Development of analytical and preparative chromatographic separations of novel growth hormone secretagogue compounds

Chromatographic separations of new growth hormone secretagogue compounds were developed to support structure-activity relationship (SAR) studies in conjunction with lead optimization. These new compounds differed from Merck's MK-677 by having two chiral centers and thus diastereomeric mixtures were generated. Separation of initial compounds in the SAR was achieved on a Kromasil C18 column using an ammonium acetate buffer and acetonitrile. However, additional candidates were not separable on C18 columns and a chiral Kromasil CHI-DMB column was used to resolve the diastereomeric compounds. The Kromasil CHI-DMB packing was also used in a preparative chromatographic system to resolve multigram quantities of secretagogue candidates for testing. Chiral separations of different intermediates were also developed in support of evolution of an asymmetric synthetic route. This report summarizes development of the preparative chromatographic system used to purify diastereomeric mixtures and chiral separations of intermediates in the synthesis.     

Apparate zur Gasanalyse

Ohne Zusammenfassung     

Passive acoustic bubble sizing in sparged systems

Passive acoustic bubble sizing was investigated in both controlled tests and in a stirred, sparged tank typical of the biotechnology or minerals processing industries. Acoustic techniques have promise for industrial systems where other bubble analysis methods are impractical. Acoustic signals were studied for bubbles precisely formed at higher airflow rates. Acoustic pulses varied with bubble production rate as well as with bubble size. A technique of windowing pulses is proposed. Two alternative versions of this windowing technique were applied to a stirred, sparged tank, giving good agreement. It was shown that, in some cases, it may also be possible to acoustically estimate the spatial distribution of void fraction. Received: 13 June 2000/Accepted: 19 October 2000     

Compact, high-pulse-energy, picosecond optical parametric oscillator

We report a high-energy optical parametric oscillator (OPO) synchronously pumped by a 7.19 MHz, Yb:fiber-amplified, picosecond, gain-switched laser diode. The 42-m-long ring cavity maintains a compact design through the use of an intracavity optical fiber. The periodically poled MgO-doped LiNbO(3) OPO provides output pulse energies as high as 0.49 μJ at 1.5 μm (signal) and 0.19 μJ at 3.6 μm (idler). Tunability from 1.5 to 1.7 μm and from 2.9 to 3.6 μm is demonstrated, and typical M(2) values of 1.5 × 1.3 and 2.8 × 1.9 are measured for the signal and idler, respectively, at high power.     

Novel integrated paired emitter-detector diode (PEDD) as a miniaturized photometric detector in HPLC

A novel low power, low cost, highly sensitive, miniaturized light emitting diode (LED) based flow detector has been used as optical detector for the detection of sample components in high performance liquid chromatography (HPLC). This colorimetric detector employs two LEDs, one operating in normal mode as a light source and the other is reverse biased to work as a light detector. Instead of measuring the photocurrent directly, a simple timer circuit is used to measure the time taken for the photocurrent generated by the emitter LED (lambda(max) 500 nm) to discharge the detector LED (lambda(max) 621 nm) from 5 V (logic 1) to 1.7 V (logic 0) to give digital output directly without using an A/D converter. Employing a post-column reagent method, a Nucleosil 100-7 column (functionalized with iminodiacetic acid (IDA) groups) was used to separate a mixture of transition metal complexes, manganese(II) and cobalt(II) in 4-(2-pyridylazo)-resorcinol (PAR). All optical measurements were taken by using both the in-built HPLC variable wavelength detector and the proposed paired-emitter-detector-diode (PEDD) optical detector configured in-line for data comparison. The concentration range investigated using the PEDD was found to give a linear response to the Mn(II) and Co(II) PAR complexes. The effects of flow rate and emitter LED light source intensity were investigated. Under optimised conditions the PEDD detector offered a linear range of 0.9-100 microM and LOD of 0.09 microM for Mn-PAR complex. A linear range of 0.2-100 microM and LOD of 0.09 microM for Co-PAR complex was achieved.     

Charge-transfer excited state in tris(acetylacetonato) europium(III)

The extremely low efficiency of the intramolecular energy transfer process in europium acetylacetonate compared with the corresponding terbium acetylacetonate is attributed to the presence of a charge-transfer excited state lying below the ligand singlet states. This is supported by the anomalous absorption spectrum of the Eu³⁺ complex and the effects of added anions in other ligand systems.     

Electrochemical detection of the oxidation of alcohols byN-hydroxyethylethylenediamminetriacetatoruthenate(IV)

The oxidation of [Ru^III(hedta)(H₂O)]=(1) to its Ru^IV monomeric complex at a glassy carbon electrode is abserved to promote oxidation of alcohols bearing an a-hydrogen (i-PrOH benzyl alcohol,sec-phenethyl alcohol). Tertiary substitution blocks the oxidation (t-BuOH). The oxidation of the alcohols is detected by an enhancement in the current of the Ru^IV/III waves at potentials above 0.96V, caused by scavenging (reduction) of Ru^IV by the alcohols. Binuclear complexes which possess Ru^IV bridged by oxo to either a second Ru^IV or to Ru^III in species of composition [LRuORuL]ⁿ⁻, L=hedta³⁻, fail to oxidize the alcohols. The terminal oxo moiety attached to Ru^IV is postulated to facilitate the oxidation of primary and secondary alcohols in a manner analogous to Meyer's [RuO(trpy)(bpy)]²⁺ catalyst. The dissociation of the (III,IV) binuclear complex into its monomers provides a pathway which increases catalytic activity at the expense of the inactive (III, IV) binuclear complex's concentration. TMC 2531     

Coordination of RuCl3(NO)(H2O)2 by imidazole,histidine and iminodiacetate ligands: a study of complexation of 'Caged NO' by simple bio-cellular donors

The 'caged NO' reagent, RuCl₃NO(H₂O)₂, has been studied by n.m.r. and i.r. methods with imidazole, histidine, histamine, and N-methyliminodiacetate as complexing ligands. These ligands are representative of cellular donors that would be encountered as RuCl₃NO(H₂O)₂ migrates through biological cells. [RuCl₃NO(imH)(H₂O)], [RuCl₃(NO)(imH)₂] and [RuCl₂(NO)(imH)₃]⁺ complexes (imH = imidazole) have been detected by ¹H-n.m.r. and i.r. and electrospray mass spectrometry (e.s.i.–m.s.) methods. Based upon the effect of cis ligand addition on the (NO) frequency causing a decrease in frequency, the 1:1 and 1:2 complexes have the imidazole donors in the plane cis to the NO⁺ moiety, whereas the 1:3 species has the third imidazole trans to the NO⁺. The trans imidazole donor causes 'trans-strengthening' of the N–O bond of the {RuNO}⁶ chromophore. ¹H-n.m.r. shows that the monodentate imidazole donor(s) is (are) in rapid exchange with free imidazole in solution for each of the n = 1–3 species. Histidine and histamine make kinetically more stable 1:1 complexes with the major isomer having an axially-coordinated histidine imidazole donor, but in-plane donation for histamine. The carboxylate of coordinated histidine remains pendant according to i.r. and ¹³C-n.m.r. data. From syntheses carried out at pH ca. 5, the amino donor is H-bonded to an in-plane H₂O in the major species (ca. 75%) and coordinated with displacement of the in-plane H₂O in the lesser isomer (25%). By contrast, the histamine ligand binds with an in-plane bound imidazole and a pendant protonated amino group (94%). The remaining 6% has an in-plane chelated histamine, analogous to the bis imidazole species and the known fac, cis-[RuCl₃NO(en)] complex. N-Methyliminodiacetate is observed to form one main [RuCl(NO)(mida)(H₂O)] complex (85%) with two chelated glycinato donor groups with RuCl₃NO(H₂O)₂, one glycinato group chelated 'in-plane' with the central amine donor and one axial coordinated glycinato donor. A second [RuCl(NO)(mida)(H₂O)] complex (the remaining 15%) has the amine donor trans to NO⁺ and chelated glycinato groups which coordinate in the RuClO₂(OH₂) plane, either cis or trans to each other, in a 60:40 split (ca. 9% and 6%). The presence of one Cl^– and one H₂O in the [RuCl(NO)(mida)(H₂O)] complexes was established by e.s.i.–m.s. These results show that RuCl₃NO(H₂O)₂ is likely to be freely mobile within a cellular environment, forming stable complexes via bidentate chelation with 'two-point' nitrogen donors (en, his, etc).     

The effect of zinc(II) on the formation of 2,2′- and 2,3′-bipyridine complexes of [Ru2II(ttha)]2−(ttha6−=triethylenetetraminehexaacetate)

Ru2II(ttha)(H2O)2]2– (ttha6–= triethylene tetramine hexa-acetate), prepared by the reduction of the ruthenium(III) precursor, reacts with 2,2-bipyridine (2,2-bpy) in a multi-step fashion. The first 2,2-bpy equivalent (1:1) adds with bidentate chelation at one ruthenium(II) site as revealed by separate ruthenium(II)/(III) waves at 0.03 and 0.54V vs. n.h.e. A second equivalent of 2,2-bpy (1:2) is initially stored and retained as the [Zn(2,2-bpy)]2+ complex. Further addition of 2,2-bpy initiates coordination at the second ruthenium(II) site. [Ru2(ttha)-(2,2-bpy)(H2O)]2– forms a strong ion-pair with zinc(II) that is in rapid equilibrium with the Zn(H2O)62+/Zn(2,2-bpy)]2+ pool. The solubility of the ion-pair is low. The ion-pair exhibits a shifted ruthenium(II)/(III) wave at 0.60V. Higher amounts of 2,2-bpy recomplex the zinc(II), solubilizing the complex and returning the E1/2 value to 0.54V. Other ligands which either have a higher affinity for ruthenium(II) centres than for zinc(II) as bidentate donors (1,10-phenanthroline), or ligands that cannot form bidentate zinc(II) complexes [(2-methylpyrazine, 4,4-bipyridine (4,4-bpy), and 2,3-bipyridine (2,3-bpy)] do not exhibit the unusual competition by zinc(II). These ligands all add statistically to the ruthenium(II) centres forming 1:2 complexes with 1:2 stoichiometries. 1H-n.m.r. studies of the Ru(II)polyaminopolycarboxylate complexes [RuII(hedta)(H2O)]– complex, and [Ru2(ttha)(H2O)2]2– itself, reveal that substitution of 2,3-bpy at ruthenium(II) sites occurs with an initial kinetic split between the pyridyl rings of the 3- less-hindered and 2-more-hindered ring. A slower rearrangement occurs, producing the isomer of the more-hindered 2-substituted ring. A process is driven by forming a more -accepting system when ruthenium(II) binds to the 2-ring of 2,3-bpy. Understanding the unusual influence of zinc(II) on the substitution of 2,2-bpy with [Ru2(ttha)(H2O)2]2– clarifies the nature of the 1:1 complex – namely that the 2,2-bpy becomes bidentate at one ruthenium(II) centre rather than serving as a trans-bridging ligand between both ruthenium(II) centres within one [Ru2(ttha)]2– unit.