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The new home of
8 reviews
Close to the Snowdonia National Park, these three spacious, attractive and comfortable barn conversions boast stunning views over beautiful countryside. The holiday cottages (refs OZ3, OZ4 and OZ5) are located just metres away from the Wales Coast Path, which leads to a lovely pebble backed sandy beach whilst affording wonderful bird watching opportunities on the way. Within easy driving distance there are a myriad of exciting activities including mountain biking, surfing, zip wires, sailing, excellent walking, riding and golf. Caernarfon, with its magnificent castle, Welsh Fusiliers Museum and a fine selection of shops, restaurants and pubs, is just 4 miles away, and is also the start of the Welsh Highland Railway for scenic journeys into the heart of Snowdonia. Tai Coch Chatham is a perfect location for relaxing winter escapes and summer beach holidays. Shop and restaurant 2½ miles, pub serving food 1 mile.
Step to entrance. Open-plan living/dining room with wood-burning stove, patio doors and kitchen area with wooden floor. Double bedroom with 5ft bed. Shower room with toilet.
14 reviews
This stunning 'upside down' barn conversion, standing detached, has been finished to a high standard and has a delightful living room/kitchen on the first floor with French doors opening to a decked balcony with great views of the surrounding countryside. From the living room, a wonderful wrought iron spiral staircase leads down to the bedroom and shower room, and can be illuminated at night for a special effect! The unobtrusive and friendly owners live opposite, and are on hand should you need advice or assistance during your stay. Their very well behaved family pets are also friendly and welcoming, but do not present a nuisance.This is an excellent, central location on the Llyn Peninsula, giving easy access to all corners, with mountains, beaches and many attractions. The bustling market town of Pwllheli is within a few minutes' drive and has a good selection of shops, bars and restaurants, not to mention the stunning beach which seems to go on for miles, topped with grassy sand dunes. Criccieth with its historic, hilltop castle and beach is a little further to the east, as is Porthmadog with the famous Ffestiniog and Welsh Highland railways. Take the steam train on a nostalgic journey through the breath taking scenery of the Welsh mountains from Porthmadog in the south, to the delightful town of Caernarfon in the north, with its majestic castle standing proudly at the harbour.The seaside villages of Nefyn, Abersoch and sleepy Aberdaron are within easy reach and worth a visit, as is the stunning village of Porthdinllaen, situated literally on the beach. Nant Gwrtheyrn on the north coast is a disused quarry village, and a café provides refreshments after a bracing walk down to the beach.The area is also great for walkers with the Llyn coastal footpath to explore, or take a boat trip to Bardsey Island. There is so much to see and do, you are sure to return. Shop 1 mile, pub 2 ½ miles.
Double bedroom. Shower room with toilet. Narrow, open-tread, spiral stairs to first floor: Open-plan living room with French doors and gas woodburner/kitchen with breakfast area and wooden floor.
14 reviews
Step to entrance. Large open-plan living/dining room with wood-burning stove, French doors and kitchen area with wooden floor. Double bedroom with 5ft bed. Shower room with toilet.
22 reviews
These detached, converted barns (refs OK7 and OK8) are located in the hills between Pentrefelin and Criccieth, with spectacular views out to sea. From the garden, guests can enjoy wonderful 360 degree views of Cardigan Bay, together with a picturesque backdrop of the Snowdonia Mountain range. These 500-year-old cottages, once owned by Henry VIII, have been lovingly restored by the owners to provide beautiful holiday accommodation. Little Barn (OK8) is equipped with a 4-person hot tub for exclusive use, which is ideally positioned to enjoy the stunning views over Cardigan Bay and the mountains. These are idyllic cosy and romantic cottages. This is an ideal location for walkers, bird watchers and golfers, with many activities just on the doorstep, including horse riding, fishing, climbing and sailing, being located just 10 minutes from Pwllheli Marina and having a spectacular golf course and fishing lake within walking distance from the properties. These properties are also popular with artists, due to the spectacular views and abundance of light. With many lovely walks from the doorstep, as well as the beaches at Criccieth and Black Rock Sands at Morfa Bychan all within easy reach. Criccieth has a good selection of restaurants and pubs, and Criccieth Castle, standing proud over the bay, is well worth a visit. In the other direction is Porthmadog, home to the famous Ffestiniog and Welsh Highland railways. Portmeirion is close by, and the mountain ranges of northern Snowdonia, the village of Llanberis and the Llyn Peninsula are also within easy reach. Shop, pub and restaurant 1½ miles.
Open-plan living room with galley-style kitchen and slate floor. Bunk bedroom with TV and en-suite bathroom with over-bath shower and toilet. First floor: Double bedroom with TV and original, low character beams.
Note: Properties OK7 and OK8 can be booked together to sleep up to 8 guests
21 reviews
Open-plan, beamed living room with wood-burning stove, patio doors and kitchen with breakfast area and slate floor. Utility. Two double bedrooms, each with TV/DVD and patio doors; one with en-suite bathroom with Jacuzzi bath and toilet, one with en-suite shower room and toilet. Wet room with toilet. First floor: Galleried sitting room with double sofa bed (for alternative sleeping only).
22 reviews
Situated in the heart of the Snowdonia National Park in a beautiful location with valley and sea views, these semi-detached barn conversions have been sympathetically converted and restored to retain character and charm. Located on a working farm, it is an ideal holiday base to explore some of the best beaches in Wales and is just 2 miles from the coast. Visit the slate caverns and castle and Portmeirion and Snowdonia are easily accessible. St David's Royal Golf Course and theatre at Harlech. Local tourist attractions include Harlech Castle, Llanfair Slate Caverns and Portmeirion, while all areas of the Snowdonia National Park are easily accessible. Shop, pub and restaurant 1½ miles.
Ground floor: Living room with beams and wooden floor. Dining area/kitchen with beams and slate floor. 1 double bedroom. Bathroom with bath, shower cubicle and toilet. First floor: 1 twin bedroom with additional pull-out single. Separate toilet.
Woodburner in living room (initial fuel included). Cot and high chair on request. Electric range. Enclosed garden with patio and sitting-out area. Parking (3 cars).
Both properties: Oil central heating, electricity, bed linen and towels included. DVD. Microwave. Washing machine. Dishwasher. Fridge/freezer. Wi-fi. Two pets welcome (to be kept on leads at all times - working sheep farm). No smoking.
0 reviews
Ground Floor: The property has electric central heating and comprises:Lounge: Large with wooden floor, beams, wood burner, 28" Smart TV, DVD player and CD player.Kitchen: Beamed with breakfast area, double electric oven, electric hob, microwave, dishwasher, fridge, freezer and washing machine.Bedroom 1: With super kingsize bed, double French windows and en-suite with shower and WC.
First Floor: Bedroom 2: With kingsize bed.Bedroom 3: With zip and link twin beds (which can be made to super kingsize - please advise when booking).Bathroom: With bath, shower cubicle, WC and heated towel rail.
All electric, bed linen and towels included. Initial fuel for wood burner provided, remainder at cost. Highchair available. Wi-Fi. Welcome pack. Tumble dryer in garage.
Large landscaped, organic garden with patios and furniture. BBQ. Parking (2 cars). Cycle store. No smoking. NB: Unfenced pond in garden.
2 reviews
On the fringes of the Snowdonia National Park, this very charming and well-presented, detached holiday home enjoys lovely views of the surrounding mountains and countryside. The location is perfect for exploring the very best of North Wales and outdoor enthusiasts will certainly delight in the myriad of activities on offer nearby, including Zip World, the longest zip wire in the northern hemisphere, superb mountain bike trails, canoeing, sailing and excellent walking opportunities with the foot of Snowdon being just 3 miles away, in the mountain village of Llanberis. This is also the starting point for the Snowdon Mountain Railway which takes on an unforgettable trip to the summit of Snowdon, at 3,560ft. The property is also convenient for visiting the National Trusts Neo-Norman Penrhyn Castle, in the university city of Bangor and mighty Caernarfon Castle, along with a host of family attractions including Greenwood Forest Park. A fantastic selection of golden sandy beaches can be found on the beautiful Isle of Anglesey which is only a short drive away. There is a good selection of shops and restaurants to be found in both Bangor and Caernarfon, both 8 miles away. Beach 12 miles. Shop ½ mile, pub 2 miles and restaurant 3 miles.
Large living room with wood-burning stove. Sitting room with TV and patio doors to decked area. Spacious well-equipped kitchen with dining area, TV and tiled floor. Wet room with toilet. First floor: Two double bedrooms; one with 5ft bed and TV. Twin bedroom. Bathroom with over-bath shower and toilet
14 reviews
Kris Kin is a charming detached barn conversion situated in a quiet hamlet with mountain views and country walks. The surroundings are alive with snowdrops in winter and bluebells in spring. This holiday property is a short drive away from the beach and castle at Criccieth, Black Rock Sands, the Ffestiniog and Welsh Highland Railways at Porthmadog, and the Llyn Peninsula.
Spacious living/dining room with wooden floor, oak beams and patio doors to garden. Large well-equipped kitchen with breakfast bar and slate floor. Wet room and toilet. First floor: Two double bedrooms, each with additional single bed (for +1) and Velux window; one en-suite bathroom with over-bath shower and toilet. Single bedroom with Velux window. Bathroom with over-bath shower and toilet.
1 reviews
Just a short stroll from a beautiful sandy beach and the famous Morfa Nefyns Golf Course, this detached holiday property is perfect for summer seaside holidays and relaxing winter escapes. Bring your own boat and make the most of the public slipway just 500 yards from the property, where you can launch your boat onto the Porth Morfa Nefyn. Surrounded by a large lawned garden it's situated in the former fishing village of Morfa Nefyn on the northern coast of the beautiful Lleyn Peninsula. An ideal location for both bird watchers and walkers with the Wales Coastal Path passing close to the cottage. Porthdinllaen with its inn on the beach is a short walk and within easy reach is Caernarfon with its magnificent castle, Portmeirion, and the exhilarating outdoor pursuits of the Snowdonia National Park. Lively Pwllheli's many amenities, including a weekly market, and fashionable Abersoch, home of the annual Wakestock Festival are a short drive away. Shop, pub and restaurant 500 yards.
2 steps to entrance. Ground Floor:Open plan living space: With wooden floor and patio doors to garden.Living area: With Freeview TV and DVD player.Dining area.Kitchen area: With electric range oven, gas hob, microwave, fridge and freezer.Utility room: With washing machine and tumble dryer.Sun room.Bedroom 1: With kingsize bed.Bedroom 2: With twin beds.Bathroom: With bath, shower cubicle and toilet.First Floor:Bedroom 3: With double bed and single bed (for flexible sleeping arrangements), and en-suite with shower cubicle and toilet.Bedroom 4: With kingsize bed and en-suite with shower cubicle and toilet.
Ground source air pump central heating (underfloor on ground floor), gas, electricity, bed linen, towels and Wi-Fi included. Welcome pack. Large lawned garden with patio and garden furniture. Boat storage available. Bike store. Private parking for 6 cars. No smoking.
9 reviews
These adjoining stone-built properties sit in a prominent position in the tiny hamlet of Llangower on the south side of Llyn Tegid, the largest natural lake in Wales. With views towards the lake, the shoreline is only about 200 yards away, they provide an enchanting holiday location for those looking for a relaxing stay or ample opportunities for the more energetic. The Grade II Georgian listed Rector's House (ref OP7) was home to the rector of the ancient chapel at Llangower which was built in the 13th-century but modernised at about the same time as the rectory was built. Retaining its character, the house catches the sun from all directions, with glimpses of the lake, valley and mountains. Rectory Cottage (ref IWA) has a romantic feel with its wood-burning stove and views. On the southern edge of Snowdonia National Park, this is an ideal place for walking, sailing or just lazing. The well-known market town of Bala is a short drive away (3 miles) where most facilities are available including several good pubs and restaurants, a supermarket, butcher, bakers, post office, chemist, curiosity and gift shops. The local leisure centre provides swimming and fitness facilities and there is a small ten-pin bowling alley nearby and single-screen cinema in the town. The coast at Barmouth is 40-minute drive, and the towns of Dolgellau and Portmadoc, both a 25-minute trip away, provide interest for all the family. Shop and pub 3 miles.
This elegant, Georgian house has a light, homely feel about it and makes a comfortable retreat, whether for a main holiday or a relaxing short break. All the main rooms have two windows, affording glimpses of the lake at the front, the valley each side and mountains at the back. It is elegantly furnished with many valuable paintings and antiques. Much attention has been paid to the detail of the restoration of the house, so as to preserve the property's features whilst providing the comforts of modern-day living. Approached over a flat, slightly inclined tarmac drive, the house is decorated and furnished to a good standard. The front and rear door are without steps.Living room with open fire. Large dining room with open fire. Kitchen with oil Rayburn (for cooking) and breakfast area. Library twin bedroom with zip & link beds. Separate toilet. First floor: Double bedroom with 5ft bed. Further double bedroom with en-suite bathroom with over-bath shower and toilet. Twin bedroom, basin. Bathroom with walk-in shower cubicle and toilet.
Both properties:
7 reviews
Step to entrance. Large living room with wood-burning stove and wooden floor. Large kitchen/dining room with range, electric cooker and wooden floor. Double bedroom with 5ft bed. Twin bedroom. Shower room with toilet. First floor: Double bedroom with 5ft bed. Twin bedroom. Bathroom with separate shower cubicle and toilet.
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cottages.com is a trading name of Wyndham Vacation Rentals (UK) Ltd. Registered in England and Wales.
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Working!! – 09May 31, 2010 at 10:18 pm | Posted in Anime | 2 Comments
Traps are really becoming more and more frequent in anime nowadays. Totally a welcome change especially if they're all as kyaaaa as this blushing "bishoujo".
And this definitely deserves the title of "Quote of the Year":
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also this trap is more win cause he is voiced by a MALE seiyuu not an obvious female seiyuu as others. too bad fukuyama jun can't pull off a female like kamiya hiroshi or akira ishida.
Comment by Mariejoanna— June 1, 2010 #
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Mar 26, 1985
|Filing date
|Oct 7, 1983
|Priority date
|Oct 7, 1983
|Fee status
|Publication number
|Paul S. Miller, Paul O. P. Ts'o
|Original Assignee
|The Johns Hopkins University
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (2), Non-Patent Citations (2), Referenced by (91), Classifications (8), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
The work disclosed herein was supported in part by a grant from the National Institute of Health.
The present invention is concerned with certain improvements in the preparation of oligodeoxyribonucleoside alkyl or arylphosphonates.
Novel and useful oligodeoxyribonucleoside methylphosphonates are described in our U.S. application Ser. No. 206,297, filed Nov. 12, 1980. Further uses of such oligonucleotides are also disclosed in our U.S. application Ser. No. 363,230, filed Mar. 28, 1982. The disclosures of these two applications are incorporated herein by reference.
The instant invention is concerned, inter alia, with improved methods for preparing oligodeoxyribonucleoside alkyl or arylphosphonates, e.g. the methylphosphonates, disclosed in Ser. No. 206,297 and Ser. No. 363,230, and related products.
Oligonucleoside methylphosphonates have been used to study the function of specific RNA sequences in biochemical and intact cellular systems. See Miller et al, Biochemistry 20, 1874-1880 (1981) and Jayaraman et al, Proc. Nat'l Acad. Sci. 78, 1537-1541 (1981). Since these nonionic nucleic acid analogs can be taken up intact by mammalian cells and certain bacterial cells in culture, these compounds promise to be useful reagents for exploring and regulating the function of nucleic acids within living cells. See, in this regard, the above-mentioned U.S. applications Ser. No. 206,297 and Ser. No. 363,230.
While various procedures have previously been described for synthesizing oligonucleoside methylphosphonates and the like, there is a real need for a synthesis method which will efficiently and effectively provide oligomers of the desired sequence and length in the yields and amounts required for use.
The synthesis of the methylphosphonate analogs on a silica gel support has previously been described (Miller et al (1983) Nucleic Acids Res.). In that work, protected nucleoside 3'-methylphosphonic chlorides or tetrazolides were used as synthetic intermediates. While oligothymidine methylphosphonates could be efficiently synthesized by this procedure, low yields were encountered when other nucleosides, particularly d-[(MeO)2 Tr]ibuG, were used. Recently the preparation of oligonucleoside methylphosphonates on a glass support using nucleoside 3'-methylphosphine chlorides as reactive intermediates has been described (Sinha et al, Tetrahedron Letters, 24, 877-880, 1983). However, there continues to be a need for an efficient and convenient method for synthesizing oligonucleoside methylphosphonates of a desired sequence.
The principal object of the present invention is to obviate or minimize previous synthesis problems and provide an improved and efficient method for synthesizing oligodeoxyribonucleoside alkyl or arylphosphonates, notably methylphosphonates, of preselected sequence and length in high yield. Other more specific objects will also be hereinafter apparent.
Broadly stated, the objects of the invention are realized, at least in part, by the provision of a synthesis method which comprises condensing (1) a 5'- protected nucleoside 3'- alkyl or arylphosphonate with (2) a nucleoside or oligomer thereof bound to a solid or insoluble polymer support, e.g. a polystyrene support. The condensation can be repeated as many times as necessary, with the same nucleoside (1) or a different one, to ultimately obtain an oligonucleoside phosphonate having the desired sequence and length. Typically the invention may be used to prepare an oligomer containing up to nine nucleoside units or even more.
The invention also provides convenient means for removing base protecting groups and for separating the final oligonucleoside product from the polymer bound thereto. Additionally the invention includes means for cleaving the oligonucleoside phosphonate product at apurinic sites to obtain individual nucleoside components or shorter length oligomers of the analog subjected to cleavage. This is useful in confirming the sequence of the product.
The invention is hereinafter described by reference to the synthesis of deoxyribonucleoside methylphosphonates by condensing a 5'-dimethoxytrityldeoxyribonucleoside 3'-methylphosphonate alkylamine salt with a deoxynucleoside bound via the 3'- position to a polystyrene support. The dimethoxytrityl (DMTr) groups serves to protect the 5'-hydroxy groups. Other protecting groups (e.g. benzoyl or isobutyl) are used to protect the base portion of the nucleoside throughout the process for preparing the desired product. However, it will be recognized that other protecting groups than dimethoxytrityl and the indicated base protecting groups may be used. Other modifications will also be evident to those in the art.
The invention contemplates the use of any polymer support which is insoluble in the reaction medium and to which the nucleoside can be removably linked. Polystyrene is the preferred support, particularly if it includes a degree of cross-linking, for example, in the order of 1-5% by weight.
According to another preferred aspect of the invention, the final oligomer product which results from the indicated condensation or repetitions thereof to obtain the desired length and sequence, is separated from the polymer support simultaneously with the removal of the base protecting groups by treatment with a diamine alkane containing, for example, up to six carbons. Representative diaminoalkanes include 1,3-diamino propane and 1,4-diamino butane dissolved in a suitable solvent. Preferably, however the diamino alkane is ethylenediamine dissolved in ethanol or the equivalent. It has been found that this treatment is effective for both separation from the polymer and removal of base protecting groups while avoiding hydrolysis of the methylphosphonate linkages.
A unique advantage of the present process is that the condensation cycle, separation from the support and removal of base protecting groups and purification can be carried out in the same vessel thus obviating the need for transferring the product from one vessel to another for processing.
The examples which follow illustrate the synthesis of two types of oligomers, namely, those containing only methylphosphonate linkages, d-Np(Np)n N, and those which terminate wit a 5' nucleotide residue dNp(Np)n N. According to the invention an advantage of the latter oligomers is that they can be phosphorylated by polynucleotide kinase, and may be separated by polyacrylamide gel electrophoresis according to their chain length. This is useful in characterizing the product.
It has also been found that piperidine randomly cleaves the oligomer methylphosphonate linkages and generates a series of shorter oligomers whose number corresponds to the length of the original oligomer. Additionally, apurinic sites introduced by acid treatment spontaneously hydrolzye to give oligomers which terminate with free 3' and 5' OH groups. These reactions may also be advantageously used to confirm sequence and structure and to otherwise characterize the oligomers.
One preferred way of practicing the present invention is illustrated in FIG. 1. The process there exemplified comprises (a) removing the protecting group (DMTr) on a nucleoside or oligonucleoside bound to crosslinked or cross-linkable polystyrene (S) to provide the polymer bound reactant (I); and (b) condensing reactant (I) through its now deprotected 5'- OH group with a 5'-protected nucleoside methylphosphonate alkylamine (II) to obtain the desired oligodeoxynucleoside product (III). The reference letters B and B' represent simplified designations of protected pyrimidine or purine bases as conventional in DNA nucleosides. As is known in the art, each DNA nucleoside is composed of a purine or pyrimidine base joined to deoxyribose sugar. The DNA bases include adenine, guanine, cytosine and thymine which are generally represented by the letters A, G, C and T, respectively. The first two of these are purine bases while the last two (C and T) are pyrimidine bases.
In using the present invention, the reactant nucleosides will be selected to provide the desired base sequence in the oligonucleotide product, it being understood that condensation between product (III) and a nucleoside (II), with appropriate choice of bases (B) and (B'), may be repeated a number of times to give a final product (III') having the desired oligomer length and sequence. As noted earlier, oligomers comprising up to nine, and even more, nucleoside methylphosphonate units may be prepared in this way.
According to the process shown in FIG. 1, the 5'- DMTr protected nucleoside is bound via its 3'- position to an aminomethyl succinyl-derivatized polystyrene support. The 5'- protecting group is removed by using, for example, 1 M zinc bromide solution. The deprotected nucleoside is then condensed with a DMTr protected nucleoside 3'-methylphosphonate trialkyl ammonium salt, specifically the triethyl ammonium salt although other generally equivalent salts may be used. The condensation, as shown in FIG. 1, is carried out with mesitylenesulfonyl-3-nitrotriazole (MSNT) in the presence of acetic anhydride although it will be appreciated that other condensing agents may also be used provided these agents and the other conditions used do not cause undesired side reactions, e.g. hydrolysis, deprotection of the unsupported nucleoside reactant, etc.
Table I below lists oligomers which have been synthesized on polystyrene supports using the process described in Example 1 and FIG. 1 except as otherwise stated:
TABLE I__________________________________________________________________________ Support Average Yield crosslinking per CouplingProtected oligomer (a) (%) step (b) (%)__________________________________________________________________________1 d-[(MeO)2 Tr]bzCpbzApT--S 1 812 d-[(MeO)2 Tr]ibuGpbzCpbzCpbzApT--S 1 823 d-[(MeO)2 Tr]bzCpbzApibuGpibuGpTpbz 2 83 ApbzA--S4 d-[(MeO)2 Tr]bzCpTpTpbzApbzCpbzCp 2 85 TpibuG--S5 d-[(MeO)2 Tr]TpbzCpbzCpTpbzCpbzCp 2 86 TpibuG--S6 d-[(MeO)2 Tr]TpTpTpbzApbzCpbzCpTpT--S 1 837 d-[(MeO)2 Tr]bzApbzApbzA--S 1 838 d-[(MeO)2 Tr]bzApbzCpbzCpbzApT--S 1 819 d-[(MeO)2 Tr]bzApibuGpbzCpbzApbz 2 74 ApibuG--S10 d-[(MeO)2 Tr]bzApbzApbzApbzApibuGpbz 1 86 CpbzApbzApibuG--S__________________________________________________________________________ (a) p = methylphosphonate linkages p = pchlorophenyl phosphotriester linkage S = polystyrene support (b) Determined by analysis of the dimethoxytrityl group after each coupling step.
The invention is illustrated, but not limited, by the following examples:
Except as otherwise stated, the protected oligonucleoside methylphosphonate listed in Table I were prepared as follows:
The synthesis was carried out in a polypropylene column fitted with a Teflon 3-way valve and a rubber septum cap. During washing operations, the septum cap was removed and the 3-way valve was connected to a filter flask via a Luerer adaptor and tubing set. Generally vacuum was not required to wash the support. During the drying step, one port of the 3-way valve was connected to a cold trap and vacuum pump via the barrel of a 1 ml plastic syringes. The other port was connected to a Drierite column filled with dry argon.
The reaction cycle which was used consisted of the following steps:
(1) The polystyrene support (60 mg, 1% crosslinked) in the column was washed with three 2 ml portions of methylene chloride/isopropanol (85:15, v/v).
(2) The support was then treated with 2 ml of 1 M zinc bromide in methylene chloride/isopropanol solution. Two five-minute treatments were used when the support-bound nucleoside was d-[(MeO)2 Tr]bzA or d-[(MeO)2 Tr]ibuG, while four treatments were used when the nucleoside was d-[(MeO)2 Tr]T or d-[(MeO)2 Tr]bzC. After each treatment the orange solution was collected in a clean flask.
(3) The support was then washed with two ml portions of methyl chloride/isopropanol and the washings were collected in same flask used in step (2). The solution was diluted to 50 ml and a 0.20 ml aliquot was dissolved in 0.80 ml of perchloric acid/ethanol (3:2, v/v). The absorbance was determined at 500 nm and the amount of trityl cation was determined using a molar extinction coefficient of 89,000.
(4) The column was attached to a waste flask and the support was washed with three 2 ml portions of 0.5 M triethylammonium acetate in dimethylformamide; three 2 ml portions of anhydrous pyridine and three 2 ml portions of diethyl ether.
(5) The column was fitted with the septum cap and set up in the drying mode under house vacuum for at least 5 min.
(6) The support was dried by adding 300 1 of anhydrous pyridine via a gas tight syringe. After the support had swollen, the vacuum (oil pump) was applied and the support was warmed with a stream of air from a hair dryer. Evaporation was continued for 10 min. Dry argon was then admitted and the drying operation was repeated two more times.
(7) The coupling mixture was prepared by dissolving 0.20 mol of MSNT in 320 1 of anhydrous pyridine. The solution was then transferred to the vial containing 0.06 mmol of d-[(MeO)2 Tr]Np.Et3 NH. The nucleotide was dissolved by vortexing and the coupling solution was then added dropwise to the support. It was deemed important to add the solution slowly and to allow the support to swell. Trapped gas bubbles could be removed by gently tapping the column. The entire operation was carried out using one predried syringe. The syringe was left in the V-vial while the reagents were being dissolved.
(8) The reaction mixture was kept at room temperature (22� C.) for two hours.
(9) The column was set up in the washing mode and the support was washed with three 2 ml portions of anhydrous pyridine. The solution, which contained unreacted nucleoside 3'-methylphosphonate and MSNT, was collected in a separate flask. A 50% aqueous pyridine solution (1 ml) was added and the solution was kept at 4� C. for later purification and recovery of d-[(MeO)2 Tr]Np.
(10) The support was treated with a solution containing 2 ml of anhydrous pyridine, 1 ml of acetic anhydride and 20 mg of dimethylaminopyridine for 30 min.
(11) The process was then repeated as necessary beginning with step (1) in order to obtain the oligomer products listed in Table I.
Oligomers 7, 8 and 10 (Table I) were synthesized as described above with the exception that step (10) was not used for oligomer 10. The other oligomers of Table I were synthesized in essentially the same manner except the condensation reactions were run in 1 ml V-vials or in glass reaction columns. When the reactions were run in V-vials, the support was dried by several evaporations with anhydrous pyridine in the V-vial. When the reactions were run in the glass reaction column, the support was dried by a single evaporation with anhydrous pyridine overnight at room temperature.
This example illustrates the methods found to be the most effective for cleaving the oligonucleotide product from the 1% crosslinked support and for removing the base protecting group with minimal hydrolysis of the phosphonate backbone.
The following steps were used for deprotecting oligomers 7, 8 and 10 (Table I):
(1) After the final condensation step the support (60 mg) was washed with three 2 ml portions of anhydrous pyridine; three 2 ml portions of methylene chloride/isopropanol and three 2 ml portions of diethyl ether. The support was then dried under house vacuum.
(2) The support was then swollen by addition of 2 ml of pyridine and the excess was removed under house vacuum.
(3) The support was treated with 3 ml of 0.017 M tetra-n-butylammonium fluoride in tetrahydrofuran/pyridine/water (8:1:1, v/v) for 40 hours at room temperature (20�-25� C.). No shaking was required. The support was then washed with three 2 ml portions of 50% pyridine/water; three 2 ml portions of pyridine; and three 2 ml portions of methylene chloride/isopropanol.
(4) The support was thereafter treated with 3 ml of ethylenediamine/ethanol (1:1, v/v) for 7 hours at room temperature without shaking. The solution was collected and the support was washed with four 2 ml portions of pyridine/ethanol (1:1, v/v) and four 2 ml portions of N,N-dimethylformamide. The combined eluate and washings were evaporated at 25� C. and the oily residue was coevaporated several times with 50% aqueous ethanol. The resulting product was the oligonucleoside separated or cleaved from the support and with the base protecting group removed. Basically this required treatment of the supported oligomer with ethylenediamine/ethanol mixture and washing of the support with anhydrous pyridine.
Oligomer 9 was deprotected in a similar fashion to that set forth above except that step (4) was carried out at 65� C. for 3 hours. For oligomers 1-6, step (3) was not included and step (4) was carried out at 65� for 3 hours.
In each instance, effective separation from the support and deprotection without any undesired effect on the phosphonate linkage or other portions of the nucleoside, were obtained.
The composition of the ethylene diamine/ethanol mixture can be varied (for example, 1-5 parts by volume of diamine can be used per part of ethanol) although a 1:1 mixture has been found particularly effective.
The temperature of the cleavage and deprotecting treatment can also be varied although it is generally adequate to operate at room temperature (20�-25� C.). If necessary or desirable, higher temperature (e.g. up to 100� C.) can be used although, in any case, care should be taken to avoid temperatures which will cause undesired side reaction, e.g. hydrolysis or the like effecting the phosphonate linkages.
This example describes the purification of the separated oligonucleoside methylphosphonates obtained in the manner described in Example 2.
For oligomers which contain only methylphosphonate linkages (1-6), the residue from step (4) of Example 2 was dissolved in a small volume of 50% aqueous ethanol. The solution was chromatographed on a C-18 reversed phase column (0.46 cm�25 cm for 100 A254 units or less; 0.9 cm�50 cm for more than 100 A154 units) using a linear gradient of 0% to 25% (8-mer or less) or 0% to 35% (9 mer) acetonitrile in water to remove non-tritylated oligomers. The desired tritylated oligomer was eluted with 50% acetonitrile in water. The solvents were evaporated and the residue was treated with 1 ml of 80% acetic acid in water for 1 hr at room temperature. The solvents were evaporated and the residue was repeatedly evaporated with ethanol to ensure complete removal of acetic acid. The oligomer was then purified by C-18 reversed phase HPLC using a 0% to 25% or 30% acetonitrile in water gradient.
For oligomers which terminate with a 5'-nucleoside phosphodiester linkage (7-10) the residue from step 4) described in Example 2 was treated with 80% acetic acid in water for 15 min. at room temperature. After removal of the acetic acid by evaporation, the residue was dissolved in 20 ml of 50% aqueous ethanol and the solution was passed through a DEAE cellulose column (2.5�8 cm, bicarbonate form) which had been previously washed with 50% aqueous ethanol. The column was monitored at 254 nm and washed with 50% aqueous ethanol until the pen returns to the baseline. The desired oligomer was then eluted with 0.15 M triethylammonium bicarbonate in 50% aqueous ethanol. The buffer was removed by evaporation and co-evaporated with 50% aqueous ethanol. The oligomer was then further purified by C-18 reversed phase HPLC using a 0% to 30% acetonitrile in 0.10 M ammonium acetate (pH 5.8) gradient. The oligomer was freed of ammonium acetate by desalting on a Bio-Gel P-2 column (1.5�20 cm).
As will be evident, this example (Example 3) illustrates two methods for purifying oligomers following cleavage from the support. For oligomers which contain only methylphosphonate linkages, the tritylated oligomer was isolated by preparative reversed phase HPLC. This separation is based upon the greater affinity of the tritylated oligomer for the hydrophobic C-18 matrix of the column. Shorter, non-tritylated oligomers were first eluted with a 0% to 25% or 0% to 35% acetonitrile in water gradient. The tritylated product was then eluted with a step gradient of 50% acetonitrile in water.
The dimethoxytrityl group was removed from the material in the 50% acetonitrile fraction by treatment with 80% acetic acid at room temperature. Because these oligomers were originally cleaved from the support by ethylenediamine/ethanol (1:1, v/v) at 65� C., it was usually found that the tritylated oligomer fraction contained shorter oligomers in addition to the desired product. These were easily separated by preparative reversed phase HPLC. While this purification procedure is qualitatively satisfactory, recoveries from the reversed phase columns varied from 50% to 90%. These losses which appeared to be due to irreversible absorption of the oligomers to the column varied depending on the base composition of the oligomers.
For the oligomers which terminate with a 5'- nucleoside phosphotriester group, the support was first treated for 40 hrs with tetra-n-butylammonium fluoride to remove the p-chlorophenyl protecting group. These oligomers were then cleaved from the support and the base protecting groups were removed by treatment with ethylenediamine/ethanol (1:1, v/v) for 7 hrs at room temperature. For example, the reversed phase HPLC profile for products obtained after cleavage of the nonamer, d-[(MeO)2 Tr]ApApApApGpCpApApG, is shown in FIG. 2a. The peaks marked with (X) are from impurities in the solvents used to elute the oligomer from the support. The tritylated nonamer appears at 22 min in the chromatogram and is eluted with 50% aqueous acetonitrile. After removal of the dimethoxytrityl group, the nonamer elutes at 15.2 min (FIG. 2b). The peaks which appear between 19 and 25 min are shorter oligomers which contain a 5'-mesitylenesulfonoate group.
At this stage, the nonamer was partially purified by ion exchange chromatography on the DEAE cellulose column. This step, which resulted in 84% recovery of material loaded onto the column, removed most of the shorter oligomers as shown in FIG. 2c. After removal of the buffer, the nonamer was purified by preparative reversed phase NPLC using a 0% to 30% acetronitrile in 0.1 M ammonium acetate gradient. The recovery of material from the column (84%) appears to be higher when ammonium acetate is used. The pure nonamer (FIG. 2d) was obtained in 10% overall yield based on the amount of d-[(MeO)2 Tr]ibuG originally bound to the polystyrene support. The oligomer was desalted on a Bio-Gel P-2 gel filtration column. Similar results were obtained for the other oligomers purified by this method. Thus, for example, dApApA and d-ApCpCpApT were obtained in 44% and 22% isolated yields respectively based upon the amount of d-[(MeO)2 Tr]N.
Oligomers which terminate with a 5'-nucleotide phosphodiester may be treated with spleen phosphodiesterase to give the oligonucleoside methylphosphonate as follows:
The oligomer (3 A254 units) is dissolved in 40 μl of water and treated at 37� C. for 2 hrs with 10 μl (1-2 units) of spleen phosphodiesterase dissolved in water. The completeness of the reaction is determined by reversed phase HPLC. The solution is then diluted with 50 μl of water and passed through a DEAE cellulose column (0.5�1 cm). The column is washed with 500 μl of water and the oligomer is recovered by lyophilization.
This example describes the recovery of protected nucleoside 3'-methylphosphonate from the reaction mixture of Example 1.
The aqueous pyridine solution from step (9) of Example 1 was evaporated after addition of 0.1 ml of triethylamine. The residue was dissolved in 50 ml of chloroform and the solution was extracted twice with 50 ml of 1 M ammonium bicarbonate. The chloroform layer was dried over anhydrous sodium sulfate. Several drops of triethylamine were added to clarify the solution. After filtration the solvents were evaporated and the residue was evaporated with three 2 ml portions of anhydrous pyridine on an oil pump. The foamy residue was dissolved in 2 ml of dry methylene chloride and the solution was added dropwise to a stirred solution of 1% triethylamine in hexane. The resulting precipitate was collected via filtration on a sintered glass filter, washed with hexane and dried in a vacuum desiccator. The product thus recovered was the protected nucleoside 3'-methylphosphonate, d-[(MeO)2 Tr]Nlp.
This example describes the removal of base protecting groups, specifically benzoyl (bz) and isobutryl (ibu), from nucleosides by using ethylene diamine, specifically ethylene diamine in ethanol.
Several mg of d-[(MeO)2 Tr]bzA, d-[(MeO)2 Tr]bzC and d-[(MeO)2 Tr]ibuG were each dissolved in 250 μl of ethylenediamine/ethanol (1:1, v/v). The solutions were incubated at room temperature. At various times aliquots were chromatographed on silica gel TLC plates which were eluted with 10% methanol in chloroform. The spots corresponding to the starting material and the product were cut out and treated with 1.5 ml of perchloric acid/ethanol (3:2, v/v)1 for 30 min. The absorbance of each solution was measured at 500 nm and the percent reaction was determined. The half-lives of the reactions, in terms of the time required to remove 50% of the base protecting group (DMTr) are given in Table II.
TABLE II______________________________________ Times required to Times required to cleave 50% of the remove 50% of the nucleoside from theProtected base protecting 1% crosslinkedNucleoside groups (min) support (min)______________________________________d-[(MeO)2 Tr]bzA 10 105d-[(MeO)2 Tr]bzC 20 105d-[(MeO)2 Tr]ibuG 40 54d-[(MeO)2 Tr]T -- 105______________________________________
The above results show that ethylenediamine may be effectively used to remove the base protecting groups (bz and ibu). It is an important feature of the invention that this removal with a base may be accomplished without cleaving or otherwise undesirably effecting methylphosphonate linkages.
This example illustrates the removal of protected nucleosides from polystyrene supports.
Five mg of d-[(MeO)2 Tr]TS (1% crosslinked), d-[(MeO)2 Tr]bzAS (1% crosslinked), d-[(MeO)2 Tr]bzCS (1% and 2% crosslinked) and d-[(MeO)2 Tr]ibuGS (1% and 2% crosslinked), where S represents the polystyrene support, were each treated with 500 μl of ethylenediamine/ethanol (1:1, v/v) solution at room temperature. At various times, 10 μl aliquots were removed, the solvent was evaporated and the residue was dissolved in 1 ml of perchloric acid/ethanol solution (3:2, v/v). The amount of dimethoxytrityl cation and hence the amount of nucleoside cleaved from the support was determined by measuring the absorbance at 500 nm. The half lives for cleavage of protected nucleosides from the 1% crosslinked support are given in Table II.
As shown in Table II, the deoxyguanosine nucleoside was cleaved most rapidly while the other three nucleosides had essentially the same rates of hydrolysis. All the nucleosides are completely removed from the support within 7 hrs. In contrast, very different rates were observed for the 2% cross-linked supports. In these cases, very little cleavage occurred during the first 4 hrs of incubation after which increasing amounts of nucleoside were released over a 24 hr period. This effect may have been due to the slower swelling rate of the 2% crosslinked support versus the 1% support. The results of these experiments indicate that, although condensation reactions occur with equal efficiencies on both the 1% and 2% crosslinked supports, the 1% support is preferred for synthesis since the oligomer can be removed more readily under mild conditions.
The stability of the methylphosphonate linkage in a number of oligomers of varying chain length and base composition was examined by measuring the degree of hydrolysis of the methylphosphonate linkage following prolonged exposure to ethylenediamine/ethanol at room temperature. This was accomplished as follows:
Oligonucleoside methylphosphonates (1.25 A254 units each) were treated with 50 μl of ethylenediamine/ethanol (1:1, v/v) at room temperature (22� C.). Aliquots (10μl) were withdrawn at various times and the solvents were evaporated. The residue was dissolved in 20 μl of 50% aqueous ethanol and the solution examined by C-18 reversed phase HPLC using a 0% to 25% acetonitrile in water gradient. The mole percent of starting oligomer remaining after 100 hrs of treatment is given in Table III.
TABLE III______________________________________Hydrolysis of Oligonucleoside Methylphosphonatesby Ethylenediamine at 22� C. Mole percent of oligomer remaining after treatment forOligomer 24 hrs 48 hrs 96 hrs______________________________________d-TpA 100 100 100d-ApT 100 100 100d-TpT 100 100 100d-TpTpT 95 97 91d-GpGpT 100 100 100d-ApApA 93 87 75d-CpCpApT 92 83 67______________________________________
As shown in Table III, some of the oligomers, most notably the dimer and d-GpGpT, were completely stable over a 96 hr period. The maximum rate of hydrolysis for a methylphosphonate linkage in those oligomers which were hydrolyzed is estimated to be 0.13 mole %/hr. The results of these studies show that the oligonucleoside methylphosphonates can be cleaved from the 1% crosslinked support and completely freed of base protecting groups with little or no hydrolysis of the methylphosphonate linkage by treatment with ethylendiamine/ethanol (1:1, v/v) at room temperature for 7 hrs. These conditions can, therefore, be used for cleavage from the support and for removal of base protecting groups.
This test was used to determine hydrolysis of the oligonucleoside methylphosphonate linkage in acid. Six A254 units of oligomer were dissolved in 100 1 of 0.01 M hydrochloric acid solution. The solution was heated at either 45� or 65� C. Aliquots (10 1) were withdrawn at various times and added to 10 1 of 0.015 M ammonium hydroxide at 0� C. The samples were then injected directly onto a C-18 reversed phase column which was eluted with 50 ml of a linear gradient of 0% to 25% acetronitrile in water at a flow rate of 2.5 ml/min. The products of the reaction was determined by comparison with authentic samples.
The 5' end of oligonucleoside methylphosphonates was labelled with T4-polynucleotide kinase as follows:
Oligonucleoside methylphosphonate (2 nmol) were dissolved in a buffer solution containing 50 mM Tris-HCl (pH 9.0), 10 mM MgCl2, 5 mM dithiothreitol, 20 μM spermidine, and [μ-32p ]ATP (8 Ci, 4 Ci/mmol). T4-polynucleotide kinase (4 units) was added to the solution, which was made up to 50 μ ml by adding water. The solutions were incubated at 37� C. for 2 hrs. The reaction was checked by PEI-cellulose TLC. The PEI-cellulose plate (2�20 cm, Merck) was preactivated by elution with 1.2 M pyridinim formate (pH 0.5). Small aliquots (0.1 μl) of PNK reaction solutions were applied and developed with 1.5 M pyridinium formate (pH 3.5). The TLC plate was dried and autoradiographed using an intensifying screen at room temperature.
Gel-electrophoresis of 5' labelled oligonucleoside methylphosphonates as obtained in Example 10 was carried out as follows.
Polyacrylamide gels were prepared by polymerizing a solution containing 18% (v/v) acrylamide, 0.8% (w/v), N,N'-methylenebisacrylamide. 7 M urea, 89 mM tris-borate (pH 8.2), 2 mM EDTA, 0.07% (w/v) ammonium persulfate, in the presence of N,N,N',N'-tetramethylethylenediamine (20μ1/20 ml solution). The solution was poured into a 0.75�140�150 mm mold and allowed to polymerize for 2 hrs at room temperature. 5'-labelled oligonucleoside methylphosphonates were dissolved in 10% aqueous glycerol solution containing 0.04% bromophenol and applied to the gel. The electrophoresis was run using 89 mM trisborate buffer containing 10 mM EDTA. After electrophoresis the gel was dried using a gel-dryer and autoradiographed using intensifying screens at room temperature.
Partial cleavage of oligonucleoside methylphosphonates with hydrochloric acid or piperidine was carried out as follows.
Aliquots (1 μ1) from the polynucleotide kinase reaction mixture were incubated with 1 μ1 of 1 M hydrochloric acid at 37� C. for 30 min. After incubation the solution was neutralized with 1 μ1 of 1 M ammonium hydroxide and then allowed to stand at room temperature for 10 min before cooling to 0� . Alternatively, aliquots (1 1) from the phosphorylation reaction were incubated with 1 μ1 of 1 M aqueous piperidine at 37� C. for 10 min after which the solution was cooled to 0� and evaporated. The treated samples were then subjected to polyacrylamide gel electrophoresis as described above without further purification.
The experimental work referred to above can be summarized as follows:
Table I shows representative protected oligonucleoside methylphosphonates which have been synthesized on aminomethyl succinyl-derivatized polystyrene supports according to the invention. The basic synthetic steps which are shown in FIG. 1 are described in detail in Example 1. These steps are: (1) removal of the dimethoxytrityl group with 1 M zinc bromide solution; (2) drying the support in a co-evaporation with anhydrous pyridine; (3) reaction of the support-bound nucleoside or oligonucleotide with a coupling mixture containing 0.15 to 0.2 M d-[(MeO)2 Tr]-Np.Et3 NH and 0.3 to 0.6 M mesitylenesulfonyl-3-nitrotriazole in anhydrous pyridine and (4) acetylation of unreacted 5'-OH groups with acetic anhydride. The reactions and washing steps were most conveniently carried out in a polypropylene Econo Column fitted with a septum cap and a 3-way Teflon stopcock. Thus no transfers of the support are required using this apparatus.
The synthetic procedure illustrated above is analogous to the phosphotriester approach used to prepare protected oligonucleotide phosphotriesters on polystyrene supports.
However, it has been found that the methylphosphonate coupling reaction is extremely sensitive to moisture. Thus drying procedures such as washing with anhydrous solvent or blowing dry nitrogen gas through the support which are satisfactory for phosphotriester synthesis result in low yields of the methylphosphonates. According to the invention, the best yields are obtained when the support is co-evaporated with anhydrous pyridine by directly attaching the reaction column to a vacuum pump and dry-ice trap. Three 10 min co-evaporations were sufficient to render the support anhydrous.
The protected nucleoside 3'-methylphosphonate triethylammonium salts used in the coupling reactions were prepared from their cyanoethyl ester derivatives in known manner. These monomers were recovered after each coupling step and were freed of unreacted MSNT by a simple extraction step. After precipitation and drying, the monomers could be reused for other syntheses. The ability to recover the unreacted monomers is particularly important for large scale synthesis since large excesses of these materials are employed in the coupling step. This recovery step represents an advantage over the phosphine synthetic method, since it is not clear that nucleoside 3'-methylphosphine chlorides can be recovered after reaction.
As will be evident from the foregoing, two types of methylphosphonate oligomers were prepared in Example 1: those which contain only methylphosphonate linkages (oligomers 1-6) and those which terminate with a 5'-nucleoside 3'-p-chlorophenyl phosphotriester moiety (oligomers 7-10). Oligomers were synthesized on both 1% and 2% crosslinked supports. As shown in Table I, the average yield per coupling step which was determined by trityl group analysis was approximately 82%, for both types of support. This yield is adequate to allow preparation of protected octamers or nonamers in 25% and 20% overall yield, respectively.
Previous work has shown that methylphosphonate linkages are cleaved by base. It has been found this hydrolysis reaction depends to some extent upon the nucleoside sequence of the oligomer. Hydrolysis of methylphosphonate linkages by concentrated ammonium hydroxide in pyridine (1:1, v/v), the reagent commonly used to remove oligonucleotide base protecting groups, can be largely suppressed if the reactions are run at 0� C. However, these conditions are unsatisfactory for removal of oligonucleoside methylphosphonates from the polystyrene support.
Recently Barnett and Letsinger (Tetrahedron Letters 22; 991-994 (1981)) described removal of base protecting groups from oligonucleotide β,β,β-trichloroethylphosphotriesters using a mixture of ethylenediamine in phenol. According to the present invention, it has been found that ethylenediamine in ethanol (preferably 1:1, v/v) rapidly and cleanly removes benzoyl and isobutyryl protecting group from dA, dC and dG nucleosides. This reagent is particularly attractive since it can be easily removed by evaporation.
Oligonucleoside methylphosphonate can be cleaved from the support and base protecting groups removed by treatment with ethylenediamine/ethanol (1:1, v/v) at 65� C. for 3 hrs. It apears that the methylphosphonate linkages of at least some dimers are stable to these conditions. However, the linkages of longer oligomers appear to be hydrolyzed to various extents by this procedure at the indicated time and temperature conditions. This can result in loss of product as evidenced by the presence of shorter oligomers in amounts greater than might be expected based upon the coupling yields. To avoid this potential problem, it is preferred to carry out the deprotection reactions at lower temperatures and it has been found that these can be run effectively at room temperature (20-25� C.). Table II shows the half-lives for removing the base protecting groups from nucleosides at room temperature. While the isobutryl protecting group is removed at the slowest rate, all the groups are completely removed within 240 min. The sole product of each reaction is the 5'-O-dimethoxytritylnucleoside as shown by TLC.
It is also noted that the 5'-terminal nucleotide may be removed enzymatically to give an oligomer which contains only methylphosphonate linkages. Thus treatment of the nonamer, d-ApApApApGpCpApApG, with spleen phosphodiesterase gave d-Ap and the octamer d-ApApApGpCpApApG whose retention time is 16.4 min on the reversed phase column. The noncharged octamer was freed of d-Ap and enzyme by simply passing it through a small DEAE cellulose column.
Methylphosphonate linkages can be cleaved by base and according to the invention, it has been found that piperidine is particularly effective for this purpose. The cleavage occurs in a random manner giving nucleosides, nucleoside 3'- or 5'-methylphosphonates and nucleoside 3'-,5'-bis-methylphosphonates. This hydrolysis reaction may be used to characterize dimers, since the identity and ratio of the products formed are easily determined by reversed phase HPLC. However, the method becomes less satisfactory for longer oligomers.
Purine-containing oligonucleoside methylphosphonates may be depurinated by treatment by hydrochloric acid at 65� C. The methylphosphonate linkage is resistant to hydrolysis by these conditions as shown by the stability of d-TpT and d-TpTpT. On the other hand, dimers or trimers containing purine bases such as d-ApA, dApT, dTpA or d-ApApA have half-lives between 92 and 144 min in 0.01 M hydrochloric acid. Following neutralization with ammonium hydroxide, the products of these reactions were characterized by reversed phase HPLC. d-ApA gave adenine, while both d-ApT and d-TpA gave adenine and thymidine as the sole products of the reaction. Adenine and d-ApA were the only products observed when d-ApApA was partially hydrolyzed. When d-CpCpApT was completely hydrolyzed in acid three products, d-CpC, adenine and thymidine, were observed in a molar ratio of 1:1:1.
In contrast to the base hydrolysis of the methylphosphonate group, apurinic sites produced by acid treatment are further hydrolyzed to nucleosides or oligomers with free 5'- and 3'- OH groups. The absence of terminal phosphonate residues suggests hydrolysis occurs as shown in FIG. 3. The OH group on the 4'-carbon generated by opening the ribose at the apurinic site may participate in an intramolecular attack on the adjacent phosphonate linkages which results in removal of the phosphonate residues from the neighboring nucleoside hydroxyls.
Previous studies by others have indicated dithymidine methyl- or phenylphosphonates can be phosphorylated by polynucleotide kinase. Sinha et al supra reported that dimers but not trimers or tetramers served as substrates for this enzyme. It was not possible to phosphorylate tetramers or longer oligomers which contain only methylphosphonate linkages. However, those oligomers which terminate with a 5'-dAp-residue are readily phosphorylated by polynucleotide kinase. The phosphorylation reaction is easily followed by PEI-cellulose TLC. As shown in FIG. 4a, the phosphorylated oligomers have a higher Rf values than does ATP.
The phosphorylated methylphosphonate oligomers can be separated according to their chain lengths by polyacrylamide gel electrophoresis on an 18% gel containing 7 M urea as shown in FIG. 4(b). Lane 1 shows the separation of a trimer, hexamer and nonamer. The band which appears directly below the nonamer may arise from traces of contaminating octamer which were not removed during the purification of d-ApApApApGpCpApApG.
When the oligomers were partially hydrolyzed with piperidine, a series of new bands appears corresponding to shorter oligomers produced via hydrolysis of the methylphosphonate linkages (Lanes 2-4). Because of the random nature of this cleavage reaction, the oligomers should terminate with either a 3'-OH or a 3'-methylphosphonate group. In the case of the trimer, 32 pApApA, hydrolysis gives two bands which correspond to d-32 pApA and d-32 pApAp (Lane 2). The same products are observed for hydrolysis of the nonamer, d-32 pApApApApGpCpApApG (Lane 4). A similar situation is observed for d3 pApGpCpApApG although the dimers d-32 pApG and d-32 pApGp have somewhat different mobilities (Lane 3). These dimers cannot be separated when the gel is run in the absence of urea.
It is possible to determine the chain lengths of the original oligomers by counting the number of oligomers produced by partial piperidine hydrolysis. For example, starting with the trimer band, one can observe 3 bands corresponding to tetramer, pentamer and hexamer for the hydrolysis products of d-32 pApGpCpApApG (Lane 3) and 6 bands corresponding to tetramer, pentamer, hexamer, heptamer, octamer and nonamer for the hydrolysis products of 32 pApApApApGpCpApApG (Lane 4). The positions of the oligomers of the same chain length appear to be very similar which suggests that the mobilities are not greatly affected by base composition on the urea-containing gel. This procedure thus provides a rapid and convenient method for characterizing the methylphosphonate oligomers.
Treatment of the phosphorylated oligomers with acid produces shorter oligomers which result from chain cleavage at apurinic sites. In this case the oligomers terminate with a 3'-OH group. Treatment of 32 pApGpCpApApG produces an intense band corresponding to the pentamer, d-32 pApGpCpApA (Lane 5) while treatment of 32 pApApApApGpCpApApG produces an intense band corresponding to the octamer d-32 pApApApApGpCpApA and fainter bands corresponding to the heptamer, hexamer, tetramer and trimer. These results indicate that under the conditions used, hydrolysis occurs preferentially at the 3'-terminal G residue of both oligomers. It should be possible to extend this methodology to other base specific depurination or depyrimidination reactions similar to those employed in the Maxam-Gilbert sequencing method.
It will be appreciated that modifications may be made in various aspects of the invention without departing from the scope and essence thereof. Thus, it is evident that any suitable nucleoside or oligonucleoside bound to an appropriate polymer, preferably polystyrene, which is at least partially crosslinked, may be used for present purposes. Such nucleosides are commercially available or readily prepared by conventional or known means, and may be selected to provide the desired oligonucleoside sequence.
Likewise, any protected nucleoside or oligonucleoside alkyl or arylphosphonate may be employed. While the invention has been illustrated using the methylphosphonates, other alkyl or arylphosphonates, e.g. the phenyl, ethyl, propyl, or butylphosphonates or the like, may be used. Preferably the reactant is in the form of a methylphosphonate lower alkylamine, e.g. the triethylamine salt, but other equivalent materials may be used. The essential feature is to select a protected nucleoside alkyl or arylphosphonate for use with the polymer supported nucleoside, which will give the desired oligomer sequence.
In the examples given above, the protected nucleosides d-[(MeO)2 Tr]N and 5-dimethoxytrityl nucleosides esterified to 1% to 2% crosslinked polystyrene were obtained from commercial sources. The protected nucleoside 3'-methylphosphonate triethylammonium salts, d-[(MeO)2 Tr]Np.Et3 NH, was prepared in known manner.
The nucleoside loading level was 40 to 120 mol of nucleoside/g of support. Mesitylenesulfonyl-3-nitrotriazole (MSNT) was also obtained commercially while the anhydrous pyridine was prepared by refluxing previously purified pyridine over calcium hydride chips for several hours followed by distillation onto calcium hydride chips in 5 ml V-vials fitted with Teflon-lined septum caps. Lyophilized spleen phosphodiesterase, T-4 polynucleotide kinase and [μ-32 P]-ATP were obtained commercially. Reversed phase high performance liquid chromatography (HPLC) was carried out on Whatman Inc. C-18 (ODS-3) columns (0.4�25 cm). Analytical columns were eluted with 50 ml linear gradients of acetronitrile in water or acetonitrile in 0.1 M ammonium acetate (pH 5.8) at a flow rate of 2.5 ml/min. The eluate was monitored at 254 nm. The following molar extinction coefficients were used at 254 nm: d-T 7,250, dA 13,270, dC 6,260, dG 13,700. Unless otherwise noted, all reactions and operations were carried out at room temperature.
The specific conditions, e.g. time and temperature, selected for the condensation reaction and for separating the nucleotide from the polymer support and for removal of protecting groups can be widely varied. The essential point in selecting such conditions is to avoid undesired hydrolysis or side reactions which will reduce the yield of the desired oligonucleoside methylphosphonate.
It will be appreciated from the foregoing that the invention contemplates the condensation of protected nucleoside 3'-methylphosphonate triethylammonium salts with nucleosides or oligomers linked to a polystyrene support to obtain oligomers of desired length and sequence. The method involved is easy to use and allows relatively rapid synthesis of oligomers of defined sequence up to nine nucleosides in length or even more. Conditions for the efficient removal of base protecting groups and subsequent purification of the oligomers are also disclosed. Additionally contemplated are novel hydrolysis reactions which can be used as a basis for characterizing and sequencing these oligonucleotide analogs including, for example, hydrolysis at apurinic sites created by acid treatment of the oligomers.
While the accompanying drawings have been referred to above, the substance thereof is summarized below for convenience:
FIG. 1 shows the preparation of protected oligonucleoside methylphosphonates on a polystyrene support.
FIG. 2 comprises the reverse phase HLPC profile obtained in the purification of d-ApApApApGpCpApApG as follows: (a) oligomers obtained after treatment of support with ethylenediamine/ethanol (1:1, v/v). (b) oligomers obtained after treatment of the mixture in (1) with 80% acetic acid. (c) oligomers eluted from DEAE cellulose column with 0.15 M triethylammonium bicarbonate. (d) nonamer obtained after preparative reversed phase HPLC. Peaks marked with the symbol (X) are derived from impurities in the solvent used to elute the oligomer from the polystyrene support.
FIG. 3 illustrates the hydrolysis of the internucleoside methylphosphonate linkages at an apurinic site produced by treatment of the oligomer with hydrochloric acid at pH 2.
FIG. 4(a) shows the results obtained with PEI-cellulose TLC of T4 polynucleotide kinase reactions for: (1) [γ-32 p]ATP; (2) d-ApApA reaction mixture; (3) d-ApGpCpApApG reaction mixture; (4) d-ApApApApGpCpApApG reaction mixture. The chromatogram was eluted with 1.5 M pyridinium formate, pH 3.5.
FIG. 4(b) shows the results obtained with polyacrylamide gel electrophoresis of phosphorylated oligonucleoside methylphosphonates as follows: (1) markers: d-32 pTpT (2 mer); d-32 pApApA (3 mer); d-32 pApGpCpApApG (6 mer); d-32 pApApApApGpCpApApG (9 mer). Lanes 2-4: products obtained after partial hydrolysis of d32 pApApA (2); d-32 pApGpCpApApG (3) and d-32 pApApApApGpCpApApG; (4) with 0.5 M piperidine at 37� C. for 10 min. Lanes 5-6: products obtained after partial hydrolysis of d-32 p-ApGpCpApApG (5) and d-32 p-ApApApApGpCpApApG (6) with 0.5 M HCl at 37� for 30 min. Electrophoresis was carried out at constant voltage (800 v) until the bromphenol blue marker dye had migrated halfway down the gel.
The scope of the invention is defined in the following claims wherein:
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|Feb 21, 1995
|Isis Pharmaceuticals
|Copolymers of N-vinyl-lactams suitable for oligomer solid phase synthesis
|Feb 25, 1994
|Nov 28, 1995
|Neu-Gene Development Group
|Uncharged polynucleotide-binding polymers
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|Torrey Pines Institute For Molecular Studies
|Equimolar multiple oligomer mixtures, especially oligopeptide mixtures
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|Antivirals Inc.
|Polynucleotide reagent containing chiral subunits and methods of use
|Sep 11, 1992
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|Jun 7, 1995
|Jul 29, 1997
|Sloan-Kettering Institute For Cancer Research
|Complementary DNA and toxins
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|Isis Pharmaceuticals, Inc.
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|Affymetrix, Inc.
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|DNA molecules stabilized by modifications of the 3′-terminal phosphodiester linkage
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|Printing molecular library arrays
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|Ajinomoto Co., Inc.
|Process for producing and purifying 2′,3′-dideoxynucleosides, and process for producing 2′,3′-dideoxy-2′,3′-didehydronucleosides
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|Oct 23, 2001
|Affymetrix, Inc.
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|Printing oligonucleotide arrays
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|Substrate preparation process
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|Mar 3, 2004
|Mar 12, 2013
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|Positionally modified siRNA constructs
|Mar 4, 2005
|Oct 29, 2013
|Isis Pharmaceuticals, Inc.
|Double stranded constructs comprising one or more short strands hybridized to a longer strand
|Nov 4, 2003
|Dec 10, 2013
|Isis Pharmaceuticals, Inc.
|Compositions comprising alternating 2′-modified nucleosides for use in gene modulation
|Sep 28, 2012
|Jan 28, 2014
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|System for photolithographic synthesis of polymer arrays
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|Kyushu University, National University Corporation
|Production method for compound comprising amino group and/or hydroxyl group
|Feb 20, 2002
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|Crooke Stanley T.
|Oligoribonucleotides and ribonucleases for cleaving RNA
|Oct 25, 2002
|May 22, 2003
|Crooke Stanley T.
|Oligoribonucleotides and ribonucleases for cleaving RNA
|Oct 25, 2002
|May 22, 2003
|Crooke Stanley T.
|Oligoribonucleotides and ribonucleases for cleaving RNA
|Oct 25, 2002
|May 22, 2003
|Crooke Stanley T.
|Oligoribonucleotides and ribonucleases for cleaving RNA
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|Crooke Stanley T.
|Oligoribonucleotides and ribonucleases for cleaving RNA
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|Printing molecular library arrays
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|Substrate preparation process
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|Structural motifs and oligomeric compounds and their use in gene modulation
|Nov 4, 2003
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|Baker Brenda F.
|2'-methoxy substituted oligomeric compounds and compositions for use in gene modulations
|Nov 4, 2003
|Jul 29, 2004
|Baker Brenda F.
|Chimeric oligomeric compounds and their use in gene modulation
|Nov 4, 2003
|Aug 19, 2004
|Baker Brenda F.
|Modified oligonucleotides for use in RNA interference
|Nov 4, 2003
|Sep 2, 2004
|Baker Brenda F.
|Phosphorous-linked oligomeric compounds and their use in gene modulation
|Nov 4, 2003
|Sep 2, 2004
|Prakash Thazha P.
|2'-Fluoro substituted oligomeric compounds and compositions for use in gene modulations
|Nov 4, 2003
|Sep 2, 2004
|Baker Brenda F.
|2'-substituted oligomeric compounds and compositions for use in gene modulations
|Nov 4, 2003
|Oct 14, 2004
|Baker Brenda F.
|Modified oligonucleotides for use in RNA interference
|Nov 4, 2003
|Feb 3, 2005
|Charles Allerson
|Compositions comprising alternating 2'-modified nucleosides for use in gene modulation
|Jun 3, 2004
|Feb 24, 2005
|Baker Brenda F.
|Phosphorous-linked oligomeric compounds and their use in gene modulation
|Jun 3, 2004
|Mar 10, 2005
|Baker Brenda F.
|Chimeric oligomeric compounds and their use in gene modulation
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|Muthiah Manoharan
|Conjugated oligomeric compounds and their use in gene modulation
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|Sep 13, 2005
|Jan 12, 2006
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|Substrate preparation process
|Sep 7, 2005
|Apr 20, 2006
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|Dec 16, 2004
|Jul 20, 2006
|Affymetrix, Inc.
|Substrate preparation process
|Jun 18, 2004
|Oct 26, 2006
|Isis Pharmaceuticals, Inc.
|Oligomeric compounds for use in gene modulation
|Dec 1, 2006
|May 31, 2007
|Balkrishen Bhat
|Double strand compositions comprising differentially modified strands for use in gene modulation
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|Jul 19, 2007
|Balkrishen Bhat
|Double strand compositions comprising differentially modified strands for use in gene modulation
|Dec 1, 2006
|Jul 26, 2007
|Balkrishen Bhat
|Double strand compositions comprising differentially modified strands for use in gene modulation
|Dec 1, 2006
|Jul 26, 2007
|Balkrishen Bhat
|Double strand compositions comprising differentially modified strands for use in gene modulation
|Dec 1, 2006
|Aug 2, 2007
|Balkrishen Bhat
|Double strand compositions comprising differentially modified strands for use in gene modulation
|Dec 1, 2006
|Aug 2, 2007
|Balkrishen Bhat
|Double strand compositions comprising differentially modified strands for use in gene modulation
|Dec 1, 2006
|Aug 9, 2007
|Balkrishen Bhat
|Double strand compositions comprising differentially modified strands for use in gene modulation
|Dec 1, 2006
|Aug 9, 2007
|Balkrishen Bhat
|Double strand compositions comprising differentially modified strands for use in gene modulation
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|Isis Pharmaceuticals, Inc.
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|Substrate preparation process
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|Antonio Giordano
|Genetic and Epigenetic Alterations In the Diagnosis and Treatment of Cancer
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|Isis Pharmaceuticals, Inc.
|Oligoribonucleotides and ribonucleases for cleaving rna
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|Sep 16, 1997
|Ajinomoto Co., Inc.
|Process for purifying 2',3'-dideoxynucleosides
|Mar 14, 1986
|Apr 1, 1987
|James Summerton
|Polynucleotide assay reagent and method.
|Oct 14, 1986
|Apr 22, 1987
|Genentech, Inc.
|Method and reagents for in vitro oligonucleotide synthesis
|Dec 22, 1988
|Jul 5, 1989
|Applied Biosystems, Inc.
|Method of synthesizing oligonucleotides
|Mar 14, 1986
|Sep 25, 1986
|James Summerton
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|Mar 14, 1986
|Sep 25, 1986
|James Summerton
|Polynucleotide assay reagent and method
|Sep 20, 1986
|Mar 24, 1988
|Applied Biosystems, Inc.
|Amino-derivatized phosphite and phosphate linking agents, phosphoramidite precursors, and useful conjugates
|Apr 9, 1993
|Nov 11, 1993
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|Production method for compound comprising amino group and/or hydroxyl group
|U.S. Classification
|536/24.5, 536/25.3, 525/54.11, 536/25.33
|International Classification
|Cooperative Classification
|European Classification
|Mar 21, 1984
Owner name: JOHNS HOPKINS UNIVERSITY THE, BALTIMORE, MD
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:MILLER, PAUL S.;TS O, PAUL O. P.;REEL/FRAME:004234/0618;SIGNING DATES FROM 19840305 TO 19840306
|Aug 22, 1988
|Fee payment
Year of fee payment: 4
|Aug 13, 1992
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|Jun 24, 1996
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Year of fee payment: 12
|Feb 28, 1997
Owner name: PARAMOUNT CAPITAL, INC., NEW YORK
Free format text: SECURITY INTEREST;ASSIGNOR:GENTA INCORPORATED;REEL/FRAME:008366/0717
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Effective date: 20040310


text:
Definition of en masse in English:
en masse
In a group; all together: the cabinet immediately resigned en masse
More example sentences
- They came en masse to the city square to demonstrate against the Communist government.
- Baked eggs are a lovely, easy dish to serve to company especially when the company has arrived en masse.
(all) together, as a group, in a body, as one, as a whole, in a mass, wholesale;
simultaneously, all at once, at the same time, at one and the same time, at the same instant, at the same moment, contemporaneously;
in unison, in concert, in chorus;
French en bloc, ensemble
rare synchronously
Late 18th century: French, 'in a mass'.
Words that rhyme with en massealas, Alsace, amass, Bass, chasse, crass, crevasse, gas, Hamas, lass, mass, morass, sass, tarantass, tass, wrasse
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2015-6 Radio Drama Rates (RDA and RAC4 contracts)
2% was applied to all rates from 1 August 2015. If you would like to contribute to negotiations or have any queries please contact the Broadcasting Group Secretary, Jo McCrum.
Note that the New Public Services Fee applied to the one transmission rate is 12.5%. Rates are calculated per minute on the slot length with the exception of the '15 minute' drama broadcast in Woman's Hour which is paid on the actuality of 14 minutes. Slot lengths are:
Friday Drama, Saturday Drama, Classic Serial: 60 minutes
Afternoon Drama: 45 minutes
15 Minute Drama (note fees paid on actual written length of 14 minutes)
11.30 and 23.00 Comedy: 30 minutes
Attendance payment
Attendance payment: £67.00
Original Drama (per minute)
- Beginners' rate: £62.05 (2 tx) / £37.23 (1 tx)
- Established writers' rate: £94.51 (2 tx) / £56.68 (1 tx)
An established writer is one where the Writer has had 120 minutes of dramatic works broadcast provided that this has been achieved over more than at least two Plays, Dramatisations, Dramatised Features, Adaptations, Series or Long Running Series.
A writer who qualifies as 'established' for the purposes of the BBC's Agreement with the WGGB and the PMA for Television Drama shall also be regarded as established for radio.
Code of Practice
You can read a guide to working relationships between writers and BBC in-house producer/directors here.
The Archers
- £948 per episode
Dramatisations are paid at 65%, 75% or 85% of the rates depending on the context and level of difficulty.
Commissioned short stories (per 15-minutes)
- 1st and 2nd: £189
- 3rd – 5th: £224
- 6th – 9th: £254
- 10th onwards: £325
- Generally established outside the BBC: £325
- Generally established inside the BBC: £386
Abridgements (per minute)
- Beginners' rate: £7.32
- Established writers' rate: £10.80
- Radio Drama Agreement (original drama, series, dramatisations and special adaptations).
- RAC4 (short stories, abridgements and other scripted contributions)
- BBC7 (now 4Extra) service licence
- 2013-14 RDA/RAC 4 Rates
Radio 4Extra
Under the current RDA a New Public Services Fee of 12.5% of the single transmission fee covers broadcasts on 4Extra (as well as simulcast etc).
Archive material is contracted and paid for by the BBC under the terms of the Radio 4 Extra GP agreement (until March 2018). In return the BBC has the right to broadcast a writer's/author's work for 6 transmission days (up to four transmissions per day) over three years on the Service and rights acquired include to make an audiostream or temporary download for up to 30 days plus a further 7 days to listen (if downloaded) following each transmission:
Original script material: £3.37 per minute
Dramatisations (including specially commissioned translations): £2.18 per minute
Source work material (including translations) £1.30 per minute
Abridgements £0.78 per minute
To view advance schedules go here using the password 'writer'. Writers will have to be sent the new link once. The file within the link will change with new week number parameters. Clicking on the "download" button in top right hand corner of screen will allow a user to open up the document in an Excel format.
BBC/Society of Authors Published Material Rates
- PA5 Complete Agreement
- PA5 Extracts Agreement
- Tel Lit 3 Extracts Agreement
- TV Source Option
- TV Drama Source Agreement for Existing Literary Works
- Television Extracts Agreement
These rates were due to increase on 1 August 2015 and are still under negotiation. If you would like to contribute to negotiations or have any queries please contact Jo McCrum.
These rates are calculated on a one transmission basis. Two transmissions, if paid upfront, are 180%.
Agreements now include automatic Public Service podcast rights for 30 days provided that any podcast could be taken down on immediate request of the rights-holder. Reasons may include any offer of an audio-publication, stage or film deal.
Translations of plays, prose and verse is payable at two thirds of the rates due to each of translator and original author. If you are dramatising an English translation a producer will have to clear rights to the translation and the underlying work. 2/3 of the prose for dramatisation rate is paid to both the original author/publisher and the translator/UK publisher.
- Prose (per minute): £31.63
- Poetry (per 1/2 minute): £37.96
- Download the TV agreement
Core Service Radio
- Plays/Prose (per min): £18.38
- Poetry (per ½ min): £18.38
- Prose for Dramatisation (per min): £14.33
- Prose Translation (per min): £12.25
World Service Radio (English)/BBC Digital Radio Originations
The rates for R4Extra originations are currently under review.
- Plays/Prose (per min): £9.19
- Poetry (per 1/2 min): £9.19
- Prose for Dramatisation (per min): £7.15
- Prose translation (per min): £6.13
Local Radio
- Plays/Prose (per min): £4.59
- Poetry (per 1/2 min): £4.59
- Prose for Dramatisation (per min): £3.57
- Prose translation (per min): £3.06
Radio Features
Features are commissioned at a minimum fee of £45.48 per minute with a baseline threshold of £318.30. The minimum fee equates to seven minutes duration. Rates increased on 1 August 2015. No residuals or repeat fees are paid for further broadcasts and the author is able to re-use their script (but not the recording) once an exclusive period lapses. The option to engage radio features contributors on a daily rate remains.
A feature writer has to provide the following services: research in depth; collection and selection of insert material (e.g. actuality recordings, literary material); scripting; presentation; interviewing as may be required by the BBC to make a radio programme, or self-contained item within a radio programme, which is a creative treatment of its subject but which does not involve actors in character or original material in dramatic form. Such programmes or items would normally be fully or partly edited or compiled by the contributor but these special terms applicable to 'features' work may still apply if the BBC carries out editing subject to this being taken into account in assessing the fee.
Some BBC producers offer authors a lower non-edit rate with the reasoning that authors are required to submit a fully edited programme. This may be reasonable if a substantial edit is necessary, but a lower rate has not been negotiated or approved the Society, Writers' Guild or NUJ.
- Download a sample Feature agreement
Radio Talks Contributions
These rates were increased on 1 August 2015. Talks are commissioned on a flat-fee basis with no residuals paid for further broadcasts. There is a limited exclusive period, after which the author is able to use their script elsewhere. Minimum rates are:
- Script and Read: £23.73 per minute with a minimum fee of £47.46.
- Script only £18.40 a minute with a minimum fee of £36.78
- Daily rate Reporting £133 per day. Minimum fee of £66.50 (per half day).
- Download a sample Talks contract
Contributions bought on a daily rate basis are bought out on an assignment basis. See a sample assignment.
BBC/PMA/WGGB TV Rates (per minutes, on slot length)
- Teleplays: £188
- Series/Serials: £171
- Dramatisations: £124
- Adaptations: £76
- Educational Drama: £115
- Shorter Scripts and Children's Drama: £188
Online Commissions
a) Teleplays £94
b) Series/Serials £86
- Attendance fees: £100
- Pension ceiling: £16,920
Established and in-demand writers may be able to negotiate a higher rate. Rates will apply to 1 November 2015.
An established writer is one where the Writer has had 95 minutes of dramatic works broadcast provided that this has been achieved over more than at least two Plays, Dramatisations, Dramatised Features, Adaptations, Series or Long Running Series.
A writer who qualifies as 'established' for the purposes of the BBC's Agreement with the WGGB and the PMA for Television Drama shall also be regarded as established for radio.
For full details see the Writers' Guild website
BBC Archive
As it is not economical for the BBC to clear every contribution individually, it has been sending out letters to writers (at a rate of 500 a week from August 2014) requesting that they give a general permission covering that writer's entire BBC radio and TV production catalogue available on similar (paid) terms to those that apply to contemporary BBC programme commissions. Consents from (and payments to) the owners of underlying rights will continue to be dealt with on a case-by-case basis. The payments for any new commercial uses will be negotiated in the relevant Radio or TV Writers' Forums. If you do not sign the BBC's request, your original contracts will continue to apply, but your work cannot be made available for any new uses which may be approved by the relevant Forums (in practice, your work is unlikely to be re-used at all). Note that you can withhold consent to individual works, withdraw or re-include works previously withdrawn at any time upon reasonable notice.
Members often complain that their past work has become inaccessible, so we highly recommend that you sign up to this deal. For full information see www.bbc.co.uk/writerslicence which has full FAQs and a downloadable licence, or contact email@example.com if you have queries. It would also be helpful if you could pass this information on to other authors (or their Estates) to ensure they don't risk missing out.
See the Broadcasting Group noticeboard for further details.
Contacts at BBC Literary Copyright
Need to speak to someone? Here are details of http://www.societyofauthors.org/sites/default/files/BBC Literary Copyright contact details.pdf">personell working in BBC Literary Copyright.


text:
Another restaurant will occupy the space where the Hidden Cypress Grille used to be in the Hidden Cypress clubhouse.
Walk with Ease is a new neighborhood-based walking program coming to Sun City neighborhoods in September in association with the Arthritis Foundation and the South Carolina Department of Health and Environmental Control's Healthy Aging division.
The tennis courts in town square reopened Sunday. Shrubbery and other landscaping around the courts was renovated to allow for the courts to dry quickly after a rain or a frost.
Pinckney Hall reopened Tuesday after a two-week renovation.
The building received new paint, carpeting and décor. Lifestyle services is open its normal hours, 8 a.m.-5 p.m. Monday through Friday and 9 a.m.-1 p.m. Saturday.
The 2012 Men's Sun City Open returns Sept. 8-9 beginning at 8:30 a.m.
Round 1 will take place at Hidden Cypress and Round 2 at Okatie Creek. It is a 36-hole individual gross (Flighted by Handicap).
Hidden Cypress Fitness Center and pool will close for a month-long renovation Monday.
Just like at the Purrysburg fitness center, Hidden Cypress will get all new cardio and weight equipment, lockers, carpeting and a fresh coat of paint.
"Between The Lines with Charlie Mitchell" opens this month with a one-on-one conversation with Bobbie Callaway, Sun City resident and president of the Women's Golf Association.
A program for preserving the community's roads by using periodic applications of a thin seal was proposed this year by staff and approved by the board of directors.
The Argent Lakes golf course, an executive course in the Lowcountry, opened as an 18-hole course for residents last week. Residents can secure tee time there by using the Chelsea Reservation System at www.schhreservations.com or 705-6234.
Damon Williams has been named executive director of Sun City Hilton Head after a nationwide search involving Wentworth Property Management's executive team with the support of Sun City Hilton Head's board of directors.
Subscriber Services:
Digital Network:
Social Networking:
Bluffton Today ©2016. All Rights Reserved.


text:
Pirates of the Caribbean
'Pirates of the Caribbean 5': Paul McCartney joins cast
Posted on Thursday, Mar 24, 2016
Now Paul McCartney has a ticket to ride with the Pirates of the Caribbean ...
26 years ago today: Bruce Springsteen surprised an Arizona bar with an impromptu jam sesh and a big tip
Posted on Tuesday, Sep 29, 2015
Birthdays: "Chuck" actor Zachary Levi (turns 35 today), "Pirates of the Caribbean" actor Mackenzie Crook (44), singer and "RuP ...
Please let these movie franchises die a peaceful death
Posted on Saturday, Jun 13, 2015
Do we really want more 'Terminator,' 'G.I. Joe' or 'Die Hard' movies?
Cosplay Spotlight - Pirates of the Caribbean's Jack Sparrow by AlysonTabbitha
Posted on Tuesday, Mar 31, 2015
What made you want to cosplay as this? I started making the costume after seeing the first "Pirates of the Caribbean" movie ...
Will Orlando Bloom return in the next 'Pirates of the Caribbean'?
Posted on Wednesday, Sep 10, 2014
The actor sat down with Yaho! Movies and revealed he's very much interested in making a return for the fifth installment of Disney's "Pirat ...
Film Nerd 2.0: How to fight parental franchise fatigue when raising kids in an endless summer
Posted on Tuesday, Sep 2, 2014
I love 'Star Wars' and 'Harry Potter' as much as anyone, but there has to be more
12 directors stuck in franchise mode: Michael Bay, James Cameron, Zack Snyder
Posted on Tuesday, Jun 24, 2014
Just how many 'Transformers' or 'Avatar' movies can one director make?
The Snap: Good God, Memorial Day Movies are Terrible
Posted on Thursday, May 22, 2014
Memorial Day is a time for reflection, but here are the bad Memorial Day movies you may have already forgotten.
Disney sets next D23 Expo for 2015
Posted on Saturday, Mar 22, 2014
" At past D23 Expos, Disney has brought out everyone from Johnny Depp, dressed as Captain Jack Sparrow to announce the fourth "Pirates of the Caribbe ...
10 movie and TV sidekicks who are way cooler than the leads
Posted on Wednesday, Feb 19, 2014
These guys aren't secondary to us


text:
Definition of judge advocate general in English:
judge advocate general
An officer in supreme control of the courts martial in the armed forces, excluding (in the UK) the navy.
Example sentences
- Layfield is now assigned to the Office of the Judge Advocate General, Washington, D.C.
- After consulting with Conner, Chief of Staff General James W. McAndrew, Judge Advocate General Walter Bethel, and Pershing met with coalition commanders-in-chief at Senlis, France.
- May I introduce to you Rear Admiral Harmon Rabb, Judge Advocate General.
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- Report: #1090361
Report - Rebuttal - Arbitrate
Complaint Review: Zha Zha Collars
Zha Zha Collars1500 North Wold Drive Atlanta, Georgia USA
Zha Zha Collars Dorita Arnold Completely Dishonest and a deceptive Crook! Atlanta Georgia
*REBUTTAL Owner of company: Zha Zha Collars is honest company
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Corporate Advocacy Program™
Woman is a thief! Dorita Arnold orderd from us and after sending her all of the materials she then called her credit card company and disputed the charges. Not only did she get our products and services she stole from us by not paying too. Stay far and clear from this dishonest, unethical, business owner.
This report was posted on Ripoff Report on 10/08/2013 10:04 AM and is a permanent record located here: http://www.ripoffreport.com/r/Zha-Zha-Collars/Atlanta-Georgia-30350/Zha-Zha-Collars-Dorita-Arnold-Completely-Dishonest-and-a-deceptive-Crook-Atlanta-Georgia-1090361. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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#1 REBUTTAL Owner of company
Zha Zha Collars is honest company
AUTHOR: dorita arnold Zha Zha Collars - ()
SUBMITTED: Tuesday, April 29, 2014POSTED: Tuesday, April 29, 2014
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