization method. 23 Cl-NBD was purchased from Alfa Aesar (99% purity). N 1 ,N 3 -dimethylpropane-1,3-diamine (N2) and N 1 ,N 3 -dimethyl-N 1 -[3-(methylamino)propyl]-propane-1,3-diamine (N3) were synthesized according to Ref. 6 ; N,N-diethyl-7-nitro-2,1,3-benzoxadiazol-4-amine (diethylamino-NBD) was obtained according to Ref. 17 , NBD-N3 and NBD-N2 were synthesized according to Ref. 4 Other inorganic salts and chemicals were purchased from Sigma Aldrich, Fisher or Acros Chemicals and used without further treatment. Purity estimation. Mass spectrometric analysis. The apparatus used was an Agilent 6210 TOF LC/MS (time-offlight liquid chromatography/mass spectrometry) System. The samples were dissolved in deionized water at concentrations of 10 mg/L. Water and acetonitrile with 0.1% (v/v) formic acid were used as solvents A and B, respectively. A solvent gradient of 50% B to 100% B in 5 min was applied. The flow rate of the mobile phase was set at 0.2 ml/min, whereas the injection volume of sample solution was 20 µL. The conditions for TOF MS were as follows. The mass range was m/z 100 to 1000, and the scan time was 1 s with an interscan delay of 0.1 s.

Cultivation of the diatom cells in the presence of NBD-tagged OPAs and synthesis of fluorescent stained silica in vitro
These experiments were done according to Ref. 4,24 Briefly, a clonal culture of the diatom Synedra acus subsp. radians (Kützing) Skabichevsky was cultivated in DM medium. 25 After three day starvation in silicon-free medium, NBD-tagged OPAs were added as 1 mM stock solutions to give 0.5 µM final concentration and sodium silicate was added at a concentration of 100 µM. The cells were observed with epifluorescent microscope after 24 h. Silica valves were cleaned from organic components by the successive treatment with CH2Cl2 -2-propanol mixture (1:1 v/v) and H2O2 (30%) -concentrated sulfuric acid mixture (1:1 v/v). To obtain silica in vitro, we prepared solution of 10 mM sodium silicate and N1,N3-dimethyl-N1-(3-(methylamino)propyl)propane-1,3-diamine as a catalyst of silicic acid condensation. 6 The concentration of the NBD-tagged OPAs in these solutions was 1 mM, pH was adjusted to 7.0 using 1 M HCl and precipitate was collected after 24 h. N 1 ,N 3 -Dimethyl-N 1 ,N 3 -bis(7-nitro-2,1,3-benzoxadiazol-4-yl)propane-1,3-diamine (2NBD-N2)

Synthetic procedures
A solution of 157 mg (0.8 mmol) of NBD-Cl in 8 ml of ethanol cooled to 4 C was mixed with a stirred ice-cooled suspension of 67.04 mg (0.8 mmol) of sodium hydrocarbonate NaHCO3 and 32.38 mg (0.32 mmol) of N2 in 4 ml of ethanol in an argon atmosphere. The resulting dark brown mixture was stirred in an ice bath for 1 h, then 2 h at room temperature, and finally 7.5 h at 40-45 C. After that the volatiles were removed under aspirator vacuum while keeping the bath temperature at ~ 25 C. The residue was thoroughly mixed with 1 ml of an aqueous solution of K2CO3 (50%, w/w) and extracted with dichloromethane (6×5 ml). The combined extracts were dried with anhydrous potassium carbonate and filtered. The filtrate was evaporated to dryness under aspirator vacuum followed by keeping under oil pump vacuum for 2 h at room temperature. The product was a carmine red powder whose yield was 45.9 mg (34%), mp 210-215 o C (with destruction). 1   The residue was thoroughly mixed with 1 ml of an aqueous solution of K2CO3 (50%, w/w) and extracted with dichloromethane (5×2 ml). The combined extracts were dried with anhydrous potassium carbonate and filtered. The filtrate was concentrated to a volume of 3 ml under reduced pressure and saturated with gaseous hydrogen chloride. Then 3 ml of ether were added to the solution to obtain an orange precipitate which was filtered off, washed with ether (3×2 ml), and kept in oil pump vacuum at room temperature for 2 h. The product was a carmine red powder whose yield was 48.42 mg (34%), mp 145-150 o C (with destruction). Hydrogen chloride content was determinated by potentiometry titration. 1   A solution of 301.6 mg (1.51 mmol) of NBD-Cl in 50 ml of ether cooled to 4 o C was added dropwise to an ice-cooled solution of 3.08 g (30.14 mmol) of N2 in 50 ml of ether in an argon atmosphere during 2 h. The resulting mixture was stirred in an ice bath for 1 h and then for 2 h at room temperature. Then the ether was evaporated to dryness under reduced pressure. The residue was thoroughly mixed with 1 ml of an aqueous solution of K2CO3 (50%, w/w) and extracted with dichloromethane (4×2 ml). The combined extracts were dried with anhydrous potassium carbonate and filtered. The filtrate was evaporated to dryness under reduced pressure to give sticky mass which was triturated with n-hexane (3×6 ml). After every trituration the solution over the mass was discarded. The residue was dried under reduced pressure and dissolved in 2.5 ml CH2Cl2. Then this solution was poured to 15 ml of n-hexane. The precipitated product was filtered off and kept in oil pump vacuum at room temperature for 2 h. The product yield was 0.2154g (53.7 %). 1