问题：发一个国外博士论文（我是偷别人的）
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提问：robert2002
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版块：高分子科学(kevlar,wmx,lby010,)
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时间：2005-12-07 11:41:05 编辑加入/取消收藏订制/取消短消息举报该贴

讨论讨论这种水平的文章与国内的博士有多大的差距？如何上传？

回复人：wza0606,▲ (热爱化学) 时间：2006-01-04 20:58:10 编辑	1楼
楼主可以发到我的邮箱吗? wza0606@sina.com,谢谢!

回复人：muyi,▲ (tianjin university,applied chemistry) 时间：2006-01-06 15:00:48 编辑	2楼
能不能发到我的邮箱理，chengfa@tju.edu.cn 谢谢

回复人：starforce,▲ (热爱化学) 时间：2006-01-07 17:09:42 编辑	3楼
我想看看啊

回复人：tobby,▲▲▲ (一个热爱化学工作的女生！) 时间：2005-12-07 15:00:18 编辑	4楼
你的论文在哪里呀，发过来看看呀

回复人：tt777_1028,▲▲▲ (专业二氧化硅生产销售) 时间：2005-12-07 21:40:10 编辑	5楼
>????

回复人：蓝宝石,▲ (滴水之恩，涌泉相报) 时间：2005-12-07 23:53:53 编辑	6楼
怎么找不到呢

回复人：robert2002,▲ () 时间：2005-12-08 08:28:04 编辑	7楼
不知如何上传呀？

回复人：zhp810313,▲ () 时间：2005-12-10 21:35:20 编辑	8楼
你不发文章，怎么讨论呢发到ftp里，或者就是放到56的邮箱共享资源里

回复人：流氓太子,▲ (功能高分子材料以及乳液聚合方面的研究) 时间：2005-12-26 13:00:17 编辑	9楼
什么方向的？在哪里啊？

回复人：liutg137,▲ (有志者,事竞成!) 时间：2005-12-26 21:22:45 编辑	10楼
看不到文章在那里!

回复人：robert2002,▲ () 时间：2005-12-27 11:18:18 编辑	11楼
刚才想发到文档中心，文件不能接受，在这里不知是否可行，看运气了。

回复人：robert2002,▲ () 时间：2005-12-27 11:20:13 编辑	12楼
请大虾告诉我FTP的相关连接。谢谢。我再传上去。

回复人：Robert2002,▲ () 时间：2008-01-22 17:34:10 编辑

13楼

Duplex Molecular Strands Based on the 3,6-Diaminopyridazine Hydrogen Bonding Motif: Amplifying Small Molecule Self-Assembly Preferences through Preorganization and Iterative Arrangement of Binding Residues

Hegui Gong and Michael J. Krische*

University of Texas at Austin
Department of Chemistry and Biochemistry
Austin, TX 78712, USA

Abstract: Structural parameters obtained through single crystal x-ray diffraction analysis of the 1-dimensional H-bonding motif expressed by 3,6-diaminopyridazine are applied to the design of related monomeric, dimeric and trimeric duplex molecular strands. The mode of assembly and interstrand affinity of the oligomers are established in solution by 1H NMR dilution experiments, isothermal titration calorimetry (ITC), and vapor pressure osmometry (VPO). Single crystal x-ray crystallographic analysis of the dimeric diaminopyridazine 3a corroborates the intended duplex mode of assembly. Binding free energy per unimer (-G/n) increases upon extension from monomer to dimer to trimer, signifying a positive cooperative effect. Micromolar binding affinity (Kd = 1.25 ± 0.1 M) was determined for the duplex trimer by ITC in 1,2-dichloroethane at 20º C. These data provide further insight into the structural and interactional features of synthetic duplex oligomers required for high-affinity, high-specificity binding and define new recognition elements for use in nanoscale assembly.

Introduction

The design of functional synthetic oligomers and polymers requires reliable methods for the control of secondary and tertiary structure, as well as a means of directing intermolecular aggregation. As part of a program in hydrogen bond mediated self-assembly, we recently introduced a strategy for the preparation of duplex molecular strands. , Specifically, noncovalent connectivities of monomers composing one dimensional hydrogen bonding motifs are used to define structural parameters for the design of related oligomers. If a commensurate relationship between the covalent connectivities of the oligomers and the noncovalent connectivities of the parent hydrogen bonding motif is established, self-association of the oligomer should occur through the formation of interstrand hydrogen bonds. In this way, the iterative presentation of binding residues along an oligomeric scaffold should amplify the inherent self-assembly preference of the monomeric subunits through preorganization and cooperative binding.
This strategy for oligomer assembly was successfully applied to the design of high-affinity duplex molecular strands based on the 2-amino-4,6-dichlorotriazine hydrogen bonding motif. As anticipated, interstrand affinity was found to be critically dependant on the structural features of the intertriazine linkage. For example, neopentyl amino alcohol linked dimers exhibit association constants three orders of magnitude greater than the corresponding neopentyl glycol linked systems, due to preorganization of the oligomer backbone induced through formation of an intramolecular hydrogen bond. Trimeric strands possessing neopentyl amino alcohol linkages exhibit nanomolar binding affinities in dichloroethane, as determined by isothermal titration calorimetry (ITC).
Ultimately, through the “covalent casting” of alternative one dimensional hydrogen bonding motifs, a family of synthetic duplex molecular strands that embody orthogonal recognition motifs may be developed. With this broad goal in mind, covalent casting of the previously undescribed 3,6-diaminopyridazine hydrogen bonding motif was explored. Here we disclose the design, synthesis and characterization of homologous duplex oligomers based on 3,6-diaminopyridazine. These data provide further insight into the structural and interactional features of synthetic duplex oligomers required for high-affinity, high-specificity binding in solution and define new recognition elements for use in nanoscale assembly (Figure 1).

Figure 1. Left: One dimensional hydrogen bonding motifs based on 2,4-dichloro-6-aminotriazine and 3,6-diaminopyridiazine. Right: Corresponding duplex molecular strands.

Design of 3,6-Diaminopyridazine Based Oligomers

The free energy of formation of a single hydrogen bond is generally smaller than the energies required to partition distinct aggregation states under equilibrium conditions. Hence, one strategy for directing formation of noncovalent constructs via hydrogen bond mediated self-assembly involves amplifying the self-assembly preferences of small molecules through preorganization and iterative presentation of binding residues, such that positive cooperative effects come into play. As described above, this general approach was successfully applied to the construction of high-affinity duplex oligomers based on the aminotriazine hydrogen bonding motif. For the aminotriazine subunit, the hydrogen bond donor (D)-acceptor (A) sites are arranged in an “ADDA” array. The juxtaposition of hydrogen bond donor-acceptor sites in 3,6-diaminopyridazine, “DAAD,” is inverted with respect to that of aminotriazine. Hence, duplex oligomers based on 3,6-diaminopyridazine were sought, as such strands should possess orthogonal recognition characteristics with respect to the related aminotriazine based systems.

3,6-Diaminopyridazine was prepared and crystallized from ethanol. The anticipated one dimensional hydrogen bonding motif was corroborated by single crystal x-ray diffraction analysis (Figure 2, Top). Based on this crystallographic data, the distance between amine nitrogens DN,N for alternate adjacent pyridazines of the hydrogen bonded tape was determined to be 5.05 Å (Figure 3). This distance is roughly commensurate with the inter-nitrogen distance D’N,N of 4.88 Å observed for the bis(Cbz-carbamate) 8 derived from cis-1,3-diaminocyclopentane as determined by single crystal x-ray diffraction analysis (Figure 3). These distances are taken from molecules in the solid state and do not take into account distortions due to crystal packing forces. Nevertheless, these data suggest the cis-1,3-cycopentanyl moiety should serve as a suitable interpyridazine linkage. Additionally, the rigidity of the cis-1,3-cycopentanyl linkage should discourage formation of intramolecular hydrogen bonds, thus promoting the desired duplex mode of assembly.

Figure 3. Defining a suitable interpyridazine linkage using interatomic distances (Å) taken from single crystal x-ray diffraction data.

The veracity of this analysis is borne out by single crystal x-ray diffraction analysis of the cis-1,3-diaminocyclopentane linked bis(diaminopyridazine) 3a, which reveals the intended duplex mode of assembly (Figure 2, Bottom). Notably, the supramolecular connectivities evident in parent 3,6-diaminopyridazine hydrogen bonding motif persist upon introduction of cis-1,3-cycopentanyl linkage. The “goodness-of-fit” of the covalent linkage may be approximated by comparing the geometries and interatomic distances found in the crystal structure of bis(diaminopyridazine) 3a with those of the parent hydrogen-

Figure 2. Top: Anticipated one dimensional hydrogen bonding motif based on 3,6-diaminopyridazine as corroborated by single crystal x-ray diffraction analysis. Bottom: Anticipated duplex assembly of bis(3,6-diaminopyridazine) 3a as corroborated by single crystal x-ray diffraction analysis. The noncovalent connectivities persist upon introduction of the cis-1,3-cycopentanyl linkage.

bonded tape. For 3a, the distance D’’N,N between amine nitrogens of adjacent pyridazines is 4.26 Å. This distance is slightly shorter than that found in the parent hydrogen bonding motif (DN,N = 5.05 Å). The non-commensurate relationship between the covalent and noncovalent frameworks is expected to cause strain to accumulate in higher oligomers, compromising positive cooperative effects. Again, this analysis is founded upon solid state data and does not account for the contribution of crystal packing forces (Figure 3).

Synthesis of 3,6-Diaminopyridazine Based Oligomers

For the preparation of homologous oligo(diaminopyridazines), divergent synthetic approaches were envisioned. Concise access to bis(diaminopyridazines), 3a and 3b is achieved through the condensation of 6-chlorotetrazolo[1,5-b]pyridazine 1 with cis-1,3-diaminocyclopentane to provide bis(6-chlorotetrazolo[1,5-b]pyridazine) 2. Treatment of 2 with tributylphosphine, followed by exposure to the isolable bis(iminophosphorane) to p-tert-butylbenzoic acid or 3,4,5-tributoxybenzoic acid, provides the bis(3,6-diaminopyridiazines 3a and 3b, respectively (Scheme 1).

Scheme 1. Synthesis of bis(3,6-diaminopyridazines) 3a and 3b.

Reagents: (a) cis-1,3-diaminocyclopentane hydrochloride, K2CO3, CH3CN, 180 oC, sealed tube, 85%. (b) n-Bu3P, CH3CN, 180 oC, sealed tube, 80%. (c) RCO2H, PhCH3, 150 oC, sealed tube, R = p-tert-butylphenyl, 30%, R = 3,4,5-tri-n-butoxyphenyl, 27%.

A key step in the preparation of the homologous tris(3,6-diaminopyridazine) 6 involves double substitution of a 3,6-dihalopyridazine using a mono-protected cis-1,3-diaminocyclopentane derivative. Under classical SNAr conditions, the reaction of 3,6-dichloropyridazine with an excess of the mono-tert-butoxy-carbamate (Boc) of cis-1,3-diaminocyclopentane provides the product of mono-substitution. Presumably, the initially introduced alkylamino residue deactivates of the pyridazine toward further substitution, and even under forcing conditions (heating at > 200 oC) only trace quantities of the desired disubstituted product are observed. Recently reported conditions for the palladium-catalyzed amination of resin-bound 3-aminoalkyl-6-chloropyridazines with aniline were examined next, but proved to be ineffective when applied to the double substitution of 3,6-dichloropyridazine using the mono-Boc-carbamate of cis-1,3-diaminocyclopentane. A promising result was obtained using Buchwald’s method for copper-catalyzed N-arylation of primary amines with aryl halides, which employs ethylene glycol as ligand. Here, using 3,6-diiodopyridazine, a 9% yield of the desired bis(adduct) 4 is obtained, along with substantial quantities of O-arylation products derived from ethylene glycol addition. However, direct coupling of unprotected cis-1,3-diaminocyclopentane followed by treatment of the crude reaction product with Boc-anhydride affords a 74% isolated yield of the bis(adduct) 4 over the two step sequence. Elaboration of 4 to the trimer 6 is achieved by acid promoted cleavage of the Boc-protecting groups followed by condensation of the free diamine with 6-chlorotetrazolo[1,5-b]pyridazine 1 to provide the bis(tetrazolopyridazine) 5. Exposure of 5 to tributylphosphine, followed by exposure to the isolable bis(iminophosphorane) to 3,4,5-tributoxybenzoic acid, provides the tris(3,6-diaminopyridiazine 6. Although compounds 4-6 undoubtedly exist as equimolar mixtures of stereoisomers, that is a chiral racemic isomer of

Table 1. Thermodynamic parameters of duplex binding as determined by ITC in anhydrous 1,2-dichloroethane at 20 oCa
Compound n NHB Kd Ka (M-1) Go Ho TSo Go/NHB
7 1 2 0.19  0.01 M 5 -0.95 -7.5  0.3 -6.5 -0.48
3b 2 6 1.14  0.1 mM 870 -3.94 -8.8  0.1 -4.9 -0.66
6b 3 10 1.25  0.12 M 8.0  105 -7.92 -20.2  0.7 -12.3 -0.79
an = oligomer length; NHB = number of H-bonds per duplex; Kd = dissociation constant; Ka = 1 / Kd ; G / n = binding free energy per aminotriazine; G / HB = binding free energy per H-bond. Units of G°, H°, and TS° are kcal mol-1. Ethanol-free 1,2-dichloroethane was used.

Table 2. Vapor pressure osmometry (VPO): results based on calibration with sucrose octaacetate (SO).a
Compound Conc. Range
(mM) MW
(Duplex) R2 Mobs Mobs/MW
3b 6.59-65.9 1854.36 0.928 1821.6 0.98
6b 3.3-9.2 2206.8 0.996 2405.6 1.09
aVPO analysis was conducted at 40C in anhydrous, ethanol-free 1,2-dichloroethane.

Scheme 2. Synthesis of tris(3,6-diaminopyridazines) 6a and 6b.

Reagents: (a) CuI (5 mol%), cis-1,3-diaminocyclopentane (600 mol%), HOCH2CH2OH (200 mol%), 1,3-K3PO4 (410 mol%), DMF (0.3 M), 95 oC. (b) Boc2O (1200 mol%), 25 oC, 74% over two steps. (c) trifluoroacetic acid, 0 oC (d) chlorotetrazole 1 (210 mol%), K2CO3 (980 mol%), 95 oC, 80% over two steps. (e) PBu3, CH3CN, 180 oC, 60%. (f) tri-n-butoxybenzoic acid (460 mol%), PhCH3, 150 oC, sealed tube, 26%.

C2-symmetry and a corresponding meso-isomer, these materials appear to have identical 1H and 13C spectra in all cases (Scheme 2).
Finally, the monomeric compounds 3,6-diaminopyridazine and 7 were prepared for the purpose of studying their modes of aggregation in the solid state and in solution, respectively. Although 3,6-diaminopyridazine is a known compound, a more convenient preparation involves the condensation of 6-chlorotetrazolo[1,5-b]pyridazine 1 with ammonia followed by Staudinger reduction.5 Monomer 7 was prepared in an analogous fashion (Scheme 3).

Scheme 3. Synthesis of 3,6-diaminopyridazine and monomer 7.

Reagents: (a) NH4OH, 95 oC, 69%. (b) PBu3, 180 oC, then HCl (aq), 100 oC, 40%. (c) HN[CH2CH(CH3)2], 130 oC, 90%. (b) PBu3, CH3CN, 180 oC, then HCl (aq), 100 oC, 80%.

Self Assembly in Solution

Isothermal Titration Calorimetry (ITC): The association of monomeric diaminopyridazine 7, bis(diaminopyridazine) 3b, and tris(diaminopyridazine) 6 were investigated by isothermal titration calorimetry (ITC). ITC experiments were performed in 1,2-dichloroethane rather than chloroform to circumvent error incurred by evaporation. The dipole moment of CDCl3 (m = 1.1 D) is lower than that of 1,2-DCE (m = 1.8 D). Consequently, hydrogen bond interactions are somewhat stronger in CHCl3 than 1,2-dichloroethane. Concentrated analyte solutions were injected into a reservoir initially containing neat 1,2-dichloroethane and enthalpy changes were monitored. A nonlinear least-squares minimization protocol was used to fit the experimental curve to a two-fold self-association model. Experiments were repeated a minimum of three times to ensure reproducibility. The average values obtained for Kd and H° were used to calculate binding free energy (G°) and enthalpy (TS°).
The ITC analysis of monomeric diaminopyridazine 7 reveals an association constant of 5 M-1. Upon strand extension from monomer 7 to dimer 3b, an association constant of 870 M-1 is observed, representing a significant increase in binding affinity. Finally, for the homologous trimer 6, ITC analysis indicates a binding constant of 8.0  105, representing an increase in binding affinity of over three orders of magnitude. As anticipated for hydrogen bond mediated aggregation in organic media, duplex binding is enthalpically driven. The binding free energy per unimer (-G/n) increases substantially upon extension from monomer to dimer to trimer, indicating a strong positive cooperative effect.

1H NMR Dilution Studies: To corroborate the association constants obtained by ITC analysis, the monomeric diaminopyridazine 7, bis(diaminopyridazine) 3b, and tris(diaminopyridazine) 6 were subjected to 1H NMR dilution analysis in anhydrous ethanol-free CDCl3 using a 300 MHz NMR spectrometer. Association constants were calculated by fitting the observed data to a two-fold self-association model using an iterative minimization protocol.
1H NMR dilution analysis of monomeric diaminopyridazine 7 reveals an association constant of 6.0 M-1 (Ka = 6.0 M-1, log K = 0.78  0.3), which is in good agreement with that determined by ITC. An association constant of 3.4  103 M-1 is calculated for the corresponding dimeric diaminopyridazine 3b (Ka = 3.4  103 M-1. log K = 3.5  0.8). This value is somewhat higher than that determined by ITC, which is likely due to the aforementioned considerations of solvent polarity (CDCl3, m = 1.1 D; 1,2-DCE, m = 1.8 D). Finally, tris(3,6-diaminopyridazine 6 was subjected to 1H NMR dilution analysis. For 6, no changes in chemical shift were observed upon dilution, suggesting persistence of the duplex at the limits of 1H NMR detection.

Vapor pressure osmometry (VPO): To corroborate the intended mode of assembly, the binding stoichiometry of bis(diaminopyridazine) 3b, and tris(diaminopyridazine) 6 were investigated by vapor pressure osmometry (VPO) in anhydrous, ethanol-free 1,2-dichloroethane. Sucrose octaacetate (SO; MW = 678.6 g/mol) was used as the calibration standard. The ratio of observed molar mass (Mobs) to molecular weight (MW) was calculated. For both bis(diaminopyridazine) 3b and tris(diaminopyridazine) 6, Mobs/MW values consistent with the intended duplex mode of aggregation were observed. That is, for both 3b and 6 Mobs/MW equaled approximately two, suggesting formation of a 1:1 complex in solution. The correlation coefficient R2 (from the linear regression used to analyze VPO data) is high for both 3b and 6, suggesting that the aggregation state does not vary over the concentration range studied.

Discussion and Summary

The previously unknown 1-dimensional hydrogen bonding motif based on 3,6-diaminopyridazine has been utilized as a template for the construction of related dimeric and trimeric duplex molecular strands 3a,b and 4a,b, respectively. In solution, ITC, 1H NMR, and VPO data strongly support the intended duplex mode of assembly for monomer 7, dimer 3b and trimer 4b. Corroborative evidence for the duplex mode of assembly stems from single crystal x-ray crystallographic analysis of the dimeric diaminopyridazine 3a, which clearly reveals the intended duplex mode of assembly. Higher oligomers were not investigated due to solubility issues.
It should noted that isomeric “head-to-head” and “head-to-tail” binding modes are possible for bis(diaminopyridazine) 3b and the meso-form of tris(diaminopyridazine) 6. Additionally, the chiral racemic form of trimer 6 may form diastereomeric duplex aggregates. The observance of a single apparent association constant for 3b and 6 by ITC, and in the former case by 1H NMR as well, suggest these isomeric binding modes are roughly equienergetic.
As anticipated for hydrogen bond formation in organic media, duplex binding is enthalpically driven. The binding free energy per unimer (-G/n) increases significantly upon extension from monomer to dimer to trimer, indicating a strong positive cooperative effect. Thus, the cis-1,3-cycopentanyl linking group of the oligomer backbone preorganizes 3,6-diaminopyridazine residues, amplifying the inherent self-assembly preference of the monomers. While the cis-1,3-cycopentanyl linkage is sufficiently rigid to mitigate or preclude intramolecularly folded states, it is of sufficient flexibility that compensation for any non-commensurate relationships between the covalent and noncovalent connectivities is possible.
In conclusion, dimeric and trimeric duplex molecular strands based on the hitherto unknown 3,6-diaminopyridazine hydrogen bonding motif have been developed and their duplex mode of assembly has been corroborated in solution and in the solid state. Future work will focus on the “covalent casting” of alternative one dimensional hydrogen bonding motifs, as well as studies that establish the orthogonality of the diaminopyridazine molecular strands in relation to the previously developed aminotriazine based strands.

Experimental Section

General. Reagents were reagent grade quality and used as received from Aldrich unless otherwise indicated. All reactions were carried out under an atmosphere of argon unless otherwise indicated. Anhydrous tetrahydrofuran (THF) and diethyl ether were distilled from sodium/ benzophenone ketyl prior to use. Dry toluene was distilled over CaH before use. Acetonitrile was used as HPLC grade from Fischer Scientific Company. Anhydrous DMF was used from passage through two columns of activated molecular sieves, containing less than 50 ppm H2O by Karl Fischer coulomeric moisture analysis. Copper(I) iodide was purchased from Strem Chemical. Anhydrous potassium phosphate was purchased from Fluka. Tri-n-butylphosphine (97%) was purchased from Aldrich. All other solvents were technical unless noted. Column chromatography was performed using Merck silica gel 60 as the solid support. All NMR spectra were recorded on Varian 300 MHz, 400 MHz or Bruker ACF-250 at STP. Deuterium solvents were used as purchased from Cambridge Isotope Laboratories, Inc., and used as received. IR spectra were recorded on a Nicolet Avatar 380 FT-IR spectrometer. Low-resolution chemical ionization (CI) mass spectra were performed on Finnigan MAT TSQ-70 instrument; High-resolution mass spectra (HRMS) were conducted with a VG Analytical ZAB2-E instrument, and ESI mass spectra were performed on Finnigan LCQ instrument. Melting points were obtained on a Thomas-Hoover UniMelt apparatus and are uncorrected.

Vapor Pressure Osmometry (VPO). VPO analyses were performed on a Jupiter 833 Vapor Pressure Osmometry thermostated at 40 oC. Sucrose octaacetate used for VPO calibration was purchased from Jupiter Instrument Co., Inc. 1,2-Dichloroethane was freshly distilled from P2O5 prior to use. VPO data were analyzed by linear regression using EXCEL software. Reported results represent single experimental runs.

Isothermal Titration Calorimetry (ITC). Analyses were performed on a MicroCal VP-ITC instrument thermostated to 20º C. Reported results represent the average of three or more runs. 1,2-Dichloroethane was prepared by distillation from P2O5, storage over molecular sieves, and filtration through a 0.45 M nylon membrane. The solvent was degassed by sonication in vacuo prior to each run. Following is a summary of the mathematical model used for data analysis.

fmonomer + fdimer = 1
fdimer = 1- Kd[(1 + 8A0/Kd)½ – 1] / 4A0 (1)

fdimer = mole fraction in associated state
Kd = dissociation constant (mol / L)
A0 = analyte concentration

A0(i - 1) = A0(i) – (syr  Vinj) / (Vinj + Vo) (2)
qobs(i) = ½H[n(i)fdimer(i)-n(i-1)fdimer(i-1)-n(inj)fdimer(inj)] (3)

H = molar enthalpy of binding
qobs(i) = integrated heat observed at injection i
n(i) = moles of analyte after injection i
n(i-1) = moles of analyte prior to injection i
n(inj) = moles of analyte in Vinj
syr = concentration of analyte in syringe
Vinj = injection volume
Vo = sample cell volume

Substitution of (1) and (2) into (3) yields an expression from which Kd and Ho were computed from raw ITC data by nonlinear least squares curve fitting in the Origin software package.

NMR Dilution Experiments. Dilution studies were carried out by incrementally diluting concentrated analyte samples while obtaining 300 MHz 1H NMR spectra. For each experiment, if significant concentration-dependent shifting of spectral signals was observed, the data was analyzed in terms of a two-fold self association model to obtain a value for log(K). For 7, data from the amine hydrogens were processed. For 3b, the internal pyridazine NH signals were processed.

Diisobutyl-tetrazolo[1,5-b]pyridazin-6-yl-amine. To a round bottomed flask (25 mL) charged with 6-chlorotetrazolo[1,5-b]pyridazine 1 (0.2 g, 1.3 mmol) was added diisobutylamine (1mL). The reaction mixture was stirred for 3.5 hrs at 130 oC. After the mixture was cooled to 25 oC, water (20 mL) was added. The aqueous phase was extracted with EtOAc (350 mL). The combined organic phases were (MgSO4), filtered and evaporated directly onto silica. The crude material was subjected to flash chromatography (SiO2: 20% EtOAc in hexanes) to give the title compound as a white solid (0.29g, 1.2 mmol, 90%). 1H NMR (300 MHz, CDCl3)  7.93 (d, J = 9.9 Hz, 1H), 7.23 (d, J = 9.9 Hz, 1H), 3.26 (d, J = 7.5 Hz, 4H), 1.90-2.00 (m, 2H), 0.75 (d, J = 6.6 Hz, 12H). 13C NMR (75 MHz, CDCl3):  155.5, 139.6, 123.3, 117.5, 58.2, 27.0, 20.2. IR (KBr): 3056, 2960, 2869, 1620, 1562, 1507, 1466, 1428, 1367, 1237, 1089 cm-1. HRMS (CI): m/z [M+1]+ found 249.1835, calcd 249.1828 for C12H21N6. MP = 108-110 oC.

N,N-Diisobutyl-pyridazine-3,6-diamine (7). To a sealed tube apparatus charged with 6- diisobutyl-tetrazolo[1,5-b]pyridazin-6-yl-amine (0.5 g, 2.0 mmol, 100 mol%) and tri-n-butylphoshine (0.82 g, 4.0 mmol, 200 mol%) was added CH3CN (2 mL). The reaction vessel was sealed, placed in 180 oC oil bath, and was allowed to stir for 3 hrs. The reaction mixture was removed from the heating bath and was allowed to cool to 25 oC. An aqueous solution of HCl (37%, 10 mL) was added and the reaction mixture was allowed to stir at 100 oC for 6 hrs. After cooling to 25 oC, the reaction mixture was transferred to a beaker and the mixture was neutralized with solid K2CO3. The aqueous phase was extracted with EtOAc (250 mL) and the combined organic extracts were (MgSO4), filtered and evaporated directly onto silica. The residue was loaded onto a silica gel column and subjected to flash chromatography (SiO2: 10% iPA in DCM) to afford the desired product 7 as sticky oil (0.36g, 1.60 mmol, 80%). 1H NMR (400 MHz, CDCl3):  6.76 (d, J = 6.9 Hz, 1H), 6.72 (d, J = 6.9 Hz, 1H), 4.32 (bs, 2H), 3.28 (d, J = 7.2 Hz, 4H), 2.00-2.14 (m, 2H), 0.88 (d, J = 6.6 Hz, 12H). 13C NMR (100 MHz, CDCl3):  155.2, 152.4, 118.5, 116.7, 58.1, 27.1, 20.5. IR (NaCl): 3254 , 2963, 1570, 1500, 2863, 1460, 1006 cm-1. HRMS (CI): m/z [M+1]+ found 223.1922, calcd 223.1923 for C12H23N4.

N,N'-Bis-tetrazolo[1,5-b]pyridazin-6-yl-cyclopentane-1,3-diamine (2). To a sealed tube apparatus charged with cis-1,3-cyclopentyldiamine dihydrochloride salt (0.5 g, 2.89 mmol, 100 mol%) and potassium carbonate (1.6 g, 11.56 mmol, 400 mol%) was added 6-chlorotetrazolopyridazine (0.95 g, 6.07 mmol, 210 mol%) followed by CH3CN (2 mL). The reaction vessel was sealed and placed in a 130 oC oil bath, and was allowed to stir for 6 hrs. The reaction mixture was removed from the heating bath and was allowed to cool to 25 oC. To the reaction mixture was added water and EtOAc. The reaction mixture was filtered and the solid was collected and dried in vacuo to afford 2 as a brown solid (0.83 g, 2.46 mmol, 85%). 1H NMR (300 MHz, CDCl3):  8.21 (s, 2H), 8.21 (d, J = 9.6 Hz, 2H), 7.17 (d, J = 9.6, 2H), 4.0-4.4 (m, 2H), 2.64-2.73 (m, 1H), 2.00-2.30 (bm, 2H), 1.70-2.00 (bm, 2H), 1.57-1.66 (m, 1H). 13C NMR (62 MHz, CDCl3):  155.2, 139.9, 122.9, 121.0, 50.9, 38.2, 30.2. IR (KBr): 3088, 3056, 2924, 1627, 1581, 1507, 1360, 1289, 1237, 1100, 838 cm-1. HRMS (CI): m/z [M+1]+ found 339.1541, calcd 339.1543 for C13H15N12. MP = 280 oC (decomp.).

bis(Iminophoshorane) en route to 3a and 3b. To an oven-dried sealed tube apparatus charged with bis(tetrazolo[1,5-b]pyridazine) 2 (0.4 g, 1.18 mmol, 100 mol%) was added tri-n-butylphosphine (1.43 g, 7.08 mmol, 600 mol%) followed by CH3CN (4 mL). The reaction vessel was sealed and placed in a 180 oC oil bath, and was allowed to stir for 3 hrs. The reaction mixture was removed from the heating bath and was allowed to cool to 25 oC. A precipitate was formed and was collected by filtration. The precipitate was then washed with EtOAc (520 mL). The residue was dissolved in DCM and the solid impurities were removed by filtration. Upon removal of the DCM in vacuo, the title compound was obtained as a light brown solid (0.65 g, 0.94 mmol, 80%). 1H NMR (300 MHz, CDCl3):  6.75 (d, J = 9.6 Hz, 2H), 6.51 (dd, J = 2.1 Hz, J = 9.6 Hz, 2H), 4.23 (bs, 4H), 2.46-2.55 (m, 1H), 2.00-2.20 (m, 14H), 1.60-1.80 (m, 2H), 1.30-1.60 (m, 25H), 0.91 (t, J = 7.2 Hz, 18H); 31P (121.5 MHz, CDCl3):  33.00. 13C (75 MHz, CDCl3):  160.9, 152.8, 125.9, 125.6, 118.1, 52.6, 41.6, 32.2, 24.6, 24.5, 24.4, 24.3, 24.2, 23.8, 13.9. IR (KBr): 3115, 3055, 2957, 2933, 2872, 1574, 1448, 1377, 1293, 1002, 837 cm-1. HRMS (CI): m/z [M+1]+ found 687.5113, calcd 687.5120 for C37H69N8P2. MP = 218-222 oC.

bis(3,6-Diaminopyridazine) (3a). To an oven-dried sealed tube apparatus charged with the bis(iminophosphorane) derived from 2 (1.0 g, 1.46 mmol, 100 mol%) and 4-tert-butylbenzoic acid (1.04 g, 5.84 mmol, 400 mol%) was added dry toluene (10 mL). The reaction vessel was sealed and placed in a 150 oC oil bath, and was allowed to stir for 24 hrs. The reaction mixture was removed from the heating bath and was allowed to cool to 25 oC. The resulting mixture was directly subjected to flash column chromatography (SiO2: 3% iso-propanol in DCM) to afford the title compound as a white solid (0.27 g, 0.44 mmol, 30%). 1H NMR (300 MHz, DMSO-d6):  10.80 (s, 2H), 8.00 (d, J = 7.5 Hz, 4H), 7.90 (d, J = 9.6 Hz, 2H), 7.53 (d, J = 6.6 Hz, 4H), 6.93 (d, J = 7.5 Hz, 4H), 4.25 (s, 2H), 2.62 (bs, 1H), 2.08 (bs, 2H), 1.69 (bs, 2H), 1.46 (bs, 1H), 1.31 (s, 18H). 13C (75 MHz, DMSO-d6):  165.5, 157.1, 154.7, 148.0, 131.1, 127.8, 125.1, 122.9, 116.1, 50.7, 24.7, 30.9, 30.7. IR (KBr): 2960, 2867, 1666, 1608, 1497, 1365, 1314, 1270 cm-1. HRMS (CI): m/z [M+1]+ found 607.3500, calcd 607.3509 for C35H43N8O2. MP = 260 oC (decomp.).

bis(3,6-Diaminopyridazine) (3b). To an oven-dried sealed tube apparatus charged with the bis(iminophoshorane) derived from 2 (0.5 g, 0.73 mmol, 100 mol%) and 3,4,5-tri-n-butoxybenzoic acid (0.99 g, 2.92mmol, 400 mol%) was added dry toluene (5 mL). The reaction vessel was sealed and placed in a 150 oC oil bath, and was allowed to stir for 24 hrs. The reaction mixture was removed from the heating bath and was allowed to cool to 25 oC. The resulting mixture was directly subjected to flash column chromatography (SiO2: 40% EtOAc in hexanes) to afford the title compound as a light yellow solid (0.18 g, 0.22 mmol, 27%). 1H NMR (300 MHz, CDCl3):  9.99 (bs, 2H), 8.28 (d, J = 9.9 Hz, 2H), 8.16 (bs, 2H), 7.19 (s, 4H), 6.88 (d, J = 9.9 Hz, 2H), 3.80-4.10 (m, 14H), 2.70-2.90 (m, 1H), 1.80-2.10 (m, 5H), 1.60-1.80 (m, 12H), 1.25-1.60 (m, 12H), 0.80-1.00 (18H). 13C NMR (100 MHz, CDCl3):  166.4, 159.1, 153.1, 148.5, 141.7, 128.7, 114.0, 106.8, 73.3, 69.0, 52.6, 39.3, 32.5, 32.0, 31.5, 19.4, 19.3, 14.1, 14.0. IR (KBr): 2958, 2872, 1669, 1583, 1495, 1427, 1332, 1210, 1110 cm-1. HRMS (CI): m/z [M+1]+ found 927.5707, calcd 927.5708 for C51H75N8O8. MP = 98-100 oC.

bis(3,6-Diaminopyridazine) (4). To an oven-dried sealed tube apparatus charged with cis-1,3-cyclopentyldiamine free base (0.19 g, 1.8 mmol, 600 mol%), 3,6-diiodopyridazine (0.1 g, 0.3 mmol, 100 mol%), CuI (2.9 mg, 0.015 mmol, 5 mol%), and K3PO4 (0.27 g, 1.27 mmol, 410 mol%) was added anhydrous ethylene gylcol (0.037 g, 0.6 mmol, 200 mol%) followed by DMF (1 mL). The reaction vessel was sparged with argon, sealed and placed in a 95 oC oil bath for 12 hrs. The reaction mixture was removed from the heating bath and was allowed to cool to 25 oC, at which point Boc-anhydride (0.79 g, 3.6 mmol, 1200 mol%) was added to the reaction mixture followed by EtOAc (5 mL). The reaction mixture was stirred for 1 hrs at 25 oC. The reaction was then partitioned between dilute aqueous NH4OH solution (5 mL) and EtOAc (25 mL). The organic layer was collected and the aqueous layer was washed with EtOAc (210 mL). The combined organic extracts were dried (MgSO4), filtered and evaporated onto silica in vacuo. The resulting residue was subjected to flash chromatography (SiO2: 50% EtOAc in hexanes → neat EtOAc) to afford the title compound as a pale yellow solid (0.11 g, 0.22 mmol, 74%). 1H NMR (300 MHz, DMSO-d6):  6.89 (d, J = 7.8 Hz, 2H), 6.61 (s, 2H), 5.90 (d, 6.6 Hz), 3.90-4.00 (m, 2H), 3.70-3.80 (m, 2H), 2.20-2.40 (m, 2H), 1.70-2.00 (m, 4H), 1.40-1.70 (m, 4H), 1.38 (s, 18 H), 1.20-1.40 (m, 2H). 13C NMR (75 MHz, DMSO-d6):  155.7, 153.8, 119.1, 78.1, 51.4, 50.7, 31.4, 29.0. IR (KBr): 2972, 2868, 1687, 1528, 1476, 1390, 1365, 1291, 1250, 1170, 1012, 828 cm-1. HRMS (CI): m/z [M+1]+ found 477.3174, calcd 477.3189 for C24H41N6O4. MP = 180 oC (decomp.).

bis(Tetrazolopyridazine) (5). To a 50 mL dry round bottom flask in an ice-water bath charged with bis(adduct) 4 (0.11 g, 0.23 mmol, 100 mol%) was added trifluoroacetic acid (1 mL). The reaction mixture was allowed to stir for 0.5 hrs at 0 oC, at which point the trifluoroacetic acid was removed in vacuo. The residue was dissolved in dry DMF (1 mL) potassium carbonate (0.31 g, 2.25 mmol, 980 mol%) was added followed by 6-chlorotetrazolo[1,5-b]pyridazine 1 (0.077 g, 0.49 mmol, 210 mol%). The reaction mixture was placed under an argon atmosphere and was allowed to stir for 8 h at 95 oC. The reaction mixture was removed from the heating bath and was allowed to cool to 25 oC. A precipitate was formed. Water and EtOAc were added to the mixture was filtered. The precipitate was collected and dried in vacuo to afford 5 as a brown solid (0.094 g, 0.184 mmol, 80%). 1H NMR (300 MHz, DMSO-d6):  8.23 (d, J = 9.6 Hz, 2H), 8.23 (s, 2H), 7.16 (d, J = 9.6 Hz, 2H), 6.67 (s, 2H), 6.09 (d, J = 6Hz, 2H), 2.56-2.70 (m, 2H), 2.00-2.20 (m, 4H), 1.60-1.80 (m, 4H), 1.40-1.60 (m, 2H). 13C NMR (63 MHz, DMSO-d6):  155.2, 153.0, 139.9, 122.7, 121.1, 118.8, 51.1, 51.0, 30.6, 30.3. IR (KBr): 3089, 2960, 1623, 1577, 1496, 1360, 1289, 1241, 1099, 826, 755 cm-1. HRMS (CI): m/z [M+1]+ found 515.2598, calcd 515.2605 for C22H27N16. MP = 179-181 oC.

bis(Iminophoshorane) en route to (6). To an oven-dried sealed tube apparatus charged with bis(tetrazolo[1,5-b]pyridazine) 5 (0.25 g, 0.36 mmol, 100 mol%) and tri-n-butylphosphine (0.73 g, 3.62 mmol, 1000 mol%) was added CH3CN (5 mL). The reaction vessel was sealed and placed in a 180 oC oil bath, and was allowed to stir for 3 hrs. The reaction mixture was removed from the heating bath and was allowed to cool to 25 oC. A precipitate was formed and was collected by filtration. The precipitate was washed with EtOAc (520 mL). The residue was dissolved in DCM and the solid impurities were removed by filtration. The DCM was removed in vacuo to afford the title compound as a light brown solid (0.19 g, 0.22 mmol, 60%). 1H NMR (300 MHz, CDCl3):  6.81 (d, J = 9.6 Hz, 2H), 6.60 (s, 2H), 6.55 (d, J = 9.6 Hz, 2H), 4.85 (bs, 2H), 4.60 (bs, 2H), 4.20 (m, 4H), 2.56-2.63 (m, 2H), 2.0-2.2 (bm, 18H), 1.6-1.9 (4H), 1.3-1.6 (m, 24H), 0.90 (t, J = 6.9 Hz, 18H). 31P (121.5 MHz, CDCl3):  32.90. 13C (75 MHz, CDCl3):  160.4, 154.4, 153.2, 126.0, 125.7, 118.1, 117.9, 52.6, 40.9, 32.2, 32.0, 28.3, 27.4, 24.5, 24.4, 24.2, 24.0, 23.7, 13.9. IR (KBr): 3117, 3025, 2962, 2938, 2864, 1594, 1451, 1370, 1331, 1135, 1059, 834 cm-1. HRMS (CI): m/z [M+1]+ found 863.6179, calcd 863.6182 for C46H81N12P2. MP 136-138 oC.

tris(3,6-Diaminopyridazine) (6). To an oven-dried sealed tube apparatus charged with the bis(iminophosphorane) derived from 5 (1.0 g, 1.15 mmol, 100 mol%) and 3,4,5-tri-n-butoxybenzoic acid (1.8 g, 5.3 mmol, 460 mol%) was added dry toluene (10 mL). The reaction vessel was sealed and placed in a 150 oC oil bath, and was allowed to stir for 24 hrs. The reaction mixture was removed from the heating bath and was allowed to cool to 25 oC. The reaction mixture was directly subjected to flash column chromatography (SiO2: Neat EtOAc → Et3N: iso-propanol: DCM = 2: 10: 88) to afford the title compound as a yellow solid (0.33 g, 0.30 mmol, 26%). Further purification prior to ITC studies were performed by preparative TLC eluting with 30% iso-propanol in DCM. 1H NMR (400 MHz, DMSO-d6):  10.85 (s, 2H), 7.86 (d, J = 9.6 Hz, 2H), 7.36 (s, 4H), 6.94 (d, J = 6.4 Hz, 2H), 6.90 (d, J = 9.6 Hz, 2H), 6.68 (s, 2H), 6.10 (s, 2H), 4.14-4.25 (m, 2H), 4.05-4.15 (m, 2H), 4.00 (q, J = 5.6 Hz, 8H), 3.90 (t, J = 5.6 Hz, 4H), 2.50-2.62 (m, 2H), 1.92-2.30 (m, 4H), 1.53-1.80 (m, 16H), 1.30-1.53 (m, 14H), 0.80-1.00 (m, 18H). 13C NMR (63 MHz, DMSO-d6):  164.9, 157.1, 153.0, 152.3, 148.1, 140.2, 128.5, 122.8, 118.8, 116.0, 106.2, 72.1, 68.1, 51.0, 50.9, 31.8, 30.9, 18.8, 18.7, 13.7. IR (KBr): 2956, 2932, 2871, 1666, 1582, 1492, 1427, 1331, 1210, 1112, 1023, 831, 757 cm-1. HRMS (CI): m/z [M+1]+ found 1103.6775, calcd 1103.6770 for C60H87N12O8. MP 214-216 oC.

(3-Benzyloxycarbonylamino-cyclopentyl)-carbamic acid benzyl ester (8). To a suspension of cis-1,3-diaminocyclopentane hydrogen chloride salt (0.2 g. 1.16 mmol, 100 mol%) in CH3CN (10 mL) was added potassium carbonate (0.8 g, 5.81 mmol, 500 mol%) followed by benzyl chloroformate (0.793 g, 4.65 mmol, 400 mol%). The reaction was stirred at 25 o0 for 22 hrs, at which point a precipitate was formed and was collected by filtration. The mother liquor was partitioned between water and EtOAc. The organic layer was separated, dried (MgSO4), filtered and evaporated to provide a solid residue, which was combined with the earlier precipitate. The combined solid residues were recrystallized from EtOAc to afford the title compound as a white solid (0.36 g, 1.0 mmol, 90%). 1H NMR (250 MHz, CDCl3)  7.25-7.30 (m, 10H), 5.00 (s, 2H), 3.75-3.90 (m, 2H), 2.10-2.25 (m, 1H), 1.77-1.85 (m, 2H), 1.48-1.55 (m, 2H), 1.25-1.45 (m, 1H). 13C NMR (100 MHz, DMSO-d6):  160.9, 142.7, 133.8, 133.3, 70.6, 55.7, 36.0. IR (KBr): 1645, 1262 cm-1. HRMS (CI): m/z [M+1]+ found 369.1799, calcd 369.1814 for C21H25N2O4. MP = 139-142 oC.

Duplex Molecular Strands Based on the 3,6-Diaminopyridazine Hydrogen Bonding Motif: Amplifying Small Molecule Self-Assembly Preferences through Preorganization and Iterative Arrangement of Binding Residues

Hegui Gong and Michael J. Krische*

University of Texas at Austin
Department of Chemistry and Biochemistry
Austin, TX 78712, USA

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