Solid-state NMR applied to photosynthetic light-harvesting complexes

This short review describes how solid-state NMR has provided a mechanistic and electronic picture of pigment–protein and pigment–pigment interactions in photosynthetic antenna complexes. NMR results on purple bacterial antenna complexes show how the packing of the protein and the pigments inside the light-harvesting oligomers induces mutual conformational stress. The protein scaffold produces deformation and electrostatic polarization of the BChl macrocycles and leads to a partial electronic charge transfer between the BChls and their coordinating histidines, which can tune the light-harvesting function. In chlorosome antennae assemblies, the NMR template structure reveals how the chromophores can direct their self-assembly into higher macrostructures which, in turn, tune the light-harvesting properties of the individual molecules by controlling their disorder, structural deformation, and electronic polarization without the need for a protein scaffold. These results pave the way for addressing the next challenge, which is to resolve the functional conformational dynamics of the lhc antennae of oxygenic species that allows them to switch between light-emitting and light-energy dissipating states.

Introduction
Natural photosynthesis achieves the conversion of solar energy with a remarkably small set of cofactors. Photosynthetic proteins use (bacterio)chlorophylls (BChls) and carotenoids (Car) both for light-harvesting and charge separation, implying that the functional programming of the pigment chromophores is encoded in their conformation, local environment, and dynamics and is not due to their chemical structure per se. While the architecture of the photosynthetic reaction centers that leads to directional electron transfer is common to all photosynthetic organisms, there is much to be learned about the structurefunction relations from the variability in photosynthetic antenna systems, as evolution has led to fundamentally different architectures for harvesting the light, depending on the variability of environmental sun light conditions. One intriguing puzzle that is currently attracting widespread attention is the molecular basis underlying the photophysical mechanism of nonphotochemical quenching (NPQ), a photoprotective switching mechanism that protects oxygenic species at high sun light conditions while optimally photosynthesizing at low light intensities.
During the past three decades, many structures of photosynthetic membrane proteins have been resolved at high resolution by crystallography, but the details of the structure-function interactions and how cofactors are programmed for their function remain to be elucidated. Solidstate NMR may not outperform crystallography for resolving membrane protein structures, but the technique has compelling advantages when it comes to resolving atomic details of pigment-protein interactions in a flexible protein environment. Better understanding of the structurefunction motifs across antenna complexes and photosynthetic species in an evolutionary context will provide A. Pandit (&) Faculty of Sciences, VU University Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands e-mail: a.pandit@vu.nl knowledge on common denominators of functional mechanisms in natural photosynthetic systems. This will guide the design of novel artificial constructs in which dye molecules are preprogrammed in the ground state by engineering of their scaffolding environment to perform the different tasks of light harvesting, charge separation, and photoprotection (de Groot 2012).
In this short review, we illustrate how solid-state NMR has contributed to a mechanistic picture for the functionalization of the electronic structure of photosynthetic antenna complexes. In addition, we will present an outlook on the application of NMR to light-harvesting antennae of oxygenic organisms, which may enhance our understanding of the molecular mechanisms of NPQ.

Preparation of biological samples for solid-state NMR
In NMR, the signals from nuclear spins are characterized by a parameter called the chemical shift, reflecting the variation of the induced magnetic field relative to the applied magnetic field. The dispersion of NMR frequencies is due to the diamagnetic susceptibility of the electrons in their molecular orbitals, i.e. the magnetic field at the nucleus is reduced by the electronic shielding from the surrounding electrons. The chemical shifts provide atomic selectivity for well-ordered systems and are highly sensitive to the local environment. In contrast to X-ray diffraction techniques that require long-range crystalline order, solid-state NMR can be applied to ordered systems without translation symmetry, including membrane proteins in a detergent shell or a lipid membrane (Renault et al. 2010;Alia et al. 2009;McDermott 2009).
Magnetic resonance occurs only for nuclei with a net nuclear spin and magnetic moment from an uneven number of nucleons. Commonly studied isotopes in natural systems are the spin nuclei 1 H, 13 C, 15 N, and 31 P. In the solidstate, the T 2 spin-spin relaxation time is short due do restricted motions, resulting in broad lines. With Magic Angle Spinning (MAS) and high power decoupling the signal overlap can be reduced. Since the 1 H NMR chemical shifts fall into a narrow range, indirect detection via heteronuclear coupling with e.g. 13 C or 15 N atoms is used to resolve the 1 H response. Since the nuclear spin species 13 C and 15 N have low natural abundance, sample enrichment with additional isotopes is generally required. For biological samples, these have to be incorporated biosynthetically, for instance by using recombinant proteins that are over-expressed in cell cultures grown on isotope-rich media. Antenna apo-proteins can be expressed in E. coli and re-assembled with their chromophores into functional complexes, but these reconstituted proteins are not easily produced in the milligram quantities required for NMR in the solid state. The a polypeptide of a purple bacterial antenna complex was also successfully expressed in a cellfree in vitro expression system and reconstituted with pigments afterward (Shimada et al. 2004). The advantage of cell-free systems is that isotope-labeled amino acids can be added directly to the synthesis reaction, without losses in the metabolic pathways. In addition, chromophores, membrane lipids, or detergent molecules can be added during the protein synthesis reaction to stimulate protein folding in vitro. For photosynthetic proteins, this could eventually lead to synthesis and folding in one step, with possibilities for selective pigment or amino acid labeling. The first trial of this approach was recently reported for the major light-harvesting complex of Arabidopsis thaliana, of which the lhcb1 apoprotein was expressed in a cell-free system in the presence of pigments or lipid nanodisc membranes (Pandit et al. 2011a). As an alternative for over-expression, photosynthetic organisms are grown on isotope-rich minimal media. Labeling experiments included growing of Chlamydomonas green algae cells on 13 C-enriched Na-acetate (Pandit et al. 2011b), 15 N labeling of spinach (Diller et al. 2007), and growing of Rps. acidophila purple bacteria on 13 C-15 N-labeled succinate medium or by using media enriched with 13 C-15 N-labeled algal amino acids (van Gammeren et al. 2004). Intrinsic labeling of (bacterio)chlorophylls was performed in purple and cyanobacteria through addition of isotope-labeled aminolevulinic acid (Ala), a precursor of (B)Chl (Janssen et al. 2010;Daviso et al. 2009).
The light-harvesting complex 2 as an NMR model; the protein By controlled growth of purple bacteria in the presence of [1,2,3,4-13 C]-succinic acid, [1,4-13 C]-succinic acid, [2,3-13 C]-succinic acid, or a mixture of uniformly labeled amino acids, a sequence-specific assignment was obtained for the aand b-polypeptides that build up the light-harvesting 2 complex of Rhodopseudomonas acidophila (LH2) (van Gammeren et al. 2005b;Neal et al. 2006). This is the only photosynthetic antenna complex of which an almost complete sequence-specific assignment has been accomplished. For many globular proteins in solution and for some membrane-bound proteins, a sequence-specific assignment enables to predict its secondary structure, since the backbone C a , C b , and CO chemical shifts cover different ranges for a-helical and b-sheet proteins, and these ranges are also different from the C a , C b , and CO dispersion for random coils (Neal et al. 2006;Cornilescu et al. 1999). The differences between the experimental backbone chemical shifts and their random coil values are called the secondary shifts and in general they correlate with the backbone torsion angles W, U, and x. The LH2 secondary shifts, however, showed several mismatches pointing to a b-sheet arrangement within the a-helical stretches in the crystal structure (Pandit et al. 2010b). The irregularities were attributed to localized structural distortions or electronic perturbations, induced by the rigid packing of the pigment-protein complex into a ring-shaped oligomer. Figure 1 shows the mapping of the NMR chemical shift perturbations on the available crystal structure to visualize where local points of conformational strain may occur along the protein backbone. This illustrates how the NMR data reveal information that is complementary to crystallographic data, and this provides synergy, rather than two separate methods for structure determination.
The BChl-coordinating histidines (His) of the LH2 acidophila complex were studied extensively by selective 13 C and 15 N labeling. The two neutral His that coordinate the BChls appeared to have the NMR signatures of a double-protonated, i.e. positively charged His (Alia et al. 2001(Alia et al. , 2004. This was explained by a charge transfer in the ground state between the His and their coordinating BChl, resulting in a partial positive charge on the His imidazoles. In density functional theory (DFT) modeling, the effect would disappear if the BChl-His geometry was optimized beforehand, but was clearly present when the coordinates were taken directly from the X-ray structure (Wawrzyniak et al. 2008). Running a geometry optimization would increase the distance between the His and the BChl from 2.12 to 2.31 Å . One could argue that this moderate change falls within the error of the X-ray spatial resolution, and indeed the sensitivity of the NMR chemical shifts to electronic effects, which might be induced by small spatial re-arrangements, exceeds the resolution of the X-ray crystallographic structures. The LH2 His model explained the electronic effects of charge transfer by mechanical stress, induced by the protein conformational constraints in the LH2 oligomer packing. It was speculated that the His-BChl charge transfer could have an effect on the lightharvesting properties. A more clear example how a coordinating His may control the chromophore function was found for the special pair of photosystem II. Here, the inverted electronic charge of the Chl nitrogens in the special pair was explained by a hinge model, in which the coordinating His imidazole ring hangs over the Chl macrocycle, altering its electronic structure in the ground state and its oxidation state compared to PSI (Diller et al. 2007).
The light-harvesting complex 2 as an NMR model; the BChl pigments In addition to the protein chemical shifts, NMR assignments were obtained for the BChl-conjugated macrocycles of the three types of BChl in LH2: the aand b-bound BChls that build a ring of BChl dimers, called the B850 band, and the so-called B800 BChls that form a ring of monomers (van Gammeren et al. 2005a  and B800 signals, a sample was prepared from unlabeled LH2 of which the B800 BChls were extracted and substituted with uniformly labeled BChls. The three types of BChls have a distinctive set of chemical shifts, reflecting their conformational structures and variation in the local protein environment. The differences between the NMR signals in the protein-bound BChls and free BChl in organic solvent Dr determine the electronic structures in the ground state. Recently, the data set was expanded with the BChl assignments of the acidophila LH1 complex, the core antenna that forms a ring-shaped oligomer of dimer ab subunits surrounding the photosynthetic reaction center (RC) (Pandit et al. 2010a). Due to their less-ordered oligomer structures, crystallization of the LH1 protein complexes has only resulted in low-resolution structures (Roszak et al. 2003;Karrasch et al. 1995). The LH1 structural inhomogeneity similarly induces broadening of the lines in the NMR spectra and in an earlier study on Rhodospirillum rubrum LH1 ab subunits reconstituted with 13 C-15 N-labeled BChls, only one set of BChl NMR signals was assigned without distinction between the aand b-BChl (Wang et al. 2002). In our recent work, NMR assignment of the two types of LH1 BChls was achieved in intact LH1-RC core complexes, using the LH2 spectra as a ''template'' for the assignment. Two sets of signals were observed, corresponding with the electronic structures of the aand the b-bound BChls in LH1 that also form a ring of dimers, similar to LH2. By overlay of the LH1 and LH2 2D-NMR spectra, the BChl ground-state electronic structures of the homologous LH2 and LH1 antenna rings were directly compared, revealing differences and similarities in their conformation or local protein environment with atomic selectivity (Fig. 2). This method circumvents referencing to monomeric BChl in an organic solvent, of which chemical shift values are biased by the solvent polarity. The LH1 and LH2 BChl NMR chemical shift patterns on the one hand could not be modeled by the effects of hydrogen bonding, side chain out-of-plane rotation and long-range electrostatic interactions, suggesting that the BChl electronic structures in the ground state are mainly shaped by macrocycle deformation (Pandit et al. 2010a). Chlorophyll macroaromatic cycles are readily distorted, which makes for a very flexible electronic structure of the porphyrin ring, where the electronic densities follow the distortions imposed upon the structure due to the predominant electron-phonon coupling. The effect of structural deformation of the chromophores, however, was omitted in prediction of the site energies and corresponding excitonic couplings of the pigments inside the major light-harvesting complex II (LHCII) and of the Fenna-Mathews-Olson (FMO) complex, due to uncertainties in the crystal structures used for the calculations that otherwise could lead to  (Muh et al. 2010;Adolphs et al. 2008). Also, here the NMR data thus complement the crystallographic data and eventually may be combined in a synergistic way for more accurate prediction of pigment site energies. For instance, shifting electron density over the macrocycle allows for changing the direction and magnitude of the electric dipole moment and the polarizability tensor, and the local electronic densities behind such processes can be probed through the NMR chemical shift (He et al. 2001;Wawrzyniak et al. 2008).

The chlorosome antennae
In contrast to the antenna apparatus of all other photosynthetic organisms, the heterogeneous chlorosome antennae of green photosynthetic bacteria contain rod-shaped oligomers of BChl-c/d/e molecules that self aggregate without assistance of a protein. Their structural functional features have been the inspiration for self-assembled artificial antennae (Ganapathy et al. 2009b;Oostergetel et al. 2010;Balaban et al. 2005). The p-p interactions of overlapping macrocycles from the adjacent BChls give rise to ringcurrent shifts; an effect in which the electronic ring current of the macrocycles induces a local magnetic field that affects the NMR chemical shifts of the adjacent BChl in the structure with molecular overlap. The magnitudes of the ring-current shifts together with long-range 1 H-13 C correlations provide constraints for the packing mode of the BChls in the macrostructures (van Rossum et al. 2002). In conjunction with distance constraints from diffraction techniques and computational modeling, this provided a method to solve a template for the chlorosome self-assembled structure in detail. By constructing a triple mutant, the heterogeneous BChl-c pigment composition of chlorosomes of the green sulfur bacteria Chlorobaculum tepidum was simplified to nearly homogeneous BChl d. Computational integration of solid-state NMR and cryo-electron microscopy revealed a syn-anti stacking mode and led to a structural model of BChls self-assembled into coaxial cylinders to form tubular-shaped elements. (Ganapathy et al. 2009a). The macrostructures are stabilized by C=O••H-O••Mg interactions between the 3 1 hydroxy group, the 13 carbonyl and the central magnesium, and by p-p interactions between the tetrapyrrole macrocycles (Fig. 3). Since low-lying CT states are an intrinsic property of higher aggregates of chlorophyll molecules and are likely to mix significantly with the exciton states, the polarizability effects in chlorosome aggregates are strongly enhanced compared to BChl-c monomers. The structural framework can accommodate chemical heterogeneity in the side chains for adaptive optimization of the light-harvesting functionality by optical tuning and broadening. In addition, the BChls form sheets that allow for strong exciton overlap in two dimensions, enabling triplet exciton formation for photo protection.
Outlook: the conformational switch in oxygenic light-harvesting systems Oxygenic photosynthetic organisms have a built-in photoprotection mechanism, in which under excess light conditions the antenna system switches from a light-harvesting to a light-energy dissipating state, in which the excitedstate energy is safely dissipated as heat. The photophysical mechanism of NPQ involves a change of the pigment configurations, creating an energy dissipation pathway via one of the pigments. The exact mechanism is under much debate and several models have been proposed, based on intra-or intermolecular conformational changes and/or cofactor exchange (Holzwarth et al. 2009;Ruban et al. 2007;Ahn et al. 2008;Standfuss et al. 2005;Holt et al. 2005). In vitro, fluorescence quenching occurs upon aggregation of the LHCII complexes, with spectroscopic signatures similar to the (Wawrzyniak et al. 2008) state in leaves and chloroplasts, suggesting that they underlie very similar photophysical mechanisms. In particular, Resonance Raman shows a twist of the neoxanthin (Neo) carotenoid upon quenching in vivo as well as in vitro (Ruban et al. 2007), demonstrating that conformational changes indeed occur. For the major light-harvesting complex II from plants (LHCII), conformational switching was observed without self-aggregation of LHCII proteins entrapped in gels (Ilioaia et al. 2008) and of LHCII trimer complexes studied by single-molecule fluorescence microscopy (Kruger et al. 2010). This suggests that the individual antenna complexes have a built-in capacity to switch between different functional conformational states, triggered by the protein local environment that can shift the dynamic equilibrium between the light-harvesting and the NPQ states. A shift of a dynamic equilibrium has been observed before with MAS NMR, e.g. for 7-helix membrane proteins in relation to signal transduction, and NMR is a good method to analyze the relation between structure and the triggering of function for such processes (Ratnala et al. 2007;Etzkorn et al. 2007). Despite the availability of two high-resolution LHCII crystal structures (Standfuss et al. 2005;Liu et al. 2004), the more subtle conformational dynamics related to NPQ remain to be resolved. In the LH2 NMR model it was shown that by using the X-ray structure of LH2, the NMR data could predict different aspects of conformational strain in the form of localized electronic perturbations, on the level of (1) the protein backbone, (2) the selective pigment-coordinating sites, and (3) the protein-bound chromophores. Recently, the first NMR experiments were performed on the LHCII trimer complexes of the green alga Chlamydomonas reinhardtii, which have a high degree of homology with the LHCII complexes of higher plants (Pandit et al. 2011b). The dispersion of the NMR signals is good, and possible conformational changes will be observable already in uniformly isotope-labeled samples. The NMR samples can be prepared in aggregated or detergent-solubilized conditions, modulating the photophysical state of the LHCII in vitro. By selective labeling of amino acids, for instance through the use of Chlamydomonas auxotrophic amino acid-deficient strains, the role of important amino acids in the quenching process can be followed without the need for mutational approaches. In addition, intrinsic Chl labeling is possible through the supply of isotope-labeled Ala to the cells (Janssen et al. 2010). By sparse labeling of chlorophylls, the NMR signals of these pigments can be resolved from the protein background signals, in order to identify the role of different Chls . The assignment of the Car pigments will be more difficult, since there is strong overlap between the NMR signals of their polyene chain 13 C nuclei. Characterization of the xanthophylls properties by NMR will probably rely on the use of recombinant proteins, where xanthophyll chromophores are substituted by selectively labeled isotopomers (de Groot et al. 1992).
The next challenge is to apply these NMR methods, which have been proven successful for characterization of purple bacterial antennae and of various photosynthetic reaction centers, to the more complex light-harvesting systems of oxygenic photosynthetic organisms, where subtle conformational features may have a functional role in maintaining the integrity of the photosynthetic antenna under high light and drought stress conditions.
Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


extfieldsofstudy: Chemistry Medicine

text:
Quantification of six bioactive compounds in Zhenqi Fuzheng preparation by high-performance liquid chromatography coupled with diode array detector and evaporative light scattering detector

A simple and accurate high-performance liquid chromatography (HPLC) coupled with diode array detector (DAD) and evaporative light scattering detector (ELSD) was established for the determination of six bioactive compounds in Zhenqi Fuzheng preparation (ZFP). The monitoring wavelengths were 254, 275 and 328 nm. Under the optimum conditions, good separation was achieved, and the assay was fully validated in respect of precision, repeatability and accuracy. The proposed method was successfully applied to quantify the six ingredients in 31 batches of ZFP samples and evaluate the variation by hierarchical cluster analysis (HCA), which demonstrated significant variations on the content of these compounds in the samples from different manufacturers with different preparation procedures. The developed HPLC method can be used as a valid analytical method to evaluate the intrinsic quality of this preparation.

Introduction
Traditional Chinese medicines (TCMs). used for thousands of years in China. have played an important role in clinical therapy [I.2]. Therefor, to ensure the clinical efficacy and safety, overall quality control of TCMs is of significant importance [ 3]. However, most of the published literature has reported the qualitation or quantification of one or two types of components from only one comprising herb, and it could not be responsible for the overall quality control of the composite formulae [4]. Therefore, it seems necessary to determine the marker compounds of Chinese medicine preparation (CMP) [ 5] . . Zhenqi Fuzheng preparation (ZFP), composed of Radix Astragali (Huangqi in Chinese) and Fructus Ligustri Lucidi (Niizhenzi in Chinese), which has been listed in "Ministry of Health Drug Standards" of Chinese medicine formulated prescription,' is commonly used in clinical practice for the treatment to improve immunity, protect bone marrow and adrenal cortex, increase leukocytes, promote the recovery of normal functions as an accessory of surgical operation, radiotherapy or chemotherapy, and it could also be used as an adjuvant therapy of cancer [6][7][8][9][10] .
In the present study. we developed the HPLC-DAD-ELSD method to determine six components of ZFP, including adenosine (l), rhodioside (2), chlorogenic acid (3), calycosin (4), fermononetin (5), and astragaloside IV (6), as shown in Figure 1. This approach is potentially ideal for routine analysis and quality evaluation of ZFP. In order to improve the sensitivity and selectivity of the determination, DAD at three different wavelengths was employed for the quantitative analysis of strong UV absorbing compounds such as phenolic acids and isoflavones due to their different UV characteristics, while ELSD was used to determine none or poor UV absorbing compounds such as saponins.  min. An aliquot of 10 !-LL of each sample was injected. Detection wavelengths were set at 254 nm for adenosine, calycosin, and fermononetin, 275 nm for rhodioside, and 328 nm for chlorogenic acid. The drift tube temperature for ELSD and carrier gas flow rate were 115'C and 3. OUmin, respectively.

Otemicals and samples
Compounds (

Preparation of~Ie solutions and negative control (NC) solutions
ZFPs were triturated in the particle size of 40 -60 mesh. The accurately weighed powder (2.0000 g) was extracted three times with aliquots of 30 mL methanol by sonication (400 W, 30 min). Then the pooled filtrate was concentrated to dryness, and the residue was dissolved in 5 mL methanol. The methanol solution was filtered with a 0.45 !-Lm membrane prior to HPLC analysis. According to the prescription and preparation protocol of ZFP recorded in Chinese Ministry of Health Drug Standards, two kinds of NC samples in which the formula contained no Niizhenzi (El) and Huangqi (B2), respectively, were prepared to validate the specificity of the method. The NC sample solutions were prepared using the same method as the sample solutions.

Optimization of chromatographic conditions
To optimize the chromatographic conditions, the different stationary phases, such as Kromasil ODS column (5 f-lm, 4.6 mm x 25 mm) and Hypersil ODS-l column (5 f-lm, 4.6 mm X 25 mm), and different mobile phases, such as methanol : water and acetonitrile : water, were attempted during the experiment. As a result, a Kromasil ODS column and the mobile phase of methanol: water were chosen to obtain the desired separation and acceptable tailing factor within the running time of 90 min. In our previous work (data not shown), these bioactive components have slightly different UV absorption properties, and it is difficult to simultaneously determine them by common analytical approaches. Therefore, three detection wavelengths at 254, 275, and 328 rim were used to record chromatograms for the different structural types of components, and to provide an optimum SIN for simultaneously quantitative analysis. The operating conditions for ELSD were optimized according to the data computed with the ELSD software: The gas flow rate was set at 3.0 Umin, and the drift tube temperature was determined to be 115°C. Chromatograms of ZFP under such detection conditions are shown in Figure 2 and Figure 3.
Based on the selected detection mode, all these six compounds had acceptable limits of detection (LOD) and quantification (LOQ) ( Table 1). The identity of each peak detected in ZFP samples was confirmed by comparison of retention time, UV spectrum of each peak with those of reference compound and blank samples. In addition, spiking samples with the reference compounds further confirmed the identities of the peaks.

Linearity, WD and U>Q
The construction of calibration curves was carried out according to the section 2.5. For the components by UV detection, their regression equations were expressed by the formula Y = AX + B, while by ELSD detection, their regression equations could be described as Y = aX b ; so, the calibration curves should be obtained in double logarithmic coordinates [ 11 ]. As shown in Table 1, a good linearity ( r >0.999) was observed in a relatively wide concentration range.
Working solution was diluted with methanol to get a series of appropriate concentrations, and the LOD and LOQ under the chromatographic conditions were separately determined at the signal-to-noise ratios (SIN) of 3 and 10. LOD and LOQ for each compound are also listed in Table 1.

Precision, repeatability, and accuracy
The precision of this newly developed method was studied by determining intra-day and inter-day variations. The intra-day variation was determined by analyzing the same standard methanol solution in triplicate for three times within one day. While for inter-day variation test, the solu-tion was examined in triplicate for consecutive three days. To estimate the repeatability, five different working solutions, which were prepared from the same sample, were analyzed. As indicated in Table 2, the overall RSDs of the precision test and the repeatability test were lower than 2.63%, which demonstrates good precision and repeatability of the method. Figure 2 The typical HPLC-DAD chromatographic profiles. A, B, C, and D stand for standard solution, ZFP in absence of Niizhenzi, ZFP in absence of Huangqi, and ZFP in sequence. Peaks 1, 2, 3, 4, and 5 represent adenosine, rhodioside, chlorogenic acid, calycosin, and fermononetin, respectively. Blank sample analysis confirms that no impurities were co-eluted with the compounds determined .
J Pharm Anal Vol  The overall RSD of the intra-and inter-day is in the range of 0.95% -1. 36% and 1.18% -2. 41 %, respectively. And the RSD of repeatability test is <3%. Figure 3 The typical HPLC-ELSD chromatographic profiles. A, B, and C stand for standard solution, ZFP in absence of Niizhenzi, and ZFP in sequence. NC sample analysis confirms that no impurities were co-eluted with the compounds determined.
The accuracy of the method was evaluated by spiking known amounts of standard samples into a ZFP sample and comparing the amount determined of these standards with the amount originally added. And as shown in Table 3, the overall RSD of the recovery of ZFP was lower than 3% for all these analyses. The recovery obtained was in the range of 95.4% -103.5%, indicating that the developed method was sufficiently accurate for determining the six bioactive components in ZFP, and analysis of NC samples confirmed that no impurities were co-eluted with the compounds determined ( Figure 2 and Figure 3).  Table 4. The results showed that the quality of this preparation was relatively uniform and demonstrated that the chemical variation was obvious among the different companies and! or preparations of ZFP. The results showed that the content of astragaloside IV (6), a characteristic and active constituent whose presence forms part of the quality assurance of ZFP, is relative high in samples compared with the other target compositions. The contents of the compounds were found lower in granule, and higher in capsule, except compound 2. Therefore, the analytical results obtained also indicated that a multiplecomponent assay might be a rational strategy to elucidate the synergic effects and much more comprehensive control for the quality of this preparation.

Analysis of samples
The developed method was subsequently applied to simultaneous determination of the six active compounds in 31 commercial ZFP samples from different pharmaceutical companies in China. All the contents of six ingredients in To evaluate the variation of ZFP, hierarchical cluster analysis (HCA) was performed based on the investigated components' characteristics from HPLC profiles of 31 test samples. The dendrogram of HCA is shown in Figure 4, and the quality characteristics are revealed more clearly.
Supposing an appropriate distance level (Level D is chosen, the samples can be classified into seven quality groups, thereby indicating that preparation procedure is related to the quality of products. If a higher distance level (Level m is adopted, samples 1 -19 tend to be classified into the same group, which indicates that the samples from the same manufacturer are more similar, and then the samples can be divided into five quality groups, among which there is an abnormal sample (No,28), Thus, RCA is an effective way to discover the abnormal sample rapidly.

Conclusion
We reported a simple and sensitive HPLC method coupled with DAD and ELSD, and these two detectors are complementary, The proposed HPLC-DAD-ELSD method makes it possible to evaluate the quality of ZFP through a determi-nation of multi-components with different structures. This method has been applied successfully to quantify six bioactive compounds in 31 different batches of ZFP samples.
The results prove that the present method is suitable for routine quantitative analysis and intrinsic quality control of ZFP.


extfieldsofstudy:
Chemistry
Computer Science
Medicine

text:
The DINGO dataset: a comprehensive set of data for the SAMPL challenge

Part of the latest SAMPL challenge was to predict how a small fragment library of 500 commercially available compounds would bind to a protein target. In order to assess the modellers’ work, a reasonably comprehensive set of data was collected using a number of techniques. These included surface plasmon resonance, isothermal titration calorimetry, protein crystallization and protein crystallography. Using these techniques we could determine the kinetics of fragment binding, the energy of binding, how this affects the ability of the target to crystallize, and when the fragment did bind, the pose or orientation of binding. Both the final data set and all of the raw images have been made available to the community for scrutiny and further work. This overview sets out to give the parameters of the experiments done and what might be done differently for future studies.

Introduction
We approached this in a significantly different way than a 'normal' fragment screening campaign in the sense that the data set was to be complete (or as complete as physically possible). To elaborate, in a 'normal' fragment screening campaign, it is usual to have a fairly short timeline, so the project is set up to screen the fragments as quickly as possible using the most effective method first, and then use subsequent methods for verification and to determine the other parameters of value. For example, in our laboratory, we will typically screen the fragment set using SPR (taking about 1 week) and then only do protein crystallography on the hits from the SPR. We would only use ITC on those that were tight binders (better than 200 lM) and where we wanted verification of the binding energy. We would generally soak all fragments into pre-formed crystals and not attempt doing co-crystallization of compounds with the protein. In contrast, for the SAMPL project, it was one of the major goals of the project to have a complete data set for the modelling community to go back to and reference. For the DINGO data set, we systematically soaked every fragment of the set into the protein crystals and collected data sets for each of these complexes. In addition, cocrystallization of the target protein with fragments was undertaken as an orthogonal approach. The target chosen for the SAMPL challenge requires an inhibitor in order for crystallisation to occur, so the presence or absence of crystals with any given fragment in co-crystallisation trials is predictive of whether that fragment binds to the protein target. The SPR was done several times and dosage curves were also done several times on all those compounds that were 'hits'.
Bovine pancreatic trypsin [1] was used as the target for several reasons. It is easily obtainable from commercial vendors; the crystallographic community has studied it rather thoroughly; it is a protease that is similar to other proteases of human health interest; and there is a body of literature that supports a prospective challenge such as SAMPL, including known positive controls that could be used for verification of our methods [2][3][4]. The Maybridge 500 fragment library was chosen as it was commercially available and we had tested it against some other targets and knew that it had fragments that could bind to trypsin.
After starting the project, it became apparent that our choices did have some drawbacks. Trypsin is a protease that will self-proteolyze, so is unstable over time for all of our experiments (ITC, crystallization, etc.). The Maybridge 500 fragment library has some compounds that are insoluble under the conditions we used in several of the techniques where aqueous solubility has significant advantages (e.g. ITC). And finally, in our effort to be comprehensive, trying to collect X-ray crystallographic data sets of 500 different fragments soaked into trypsin crystals required the growth of well over 3,000 'production' crystals and the collection of well over 1,000 data sets at the Australian Synchrotron [5].

Methods
All SPR Experiments were performed using a Biacore T100 instrument (GE Healthcare). Trypsin was immobilized onto a CM5 chip using standard amine coupling chemistry. Benzamidine was used as a positive control to validate trypsin activity on the chip. The binding capacity of immobilized trypsin (R max ) was increased by purifying the protein using size exclusion chromatography and immobilizing the protein in the presence of 5 mM benzamidine and up to 20 mM CaCl 2 . Typically in SPR experiments, a gradual decrease in analyte binding capacity (R max ) by the immobilized protein is indicative of protein decay. CaCl 2 is a structural inhibitor of trypsin [6] and its presence was observed to prolong the activity of the immobilized surface.
For the fragment screening experiments, two of the four channels (flow cells 2 and 4) on the chip surface had trypsin immobilized. One trypsin surface was 'aged' in that it was put down 24 h prior to application of the second trypsin surface to see what the effect of this would be on binding. Our expectation, which was borne out, was that this aged protein would have less binding capacity and we should see a comparably lower response in this channel for real hits. Bovine carbonic anhydrase II (CAII) was immobilized in flow cell 3 where it served as a negative protein control. Flow cell 1 was left intact and used as a reference (blank) surface. Maybridge library fragments, previously prepared at 100 mM in neat DMSO (master stocks), were diluted into SPR running buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.05% (v/v) Tween-20, 1 mM CaCl 2 and 5% [v/v] DMSO) to 100 lM and injected over the chip surfaces. To assess the stability and reproducibility of the assay, positive controls (benzamidine and p-amino-benzenesulfonamide) were injected several times throughout the screening experiment. Three hundred and eighty-four fragments in a 384-well plate were screened within approximately 30 h. Remaining compounds were screened later using a similar screening approach. Scrubber (http://www.biologic.com.au) was utilized for data processing and analysis. SPR signals were referenced against the blank surface and further corrected for DMSO refractive index changes (excluded volume effect). Binding data were normalized for the molecular weight of the fragments. The normalization scheme of Giannetti et al. [7] was further applied to the processed data based on the maximal binding response (Rmax) determined from fitting the control compound sensorgrams. Compounds showing undesirable SPR binding characteristics similar to those described previously [7] were removed from the screening data.
The selected top 20 hits were further analysed using dosage experiments. These were performed at 20°C by injecting a concentration series in two-fold dilutions (C = 4-256 lM). To estimate binding affinities (equilibrium dissociation constant, K D ), binding responses at equilibrium (R eq ) were fit to a 1:1 steady state affinity model (available within Scrubber) which utilizes a nonlinear least squares regression method to fit the Langmuir adsorption isotherm (R eq = R max *K D /[K D ? C]) to each data set. A normalized saturation response (R max ), derived using the reference compound, was applied to the responses obtained with fragment hits that, due to solubility and chip surface artefact issues, could not be injected at or near saturating concentrations. A SPR dosage experiment for benzamidine binding to immobilized trypsin is shown in Fig. 1a. Interestingly, a marginally higher affinity was consistently estimated in the presence of CaCl 2 where the K D for benzamidine binding to trypsin was measured to be *7 lM whereas in the absence of CaCl 2 , K D was estimated to be approximately *15 lM (data not shown).
To further confirm our SPR and crystallography hits, isothermal titration calorimetry experiments (ITC) were performed using a MicroCal Auto-iTC 200 (GE Healthcare). Trypsin solutions were freshly prepared in 50 mM Tris-HCl, 10 mM CaCl 2 , pH 8.0 and dialysed overnight against the same buffer at 4°C. Prior to titration, the trypsin solution was spiked with DMSO to match the 5% (v/v) DMSO in the small molecule solution. Fragment solutions (concentration in the range 1.8-16 mM, depending on the specific inhibitor) were titrated into the stirred (1,000 r.p.m.) cell (300 lL) containing trypsin solution (0.16-1.6 mM). Data were analysed using Origin software by fitting a single-site binding isotherm that yields DH (enthalpy of binding) and K A (binding constant). These titration experiments only allowed for estimation of the tightest binding fragments from the SPR hit list (K D \ 300 lM, Table 1). Weaker binding fragments could not be accurately measured due to the very high concentrations of both protein and compound required to generate sufficient heat that can be detected in the microcalorimeter. A more detailed description of the SPR and ITC experiments, along with the PDB coordinates of the fragment hit structures, will be published in the near future (manuscript in preparation).
All of the crystallization experiments were performed at the Collaborative Crystallisation Centre (C3) at CSIRO in Melbourne, Australia. Drops were set up in two subwell sitting drop plates (SD-2, IDEX Corp) using a Phoenix robot (Art Robbins Industries) with 50 lL of crystallant in the reservoir and droplets consisting of 300 nL of the reservoir and 195 nL of the protein sample and 5 nL of seed stock [8]. Only one of the two crystallisation subwells was utilised for the initial crystallisation. A robotic procedure using a Mosquito robot (TTP) was developed to place a mixture of fragment and a cryoprotectant onto the both the crystallisation droplet and the unused subwell in the sitting drop plates [9]. The second subwell was used as part of the 2 step soaking procedure to make sure the fragments had a chance to displace the benzylamine in the crystals. After allowing the fragments to soak into the crystals for 24-48 h, the crystals were transferred manually to the fresh fragment/cryoprotectant solution in the second subwell and allowed to soak an additional 24-48 h. Crystals were gently removed using mylar loops (MiTeGen) mounted in copper pins (Crystal Positioning Systems, USA) and cryo-cooled in liquid nitrogen and placed in a 96 hole cassette that was kept submerged in liquid nitrogen until the individual pin with the crystal of interest was placed in the X-ray beam at the Australian Synchrotron. At least two crystals were harvested for each of the soaks and data sets were attempted for both in all cases. 181 frames of data, each one a 1°oscillation with 1-3 s of exposure, were taken for each crystal. All of the data sets were initially processed using a script called Jigsaw [5] (available upon request) that uses the following crystallographic programs to automatically index, scale, do molecular replacement, an initial round of refinement and then try to place a ligand in the excess density of the active site (when present): XDS [10], Pointless (CCP4) [11], SCALA (CCP4) [11], Phaser (CCP4) [11], Refmac (CCP4) [11], Flynn (OpenEye) [12]. Coot was used to visualize the model and electron density as well as manually rebuild the model where there were changes [13].

Discussion
This was a project which could not have been attempted without a lot of recent (and expensive) tools: for example, automation in crystallogenesis, X-ray data collection and computing. It is notable that the technology for one of the major techniques used in this project, surface plasmon resonance, only became available in the early 1990s. In all,  to assemble the experimental underpinnings for this project took five domain experts close to 2 years, and required equipment that was millions of dollars to purchase and run. This is excluding the cost of the Australian Synchrotron, where the equivalent of about a month of continuous beamtime was required to collect the X-ray diffraction data for this challenge. If we were to attempt this same amount of data collection on a standard X-ray home source it would take closer to a decade of continuous beamtime to collect the same amount of data. Similarly, about 200 96-well crystallisation plates were set up during the course of this experiment; by hand, assembling that many experiments would take close to 3 working months, and that is without even taking a peek at the experimental results once they were set up. The enormity of the project is quite obvious to most experimentalists, and explains why this type of challenge has not been taken on previously; the modelling community has been relying on retrospective analyses in part because the prospective data are so expensive to obtain. These experimental data are not perfect: there is 'real world' noise in the data-machines break, chemicals degrade, data get misplaced (despite best efforts) and then the reality is that data from different biophysical techniques cannot be cleanly compared to each other. The use of amine coupling techniques to prepare SPR chips precludes the use of Tris buffers to attach the protein to the chip. The requirement for cryoprotection of protein crystals results in protein structures with blobs of extra density which are from the ethylene glycol cryoprotectant rather than any fragment. There are numerous examples where the details of experimental setup are where the difficulties lie.
Looking at the differences in the techniques, we see that the pH and buffer was different for each: SPR used 50 mM Hepes pH 7.4, 150 mM NaCl, 0.05% Tween P20, 20 mM CaCl 2 ? 5% DMSO for the fragment; ITC used 50 mM Tris pH 8.0, 10 mM CaCl 2 ? 5% DMSO for the fragment; and crystallization used 22.5% w/v PEG 3350, 0.18 M (NH 4 ) 2 SO 4 , 0.12 M NaSCN, 0.09 M Bis-Tris pH 5.5, 0.01 M Tris pH 8.5, which gave a final pH of 5.8. DMSO was used in all cases as the fragments were solubilized in neat DMSO at the start. It should be noted that the crystals obtained for soaking were in space group P2 1 2 1 2 1 , whereas most of the crystal structures determined for the co-crystallization with fragments were found in P3 1 2. This is due to the fact that when DMSO is present during the crystallization process, the space group tends to fall into the trigonal space group. There may also be some influence due to a pH change-the co-crystal experiments were done at pH 7.0 instead of pH 5.8. We have typically found that SPR is a reliable method for estimating binding constants of fragments up to K D = 250-500 lM, but beyond this level the error associated with the measurements can become significant. In particular, the insolubility of fragments in SPR compatible buffers and at high fragment concentrations, can cause chip surface interaction artefacts, and this prevents fragment injections at or near the saturating concentrations required for accurate affinity estimations. As discussed previously, by applying a normalization scheme based on the saturation response from a positive control it is possible to estimate affinities without achieving saturation (Fig. 1b, c). Using this approach we attempted to estimate K D up to 1 mM values, but as can be seen in Table 1, the correspondence between the SPR and crystallography methods breaks down beyond 300 lM and we saw no fragments in the crystal structures beyond the 500 lM barrier. Although we were limited by the solubility and weak binding of the fragments in the ITC experiments, the correlation between the SPR and ITC is relatively good (see Table 1). For most of the SPR hits better than 300 lM, we have multiple X-ray data sets to confirm the position of the ligand found in the binding site. All of the ligands found to date sit in the same binding site as the benzamidine and benzylamine controls and all have a primary amine that binds to the Asp189 residue of trypsin (see Fig. 2). Trypsin is a rather rigid molecule and besides a few rotomer changes of side chains, there are no large loop or domain movements upon binding these fragments.
We conclude from looking at our experimental results that the rigidity of the target limited the hit rate of fragment binding, and that an experienced protease expert would have looked at the fragment library and picked out the likely binders simply by choosing molecules that look somewhat akin to well known protease inhibitors such as benzamidine. This would have probably taken an afternoon, rather than the 2 years to collect the experimental results! However, despite the 'obviousness' of the results in retrospect, there was no modeling technique that found or ranked all the experimental hits correctly, showing clearly the value of this work-without guides to let us know when an approach/method isn't working, that method cannot advance. We are glad that the experiments have opened up new directions for modeling development, and in future years (when the memory of this data collection has faded) we may be able to do this again to see how far modeling has progressed.


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Synthesis of novel 3-benzyl-2-phenyl-4(5H)-(substituted phenylhydrazino)-1, 3-oxazolidines and their antihyperglycemic activity

A number of 3-benzyl-2-phenyl-4(5H)-(substituted phenylhydrazino)-1, 3-oxazolidines 6a-f derivatives were synthesized and their antihyperglycemic activity was evaluated by streptozotocin (STZ) and sucrose-loaded model. Many of oxazolidine derivatives as well as their corresponding substituted phenylhydrazine showed significant reduction in blood glucose level in STZ and sucrose-loaded rat models.

INTRODUCTION
Diabetes mellitus is a main threat to human health in the 21 st century. [1] The incidence of the disease currently is estimated to reach 300 million by the year 2025. [2] Oxazolidine is an important group of heterocyclic compounds that plays a vital role in many biological activities in the areas of medicine and molecules containing oxazolidine ring systems and exhibits many activities such as anti-TB activity, [3] antibacterial activity, [4] mono-amine oxidase inhibition, [5] and GP IIb/IIIa antagonism [6] neuroleptic activity. [7] The information on oxadiazolidine as antihyperglycemic agents has been reported previously. [8] In view of the literature led to the conception that a novel series of 3-benzyl-2-phenyl-4(5H)-(substituted phenylhydrazino)-1, 3-oxazolidines 6a-f derivatives were synthesized and their chemical structure were confi rmed by IR, 1 H-NMR, mass spectral and elemental analyses. These compounds were screened for their antimicrobial activities.

Materials
Synthetic starting material, reagents and solvents were of analytical reagent grade or of the highest quality that were commercially available and were purchased from Aldrich Chemical Co (Hyderabad, AndhraPradesh, India), Merck Chemical Co (Mumbai, Maharashtra, India) and Acros Organics (Mumbai, Maharashtra, India) and were dried when necessary.
The melting points were taken in open capillary tube and uncorrected. IR spectra were recorded with KBr pellets (ABB Bomem FT-IR spectrometer MB 104 ABB Limited, Bangaloru, India). Proton ( 1 H) NMR spectra (Bruker 400 NMR spectrometer, Mumbai, India) were recorded with TMS as internal references. Mass spectral data were recorded with a quadrupole mass spectrometer (Shimadzu GC MS QP 5000, Chennai, India) and microanalyses were performed using a vario EL V300 elemental analyzer (Elemental Analysensysteme GmbH, Chennai, India). The purity of the compounds was checked by TLC on pre-coated SiO 2 gel (HF 254 , 200 mesh) aluminium plates (E. Merck) using ethyl acetate:benzene ratio of 1:3 and visualized in UV chamber. IR, 1 H-NMR, mass spectral data and elemental analyses were consistent with the assigned structures.
The in vivo antihyperglycemic activity of synthesized compounds was evaluated using male Sprague-Dawley rats in two diff erent models.

Streptozotocin model
A solution of streptozotocin (STZ) (60 mg/kg) in 100 mM citrate buff er, pH 4.5 was prepared and calculated amount of the fresh solution was dosed to overnight fasted rats (60 mg/kg) intraperitoneally. The blood sugar level was measured a er 48 h by glucometer. Animals showing 200-400 mg/dL were selected for antidiabetic screening. The diabetic animals were divided into groups of six animals each. Rats of experimental group were administered a suspension of the desired test sample (prepared in 1% gum acacia) orally (100 mg/kg body weight). Controlled group animals were also fed with 1% gum acacia. The blood glucose levels were measured at 1-, 2-, 3-, 4-, 5-, 6-, 7-and 24-h intervals. The % fall in blood glucose from 1 to 24 h by test sample was calculated according to the area under curve (AUC) method. The average fall in AUC in experimental group compared to control group provided % antihyperglycemic activity.

Sucrose-loaded (SLM) model
Overnight-fasted male Sprague-Dawley rats were used for sucrose-loaded experiment. Blood was collected initially and there a er test compounds were given to the test group consisting of five rats by oral gavage at a dose of 100 mg/ kg body weight. A er half an hour post-test treatment, a sucrose load of 10 gm/kg body weight was given to each rat. Blood was collected at 30, 60, 90 and 120 min post-sucrose load. The % fall in blood glucose level was calculated according to the AUC method.  Figure 1: 3-benzyl-2-phenyl-4(5H)-(substituted phenyl hydrazino)-1, 3-oxazolidines

Antihyperglycemic activity
Among the six screened compounds only 6e, 6a, 6b exhibit antihyperglycemic activity in STZ model and SLM model. Compound 6c displayed 27.3% blood glucose lowering activity at 100 mg/kg dose in SLM model and 6d showed 13.4% blood glucose lowering activity at 100 mg/kg dose in STZ model. In these compounds, 3-benzyl-2-phenyl-4(5H)-4'acetamidophenyl hydrazino-1, 3-oxazolidine 6e reduced blood glucose level to 54 and 71% in STZ and SLM models, respectively. It is evident from Table 1 that, the presence of benzyloxy (6f) caused a complete loss of antihyperglycemic activity. A closer inspection of data from this table indicates that compounds acetamido, bromo and chloro phenylhydrazino substitution of oxazolidine having excellent antihyperglycemic activity compared with standard. The transformation order of screened compounds is 6e>6a>6b>6c>6d>6f. Metformin and glybenclamide were used as standard antidiabetic drug in both the models.