extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Chemistry Medicine Biology

text:
Ursolic acid rich Ocimum sanctum L leaf extract loaded nanostructured lipid carriers ameliorate adjuvant induced arthritis in rats by inhibition of COX-1, COX-2, TNF-α and IL-1: Pharmacological and docking studies

Background Ursolic acid (UA) is a promising molecule with anti-inflammatory, analgesic and potential anti-arthritic activity. Methods This study was undertaken to make formulation and evaluation of Ocimum sanctum L. leaf extract (OLE) loaded nano-structured lipid carriers (OLE-NLCs) for improved transdermal delivery of UA. Different surfactants, solid lipids and liquid lipids were used for the preparation of NLCs. The NLCs were developed using emulsion solvent diffusion and evaporation method. Different physicochemical properties, entrapment efficacy, in vitro release evaluation, and ex vivo permeation studies of the prepared NLCs were carried out. The in vivo anti-arthritic activity of OLE-loaded NLC gel and control gel formulation (OLE free NLC gel) against Complete Freund's Adjuvant (CFA) induced arthritis in wister albino rats was also carried out. Results OLE-NLCs were composed of spherical particles having a mean particle size of ~120 nm, polydispersity index of ~0.162 and zeta potential of ~ -27 mV. The high entrapment efficiency (EE) of UA ~89.56% was attained. The in vitro release study demonstrated a prolonged release of UA from the NLCs up to 12 h. The developed formulation was found to be significantly better with respect to the drug permeation amount with an enhancement ratio of 2.69 as compared with marketed formulation. The in vivo biological activity investigations, studies showed that the newly prepared NLCs formulation of OLE showed excellent anti-arthritic activity and the results were found at par with standard marketed diclofenac gel for its analgesic and anti-arthritic activities. These results were also supported by radiological analysis and molecular docking studies. Conclusion The overall results proved that the prepared OLE-NLCs were very effective for the treatment of arthritis and the results were found at par with standard marketed the standard formulation of diclofenac gel.

Introduction
Rheumatoid arthritis (RA) is a most common chronic inflammatory disease of an autoimmune origin. It affects a large population of the society, especially middle-aged people. Arthritis involves the inflammation of joints (affecting one or more joints), causing pain and stiffness in the joints. It causes destruction of cartilage, bone and affects synovial joints which lead to deformity, destruction and disability of joints. This is growing clinical health problem throughout the world. There are different types of arthritis like rheumatoid arthritis, gout, ankylosing spondylitis, psoriatic arthritis, septic arthritis, pseudo-gout, osteoarthritis, and juvenile idiopathic arthritis. The osteoarthritis is a degenerative joint disease and very commonly found among people. Arthritis might lead to poor physical function due to severe joint pain, hence it may adversely disturb the quality of life. The prevalence statistics of the arthritis reveal that Rheumatoid arthritis (RA) affects approximately 1% of the world population. Arthritis is widely prevalent in the American population and a leading cause of disability. According to the Centers for Disease Control and Prevention (CDC) data on arthritis in the United States; it has been reported that 54.4 million U.S. adults (approximately 25% of the total adult population) are suffering from arthritis. These figures of arthritis in adults are expected to increase up to 67 million by 2030. The total figures of doctor-diagnosed arthritis is projected to reach up to 78.4 million adults (25.9% of all adults) and the arthritis-attributable activity limitation in adults is expected to increase to 34.6 million (11.4% of all adults) by 2040 [1][2]. These data indicated that arthritis is a serious disorder which might be more prevalent than other known diseases like diabetes, cancer and AIDS. Therefore, there is an urgent need to address these growing problems of arthritis. Recent studies report that the occurrence of RA in Saudi Arabia is approximately found to be 2.2 per thousand people. Further, it is reported to be found more commonly in females in comparison to males, and prevalence of this disease increases with age. If RA patients remain untreated, it may lead to high morbidity and mortality. It is also said that RA has also resulted from cardiovascular diseases. In another recently conducted study in Saudi Arabia, the prevalence of hyperlipidemia in patients suffering from RA and its relationship with C-reactive protein level was investigated. It is reported that hyperlipidemia is frequently found among patients suffering from RA and significantly linked to the level of C-reactive protein (CRP) and disease activity. There are continuous efforts by the medical community and researchers to remove all obstacles in the management of RA around the world, particularly in Saudi Arabia [3][4]. The mechanism underlying the inflammatory progression of arthritis involved the infiltration of inflammatory cells into the joints, which causes the proliferation of synoviocytes and damage to the bone and cartilage. Certain proinflammatory cytokines e.g., TNF-α, interleukin-1 (IL-1), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and interferon gamma (IFNγ) are considered as the primary mediators which have a vital role in the pathophysiology of RA. These pro-inflammatory cytokines promote inflammation by tissue destruction, angiogenesis, and leukocyte linkage in RA. Some new drugs have been used to target these primary mediators for the management of RA [5]. Although arthritis is considered as a disease of the joints, but abnormal immune responses may end up causing various articular manifestations. Furthermore, in certain cases, rheumatoid factor (RF) may also cause extra-articular findings. Various allopathic medications are employed in the management of arthritis. The corticosteroids (GCs) and non-steroidal antiinflammatory drugs (Commonly referred as NSAIDs) are used as first line drugs of choice for the treatment of arthritis. NSAIDs are most frequently used drugs as compared with corticosteroids. The mechanism of action of NSAIDs involves the inhibition of cyclooxygenase enzymes both COX-1 and COX-2. GCs are steroid hormones, which act by binding to the glucocorticoid receptors. GCs plays vital role in the treatment of variety of diseases like asthma, autoimmune diseases, allergies, and sepsis etc. GCs have various pleiotropic actions with several potentially harmful side effects. Glucocorticoids are potent anti-inflammatory agents which can inhibit all stages of the inflammatory reactions. GCs are frequently used to treat a wide range of inflammatory diseases. Long term use of GCs use might lead to serious adverse effects like immune-suppression, fluid shifts, psychological changes and brain changes. Mechanism of corticosteroids action also involves the inhibition to the induction of COX enzyme. In addition to this, the corticosteroids also reduce the macrophage phagocytosis and interleukin-1 (IL-1) secretion in order to inhibit the release of lysosomal enzymes and collagenase [6]. The allopathic medications suffer from certain untoward effects. For instance sulfasalazine causes gastrointestinal disturbances, malaise and headache. Use of gold compounds is associated with skin rashes, mouth ulcers and blood dyscrasias and methotrexate may cause pulmonary fibrosis. Common side effects of NSAIDs include nausea, dyspepsia, vomiting, skin reactions [7]. Herbal medications on the other hand, are devoid of such untoward effects. Natural products in the form of the conventional dosage forms have been used for a long time. Use of nanotechnology in drug delivery technique is relatively new to pharmaceutical technology. The size range of nanoparticles is <250 nm. Because of their very small size they are benefitted with various properties that could be used in drug delivery techniques. Various nanoformulations have been prepared for herbal components and their rich extracts. They have been proven to provide additional benefit over the conventional dosage forms. For example, paclitaxel provides enhanced anti-tumor activity in the form of nano-particles as compared to its conventional dosage form. Aloe vera extract, Ginkgo biloba extract, curcumin and many more phytoconstituents have been modified into nano-type so as to provide additional benefit to the users, thereby increasing patient compliance [8][9].
In the present investigation a well-known Ayurvedic herbal drug Ocimum sanctum L was chosen for the undertaken research study. Ocimum sanctum is an important aromatic plant (Family Lamiaceae). It is commonly called as tulsi or holy basil. It is also known as Ocimum tenuiflorum and is frequently used by many in the treatment of variety of disorders/diseases throughout the world. It is inhabitant to the Indian subcontinent and is abundantly found in India. It is a very popular herb in Ayurvedic system of medicine and holds the highest regard for both its medicinal properties and religious beliefs. It is said to be an "elixir of life" [10]. Tulsi is known as "The Queen of Herbs," and "Mother Medicine of Nature" [11][12]. In India, tulsi has been accepted in many religious customs and lifestyle practice for various health benefits. Daily use of tulsi is believed to promote general health, wellbeing and prevent various diseases and illness. In addition to this, several in vitro and in vivo scientific studies have been published throughout the world that provide the scientific basis of the medicinal properties of tulsi. Tulsi possess a wide range of pharmacological actions and used in the treatment for the variety of disorders/diseases like arthritis, asthma, anxiety, back pain, cardiac disorders, cough, dysentery, diarrhea, eye diseases, fever, genitourinary disorders, indigestion, gastric disorders, malaria, skin diseases, vomiting, ringworm, insect, scorpion, and snake bites. Tulsi is considered as a potent adaptogen which helps to improve the adaptation to stress and offer great support to maintain homeostasis [13][14][15][16]. The leaves have been reported to contain 0.7% volatile oil, comparing of eugenol (71%) and methyl eugenol (20%) and other constituents [17]. This is reported in the literature that leaves extract contains a higher concentration of ursolic acid [18]. Ursolic acid (UA) forms an indispensable part of Indian traditional system of medicine. Chemically speaking, UA is a triterpinoid and form an important constituent of Tulsi [19]. Moreover, it is also an important component of several herbal medicines marketed worldwide. UA (Fig 1) is known to possess analgesic, anti-inflammatory, anti-atherosclerotic, anti-cancer, anti-diabetic, anti-epileptic, hepato-protective, anti-hyperlipidemic, anti-fertility, anti-platelet aggregation, anti-tuberculosis and anti-HIV activities [20][21][22][23][24][25].
The preparation of crude extract is comparatively simple, easy, cheap and consume lesser time as compared with the manufacturing procedure of pure compounds. In addition to this, a crude extract contains natural active ingredients. The crude extracts also have potent pharmacological actions as compared to pure compound due to the synergistic outcome of the other compounds present in the extracts. Thus, in the current study, Ocimum leaf extract (OLE) was used instead of pure ursolic acid (UA). Nanostructured lipid carriers (NLCs) are lipid nanoparticles of the 2 nd generation. These lipid nanoparticles were prepared after the first generation; solid lipid nanoparticles (SLNs) [26][27]. NLCs are distinct from SLNs with respect to their solid matrix composition. SLNs contains solid lipids only, on the other hand, NLCs consist of the mixture of liquid and solid lipids. NLCs exhibit more advantages as compared with than SLNs since NLCs present a smaller amount of lipid matrix which might offer extended loading capacity and NLCs also overcome a number of possible problems encountered with SLNs like expulsion of the drug for the period of the storage, lower drug loading and higher water content of SLNs distribution [28]. Further, the lipid nanoparticles the loaded active compounds have an additional advantage to prevent the degradation of the drug and also improve the penetration to the skin. The present study was aimed to develop herbal extract based NLCs to enhance bioavailability of UA and other constituents present in the extract for better management of arthritis with lesser adverse effects.

Quantification of UA by High Performance of Thin Layer Chromatography (HPTLC)
The quantification of ursolic acid (UA) was performed using HPTLC (CAMAG, Switzerland). The samples of ursolic acid were smudged in the form of bands having width 5 mm with a CAMAG microliter (μL) syringe on pre-coated silica gel aluminum plate 60 F254 (20 cm ×10 cm with 0.2 mm thickness, E. Merck, Germany) using a Camag Linomat V (Switzerland) applicator. The mobile phase was composed of Toluene: Acetone: Formic acid (7.8:2.2:0.15 v/v/v). Densitometric scanning was carried out on CAMAG TLC scanner III in the absorbance mode at 245 nm.

Preparation of Ocimum leaf extract loaded nanostructured lipid carriers (OLE-NLCs)
The solvent evaporation method was adopted to prepare the OLE-NLCs followed by ultrasonication [29][30]. GMS as solid lipid, capryol-90 as liquid lipid and OLE were dissolved in ethanol maintained in water bath at 80 0 C to form lipid phase. Ratio of total lipid and organic solvent was kept at 1:20. The aqueous phase was formulated by dispersing 3% Tween 80 in distilled water maintained at the similar temperature as that of lipid phase. The lipid phase was scattered in a hot surfactant aqueous solution pre-heated at the equal temperature added drop by drop to the aqueous phase and stirred continuously at 80 0 C until all the organic solvent was evaporated. The hot lipid nano-emulsion so obtained was a probe syndicated at 40% amplitude for 4 minutes. Lastly, the stirring of obtained nano-emulsion (O/W) was carried out on magnetic stirrer at 600 rpm for one hour approximately and then it was made to cool down at normal room temperature with continuous stirring. Finally the nano-emulsion was mixed with 3% mannitol. Blank NLC was also synthesized without the addition of OLE into the lipid phase.

Evaluation of morphology, particle size and zeta potential
The dynamic light scattering (DLS) technique was used to measure the size and zeta potential by using Malvern Instruments (HAS-3000, Malvern, UK). OLE-NLC suspension was mixed with the suitable medium and measurements of the particles were recorded in triplicate. The poly dispersity index (PDI) was established to determine the homogeneity [31]. The determination of the morphology of OLE-NLC was carried out by transmission electron microscopy (TEM Morgagni 268-D; FEI Company, Eindhoven, Netherlands). A single drop of the dispersion was placed to a carbon-coated copper grid and kept for one minute to let the OLE-NLC to stick to the carbon substrate. The residual dispersion was eliminated by absorbing the drop with a piece of filter paper. Subsequently, a drop of 1% phosphotungstic acid was kept on the grid and this sample was then allowed to dry in the air and then scanned. The image was taken by using Soft Q3 Imaging Viewer software.

Entrapment efficiency
Entrapment efficiency (EE) was calculated in terms of UA content. The untrapped drug was removed by centrifugation of the sample at 4˚C for 30 minutes at 14,000 rpm (REMI Cooling centrifuge, C-24, Mumbai) and the supernatant liquid was collected separately, then phosphate buffer was added to make dilution. The quantity of UA was estimated by HPTLC (CAMAG, Switzerland) method as illustrated above. The entrapment percentage of the UA was determined by using the equation given below [31].
In the equation, Ct means is the concentration of total UA and Cr indicates the concentration of free UA.

Preparation of Rat's skin for permeation studies
Wistar albino rats (200-250g) were used to take their skin for permeation studies of the NLCs. Care of the rats was taken in accordance with the institutional guidelines. The rats were scarified by cervical dislocation with utmost care to avoid pain to the animals. The skin was surgically removed safely from the abdominal area. The hairs were shaved and eliminated with help of an electric clipper and subcutaneous fat was eradicated by treating the excised skin with isopropyl alcohol (IPA). The excised skin was then thoroughly washed with fresh distilled water and then, it was physically checked for any possible damage during surgical removal. This final cleaned skin was properly wrapped in a sterile aluminum foil, and carefully stored at -21˚C and was used within a week.

Ex vivo skin permeation studies
The Franz diffusion cells with 1 cm 2 area and 10 mL volume of receiver cell were used to perform the ex vivo skin permeation studies on the skin of the rat. Full-thickness skin was mounted after removal of hair and fat on diffusion cells with water jacket to measure the permeability to the skin. The stratum corneum side of the skin was placed to face upward towards the donor compartment and dermal side was placed to face downwards to the receptor compartment. The donor cell was filled with 2 mL of OLE-NLC dispersion containing (1.5% OLE) and receptor medium was filled with permeation media at 37˚C with 600 rpm. One mL of aliquot was collected from the receiver cell at specific time intervals for 24 h and an equal volume of the fresh receptor medium was filled immediately. The collected samples were diluted and were filtered using 0.45 mm membrane filter and analyzed using HPTLC (CAMAG, Switzerland). Same experiments were carried out for control formulation of UA for the comparison the permeation enhancement. The degree of permeation enhancement, enhancement ratio (ER) was determined as per the below given formula:

In vitro release studies
The OLE-NLC formulation was converted into gel based on the highest entrapment efficiency and ex vivo transport data. The OLE-NLC gel was prepared by adding Carbopol-934 (1%, w/ w) and set aside for the whole night for entire humectation of polymer chains. Some other components, e.g. 15% (w/v) PEG-400 and Triethanolamine (TEA) (0.5%, w/v) were also added to obtain the homogeneous dispersion of gel [32][33] and were taken for the assessment of analgesic and anti-arthritic activity. The control gel formulation (hydroethanolic solution 7:3) was also converted in gel as per the above procedure. The in vitro release experiments were performed for OLE-NLC gel formulations by paddle method. The release was performed with phosphate buffer (pH 5.5) and temperature was kept maintained at 32 ± 0.5˚C to pretend both, pH and temperature of the human skin. Both the formulations were precisely weighed and putted in a dialysis bag which was closed from both sides. The whole assembly was kept at the base of the USP dissolution tester (VDA-8DR; Veego Scientific, Mumbai, India). The vessel contained buffer solution (500 mL) and speed was fixed to 50 rpm [34]. Aliquots (5 mL) were taken out from the release medium at different intervals of time and substituted by an equal volume of release medium. The released amount of the drug from the gel was found out by HPTLC. The obtained data from the undertaken release studies were then kinetically analyzed and the drug release pattern from both formulations were calculated.

Animals
The healthy Wistar albino rats (100-150 gm) of both sexes were utilized for the in vivo study to assess the anti-arthritic activity of OLE-loaded NLC gel and marketed control gel formulation. All animals used in this study were acclimatized for one week by providing standard laboratory conditions. The animal rooms were maintained at 24 ± 1˚C temperature; 45-55% relative humidity with 12:12 hours dark and light cycle. The animals were supplied with standard marketed pellet diet and distilled water ad libitum. The institutional guidelines on use and care of experimental animals were observed throughout the experimental work on the animals. The study protocol was approved by the Research Ethics Committee at HIT department, Jeddah Community College, King Abdulaziz University, Kingdom of Saudi Arabia (approval number: HIT/JCC-111-1438).

In vitro anti-arthritic activity
In this study, the in vitro anti-arthritic activity of OLE-NLC gel and marketed gel formulation containing diclofenac sodium as standard were assessed by protein denaturation method. The denaturation mechanism, possibly involves modification in electrostatic hydrogen and disulphide bonding [35][36]. The reaction mixture (0.5 mL) contained 0.45 mL of bovine serum albumin (5% aqueous solution) and 0.5 gm each of both formulations. The pH was adjusted to 6.3 by using a little quantity of 1N HCl. The samples were then incubated at 37˚C for 20 minutes and heated at a temperature of 57˚C for the duration of 30 minutes. The sample was cooled and 2.5 mL of PBS (pH 6.3) was added in each sample. Similarly, the control test was performed and turbidity was precisely measured by the spectrophotometer at the wavelength of 660 nm. The percentage inhibition of the protein denaturation was then simply calculated by using the formula as given below.

In vitro COX-1 and COX-2 assay
OLE-NLC gel and marketed gel formulation containing diclofenac sodium were evaluated for COX-1 and COX-2 inhibition. The COX inhibition screening standard assay kit (Catalog No. 760111, 760700; Cayman Chemical, USA) was used to perform the inhibitory assay of COX-1 and COX-2. The instructions supplied by manufacturer were followed [2].

In silico Molecular Docking Analysis
To further strengthen the results of our in vitro and in vivo studies, we also performed in silico molecular interaction analysis on Ursolic acid and COX proteins using the Auto dock tool.

Protein setup
Due to unavailability of crystal structural of rat COX enzymes, we retrieved the crystal structure of Ovis aries COX-1 (PDB ID: 1CQE) and Mus musculus COX-2 (PDB ID: 6COX) which is having more than 90% sequence homology rat Cox enzymes obtained from the Protein Data Bank available online [www.rcsb.org] [37]. The crystal structures PDB files were manually edited by removing heteroatom's and only chain A of COX-1 and COX-2 were selected for docking studies. The protein structure's quality was further improved by energy minimization using steepest descent algorithm, in GROMACS tool [38]. During structure minimization, the hydrogen were flexible and amino acid atoms were fixed at their coordinates. After reaching the saturated gradient, the complex structure was saved for further annotations. Finally the protein prepare for docking by adding charges (gigester), H-bonds (polar only) and neutralizing the histidine charges [39].

Drug preparation and docking method
The drug Ursolic acid (CID 64945) co-ordinate was downloaded from the online PUBCHEM database [https://pubchem.ncbi.nlm.nih.gov/] [40] and atomic partial charges were then added to drug using PRODRG server [41], Audock 4.0 [42] tool used to perform molecular docking analysis in between protein and drug. Ligand parameter sets for docking analysis, as gyratory bonds kept flexible in docking with rigid protein structure. The blind docking were performed on rigid protein structure by giving the grid box dimension 70X60X60 at 0.375 Å center spacing. By using autorid4.0 the affinity maps for all the atoms (HD, C, NA, N, A, SA, Cl, S, Br, F, P and I) were calculated. Finally the Lamarckian genetic algorithm (LGA) search parameter was stimulated to activate the ligand-protein with one hundred simulations runs, with three hundred population size. The final docking results were prioritized based on the binding energies (lowest value).

Complete Freund's Adjuvant (CFA) induced arthritis in rats
The in vivo study in rats was undertaken to assess the anti-arthritic activity of OLE-loaded NLC gel and control gel formulation (OLE free NLC gel). The rats were picked randomly and divided into four experimental study groups. There were six animals in every experimental study group. Group-I (Normal control): treated with OLE free NLC gel (applied topically); Group-II (CFA. Toxic control): No drug treatment; Group-III (Treated group): Treated with OLE loaded NLC gel (1.2% applied topically); Group-IV (Positive control): Treated with marketed gel containing 1.16% diclofenac applied topically. The study was carried out for 21 days. On day 0, all the groups except group-I were inoculated by intradermal injection of CFA. The CFA suspension (0.1mL) was injected into the sub-plantar area of the left hind paw of all animals in each group. The CFA mainly contains the suspension of heat-killed Mycobacterium tuberculosis (1 mg/mL) into mineral oil. CFA causes inflammatory responses within 24 hours after injection. The volume of the hind paw was estimated by using the digital Plethysmometer. The paw volume was taken as parameter to measure the anti-arthritic activity of newly prepared NLCs and standard control marketed diclofenac gel [43].

Biochemical estimation and radiological analysis
For the analysis of biochemical parameters, the blood sample was collected from all groups of rats into anticoagulation tubes. All required reagents were precisely added to find out the Erythrocytes (Red blood cells) count, Leucocytes (White blood cells) count [44], Erythrocyte sedimentation rate (ESR) [45], and Hemoglobin (Hb) contents [1]. Radiological analysis was also done to confirm the activity. The images were taken and safely compared.

Inflammatory mediators analysis
The cytokines inflammatory mediators e.g. tumor necrosis factor alpha (TNF-α) and Interleukin-1 (IL-1) were precisely estimated using the ELISA kits methods (Biosource Int., Camarillo, CA, USA) as per the information supplied by the manufacturer of the kits [2].

Analgesic activity
Analgesic activity of the test samples was performed by the hot water immersion tail-flick method. The treatment was given by gently applying the formulations of each group on the dorsal surface of the paw with the index finger. Five-centimetre portion of the tail of animals was immersed into hot water at 55 ± 5˚C after 15 minutes treatment of gel. It was observed that in response to thermal stimuli, rats were quickly responded to withdrew their tail within few seconds. The cut off time (50 seconds) was undertaken to avoid any physical damage to the tissues of the tail of the rats. Time at which the animals withdrew their tail was noted [46].

Statistical analysis
Statistical analysis of the data was done by analysis of variance (ANOVA) using Prism software (Graph Pad, version 5.0 San Diego, CA, USA). All values in this experiment are articulated as mean ± SEM. The values were regarded as significant, if P value was found p<0.05.

Preparation and determination of Ursolic acid content of the extract
Preparation of OLE was done by adopting the method explained previously in the methods section. The extract was prepared with the yield of 8.85 ± 0.97% w/w in comparison with the weight of dried powder. The extract powder was found dark brown in color. UA was qualitatively estimated in extract of O. sanctum by HPTLC. A sharp peak was found at Rf value 0.48 (Fig 2). Based on HPTLC analysis, UA was revealed as the key constituent in the extract. The extraction method was efficiently increasing the UA content in the extract to 60.81 ± 5.23% w/w.

Preparation and characterization of OLE loaded NLC
A total of ten NLC formulations were prepared by varying the ratio of solid lipid, liquid lipid, and surfactant, keeping drug concentration constant. The prepared formulations were characterized for particle size, PDI and TEM etc.

Analysis of particle size and zeta potential
The obtained results are given in the Table 1 (Fig 3). The mean particle size of OLE-free NLC and OLC-loaded NLC was found to be smaller than 130 nm with the PDI values of lesser than 0.2 demonstrating that the system has moderately narrow size distribution.

Transmission electron microscopy (TEM)
In order to get some additional information on the morphology and particle size, TEM was also done to take photos of OLE-NLC (Fig 4). As anticipated, particles found to be a spherical shape with varying size ranging approximately from 90 to 120 nm. Compared to diameter determined by DLS, the diameter observed by TEM was smaller, which might be explained by saying that the two methods were depending on different batches of samples and diverse principles [26,47]. It was reported that diverse morphologies of NLC were observed by different microscopes [48][49]. These discrepancies might be resulted due to lipid compositions and surfactants and/or due to the photographic tools used [50].

Entrapment efficacy (EE)
The entrapment efficiency (EE) is an important physicochemical characteristic of lipid nano-particles as carriers of drugs. It is defined as their capacity for drug encapsulation. Based on the literature, a number of drugs have been effectively trapped in the SLN and NLC [51]. In comparison of the encapsulation efficiency between SLN and NLC, it was revealed that NLC showed a higher capacity of drug loading and entrapment efficiency (E. E.) [52][53]. In addition to this, it was also examined that the amount of encapsulated drug enhanced on raising the amounts of oil [29], which attributed to the better solubilization of lipophilic drugs in oils than in solid lipids. In the present investigation, EE of the best formulation was 89.56 ± 6.58%.

Ex vivo skin permeation studies
The ex vivo transport study profile of all the formulations showed enhanced skin permeation data in comparison to control formulation. The maximum transdermal flux value was 92.51 ±11.47 μg/cm 2 /h over control formulation (34.39 ±12.54 μg/cm 2 /h) with an improved ratio of 2.69 across rat's skin. The developed formulation exhibited biphasic kinetics, with an elevated initial flux of UA during the first 3 hours followed by a decreased flux over the 3-12 hours period of time. These results printed out that the type of oil and surfactant display vital role in UA permeation in the course of rat's skin. On a therapeutic Consideration, a high initial flux can be regarded as beneficial since adequate quantity of biologically active ingredients are quickly released from the gel to the skin to exhibited an primary therapeutic action followed by a desired controlled release of the remaining bioactive components from the NLC. The flux and permeation of the drug through the rat skin were calculated using the following two equations. Equation-I is J = Q/(A X t) Where J is flux, Q is the quantity of compound traversing the membrane in time t, and A is the area of exposed membrane in cm 2 and Equation-II is Kp = J/ C 0; Where J is flux, C 0 is initial concentration of drug and Kp is the permeability constant.

In vitro release evaluations
The in vitro release evaluations on both conventional OLE gel and OLE loaded NLC gel demonstrated a prolong released characteristics. The developed formulation exhibited a biphasic pattern with a surge release for the duration of the first 3 hours, followed by an extended release maximum (82%) up to 12 hours. Conversely, a rapid release rate (96%) of the UA from conventional OLE gel was found as compared to OLE loaded NLC gel. It might be due to the diverse melting characteristics between liquid lipid and solid lipid. The solid lipid having a higher melting point may possibly crystallize first, which results small or no liquid lipid trapped into the core throughout the process of cooling. Lastly, many liquid lipids were situated on the superficial surface of a shell of the nano-particles which could be credited to fast drug release at the initial stage detected above [29]. Therefore, the rupture of nano-particles demonstrated the surge release at the beginning stage and subsequently sustained extended release.

In vitro anti-arthritic activity of OLE-NLC-gel formulation
This is well documented and established fact that denaturation of protein is an important and major manifestations of arthritis. Formation of auto-antigens as in the case of rheumatoid arthritis might be due to de-naturation of proteins. Diclofenac gel (1.16%) was taken as a standard gel to compare the results with developed NLC gel formulation. The results revealed that standard gel showed percentage inhibition of 87.45%, whereas OLE-NLC showed percent inhibition of 84.67%. The above results showed that our laboratory formulation efficiency was comparable with that of the standard gel.

Analgesic activity OLE-NLC-gel formulation
The results of analgesic activity in rats showed that the thermal stimulus time (TST) in animals of the negative control group was found to be 15.43 seconds, whereas standard gel formulation showed TST of 28.85 seconds. OLE-loaded NLC gel group animals showed TST as 34.87 seconds which is higher than standard (p<0.01). The control formulation showed TST of only 19.88 seconds which is higher than that of negative control. The enhancement in analgesic activity in case of developed formulation is due to higher penetration of drug into skin as compared to control and standard treatment. This is evident from the above results that OLE-NLC-gel formulation exhibited better analgesic activity as compared with the standard control gel.

Effect of OLE-NLC-gel formulation on volume of Rat's paw in CFA induced arthritis
The Paw volume of the rats was recorded on 0, 5th, 12th and 21st day from the initiation of the study. The results indicate that there was an increment in paw volume of all groups from Day 0-5. OLE-NLC-gel formulation showed comparable results to those of the standard marketed gel formulation which supports the therapeutic potential of the test formulation (Table 2). Whereas, toxic group showed continuous increase in paw volume due to no-treatment. The CFA induced chronic inflammation is comparable to clinical situations of arthritis. The redness and swelling was shown over 24 h periods in the injected foot with irritation in the arthritis caused by CFA. The inflammatory reactions provoked by CFA induced the arthritis in a few days [54]. CFA increases the migration of monocytes/macrophage and lymphocytes into the synovial cavity of the affected area by inflammation. The arthritis induced by CFA found to develop into two types of reactions. The primary and secondary reactions. The swelling in hind paw was developed in primary reaction, while the swelling(inflammation) in front paw and nodules in tails and ear was the result of the secondary reaction. The OLE-NLC-gel formulation found to significantly (P<0.01) inhibit the immune response in CFA induced arthritic rats. This is due to the capability of OLE-NLC-gel formulation to control the acute inflammation by inhibiting the inflammatory mediator such as PGE2 and reducing the vascular permeability (Table 2) Effect of OLE-NLC-gel formulation on hematological changes in rats of CFA induced arthritis Table 3 displayed the CFA induced the hematological disturbance like reduced level of erythrocytes and hemoglobin (Hb) and increased level of leucocytes (WBC), erythrocytes sedimentation rate (ESR) in comparison with the normal negative control group. These parameters were found significantly altered by OLE-NLC-gel formulation treatment. The anemia is a main problem during arthritic condition which is due to reduction in RBC count and decreased level of Hb. The prominent causes of anemia include abnormal storage of iron in synovial tissue, reticulo-endothelial system, increased leukocyte count and the bone marrow failure to produce more red blood cells in response to the anemia. There was a reduction in RBC count, decreased level of Hb and increased WBC count and increased ESR level in the CFA induced arthritic rats which showed the anemic condition. Treatment of CFA induced arthritic rats with OLE-NLC-gel formulation was found to significant (P<0.01) decrease the leukocyte count. The mechanism of arthritic effects of OLE-NLC-gel formulation might be due to the immune system stimulation and might have the immunomodulatory effect, ESR- (Table 3) [1].

Effect of OLE-NLC-gel formulation on pro-inflammatory mediators in rats of CFA induced arthritis
The results of the treatment of OLE-NLC-gel formulation to CFA induced arthritic rats showed significant inhibition to the pro-inflammatory cytokines like IL-1 and TNF-α as shown in the Table 3. The outcome of these results indicated that the anti-inflammatory potential of OLE-NLC-gel formulation against the CFA induced inflammatory condition, which is very similar to clinical inflammatory manifestations. Since OLE-NLC-gel formulation showed the anti-inflammatory effect against CFA induced arthritis, then further, it was considered worthwhile to evaluate the effect of the OLE-NLC-gel formulation against COX-2 and COX-1 to further elucidate the potential mechanism of anti-inflammatory action ( Table 3). The level of pro-inflammatory cytokines like IL-1 and TNF-α in the knee joint and serum was found to be increased in the RA patients [55]. Numerous inflammatory biomarkers and proinflammatory molecules are reported to investigate the inflammation during the arthritis.
There is a correlation of increased serum level of cytokines and Arthritis. There are various the inflammatory mediators which play vital role in the progress of the inflammation in the joint. A variety of cytokines e.g. interleukins (IL-1, IL-6, IL-8-6, IL-12 etc.) and TNF-α are reported to be involved in the pathogenesis of RA. IL-1 and TNFα collectively responsible to engage the leukocytes into the inflammatory joint in arthritis. TNF-α is a pleiotropic cytokine, mainly secreted by activated macrophages and monocytes. TNF-α regulates the immune cells and plays a vital role in both chronic and acute inflammation. TNF promotes the inflammatory response which causes autoimmune disorders like ankylosing spondylitis, rheumatoid arthritis, and inflammatory bowel disease, etc. TNF-α is reported to be found in the serum and synovial fluid of arthritic patients and it has been linked with clinical symptoms as well as a laboratory marker of RA. The main approach to inhibit the progress of the RA by blocking the TNF-α signal by two ways. Firstly, to decrease the severity of inflammatory reactions and secondly by decreasing the damage to the bone in CFA induced arthritis in rats. IL-1 is an important inflammatory mediator which destroyed the synovial cells by promoting the prostaglandin (PGE2) synthesis and by production of fibroblast in the synovial fluid [56][57]. Our results revealed that OLE-NLC-gel formulation possess a significant antiarthritic effect as shown by the inhibition of above pro-inflammatory mediators.

Radiological studies
X-rays of the hind paw of all groups of rats were taken for the evaluation of the bone damage (Fig 7). In Fig 7(A), the radio graphical analysis indicates no arthritic condition. In Fig 7(B), This group did not receive any treatment, so severe arthritic condition was seen in this group. In Fig 7(C) and 7(D), the X-ray of rat showed good recovery from arthritis, which was treated with OLE-NLC-gel formulation and standard diclofenac gel respectively. The good recovery in the given state might be attributed to the better therapeutic efficacy of OLE-NLC-gel formulation in arthritis and due to greater augmented penetration of OLE-NLC-gel formulation into skin.

Conclusions
The preparation of OLE-loaded NLCs were successfully done by adopting a solvent evaporation method and investigated for in-vitro and in vivo anti-arthritic activity. The gelling agent known as carbopol-934 was suitably utilized to convert the optimized NLCs formulation into gel. In the present study, it was found that OLE-loaded NLC provides enhanced transdermal flux value across rat skin. The in vivo data revealed that the analgesic effect of OLE-NLC gel was comparable to that of standard gel. Rat paw volume showed a depriving effect with enhanced RBC count and decreased WBC count. The results obtained in COX-1 and COX-2 study indicated that developed formulation has inhibitory effects against these enzymes, and also reduced the IL-1 and TNF-α levels. The above observations make it evident our in-house OLE-NLC gel was effective in arthritis management. Further, corroboration was done by radio-graphical analysis, which depicted a good recovery phase in the group treated with developed formulation.


Zirconium‑Mediated Synthesis of Multi‑substituted Dibenzosilole and Benzonaphthosilole Derivatives

In this study, a new zirconium-mediated cycloaddition for preparing dibenzosilole derivatives was developed using silicon-bridged diynes and electron-withdrawing alkynes as starting materials. The preparation of silicon-bridged diynes from 1-bromide-2-iodobenzene, terminal alkynes, and dimethyldichlorosilane was also studied. Unlike in the previous synthesis methods, much higher yields of electron-withdrawing group-substituted dibenzosilole derivatives were obtained. In addition, a new synthesis strategy for preparing benzonaphthosilole derivatives using internal alkynes, 1,4-dibromobenzene, and electron-withdrawing alkynes as starting materials is proposed. Compared with previous methods, alkyl, phenyl, and electron-withdrawing groups can be successfully introduced onto aromatic rings, and the positions of these substituents can be easily controlled. The cycloaddition reactions for dibenzosilole and benzonaphthosilole derivatives are highly efficient one-pot processes, and the raw materials are available and easily prepared. Using these new methods, a series of novel multi-substituted dibenzonsilole and benzonaphthosilole derivatives were obtained effectively.

Dibenzosilole molecules have received much attention because of their applications as important building blocks of different donor units and new conjugated polymers for solar cells [5][6][7], electroluminescence materials [8], and explosives detection [9]. Many researchers have adopted the ringclosure method [10][11][12][13][14][15] for preparing dibenzosilole derivatives. However, this method has some limitations such as the difficulties in introducing substituents onto benzene rings and preparing raw materials. Matsuda et al. [16] reported an iridium(I)-catalyzed cycloaddition method for synthesizing dibenzosilole derivatives (Scheme 1), in which electrondonating groups were introduced onto benzene rings. Unfortunately, the reaction was sluggish when R 3 was a dimethoxycarbonyl group (an electron-withdrawing group, EWG), and the product could hardly be obtained (yield = 7%). Interestingly, we found that silicon-bridged diynes can react with Cp 2 ZrBu 2 and then undergo a cycloaddition reaction to yield 79% of dimethoxycarbonyl-substituted dibenzosilole derivative. In addition, various silicon-bridged diynes were subjected to a zirconium-mediated cycloaddition reaction to yield 64-79% of novel alkoxycarbonyl-substituted dibenzosilole derivatives (Scheme 1).
The synthesis of benzonaphthosilole (benzo[b]silafluorene) derivatives has seldom been reported although they are promising silole derivatives with extended π-conjugation based on dibenzosilole ( Fig. 1) [17][18][19], and no synthesis of benzonaphthosilole derivatives has been reported where alkyl, phenyl, and EWGs groups were substituted on the aromatic rings. Because substituents can effectively regulate molecular properties, lack of a synthesis method has limited the performance studies of benzonaphthosilole derivatives. Fortunately, a multi-substituted benzonaphthosilole derivative ( Fig. 1) is obtained by our strategy, which combines zirconiummediated coupling, Sonogashira coupling, and the novel zirconium-mediated cycloaddition in moderate yield. Using this strategy, propyl, phenyl, and dimethoxycarbonyl groups can be efficiently and selectively introduced onto aromatic rings.
In our previous studies, zirconacyclopentadienes have been used as intermediates for the synthesis of naphthacene derivatives, multi-substituted benzenes, and 1,2,3,4-tetraalkyl-1,4-diarylbutadienes [20][21][22]. In these studies, zirconacyclopentadienes were formed using silicon-free terminal alkynes and internal alkynes as starting materials. However, in this study, silicon-containing acetylenes, i.e., silicon-bridged diynes, were used as the starting materials for zirconium-mediated cycloaddition. A series of dibenzosilole and benzonaphthosilole derivatives were efficiently synthesized from silicon-bridged diynes by one-pot processes. The zirconium-mediated cycloaddition showed good group tolerance for silicon-bridged diynes having different alkyl and aryl groups.

Reagents and Measurements
Anhydrous tetrahydrofuran (THF) was distilled from sodium and benzophenone under dry nitrogen. Catalysts PdCl 2 (PPh 3 ) 2 and Pd(PPh 3 ) 4 were purchased from Woerjiming, China, and n-butyllithium (n-BuLi) was purchased from Ouhechem, China. All other chemicals and reagents were purchased from TCI (Japan) and Aladdin (China) and were used without further purification.
All reactions were carried out under nitrogen protection except for special instructions. 1 H and 13 C NMR spectra were recorded on a Bruker AVANCE III spectrometer (Switzerland) in deuterated chloroform using tetramethylsilane as the internal standard. Mass spectra were obtained on an LCQ Scheme 1 Iridium(I)-catalyzed cycloaddition and zirconiummediated cycloaddition synthesis of dibenzosilole derivatives Deca XP MAX (Thermo Fisher, USA) mass spectrometer system. Thin-layer chromatography (TLC) was performed using Energy GF254 plates.

General Preparation Procedure of 2-Alkynylbromobenzene (2)
First, 316 mg (0.45 mmol) of bis(triphenylphosphine)palladium dichloride (PdCl 2 (PPh 3 ) 2 ) and 95 mg (0.5 mmol) of cuprous iodide (CuI) were added to 100 mL of triethylamine, and the mixture was stirred. Then, 4.24 g (1.9 mL, 15 mmol) of 1-bromo-2-iodobenzene and 15 mmol of alkynes (1) were added, and the mixture was further stirred overnight at room temperature. The mixture was filtered, and the filtrate was evaporated under a reduced pressure. The residue was purified by flash column chromatography on silica gel with n-hexane as eluent to obtain 2-alkynylbromobenzene. [23]: 1  First, 6.96 mL (2.5 mol/L, 17.4 mmol) of n-BuLi was added dropwise to a solution of 2 (15 mmol) in 35 mL of THF at − 78 °C, and the mixture was stirred for 1 h. Then, 7.1 mL of dimethyldichlorosilane (Me 2 SiCl 2 ) in 3 mL of THF (cooled to − 78 °C first) was added to the mixture in one portion, and the obtained mixture was gradually warmed to room temperature. After stirring for 18 h, the reaction was quenched with n-hexane, the mixture was filtrated, and the filtrate was evaporated under a reduced pressure to give crude 1-chlorodimethylsily-2-alkynylbenzene (3), which was then dissolved in 3 mL of THF and protected under nitrogen. The solution of 3 was used directly in the next step without further purification. The quenching, filtration, and concentration processes of 3 were under nitrogen protection.

1-bromo-2-(phenylethynyl)benzene (2a)
In a dropwise manner, 8.64 mL (2.5 mol/L, 21.6 mmol) of n-BuLi was added to a solution of 18 mmol of alkynes (4) in THF (16 mL) at − 78 °C, and the mixture was stirred at − 78 °C for 1 h. The solution of 3 in THF was added, and the obtained mixture was gradually warmed to room temperature. After stirring for 16 h, the reaction was quenched with water and extracted with ethyl acetate. The solution of organic phase was removed under a reduced pressure, and the residue was purified by flash column chromatography on silica gel with n-hexane and triethylamine (v/v = 100:1) to give silicon-bridged diynes. [16]: 1 [16]: 1 1  First, 2.13 mL of n-BuLi (1.6 mol/L, 3.4 mmol) was added dropwise to a mixture of bis(cyclopentadienyl)zirconium dichloride (Cp 2 ZrCl 2 , 0.5 g, 1.7 mmol) and 5 mL of THF at − 78 °C, and the mixture was stirred for 1 h. Then, 5 (1 mmol) was added, and the resulting mixture was heated to 50 °C for 30-40 min (5a: 30 min, 5b-c: 40 min). The mixture was cooled to 0 °C and cuprous chloride (CuCl, 0.38 g, 3 mmol) was added, and the resulting mixture was warmed to room temperature for 10 min. Dimethyl acetylenedicarboxylate or diethyl acetylenedicarboxylate (DMAD or DEAD, 4 mmol) was added and the resulting mixture was heated to 50 °C for 15-90 min (6a: 15 min, 6b: 90 min, 6c-d: 30 min). The reaction was quenched with saturated ammonium chloride solution and extracted with ethyl acetate. The solvent of organic phase was removed under a reduced pressure, and the residue was purified by flash column chromatography on silica gel with n-hexane and ethyl acetate (v/v = 5:1) to give 6.
Spectroscopic data

Results and Discussion
In this study, new zirconium-mediated cycloaddition strategies for synthesizing several novel dibenzosilole and benzonaphthosilole derivatives are developed. Using these methods, EWG, alkyl, and phenyl groups can be selectively introduced onto the aromatic rings of these new derivatives.
The Sonogashira coupling of 1 and 1-bromo-2-iodobenzene in the presence of Pd(PPh 3 ) 2 Cl 2 and CuI produced high yields of 2 and 8. However, when Pd(Ph 3 ) 4 and CuI were used as catalysts, the reaction was sluggish and did not accelerate with an increase in temperature. This suggests that Pd(PPh 3 ) 2 Cl 2 is the suitable catalyst for this reaction. Since iodine is more reactive than bromine, the Sonogashira coupling occurred selectively at the iodine position, and the introduction of R 1 and R 2 groups could be effectively controlled.
Side reactions occurred when Me 2 SiCl 2 was added dropwise during the synthesis of 3 and 9. These reactions reduced when Me 2 SiCl 2 was added in one portion. This indicates that insufficient amount of Me 2 SiCl 2 would react with double its amount of lithiated molecules of 2 and 8 to give by-products, and adding Me 2 SiCl 2 in one portion reduced the by-products. To prevent overheating in the process of adding Me 2 SiCl 2 in one portion, Me 2 SiCl 2 and the reaction mixture were first cooled to − 78 °C. Given that Si-Cl bonds in 3 and 9 can hydrolyze easily, n-hexane was used as the quencher. In addition, the corresponding quenching, filtration, and concentration operations were carried out under nitrogen atmosphere.
The reaction mechanism of zirconium-mediated cycloaddition of silicon-bridged diynes is shown in Scheme 4. It has been reported that zirconacyclopentadienes can react with electron-withdrawing alkynes to give benzene derivatives [26,27]. It was speculated that silolozirconacyclopentadiene intermediates were formed from the reaction of silicon-bridged diynes with Cp 2 ZrBu 2 . The treatment of silolozirconacyclopentadienes with CuCl generated coppersubstituted butadiene intermediates, which was followed by an insertion reaction with EWG-substituted alkynes at the C-Cu bond, and copper-substituted 1,3,5-hexatriene intermediates were produced. Then a six-membered ring molecule was formed via Michael addition reaction. Afterward, Cu(I) at C-Cu bond was reduced to Cu(0), at which point EWG-substituted silole derivatives were formed. Through this reaction, Cu(I) was converted to Cu(0), which suggests that CuCl was not a catalyst but a reactant. Moreover, a copper mirror was observed on the inner wall of the bottle after this reaction.
As for the formation of silolozirconacyclopentadienes of 5, the conversion rate of silicon-bridged diynes was greatly affected by the temperature. As detected by TLC, the conversion rate was low after 3 h of reaction at room temperature. Surprisingly, 30-40 min of stirring at 50 °C resulted in a high conversion rate. The results showed that 50 °C was the optimum temperature for the silolozirconacyclopentadienes formation. In addition, a longer time was needed for this reaction when there was an alkyl group on the siliconbridged diynes.
To optimize the reaction temperature of the EWG-substituted alkynes cycloaddition, the reaction of 6a was investigated, and different temperatures were tried (25,35,50,60, and 70 °C). It took these reactions 6 h at 25 °C, 1 h at Scheme 4 Reaction mechanism of zirconium-mediated cycloaddition of silicon-bridged diynes 1 3 35 °C, and 15 min at 50 °C to prepare 6a, and by-products were detected by TLC when the temperature was higher than 60 °C. Based on these, 50 °C was chosen as the optimum temperature for the cycloaddition reaction of EWG-substituted alkynes.
During the synthesis of 11, TLC detected that the silolozirconacyclopentadiene intermediate formation was sluggish at 50 °C. Considering the steric effect of the naphthyl group, 55, 60, 65, and 70 °C were attempted. It took 1 h for the reaction to convert silicon-bridged diynes into intermediates even at 70 °C, and the conversion was slower for lower temperatures. Therefore, 70 °C was selected as the optimum temperature for the formation of the silolozirconacyclopentadiene intermediate of 10.
For the cycloaddition of dimethyl but-2-ynedioate to obtain 11, 50, 55, 60, 65, and 70 °C were tried. The reactions were slow when the temperatures were below 65 °C, and at 70 °C, the separation of 11 was greatly affected by the large amount of by-products. In contrast, 2 h of reaction at 65 °C moderately yielded 11; hence, it was chosen as the optimum temperature for the cycloaddition to prepare 11. The yields obtained during the preparation of benzonaphthosilole derivative are listed in Scheme 5. As noted above, there are few reports on the synthesis of benzonaphthosilole derivatives. Our report on the synthesis of 11 is unique, because to date, it is the only synthesized benzonaphthosilole derivative with substituents on the aromatic rings. The structure and synthesis strategy of 11 are rather different from those of the previous literature [17][18][19]. In contrast to the rhodium-catalyzed reaction, the synthesis strategy we designed uses readily available alkynes as the starting materials instead of dimethyl[2-(naphthalen-2-yl) phenyl]silane, which is difficult to prepare. Moreover, propyl, phenyl and dimethoxycarbonyl groups can be selectively introduced onto the aromatic rings of 11 by combining zirconium-mediated coupling reaction, Sonogashira coupling reaction, and the novel zirconium-mediated cycloaddition reported in this paper. The result of preparing 11 shows that zirconium-mediated cycloaddition has excellent adaptability to silicon-bridged diynes bearing a larger group as naphthyl. This indicates that zirconium-mediated cycloaddition reactions of silicon-bridged diynes can be used to synthesize various silole derivatives.

Conclusions
In summary, a new method of synthesizing alkoxycarbonylsubstituted dibenzosilole derivatives was developed, and the preparation of 5c, 6b, 6c, and 6d was reported for the first time. High yields of dibenzosilole derivatives were obtained, which were much higher than that of iridium(I)-catalyzed cycloaddition. In addition, we successfully designed a strategy based on zirconium-mediated cycloaddition reaction for synthesizing a moderate yield of benzonaphthosilole derivative bearing several substituents, which suggests that this reaction is suitable to silicon-bridged diynes bearing different groups. By synthesizing compound 11, we successfully accomplished the selective and multiple derivatization of benzonaphthosilole. Using the synthesis strategies we reported, alkoxycarbonyl, phenyl, and alkyl-substituted dibenzosilole and benzonaphthosilole derivatives were efficiently synthesized, where the alkoxycarbonyl, phenyl, and alkyl groups were selectively introduced onto the aromatic rings. All raw materials of the cycloaddition reactions are available or easily prepared. In addition, alkoxycarbonyl groups have potential for further derivatization, which is of great significance for expanding the range of applications of silole derivatives. These novel zirconium-mediated cycloaddition synthesis strategies can provide a wider variety of silole derivatives.


s2fieldsofstudy: Chemistry Biology

text:
Photochemical and Structural Studies on Cyclic Peptide Models

Ultra-violet (UV) irradiation has a significant impact on the structure and function of proteins that is supposed to be in relationship with the tryptophan-mediated photolysis of disulfide bonds. To investigate the correlation between the photoexcitation of Trp residues in polypeptides and the associated reduction of disulfide bridges, a series of small, cyclic oligopeptide models were analyzed in this work. Average distances between the aromatic side chains and the disulfide bridge were determined following molecular mechanics (MM) geometry optimizations. In this way, the possibility of cation–π interactions was also investigated. Molecular mechanics calculations revealed that the shortest distance between the side chain of the Trp residues and the disulfide bridge is approximately 5 Å in the cyclic pentapeptide models. Based on this, three tryptophan-containing cyclopeptide models were synthesized and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Experimental data and detailed molecular dynamics (MD) simulations were in good agreement with MM geometry calculations. Selected model peptides were subjected to photolytic degradation to study the correlation of structural features and the photolytic cleavage of disulfide bonds in solution. Formation of free sulfhydryl groups upon illumination with near UV light was monitored by fluorescence spectroscopy after chemical derivatization with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) and mass spectrometry. Liquid cromatography-mass spectrometry (LC-MS) measurements indicated the presence of multiple photooxidation products (e.g., dimers, multimers and other oxidated products), suggesting that besides the photolysis of disulfide bonds secondary photolytic processes take place.

Introduction
Near UV-light-induced photochemical reactions have several applications in biotechnology and in medical sciences. Light-induced immobilization of proteins is a novel technology which can be utilized for covalent coupling of biomolecules on thiol-reactive surfaces in spatially oriented and/or spatially localized manner. Covalent immobilization using this emerging technology has been demonstrated for various proteins, such as for hydrolytic enzymes (lipases/esterases, lysozyme), proteases (human plasminogen), etc. [1,2]. The epidermal growth factor (EGF) receptor protein, often overexpressed in cancers and in other proliferative skin disorders, is rich in aromatic residues close to the disulfide bridges. It was demonstrated that the EGF receptor could be a promising target for laser-pulsed UV light treatment [3] to reduce the proliferative potential of cancer cells. Furthermore, UV light-induced immobilization could be also used for biomolecular screening techniques for diagnostic and therapeutic purposes [4].
Exposure to near-UV irradiation induces structural changes in proteins, that may alter their biological function [5,6]. One of the well-known processes of protein degradation is the near-UV light induced splitting of disulfide bonds which yields thiyl radicals (CysS) [7]. Thiyl radicals are formed via one-electron oxidation of cysteine residues [8] or through one-electron reduction of disulfide bonds [9,10]. UV irradiation-induced alteration of protein structures was extensively studied during the last decades. Photon absorption of peptide bonds, disulfide bridges, as well as side chains of aromatic amino acids (tryptophan, Trp; tyrosine, Tyr; phenylalanine, Phe) and histidine (His) was proposed to mediate the degradation process. Indole rings of Trp residues are the strongest near-UV absorbing groups in proteins, therefore, Trp residues are considered to be the primary components responsible for the photodegradation of proteins [2,[5][6][7]11]. Light-induced, Trp-mediated breakage of disulfide bridges was investigated in detail on cutinase [11,12], bovine growth hormone [13], α-lactalbumin [6,14] and lysozyme [15] proteins. Determination of the required distances between aromatic amino acid side chains and disulfide bridges was the target of numerous studies, for example in the case of class I and class II major histocompatibility complex (MHC), mannose-binding lectin (MBL) subunit A, immunoglobulins, triosephosphateisomerase (TIM) barrel glycosidases, alkaline phosphatases and lysozymes [14][15][16]. Photodegradation of goat α-lactalbumin mutants containing substituted Trp residues revealed that the contribution of Trp residues to the photolytic breakage of the disulfide bonds depends strongly on the microenvironment of the Trp side chains and the disulfide bonds [6,14]. It was also demonstrated that thiyl radicals could be involved in several additional chemical reactions, such as recombination with other radical intermediates (Trp-, Tyr-and Phe radicals) [16], sulfenylation of the side chain of Trp (or Tyr/Phe), or recombination through interprotein cross-links, leading to dimerization [17,18]. Antibodies are also sensitive to photodegradation by near-UV light [19]. The formed thiyl radicals could generate both intra- [20] and interchain bonds which could lead to fragmentation and/or aggregation of these proteins [21].
Disulfide-bonded cystein residues with a neighboring Trp (Cys-Cys Trp structural triads) can be found in most of the protein families, but these are especially characteristic for immunoglobulins, lysozymes, and complement-associated proteins [22]. Lys and Tyr amino acids were also found to be preferred neighbors of this specific structural motif. Statistical analyses demonstrated that reduction-sensitive disulfide bonds are usually within the van der Waals distance (d ≤ 5Å) of photoactive aromatic groups (Trp, Tyr) [23,24]. However, the effect of neighboring aromatic groups could be exerted longer distances as well (d~8-9 Å) [6,25,26].
Cation-π interactions, involving positively charged amino acid side chains (Lys, Arg) and an aromatic ring of Trp, Tyr or Phe might have a significant contribution to the stability of proteins [27] when the participating moieties are within 6.6 Å distance in a well-defined geometry [27][28][29]. According to statistical data, Arg residues are involved in these interactions in a larger extent than Lys residues and over one-fourth of Trp side chains participate in cation-π interactions [28]. These interactions could also have influence on peptide structures, but the stabilizing effect is usually much smaller [30].
To investigate the structural determinants of the aromatic group-mediated photolysis by near-UV light, a series of cyclic pentapeptides was designed and analyzed by molecular mechanics (MM) calculations. The peptide models contain a Trp residue as well as a disulfide bridge which limits conformational flexibility. Control peptides were also designed, in which Trp was replaced by Val (Table 1). Selected model peptides were also synthesized and illuminated by UV light at the absorbance maximum of Trp. The free sulfhydryl groups formed during the UV-illumination were detected through their CPM (7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin) adducts, which have higher fluorescence absorbance than the CPM itself [31]. This paper reports on the MM calculations of the peptide models as well as the synthesis, photoreduction study and combined NMR and molecular dynamics (MD) based conformational analysis of the selected cyclic peptide models. In addition, LC-MS measurements were performed to estimate the amount of unreacted cyclopeptides of the studied models after the UV-illumination. LC-MS/MS experiments were also performed to identify the photolysis byproducts.

Design of Cyclic Peptide Models and Molecular Mechanics Calculations
The size and flexibility of the disulfide-linked peptide macrocycle strongly influence the orientation of Trp side chains and so their distances from the respective S-atoms of the disulfide bridge. For the present photochemical and structural study, both symmetric and asymmetric cyclic pentapeptide models, containing a Trp residue and a disulfide bridge between the terminal Cys 1 and Cys 5 residues (Table 1), have been constructed and analyzed. Trp was replaced by Val in the corresponding reference compounds.
MM calculations were performed to appoint optimal (ideal) peptide models, which fulfill structural criteria preferred in the photolytic processes. These structural parameters are the following: the distance between the Cε 2 atom of Trp side chain and the center of disulfide bridge (d 1 ) is 3-10 Å [22,24], the distance between the centroids of the benzene rings of the indole moieties of Trp and the cationic side chains of Lys (d 2 ) and Arg residues (d 3 ) are less than 6 Å [28,29]. Schematic representation of these key distances is shown on Figure 1. Corresponding structural characteristics of the designed peptides were determined by MM-based molecular modeling.   Figure 1) and analytical data of the selected synthetic peptide models. The results of molecular mechanics calculations indicated that the shortest distance (d 1 ) between Trp and the SS bridge is approximately 5 Å in the designed cyclic pentapeptide models (Table S1).
The Trp side chain and the disulfide bridge were found in the most favorable positions for interaction in Ac-c(CAWAC)-NH 2 , Ac-c(CWAKC)-NH 2 and Ac-c(CWKAC)-NH 2 peptides, in which the relevant distances (d 1 ) were between 5.5-7.9 Å. In contrast to that, in Ac-c(CWAGC)-NH 2 this distance was found to be d 1 = 9.6 Å (Table S1). Based on MM calculations, the Arg-containing models (Table S1) do not fulfill the essential distance criteria of cation-π interactions. The Lys-containing peptide models, however, match such requirements. Based on the results of these calculations, the following cyclic pentapeptides were identified as possible targets of photoactivated disulfide reduction: Ac-c(CWAGC)-NH 2 < Ac-c(CWKAC)-NH 2 < Ac-c(CAWAC)-NH 2 , where the order indicates the degree of predicted photosensitivity. These three peptides (Table 1) were then selected and synthesized for further structural studies and photodegradation experiments. The corresponding linear peptides were made by solid phase peptide synthesis (SPPS) [32], and these precursors were cyclized afterwards in solution [32,33]. Analytical data (MW) of the peptides and calculated distances are summarized in Table 1.

Determination of Key Distances by NMR and MD Analyses
Distances relevant for photolytic activity were analyzed by combined NMR and MD methods, through exhaustive conformational analysis of the three selected Trp-containing pentapeptide models: Ac-c(CWAGC)-NH 2 , Ac-c(CWKAC)-NH 2 and Ac-c(CAWAC)-NH 2 . To explore the role of the amino group close to the Trp residue and to study the cation-π interactions, peptides with and without Lys residues were also examined.

NMR Analysis
Sequential (i ± 1), medium range (i ± 2,3) rotating-frame Overhauser effects (ROEs) and the contacts of the Trp residue were assigned in the rotating-frame Overhauser effect spectroscopy (ROESY) spectra in order to determine the pertinent conformational features of the peptides and to assess the spatial proximity of the indole ring to the disulfide bridge (Trp-SS).
Besides the sequential and medium range ROEs observed for Ac-c(CWKAC)-NH 2 , the ROESY spectrum ( Figure 2) shows cross peaks between Trp and Cys residues with significant intensity (e.g., HZ2 Trp 2 /HB2 Cys 5 ) suggesting the spatial proximity of the Trp ring to the disulfide bridge. In addition, almost all of the Trp ring H-atoms show ROE cross peaks with the side chain of Lys, indicating the presence of cation-π interaction. The ROE patterns observed in the ROESY spectra of Ac-c(CAWAC)-NH 2 and Ac-c(CWAGC)-NH 2 ( Figure S1) also support the Trp-SS proximity. Besides qualitative analyses of ROESY spectra the ROE-derived proton-proton distances were utilized as restraints in the subsequent iterative structure refinement, yielding conformational ensembles shown in Figure 3.  (Table S2).

MD Analysis of Key Interatomic Distances
Analyzing the MD-trajectory in Ac-c(CWKAC)-NH 2 (Figure 4a), the distance (d 1 ) between C ε 2 atom of Trp side chain and the center of disulfide bridge is found to be mostly above 7 Å with an average of 8.55 ± 1.34 Å. The population of conformers having favorable distance (<7 Å) of the aromatic and disulfide groups was found to be around 7% for this peptide. The close contact of the respective residues is supported by the observed ROEs as well ( Figure 2). In contrast, for Ac-c(CAWAC)-NH 2 , the most flexible peptide, a significant population (33%) of conformers with distance d 1 below 7 Å was found, giving an average of 8.12 ± 2.40 Å (Figure 4c). In Ac-c(CWAGC)-NH 2 , the least flexible peptide, the indole ring did not show up in the proximity of the disulfide bridge. The corresponding disulfide-Trp C ε 2 distance d 1 was 9.21 ± 0.62 Å along the 200 ns MD trajectory (Figure 4d). The only peptide with a protonated Lys residue, in which the contribution of possible cation-π interaction could be examined, is Ac-c(CWKAC)-NH 2 . MD results demonstrated that the flexible side chain of Lys 3 is likely to be positioned near the aromatic ring of Trp 2 , which is in agreement with the ROE contacts shown in Figure 2. The corresponding average distance is 6.38 Å with a deviation of ±1.96 Å. In 18% population of conformers this distance fell in the range of 2 Å < d 2 < 5 Å (Figure 4b).
The distance between groups being putatively involved in photolysis through the formation of a super-oxide radical of the Trp carbonyl oxygen (CO) was also investigated for each peptide. In Ac-c(CWKAC)-NH 2 , Trp 2 -CO was located close to the disulfide bridge, at an average distance of 3.62 ± 1.01 Å. In Ac-c(CAWAC)-NH 2 , the corresponding average distance was 5.31 ± 1.44 Å, while in the most rigid peptide, Ac-c(CWAGC)-NH 2 , a relatively long distance (8.16 ± 0.75 Å) was observed.

Comparative MD Analysis of Conformational Ensembles in Dimethyl Sulfoxide and Water
Although the NMR measurements could be carried out only in dimethyl sulfoxide (DMSO-d 6 ) due to low water solubility of the studied peptides, but to disclose the potential impact of solvent on the conformational ensembles, MD calculations were performed in both DMSO and water.
The structure of Ac-c(CWKAC)-NH 2 was found to be similar in both water and DMSO. This is supported by the secondary structure analysis of the MD-derived structural ensembles reported in Table 2. The largest conformational families adopt unordered backbone structures, while the other conformational groups adopt type II- (Figure 5a) or type IV-β-turns. Comparing their representative structures, significant differences can be observed in the side-chain orientation of Trp.
Cluster analysis of the MD ensembles of Ac-c(CAWAC)-NH 2 with regard to backbone and side chain conformations indicated that both backbone and the Trp 3 side chain possess enhanced flexibility and the peptide adopts mainly unordered conformations. Minor conformational families of type-IV β-turn with different orientations of the Trp ring exist in both solvents (Figure 5b), but with considerably reduced population in DMSO.
In the case of Ac-c(CWAGC)-NH 2 , a large conformational family, stabilized by a type-I β-turn was observed in water besides the unordered backbone conformations. In contrast, a type-II β-turn structural family ( Figure 5d) and a type-IV β-turn family (Figure 5c) were found in DMSO, where this latter family can be further subdivided on the basis of Trp 2 side chain conformation: one adopted gauche+ rotameric state (χ 1 = 67 • ), while the other preferred trans (χ 1 = 179 • ). In comparison with the other peptides, the populations of the unordered structures in Ac-c(CWAGC)-NH 2 were lower in both solvents. Table 2. Secondary structure analysis of MD ensembles.

Unordered
Type I β-turn

Γ-Turn
- In summary, comparing the results of MD simulations performed in the two solvents (H 2 O, DMSO), it can be concluded that the conformational equilibria of Ac-c(CWKAC)-NH 2 and Ac-c(CAWAC)-NH 2 do not show significant differences, neither in the type nor in the population of the adopted secondary structures of peptide backbone. On the other hand, in the case of the most rigid Ac-c(CWAGC)-NH 2 , significant effect of the solvent was observed. Namely, type I β-turn structures were favored in water, and type II β-turns in DMSO.

Analysis of Hydrogen Bonds
NMR data and MD trajectories were also examined for structure-stabilizing hydrogen bonds. The small temperature dependence of amide proton chemical shifts predicts exchange-protected, H-bonded amide protons of the third residues in Ac-c(CWKAC)-NH 2 (Lys 3 ) and Ac-c(CAWAC)-NH 2 (Trp 3 ) with −1.7 and −1.6 ppb/K temperature gradients (Table 3), respectively. This may indicate a population of β-turn structures, stabilized through a H-bond between Lys 3 /Trp 3 (NH) and Cys 1 (CO). In contrast, all amide protons of Ac-c(CWAGC)-NH 2 show large temperature effect (ranging from −4.8 to −5.8 ppb/K), thus no H-bonded sites could be confirmed by NMR. Table 3. Temperature-dependent 1 H-NMR measurements of the studied peptides, providing the chemical shift gradients of amide protons.

NH-Chemical Shift Temperature Gradient (ppb/K) in Model Peptides
Ac-c(CWKAC)-NH 2 Cys 1 (NH) Geometrical restraint calculations for hydrogen bonds in the structures of MD ensembles were carried out in parallel, searching for donor-acceptor pairs (Table S3). In DMSO, the populations of conformers having possible H-bonds are reduced in comparison to water. However, the same pattern (donor-acceptor pairs) was observed in both solvents. The occurrence of stabilizing H-bonds between Cys 1 CO and residue 3 NH in all three peptides was confirmed. In Ac-c(CWKAC)-NH 2 and Ac-c(CAWAC)-NH 2 H-bonds between Trp 3 NH and Cys 5 CO were also established. In addition, the contribution of protecting groups to structure stabilization was also observed. The acetyl group CO likely accepts amide-NHs from the proximal residues as well as the C-terminal amide NH, which is a good donor for Cys 5 CO as well.
In conclusion, the presence of H-bond involving NH of residue 3 in Ac-c(CWKAC)-NH 2 and Ac-c(CAWAC)-NH 2 has been corroborated by both experimental NMR data and MD statistics. In the case of Ac-c(CWAGC)-NH 2 , differences were observed between the H-bond patterns predicted with the two approaches, which could be likely explained by possible conformational exchange processes resulting in enhanced temperature gradients.

Ac-c(CWAGC)-NH 2 Gauche (+) Gauche (−) Trans
According to the MD ensembles, the distributions of the χ 1 torsion angle in Ac-c(CWKAC)-NH 2 and Ac-c(CAWAC)-NH 2 show nearly the same pattern in both solvents. In contrast, a significant solvent effect was observed in Ac-c(CWAGC)-NH 2 . The most dominant rotamer with 93% propensity was gauche (+) in water, while in DMSO the rotamer distribution became more evenly balanced with contributions from all three rotamers but with definite preference toward the gauche(−) state. It should be noted that rotamer populations being available from both the NMR and MD analyses (Table 4) are generally in excellent agreement, providing further proof of the proposed structural features.
In conclusion, based on the detailed MD calculations being supported by experimental NMR data, our results indicate that the structural characteristics of the three selected Trp-containing peptides conform to the predicted features, suggested on the basis of molecular mechanics force field calculations. This means that the peptides synthesized and investigated in the present work are suitable models for studying the structural aspects and determinants of the photolytic cleavage of disulfide bonds and thereby to prove or disprove the different hypotheses proposed in the literature so far.

Quenching Effect of Disulfide Bridges in Peptide Models
To study the effect of disulfide bridges on fluorescence emission, spectra of linear and cyclic model peptides at λ ex = 280 nm were measured under similar conditions ( Figure 6). Ac-Trp-OMe derivative was used as reference compound to mimic the peptide-bound Trp residue. The intensities/amplitudes of spectra vary according to the amino acid composition and sequence of the peptides, and according to their linear or cyclic nature. The emission intensities of cyclic peptides are systematically lower compared to the Ac-Trp-OMe derivative or to the linear peptide variants with similar amino acid compositions. This phenomenon can be explained by the quenching effect of disulfide bridges present in cyclic peptides [34][35][36]. The difference in fluorescence intensities of cyclic peptide models can be explained by the different distances (d 1 ) between Trp residues and SS bonds (i.e., the quenching effect of the SS bond). We propose that the difference in Trp-fluorescence intensity could provide qualitative information about the spatial proximity of the SS bond and the Trp-chromophore ( Figure 6 and Table S4) or about a different orientation of the Trp side chain. However, quenching effect of the peptide backbone as well as of the disulfide bond could also modify the data. Linear peptide models and the reference compound Ac-Trp-OMe show higher fluorescence intensity possibly due to the lack of disulfide bridge and the conformational diversity of linear peptides.

UV Irradiation of Peptides Monitored by Measuring the Amount of Evolving Thiol Groups with Fluorescence Spectroscopy
Changes in the fluorescence spectra were registered as a function of the absorbed light energy after UV irradiation together with the determination of the amount of free thiol groups formed. To distinguish between degradation processes induced by light absorption of Trp and other less specific light-induced reactions, control measurements were performed using Val-substituted disulfide bridged cyclic pentapeptides lacking the aromatic Trp residue.
Peptides were irradiated in solution with UV light at 280 nm (see Experimental). The fluorescence emission spectra of the cyclic peptides irradiated with various time periods are shown in Figure 7 and Figure S2. Significant (15-20%) decrease in Trp fluorescence intensities was observed with increasing duration of irradiation time in the case of all Trp-containing model peptides. Extended irradiation time (2-3 h) resulted in a significant, about 30% decrease in the Trp fluorescence intensities (Figure 7 and Figure S3A). Note that similar results were obtained for goat α-lactalbumin [6,14].
The decrease of the fluorescence intensity shown on Ac-c(CWAGC)-NH 2 (Figure 7) can be explained by the photodestruction of the fluorophore, therefore 1-h illumination time was chosen for further studies. Formation of free sulfhydryl groups under UV irradiation of peptides was detected by chemical derivatization with CPM reagent. CPM reacts with free sulfhydryl groups immediately and the reaction is complete within 10 min. The formed CPM-peptide adducts are stable for a long time. Due to the high fluorescence intensity of the adducts, the reaction is suitable for the sensitive detection of sulfhydryl formation. In the experiments, a model peptide was designed and synthesized as calibration standard. This linear peptide, Ac-CWAKC(Acm)-NH 2 , contained one free sulfhydryl group, while the second sulfhydryl group was blocked by an acetamidomethyl (Acm)-protecting group [37] ( Figure S4). The impact of a protonated amino group near to Trp (cation-π interaction) was also investigated using peptides with and without Lys in the sequence. Figure 8 shows the fluorescence intensity of the CPM adducts of the selected peptides after 1 h of illumination. Interestingly, under these conditions, no significant difference was observed between the peptides after UV irradiation, as presented by the data in Table 5. The data (Table 5) verified that the photodegradation of the SS bridge occurs in the studied peptides, and the quantity of the free sulfhydryl groups evolved is between 5-6% in all case. The low amounts of detected sulfhydryl groups can be explained by their reactivity and also by a fast conversion to other products (see paragraph 2.8 below). However, fluorescence measurements do not support the structure-based predictions of the photodegradation efficacy. It is important to note that the control model peptide containing Val instead of Trp also produced free thiols in detectable amount.
Moziccionacci [38] and coworkers reported similar results earlier after illuminating nonaromatic Cys-containing hexapeptides. The UV-irradiation of a peptide disulfide bond generates thiyl radicals which form two main products, namely the cysteine and the thioaldehyde-containing peptides. This proton transfer occurs prior to the formation of final, nonradical products including free thiol, thioaldehyde, and aldehyde. Thiyl radicals in peptides and proteins may also be involved in reversible, intramolecular proton abstraction reactions from nearby Cα-H or side chain C-H bonds generating intermediary C-centered radicals. Their results demonstrate that the thiyl radicals of Cys react with the Val residues but also enter a pathway which yields a new reaction product, tentatively identified as isothiazol-3(2H)-one [39]. We suppose that these alternative photodegradation pathways also influence the formation of free sulfhydryl groups from the cyclic peptide models.

LC-MS Measurements
To investigate the possible secondary photolytic processes originating from radicals in the peptide solutions after UV-irradiation, mass spectrometric experiments were performed. The amount of unreacted cyclic peptides after the illumination was estimated by LC-MS measurements. To have a qualitative overview of the degradation of the peptides, three parallel experiments were performed for both the illuminated and non-illuminated peptides. The percentage of the unreacted peptides was calculated from the integrated mass peak intensities. The results verified the significant decrease in the amount of the cyclic model peptides. In the case of peptide Ac-c(CWAGC)-NH 2 , approximately 80% of the peptide remained intact after 1 h illumination time. Other peptides exhibited even lower survival: approx. 65% of peptide Ac-c(CAWAC)-NH 2 remained intact, while Ac-c(CVAKC)-NH 2 and Ac-c(CWKAC)-NH 2 , showed the lowest values with approx. 45% survival only. Our data therefore confirm the occurrence and importance of secondary photolytic processes, leading to alternative byproducts beside the formation of free sulfhydryls.
Beside the formation of the CPM adducts of the peptides, LC-MS experiments confirmed the presence of oxidized products, such as the oxidation of the Trp residue ( Figure 9) as well. In addition to oxidation, a major side reaction was the dimerization of the peptides through newly formed disulfide bridges. This can be explained with the fast rearrangement of the disulfide bridges under pH 7.5 upon illumination, resulting in the formation of head-tail-or head-head-oriented peptide dimers. Our results show that the indolyl side chain of Trp is highly sensitive in the near UV region, therefore, UV irradiation results in the formation of complex mixtures, even in the case of the studied simple model peptides.

LC-MS
Analytical LC-MS measurements were performed on a Waters Acquity SQD MS system equipped with Alliance 2795 separation module using reverse phase column (Herts, UK).

LC-MS/MS
LC-MS/MS tandem mass spectrometric experiments were performed on a Bruker Esquire 3000+ ion trap mass spectrometer (Bremen, Germany) equipped with electrospray ionization (ESI) source.

MM Calculation
MM calculations were performed with HyperChem 6.0 (Hypercube, Inc., Gainesville, FL, USA) using Amber 96 force field in vacuum (Newton-Raphson and Polak-Ribiere algorithms, 2000 cycles). Optimization was started from structures obtained by setting trans amide bonds and SS distance to 2.04 Å. During the conformational search ϕi, ψi, ξi and χi torsion angles were scanned.
The gradient enhanced, zero-quantum filtered, pure absorption phase 1 H-1 H TOCSY spectra were recorded using MLEV-17 sequence [40,41] with a mixing time of 60 ms. The 2D data matrix consisted of 4096 and 512 total data points. A spin-lock field of 8300 Hz was used for the TOCSY transfer. A relaxation delay of 1.7 s was allowed between scans. Zero-filling in F1 and a squared cosine function in both F1 and F2 were applied prior to Fourier-transformation.
The 1 H-1 H ROESY spectra were recorded using the conventional ROESY experiment [42,43] with a spin-lock field of 3300 Hz and 150 ms duration of mixing. The number of scans was 32 in each experiment and the 2D data matrix consisted of 2048 and 512 total data points. These matrices were zero filled and apodized by a squared cosine function in both dimensions.
The 1 H-13 C HSQC correlation maps were recorded using the standard Bruker pulse sequence [44,45]. A relaxation delay of 1.7 s was allowed between transients and the size of the matrices was 2048 x 512 complex data points. 32 scans were collected for each of the 512 experiments.
Proton-proton scalar coupling constants were extracted from 1D spectra apodized with a Gaussian function for enhancing resolution, or in case overlapping signals from the highly digitized (0.3 Hz/point) one dimensional traces of 1 H-1 H TOCSY spectra.
Bruker TOPSPIN 3.0 software was used for NMR data processing.
To study the temperature dependence of amide proton resonances, one dimensional 1 H spectra were recorded at 293, 303 and 308 K, respectively. The presence of intramolecular hydrogen bonds was assessed from the temperature gradient of amide proton resonances. NHs involved in H-bonds or pointing to the inner, sterically crowded region of the cyclic peptide are not accessible by the solvent, and therefore are exchange-protected, characterized by small temperature gradients (−1.6/−1.7 ppb/K) [46].
Complete, sequence-specific resonance assignment of proton resonances was accomplished (Table S5.) following the general strategy of Wüthrich [47], using CARA [48] (Computer Aided Resonance Assignment, Dissertation ETH Nr. 15947, by Rochus Keller, Zurich, Switzerland), a software designed for protein NMR data analysis.
Volume intensities of ROESY cross peaks were converted into structural information by distance geometry calculations using program package UNIO [49]. Automated ROESY peak picking and assignment were carried out by the ATNOS [50] and CANDID [51] algorithms. As input, the amino acid sequences, the complete sequence specific resonance assignments and the 2D 1 H-1 H ROESY spectral data were used. Structures were calculated by torsion angle dynamics and simulated annealing with MD software CYANA [52].
The final pools of structures were obtained in seven cycles of iterative calculation, in each cycle the experimental and calculated ROESY spectra and the obtained geometries were compared. By eliminating the wrong and/or ambiguous assignments and constraints, a good agreement was achieved for all peptides at the end of the iterative process. The quality of structures generated was characterized by several parameters, including the value of target function, the remaining number of ROE distance violations, and the average root mean square deviation (RMSD) of the bundle of conformers to the mean structures.
NMR structures were validated using the WHATIF [53] online service. The packing quality of peptides, quality of Ramachandran plot, planarity of aromatic rings and existence of anomalous bond lengths were checked. The results have confirmed that the obtained NMR ensembles contain valid peptide geometries.

Molecular Dynamics Simulations
Refinement of the NMR structures was performed by molecular dynamics simulations without the ROE distance restraints, allowing also to characterize the dynamic behavior of peptides. The GROMACS [54] 4.5.5 software package (Groningen University, Stockholm, Sweden) with the AMBER ff03 force field parameter set was used for calculations.
The NMR-derived structural ensembles of Ac-c(CAWAC)-NH 2 and Ac-c(CWAGC)-NH 2 were found to be uniform, therefore starting structures for MD simulations were chosen randomly from those ensembles for these peptides. For Ac-c(CWKAC)-NH2 three well defined structural states were identified by NMR data, therefore, to reach high degree of conformational sampling, those three different starting structures, corresponding to the most populated structural states of this peptide were picked as starting structures of MD simulations. N-terminal acyl and C-terminal amide groups were added to peptide termini using Pymol 1.4.1 protein builder, and then the selected models were converted into GROMACS format. The structures were centered in a cubic, periodic simulation box (3.5 × 3.5 × 3.5 Å) of pre-equilibrated TIP3P water or DMSO [55] molecules. The systems were then energy minimized (EM) to remove atomic clashes with 1000 steps of the steepest descent method, using a 0.001 kJ/mol convergence criteria for the energy gradient. To neutralize the protonated side chain of the Lys 3 residue in Ac-c(CWKAC)-NH 2 a solvent molecule in the most favorable position regarding electrostatic potential was replaced by a single chloride ion. The simulation box was then subjected to EM again with the parameters mentioned above. For calculations in DMSO as solvent, the combination of conjugate gradient and steepest descent EM was necessary to reach optimum. Solvent molecules were further equilibrated by a 500 ps long MD simulation in the NVT ensemble, where heavy atoms of the solute molecule were fixed by the LINCS algorithm by applying a force constant of 1000 kJ mol −1 Å 2 .
Production simulations were performed in the NPT ensemble for 100.000.000 steps with a step size of 2 fs, yielding 200-ns-long MD trajectories. To keep the bonds close to their equilibrium lengths, the LINCS constraint algorithm was used. Temperature was set at 300 K and regulated using the velocity rescaling method with a coupling time constant of 1 ps. The whole system was kept at constant pressure (1 bar), maintained by the Berendsen isotropic scaling method with a relaxation constant of 2 ps, and 5.25 × 10 −5 bar −1 and 4.5 × 10 −5 bar −1 isothermal compressibilities for DMSO and water, respectively. Non-bonded electrostatic interactions were calculated using the Particle-Mesh-Ewald method with all cut-off values set to 10 Å.
Coordinates of the system were stored after every 10 ps and the first 1 ns of equilibration was excluded resulting in trajectories of 19,900 individual geometries for each peptides.

MD Analysis
Data for structural refinement were obtained by the analysis of the MD trajectories, using the utilities of GROMACS package. Furthermore, softwares such as VMD [56], Pymol [57], Xmgrace and Microsoft Office Excel (Redmond, WA, USA) were used for visualization, statistics and generating plots.
Intramolecular hydrogen bonds were examined along the trajectories and evaluated with the g_hbond utility of GROMACS, using a 3.5 Å cut-off distance between donor and acceptor atoms and a 60 • angle cut-off for donor-hydrogen-acceptor angle.
To describe the conformational properties and flexibility of the peptide backbone, secondary structure assignment was performed. The occurrence of the secondary structural elements along the trajectory was examined by the STRIDE [58] method, which analyses backbone dihedral angles and the hydrogen-bond patterns during the assignment process.
The representative MD structures and main conformational families along the trajectories were identified by cluster analysis, fitting all atoms of the peptides, using an RMSD similarity cut-off between 0.07 nm and 0.15 nm using the gromos [59] method implemented in the g_cluster GROMACS utility.
Previously proposed intramolecular atomic distances that could play important role in the photolysis of disulfide bridges upon UV-radiation of peptides were examined in the structural ensembles.
The effect of aromatic residues was studied with peptides containing a Trp residue. The distances and their fluctuations between the sulphur atom of Cys 5 and the C ε 2 of the aromatic ring and the carbonyl oxygen of Trp residues have been determined along the whole trajectory.
The conformational properties and flexibility of the side chains were described by the rotameric distributions. According to the value of χ 1 dihedral angle extracted from the trajectories, side chains were divided into three conformational states (gauche+, gauche− and trans).

Synthesis of Model Peptides
Linear peptides (precursors) were synthesized using Rink-Amid-MBHA resin (0.66 mmol/g) using Fmoc/ t Bu protocol [33] in DMF. The coupling reaction efficiency was monitored by Kaiser test [60]. In the last step, the Fmoc protecting group was cleaved from the peptidyl-resin and the acetylation reaction of N-terminal α-amino group was performed on resin with Ac 2 O/DIEA in DMF. The final cleavage from solid support was achieved with TFA cleavage cocktail containing scavengers: TFA/TIS/Phenol/dist.water 92.5/2.5/2.5/2.5 v/v/ m%/V, 1h. The cleaved crude peptide was precipitated with ice cold diethyl ether, dissolved with water containing 5% acetic acid and lyophilised. Characteristics of the synthesized linear peptides are summarized in Table S6.

Cyclization of Linear Peptide Precursors through Disulfide Bond in Solution
Linear peptides were oxidized without further purification under mild alkaline conditions (0.1-0.5 mg/mL, pH 7.8-8.0 where the pH was adjusted by NH 4 HCO 3 ). The cyclization reaction was monitored by analytical RP-HPLC (5 µm, 150 mm × 4.6 mm, 110 Å, Gemini-NX, Phenomenex. After lyophilisation, the crude cyclic peptides were purified by preparative RP-HPLC. All purified peptides were shown >95% homogeneity by analytical RP-HPLC and ESI-MS. Characteristics of the synthesized cyclic peptides are summarized in Table S7.

Fluorescence Spectroscopic Measurements
Irradiation and fluorescence measurements were performed on Jobin-Yvon Fluorolog 4 Spectrofluorometer (Kyoto, Japan) equipped with a 450-W Xenon lamp at 4 • C (by thermostat) in 1 cm × 1 cm quartz cell in argon atmosphere. The photon flux was determined by Ferrioxalate actinometry [61] at 280 and 290 nm: θ photon (280 nm) = 1.92.10 18 photon/s; θ photon (290 nm) = 3.56/10 18 photon/s. The model peptides were illuminated with 280 nm light, which was centered at 280 nm with the slit set to 16 nm band pass. The peptide solution (c = 185-220 µm in 5 mM NH 4 HCO 3 buffer solution at pH~7.5) was illuminated at 4 • C in a quartz cell (path length 1cm) with magnetic stirring (at 120 rpm) for 1 h. Before the illumination, all solutions were degassed and kept under argon atmosphere.
The evolved sulfhydryl group during the illumination of the peptide solution were quantified by fluorescence spectroscopic measurements at concentration of 35 µM peptide and in the presence of 3.5 µM CPM at 4 • C. Appropriate volume of CPM solution from DMSO stock solution was added to the irradiated peptide solution in a final concentration of 3.5 µM. The detection was based on the fluorescence increase of the peptide-CPM adduct (λ ex = 387 nm, λ emmax = 481 nm) The excitation was carried out at 387 nm at the 4-nm slit. The emission spectra were detected in the region of 395-680 nm. The fluorescence increase of CPM upon binding the free sulfhydryl groups was calibrated with a dilution series of linear peptide precursor Ac-CWAKC(Acm)-NH 2 .

Conclusions
Proteins, such as glycosidases, lysozymes and immunoglobulins can exhibit disulfide bridge photosensitivity [11,16]. The abundance and relative orientation of the Trp/SS motifs could have a high impact on the photosensitivity of polypeptides.
In the present study ideal small cyclic peptide models were proposed by MM in vacuum based on the following structural characteristics: i) distance between the aromatic ring and the disulfide bridge (d 1~4 -7 Å); ii) distance between the centroid of Trp aromatic ring and the cation center of the Arg and Lys side chain (d 2 /d 3~5 -8 Å); and iii) the angle between the Trp ring plane and the line connecting the cation and the ring center (~60-90 • ). In accordance with these structural criteria of the Trp-mediated photolysis, cyclic peptides (Ac-(CAWAC)-NH 2 , Ac-(CWKAC)-NH 2 and Ac-(CWAGC)-NH 2 ) were proposed as ideal models fulfilling the required criteria. Based on the pertinent W-SS distance and the presence of Lys, the following order could be proposed for the efficacy of the photolysis: Ac-(CWAGC)-NH 2 < Ac-(CWKAC)-NH 2 < Ac-(CAWAC)-NH 2 .
The detailed MD calculations supported by experimental NMR data also validate the expected structural characteristics of the three MM-designed Trp-containing peptides. Thus the proposed models conform the structural criteria required for efficient photolytic cleavage of the disulfide bond and so the selected peptides seemed to be suitable for studying the structural aspects of the Trp-mediated photolytic cleavage of disulfide bonds, which is the proposed mechanism of photolytic cleavage in proteins [1,2,4,13].
In the present study, however, we show that such events occurred only at low levels in the selected cyclic peptide models. Specifically, the results of fluorescence measurements did not support a systematic correlation between the Trp-SS distance and the amount of the detected free thiol. Instead, our results suggest the presence of other mechanisms of the photolytic cleavage of disulfide bonds in the studied peptides which was also supported by the results of LC-MS measurements, verifying the formation of oxidative photoreaction side products as well as peptide dimers and multimers.
Supplementary Materials: The following are available online. Table S1. Characterization of the devised cyclic peptides by MM calculation, Figure S1. 1 H-1 H ROESY spectra of the cyclic peptides showing the sequential and medium range ROE cross peaks of NHs as well as the ROEs of the Trp residue, Table S2. NMR-ensembles and the summary of structure calculations, 4. Table S3. Analysis of MD trajectories with regard to the occurrence of hydrogen bonds. Numbers represent the population of structures in the MD ensemble possessing the specified H-bond, Table S4. Trp-fluorescence measurements of model peptides, Figure S2. CPM fluorescence I max at 481 nm under various UV illumination times at 280 nm, Figure S3. Trp fluorescence emission spectra (A) λ ex = 280 nm and CPM fluorescence emission spectra (B) λ ex = 387 nm of Ac-c(CWKAC)-NH 2 after irradiation for 1, 1.5, 2 and 3 h, Figure S4. CPM calibration with Ac-CWAKC(Acm)-NH 2 peptide, Table S5. Proton ( 1 H) and carbon ( 13 C) chemical shifts of the studied cyclic peptides, Table S6. Analytical characteristics of linear peptides, Table S7. Analytical characteristics of cyclic peptides.