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Blue Man Group Boston | Face painting, and drumming, and dance parties, oh my! February Vacation Week at the Charles Playhouse
As you can probably imagine, during these frigid winter months, I spend a little bit less time shooting weddings (although still meeting and planning with all of my wonderful 2014 clients!), and a little more time on back end and less glamorous work. During the past few weeks, I was able to step away from the computer, and into a new type of office... Welcome to the Charles Playhouse! I was so thrilled when Jamia over at Blue Man emailed me to see if I had some time to work with them during February school vacation week to cover some of the super fun events and shows they had planned for families looking for something fun to do while the kiddos were home. After working with Blue Man on a multimedia project a little while back (check that out here!), I knew that I was in for a great time. I can't count how many times I said "you guys, this is a WORK day!"
Before a few of the shows, any kids (or kids at heart) who had a ticket to see the show could have their face painted to look like any member of the band (or really any pattern they wanted on their face). It was so exciting to see everyone all lined up in the newly renovated Charles Playhouse Lounge! If you haven't seen the new space, definitely get over there. It's so awesome! I challenge you not to play with the new Lounge-U-Lum, an interactive musical wall that only Blue Man would dream up.
Naturally, the Blue Men were attracted to the Lounge-U-Lum and couldn't help but sneak in to play around, and I think I spotted a future Blue Man getting his groove on!
Even some dads couldn't help but get in on the face painting action.
With everyone good and painted, you can tell that this theater is prepping for some great times ahead!
You just can't help but to be pulled into the show.
Ask any Blue Man (not that they will answer!) , the most important part of the entire show is the audience. This is not a show where there are performers and onlooking audience members. It's a 100% interactive experience, and the normal 4th wall in theater just doesn't exist here.
I've been spotted!
I'll leave some surprise for you as to what happens as the Blue Men enter the audience, but here's a taste of how you can expect to react...
What was that I was saying about a dance party?
After every show, the audience has a chance to get up close and personal with the Blue Men, and of course, the occasional selfie...
This is what happens when you trust a Blue Man with your phone. You end up with 500 photos of yourself and everyone around you...
Thanks everyone for another few wonderful days! I think I've got the Blue Man Face nailed, what do you think?

subset: commoncrawl


text:
Smart people, smart process
By Donald J. Boudreaux
Published: Tuesday, March 26, 2013, 9:00 p.m.
We humans are not as smart as we humans think we are. Sure, we figured out how to split the atom, fly, vaccinate against smallpox and pipe ESPN 24/7 into our homes and onto our smartphones.
But these marvels are more the results of smart processes rather than of smart people .
Look at your cellphone. Even if your particular phone isn't as smart as an iPhone or a Samsung Galaxy S3, it's still an incredible piece of technology. By pressing just a few buttons, you can talk in real time, at little cost and with no wires, to your children across town or to your friend across the ocean. France's King Louis XIV could have offered his entire kingdom in exchange for that ability and still found himself unable to do what you do routinely with your phone.
And while lots of smart people — engineers, bankers, accountants, marketing specialists — were necessary to make your phone a reality, they weren't sufficient. Smart human beings existed for tens of thousands of years without coming close to developing any of the modern marvels that we today take for granted.
Another indispensable ingredient for our modern prosperity is a process that not only inspires people to exercise their smarts, but also leverages those smarts into a whole that is immensely larger than the sum of its parts.
The undeniably smart Steve Jobs' vision for an affordable smartphone was dazzlingly creative. But that vision would have amounted to zilch had not many other people been available to help make Jobs' vision a reality. Some of these other people were also smart. Others were of only ordinary intelligence. And almost every one of them was a complete stranger to Jobs.
Yet somehow, they all cooperated with Jobs and with each other to create the iPhone.
What has not been around, unlike smart people, for tens of thousands of years is the smart process that encourages the globe-spanning cooperation that makes goods such as the smartphone possible.
The "smart process" is a market economy. It has two essential features. First, participants in a market economy are governed by the laws of property and contract: Jones is not entitled to take, damage or destroy Smith's property without Smith's permission — and Jones doesn't get such entitlement even if he becomes a high government official.
Second, the bourgeois pursuits of innovation, entrepreneurship and industry are widely celebrated — and with little concern given to the nationalities or religious identities of other market participants.
Only in the past few centuries did this open-ended market process emerge. It, and it alone, supplies the indispensable institutional framework to leverage people's ideas into the goods and services that make our modern lifestyles possible.
Everything that is distinctive about a modern economy requires the joint, cooperative efforts of thousands or even millions of people. The process that combined Steve Jobs' vision of a smartphone with the knowledge and efforts of manufacturers of metals, glass, microchips and other components of an iPhone is far greater than the brainpower of even the smartest human being — a zillion times greater.
So we can take pride in our ability to produce airplanes and Wi-Fi signals and smartphones and all of the other amenities of modernity. But let's also be aware that the market process that produces these things is much "smarter" than any of us is as an individual.
Donald J. Boudreaux is a professor of economics at George Mason University in Fairfax, Va. His column appears twice monthly.
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What do you mean 'Ghosts'?
by Bridget Willoughby
Tote Bag
Our quality crafted Tote Bags are hand sewn in America using durable, yet lightweight, poly poplin fabric. All seams and stress points are double stitched for durability. Available in 13" x 13", 16" x 16" and 18" x 18" variations, the tote bags are washable, feature original artwork on both sides and a sturdy 1" wide cotton webbing strap for comfortably carrying over your shoulder.
I'm not sure I follow... what do you mean 'ghosts'?
- 8Promote


text:
I know that you can do Dir.entries('.', {:encoding => 'utf-8'})
and it works properly, but I can't find any way to force the encoding when doing Find.find
. On Windows, the first method correctly reads a filename of 舦舨.txt, and doing File.file?
on the value returns true
. However, doing Find.find
returns ??.txt, even though ruby says its encoding is utf-8. Also, File.file?
returns false
Is there no way to set the encoding on Find.find


text:
Queerty is better as a member
Who cares about Natasha Bedingfield? When was the last time she had a hit?
When was the first time she had a hit?
She's better then KYLIE MINOGUE who hasn't had a hit on the radio in the U.S. since the 80′s …
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First Same-Sex Wedding Takes Place In Myanmar And It's Cute As Heck
Lebanon Court Rules Being Gay Does Not Violate "Laws Of Nature"
Scott Bakula Thought It Was Great To Show Off In The Steam Room On "Looking"
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What does EAD mean in Chat?
This page is about the meanings of the acronym/abbreviation/shorthand EAD in the Internet field in general and in the Chat terminology in particular.
Find a translation for EAD in other languages:
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Use the citation below to add this abbreviation to your bibliography:
"EAD." Abbreviations.com. STANDS4 LLC, 2014. Web. 8 Mar. 2014. <http://www.abbreviations.com/term/366072>.
EAD also stands for:
- Earliest Arrival Date
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- Echelons Above Division
- Economic Affairs Division
- Electrically Alterable Device
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Arguments over money might come up, Gemini. This might not involve your own funds but money belonging to a group with which you're involved. Try to be logical. This isn't a good day to set your sights on new goals or make definite plans. Wait a day or two. If you start today, your project could be fraught with delays, interruptions, or disagreements.
Don't know your sign? Enter your birthday:
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text:
Akay Pension
Travel Blogs from Patara
Looking' for Lycia ....Hard To Find
... to the oasis of the Hotel Vanilla (highly recommended!).
The next day we prepaid ourselves for another full day of 'bus battles' only to be pleasantly pleased by what Shane called a "Different Dolmus Day". One after the other we made connections we hasn't even prepared for finding ourselves in the sleepy town of Datca at check in time. This will be our kick back before heading north to Ephesus and eventually Istanbul.
In Datca we seem ...
SCUBA Diving
... is not too bad at all! I did go to the pharmacy, said seasickness, and was handed a box of green pills. They seem to work, so I will be stocking up on them! I also decided that waiting on the boat in between dives was stupid, so I swam to the shore whenever I could and waited there.
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The Aegean Coast
... camera", "look here", etc. We were not enjoying ourselves. Once we got to the canyon we were given water shoes - while we were not forced to wear them it was strongly recommended by multiple people. Unfortunately they cut at my ankles and Christine's heels so badly that we quickly called the hike quits. Next up was the star attraction of the day, 5km of rafting on inner tubes. For the first time all day, we started ...
Bagging Biblical Munros
... We finally arrive at Patara and climb it's hill for a better view. Patara is a ruined city with Hittite remains dating from the 13th century B.C. It is sandy at the bottom because we are so close to the beach, miles of fine grain beach which is Patara's main attraction now. The tangled brush hids a trail that winds upwards steeply until we reach the clearing at the top. Beside us are some tomb arches to mark a Pataran notable's final resting place. Behind us, ...
Blue Cruise
... girls were trying on their sailor's hats. Must've been half an hour before the first real action. Anyway had a great time there, dancing to the loud music.
Tuesday 24 August, 2010
Woke up feeling not too bad considering, but I can tell you I am looking forward to sleeping inside and not having the sun burn me up at about half seven when I'm still trying to sleep! Went for a couple swims, before ...


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- The Most Powerful Photo From The Sochi Winter Games
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An Idiot Is Reportedly Suing Derrick Rose, Because The Idiot Got Fat, Because He Was Sad That Derrick Rose Is Hurt
This may not make sense, may be an unreliable report and almost certainly won't accomplish anything. Yet this dumb lawsuit is as dumb as any lawsuit we've seen, so it's worth noting. A 25-year-old guy is suing Chicago Bulls superstar cheerleader Derrick Rose, because his absence in the 2012-2013 NBA season reportedly caused this man to be sad, and since he was sad, he got fat. So, it's Derrick Rose's fault for getting hurt, and it's also his fault that this guy was sad, and it's also his fault that his sadness caused fatness.
According to reports, Thompson claims Rose displayed negligent behavior by refusing to return to the court, despite having been cleared by team doctors. He blames his health issues on the MVP's refusal to return.
No word if Peoria County Officials will take this lawsuit seriously.
No word on if other men on Earth possess fewer than four brain cells.
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text:
Call Of Duty: Ghosts Onslaught DLC New Details Revealed
New images have been revealed for the Maverick... Read More»
Looks Like We Are Getting Kraven's Help In The Amazing Spider Man 2 – @AggroGamer News
AggroGamer: "We had a tease for The Amazing Spider Man 2 a little while back and the time has come where we get the full trailer for the game. Yes it is another film tie-in game but the previous game was actually quite decent given the usual lackluster experience these kinds of games usually provide. ... Read More»
TitanFall Will "Most Definitely Have an Ending", Told From Perspective of Both Armies
Every beginning has an ending... Read More»
Call of Duty: Ghosts Onslaught Trailer Details Extinction Episode 1
Enormous alien. In a bad mood... Read More»
Call of Duty: Ghosts double XP weekend begins today
Infinity Ward has announced that this weekend Call of Duty: Ghosts players will be able to enjoy double the XP all this weekend to celebrate the release of the game's first DLC. The double XP event will begin today at 10 a.m. PST (1 p.m. EST) and will run until January 27 at 10 a.m. ... Read More»
The Amazing Spider-Man 2 Swings Back With A New Trailer
Let's hope Beenox gets it right... Read More»
New Call of Duty: Ghosts trailer highlights first episode of new Extinction campaign
The latest trailer for Call of Duty: Ghosts sets up for the first narrative chapter of the new Extinction mode, "Nightfall," included with the upcoming Onslaught DLC. The basic gist of the trailer, which you can watch below, is that before the nuke was set off in Colorado (the Extinction Mode map shipped with the game), scientists from ... Read More»
New Call of Duty: Ghosts Onslaught DLC trailer introduces the CODnapper
A new trailer for Call of Duty Ghosts' first wave of DLC, Onslaught, showcases the add-ons new maps, two new weapons, and new Extinction campaign. In the same vein as Black Ops 2's Replacer series, the Ghosts DLC footage is first introduced by the CODnapper, played by Stephen Graham (Snatch, Pirates of the Caribbean's Scrum, ... Read More»
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New Destiny screenshots showcase environments and enemies
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Call of Duty: Ghosts screenshots show off Onslaught DLC maps
Yesterday, Activision detailed Call of Duty: Ghosts' first DLC pack, Onslaught, and now the publisher has released a batch of screenshots for the new content. "We know our fans have an appetite for new gameplay content all year long and the team here is committed to delivering a full season's worth of amazing new maps, ... Read More»
Activision details Call of Duty: Ghosts Onslaught DLC
Activision has finally offered up some official details on the recently confirmed Onslaught DLC for Call of Duty: Ghosts. The DLC includes four new multiplayer maps, the first part of Extinction's campaign, a new weapon, and a few new gameplay twists, such as the ability to play as Halloween's Michael Myers on a map that ... Read More»
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Call of Duty: Ghosts Onslaught DLC Releasing January 28
Call of Duty: Ghosts' Onslaught DLC, the first of four planned content packs, is set to release January 28, Activision has confirmed. The publisher posted a short trailer on Instagram, accompanied by the message: "Something menacing is coming Jan. 28. Get ready for the #Onslaught." This announcement follows the leaking of GameStop promotional materials that confirmed ... Read More»
Call of Duty: Ghosts Onslaught DLC Releasing on January 28th
New video clip teases "something menacing"... Read More»


text:
Fred`s 202 on May 22, 2012 03:24Relooped
Fred`s 202 on May 02, 2012 09:50Paste
Fred`s 202 on April 19, 2012 21:19Crackatac
Fred`s 202 on January 07, 2012 00:00Gum
Fred`s 202 on March 15, 2011 10:36Wierdo
Fred`s 202 on February 04, 2011 09:48Oi
Fred`s 202 on January 05, 2011 19:40Bøtte
Fred`s 202 on November 15, 2010 10:44
Need a gift for a gifted musician?
The SoundCloud Premium accounts also come as virtual gifts and it takes only two minutes to get one. Head over to our Gift page and check out the different Premium accounts starting at only €29 per year.
Havn`t really got a clue if I`m taking music making seriously or not. Cus some nights I do, and others I just have fun. Beleive it`s gonna take alot of effort to come to another level, but hey! what´s the rush?


text:
ABOUT 600 volunteers are needed to help with the running of next year's Asia Pacific Outgames, which will be held in Darwin.
A variety of roles are available for those interested in helping at the event that's due to run for a week on May 10-16.
18 sports will be on offer at the Darwin Outgames, with each requiring a team of volunteers, including people with specific referee, umpiring and technical knowledge.
Other areas of the Outgames will also require assistance, such as parties and other cultural activities, accreditation, functions, transport and ceremonies.
A human rights forum to take place during the Outgames will also need the assistance of a voluntary team to ensure it meets international conference standards.
Darwin Outgames 2014 board member Olivia Birkett said volunteers would be invaluable and key to the running of the event.
"For a minimum of 10 hours voluntary assistance, you will be provided with a volunteer t-shirt, a participation pack, event accreditation and the perks of being involved with this exciting event," she said.
Registrations are now open online and will close on April 10.
INFO: To register for the Darwin Outgames please visit its website. For further volunteer enquires contact firstname.lastname@example.com


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The Great U Turn
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Do Companies Have a First Amendment Right to Track You?
Dig led by Mike Rose
Dig led by Truthdig Staff
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A new documentary by Oliver Stone called "South of the Border" follows his earlier trajectory of "Salvador" (1984), "Comandante" (2003), and "Looking for Fidel" (2004) as he talks to several Latin American leaders to understand what is happening on the continent and how U.S. perceptions of our own backyard are skewed.
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I believe in breakfast. It's the one meal that my kids usually eat without a fuss, so that's huge. As for myself, I can't function without it, and I see it as a great way to get some healthy greens in, some coffee, and on a good day, maybe even some news of the world via the newspaper.
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Living Sunlight: How Plants Bring The Earth To Life Author: Molly Bang and Penny Chisholm Illustrator: Molly Bang Ocean Sunlight is an enchanting educational book which covers complicated topics in a fun and exciting way, which children can absorb through the clear informative text and the marvellous energised illustrations. It moves forward without getting too technical and yet delivers complex information about ocean eco systems. Wearing our microscopic lens we see into the exotic world of zooplankton and phytoplankton with their primitive swimming appendages and transparent … [Read more...]
LIVING SUNLIGHT: HOW PLANTS BRING THE EARTH TO LIFE – Book by Molly Bang and Penny Chisholm
Living Sunlight: How Plants Bring The Earth To Life Author: Molly Bang and Penny Chisholm Illustrator: Molly Bang A magnificent book which explains the basic scientific principles behind life on earth. Children can explore the stages in chemical transformation as the sunlight is photosynthesised and made into sugars, the energy packs and building blocks for life on earth. Simple chemical equations are introduced for those who are interested to know more. Molly Bang's illustrations are colourful, rich and descriptive, allowing the scientific concepts to be absorbed both visually and … [Read more...]
WHEN SOPHIE GETS ANGRY, REALLY, REALLY ANGRY – Book by Molly Bang
When Sophie Gets Angry, Really, Really, Angry. Author and Illustrator: Molly Bang An excellent story about a child learning to overcoming her own anger without the assistance of adult intervention. When Sophie gets angry, really, really angry, she loses her temper with spectacular ferocity. The anger is depicted as an unrestrained fiery jet erupting from her mouth and as a palpable force radiating from her in huge orange and red waves. Sophie's intense emotions are set off when she has to share a gorilla toy with her sister. In the struggle she trips over and gaining no sympathy … [Read more...]
WE HAVE LIFT OFF – Book by Sean Taylor and Hannah Shaw
We Have Lift Off Author: Sean Taylor Illustrator: Hannah Shaw They could take no more of his environmental destruction and their mistreatment, but who could have guessed that the animals on Mr Tanner's farm would take action! While selfish Mr Tanner was busy polluting the air with smoke. filling the river with muck and junk, cutting down the trees they set about building an intergalactic rocket to escape to the clean, clear stars. Narrated by the 'first chicken into outer space' this hilarious story follows the efforts of the farmyard animals and their series of disastrous test … [Read more...]
THE BEATLES – Book by Mick Manning and Brita Granström
The Beatles Author and Illustrator: Mick Manning and Brita Granström The Beatles live on in this exciting new book for children. Told in colourful fun cartoon frames, this factually correct account follows the 'Fab Fours' history from their school days to independent careers after The Beatles disbanded. Why did the US crime rate drop to an all-time low when The Beatles appeared on the Ed Sullivan Show? Why did John Lennon returned his MBE to Buckingham Palace? Read about John's Liverpool childhood, his rebellious school years and how he got his first guitar. Friendships and band … [Read more...]
ONE WORLD TOGETHER – Book by Catherine and Laurence Anholt
One World Together Author and Illustrator: Catherine and Laurence Anholt A gentle engaging story for little ones which encourages friendship and the understanding of different global cultures. We travel to nine different countries with a little boy who is looking for a new friend. He discovers that we are all 'one world together' even though we might be separated by distance. Each child greets him in their own language and he learns several interesting things about where they live, what they like to do and a few cultural aspects of their lives. Paulo is from Brazil. He lives in … [Read more...]
COUNTING CHICKENS – Book by Polly Alakija
Counting Chickens Author and Illustrator: Polly Alakija Amid the pineapple patches and mud brick houses a group of friends share the excitement of spring. Ade's cow has a calf, Tunde's sheep has two lambs, Bisi's goat has her kids, Aduke's cat has kittens, Laolu's dog has puppies, Dapo's pig has piglets and by the end of the week Tobi's prize hen has laid seven beautiful eggs! The animals are all counted with delight. The babies grow quickly and begin to play, but Tobi's hen just sits on those eggs. When will he be counting chickens? The friends tease Tobi saying they will … [Read more...]
THE STRONGEST BOY IN THE WORLD – Book by Jessica Souhami
The Strongest Boy In The World Author and Illustrator: Jessica Souhami Not many people know that long ago in Japan, champion sumo wrestlers were famous celebrities with riches and fans like today's pop stars and football heroes. When a kid called Kaito decides to try his luck at becoming the strongest boy in the world, he sets out from his village for the big city of Kyoto. Kaito thinks he's pretty tough but how will a weedy kid defeat all those gargantuan wrestlers? Along the way he encounters a girl carrying a full water bucket on her head. Unable to resist a bit of … [Read more...]
READING RAVEN VOL 2 – Learning to Read App Review
Reading Raven Vol 2 Learning To Read App by Early Ascent Platform: IPhone & IPad Recommended Ages: 3-7 Help your kids get reading with the newest version of the best seller 'Reading Raven'. Reading Raven's 'Learn to Read App' comes to us highly recommended by parents, teachers and learning professionals. This Apple based platform app is rated as an outstanding learning tool. Building on the success of Reading Raven's first release in 2012, the newest version Reading Raven Volume 2 has even more to offer kids that have grown up enjoying and learning from this exciting … [Read more...]
READING RAVEN – Learn to Read App Review
Reading Raven Vol 1 Learn To Read App by Early Ascent Platform: IPhone & IPad (Android under development) Recommended Ages: 3-7 Finding the right tools to help your kids with reading is getting easier! This app includes five extensive lessons with hundreds of individual activities. Reading Raven's 'Learn to Read App' comes to us highly recommended by parents, teachers and learning professionals. This Apple platform app is rated as an outstanding learning tool. One of the many strengths of this application is its ability to engage, using great illustrations to capture … [Read more...]


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What to buy on Record Store Day's Back to Black Friday (slide show)
On the biggest shopping day of the year, skip the department store and support your local indie record shop instead
Mon Nov 25 2013
Every year, Record Store Day brings vinyl rarities and one-off releases to indie brick-and-mortar music stores—and this year, it'll once again capitalize on the shopping frenzy of Black Friday with a special edition of RSD on Friday, November 29. Show your love for the shiny black plastic discs and the artists who make them, and score some great tunes to boot; plus, the event will bring in-store performances and extended hours to your favorite record stores. Below, find our picks for the best releases, and for more details on what's happening in NYC, visit the Record Store Day website.
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William G. Miles | On the Move
- William G. Miles
- Date added:January 6, 2014
- Submission Type:New Hire
- Current employer:Wallace, Plese + Dreher
- Current title/position:Senior Tax Manager
- Industry:Banking & Financial Services
- Position level:Senior Manager
- Position department:Accounting & Finance
- Duties/responsibilities:Jerry Miles, CPA, CFP, joined Wallace, Plese + Dreher, as a Senior Tax Manager. With over 25 years of experience, he has worked with business owners and entrepreneurs in navigating individual and business performance through income tax planning and appropriate financial planning.
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Anyone done that this year, the trail from Angeles Crest @Baden Powell down toward Southfork CG/ Just wondering how it is. Thinking about including that in a loop this weekend. It usually is semi rough and needs a bit of hike-a-bike...used to that.
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Boris Kodjoe: B-day Bash Host
Boris Kodjoe Hosts Birthday Bash for Ehis aka Shades, Sike, and Cynthia MasasiHollywood
Hollywood Trailer: Takers
Monday, July 26th, 2010Tags: Chris Brown, Golden ICONS, Hayden Christensen, Hollywood, Idris Elba, Matt Dillon, Michael Ealy, Paul Walker, T.I, TAKERS
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Alpha Galore Films: Anchor Baby
Saturday, July 17th, 2010Tags: Alpha Galore Films, Anchor Baby, Colin Paradine, Cyrus Faird, former Tommy Hilfiger, Hollywood, Mark Cassius, Michael Scratch, Model, Movies, Nollywood, Omoni Oboli, Sam Sarpong, Santiago Lopera
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Saturday, May 22nd, 2010Tags: Arthlene Legair, Awilo Longomba, Dominica, Fashion, fashion designer, Hollywood, I Wear African, Ill-la-la Designs, Kahindo Mateene, Korto Momolu, Legair Brand, Project Runway, Sheila Denanyoh, Tangi Miller, West Indies
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My American Nurse 2 Premier
Saturday, March 13th, 2010Tags: Aloma Wright, Cloud9, golden icon, Hakeem Kae-Kazim, Hollywood, HOLLYWOOD/ NOLLYWOOD, Micheal Blackson, My American Nurse 2, Nollywood, Pascal Atuma, Vida Darko
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Exclusive dialogue with Ngo
Saturday, February 27th, 2010Tags: Actor, behind-the-scenes look at athletes modeling spring suits, Bode Ojo, Boxer, Boxing, Catherine Zeta-Jones, ESPN magazine, exclusive, Fashion, Fortune magazine, Golden Gloves Champion, golden icon, Hollywood, Interview, Kate Auletta reports, Men's Fitness, Men's Health, Model, Ngo Okafor, The Source, Vibe, Vogue, World Street Jornal, WSJ
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Lucky Ejim, World Class Actor
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Paramount, Namco Bandai Partner for 'Star Trek' Video Game
The game's release will coincide with J.J. Abrams' latest installment of the sci-fi saga, scheduled to hit theaters May 17, 2013.
Paramount and video game maker Namco Bandai announced a deal on Friday to co-publish and distribute a Star Trek game in early 2013, ahead of the sequel to J.J. Abrams' 2009 reboot of the space franchise.
The combat game is centered on the adventures of Kirk and Spock, portrayed by Chris Pine and Zachary Quinto in the Abrams incarnation, as they pair up to defeat a shared -- and so-called "legendary" -- enemy, the companies said.
"With our Namco Bandai partners leading the distribution effort, we are confident that we will meet an overwhelming demand to get this exciting console experience into the hands of gamers and Star Trek fans worldwide," said LeeAnne Stables, Paramount's executive vice president of worldwide marketing partnerships and head of the studio's video game unit.
VIDEO: George Takei Declares 'Twilight' the Enemy of 'Star Trek,' 'Star Wars' Fans
"The positive response from our initial unveiling at E3 last year was unprecedented, and we are fortunate to have the highest level of production and creative talent on our team that will bring this incredible game out to market next year," she said in a statement, referring to the annual Electronic Entertainment Expo, a video game conference and show at the Los Angeles Convention center.
Paramount debuted Star Trek video game in pre-alpha phase at 2001's E3 outing to critical acclaim; studio exec Brian Miller is overseeing production of the final result by Digital Extremes, a video game developer based in Ontario.
Marianne Krawczyk, writer of the fantasy game God of War, is co-writing an original plot for the Kirk-Spock adventures with Roberto Orci and Alex Kurtzman, who are writing and producer the upcoming Star Trek movie.
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I always wonderd why it says 'Door Mat' On my forehead!
I get taken advantage on and at the time dont even realise it, not untill its too late
Only when I pay them.
Yes I do. Everyone always says I'm so nice, kind, sweet, and beautiful. THEN they feel free to walk over me and use me for their own personal gain.
its hard to say.
yes, all the time. i don't feel destined or determined to do anything with my life right now, so if someone wants to step on me on there way up or anything like that, might as well let them, i'm not doing anything important and obviouly they think they are.
yes they do take advantage of me and i always have a lame excuse that i let it happen because it had to be that way......etc etc....
but the reality is that i dont know how to not let that happen and still not be hated or fought with or attacked and still be popular
When I was younger yes but now I am older and wiser!! NO!


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SoleRebels "Whole Rebel" Sandals
Do good while looking good! These fashionable "Whole Rebel" sandals are organic, eco-friendly, and also help support injured former soldiers in Ethiopia. Founded by Kiru Alemu, SoleRebels bring a source of hope by creating job opportunities for wounded veterans while creating something that can be enjoyed worldwide.
The soles of the sandals are made from recycled tires, and the tops of the sandals are made from recycled and organic materials in your choice of Blue, Red or Green.
Choose U.S. women's sizes 6, 6 1/2, 7 1/2, 8 1/2, 9, or 9 1/2 (European women's sizes 36, 37, 38, 39, 40, or 41). Handmade in and fairly traded from Ethiopia.
Shop Green!
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July 9, 2012 - I wish these sandals would stay on my feet! I bought red, size 71/2 which is my normal shoe size. The length of the soles is fine, but the top straps that hold them to my feet are too loose. My foot would have to be about 10" diameter for them to fit snugly. So sad because they are very cool looking sandals.
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Mexico's auto industry is set to ship more product north to the United States than Japan and Canada by the end of 2015, in part due to the effects the North American Free Trade Agreement has had on the country since its signing two decades ago.
Tag: united states
According to the University of Michigan Transportation Research Institute, the fleetwide fuel economy in the United States increased for the second consecutive month to 24.9 mpg during the month of January 2014.
Though Nissan remains Japan's second-biggest automaker with a wide gap ahead of Honda, the latter continues to outsell the former in the United States and at home, much to Nissan's dismay
For the past few months, sanctions against Iran for their nuclear ambitions have sidelined PSA and Renault from the Persian market. Behind the scenes, General Motors outmaneuvered PSA despite their one-time alliance allowing them to muscle their way into aan emerging market via loophole abuse and an unknown quantity of Camaros. With GM out of the way, however, PSA would now be free to regain their footing once sanctions were lifted.
PSA won't be alone in the upcoming battle, of course, as their compatriots at Renault have plans to return to Iran to reclaim what was lost, and then some.
Should General Motors new product boss Mark Reuss have his way, there may come a day when a new affordable wagon could be driven off the lot onto the highways and driveways of America.
In a sign that the 21st Century could belong to China after all, Porsche's head of sales and marketing Bernhard Maier predicts that the United States will finish second on the podium to China as far as 911s and Macans are concerned by the end of 2014 at the earliest.
2014 may only be a day old, but it's already shaping up to be a rough year for Hyundai and Kia as they prepare to increase global sales by just 4 percent this year, the lowest and bleakest forecast for the Korean duo since 2003.
Barring a last minute campaign from another manufacturer, Toyota will be number one in recalls on the American market for 2013. This will be the second year in a row that Toyota has topped the recall rankings. Since the 2009 sudden unintended acceleration controversy, Toyota has led the nation in recalls every year except 2011.
The green warriors who hoped EVs and hybrids would be the dominate force on the highways of America may need to wait a bit longer: the United States Department of Energy predicts gasoline will be the fuel of a generation until at least 2040.
Following on the heels of General Motors CEO Dan Akerson's recent resignation, Chevrolet's chief marketing officer for the United States Chris Perry has called it a day effective immediately.
Due to high demand from customers jumping aboard the CLA bandwagon, Mercedes-Benz has warned dealers in the United States that supply of the new four-door coupe will be limited for the first half of 2014.
The land of Canadian Tire, Tim Hortons and Michelle Creber has yet another thing going for it: Their auto workers have a cost advantage over their two-tiered brothers and sisters down south according to a study from Toronto, Ontario-based Scotiabank.
September was a great month for Subaru, with the brand up 15 percent. Ford, Chrysler, Jaguar Land Rover and BMW also posted solid gains. General Motors,Hyundai/Kia, Toyota, Nissan and Honda saw declines, as the SAAR dipped to 15.3 million units amid decreased consumer and business confidence. Table below the jump.
Weaker than expected growth in the United States has led Carlos Ghosn to issue an even more ambitious goal; double Nissan's sales by 2017.


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US 7351797 B1 Abstract The invention provides intermediates and methods that allow for site-specific modification of peptides after synthesis. Accordingly, functional molecules can be selectively linked to a peptide to provide a peptide conjugate having altered biological, chemical, or physical properties. For example, functional molecules (e.g. biophysical probes, peptides, polynucleotides, and therapeutic agents) can be linked to a peptide to provide a peptide conjugate having differing and useful properties. The invention also provides a compound of formula (III):
Claims(21) 2. The peptide conjugate of
3. The peptide conjugate of
4. The peptide conjugate of
5. The peptide conjugate of
6. The peptide conjugate of
7. The peptide conjugate of
8. The peptide conjugate of
9. The peptide conjugate of
10. The peptide conjugate of
11. The peptide conjugate of
12. The peptide conjugate of
13. The peptide conjugate of
14. The peptide conjugate of
15. The peptide conjugate of
16. The peptide conjugate of
1,1 ′dioctadecyl-3,3,3′,3′-tetramethlindocarbocyanine perchlorate,
dichlorofluorescin, dihydrorhodamine, monochlorobimane, or calcein.
17. The peptide conjugate of
R6 is SEQ ID NO: 1;
X is Ra�C(═O)CH(NH2)CH2N(H)C(═O)CH2�Rb; wherein Ra is a direct bond to the amino terminus of R6 and wherein Rb is a direct bond to the oxygen of the aminooxy group of formula (III);
R7 is methyl;
Y is a direct bond; and
D is Alexa-532.
19. An isolated peptide consisting essentially of a backbone and one or more protected secondary aminooxy groups, wherein the protected aminooxy group is on an amino acid side chain, the protected aminooxy group is protected with an amino protecting group and is unreactive during Boc solid-phase peptide synthesis, and a substituent on the aminooxy group is (C1-C6)alkyl; and wherein the backbone of the peptide further comprises �C═N�O�CH2�C(═O)�.
20. The peptide of
21. A method for preparing a peptide conjugate comprising reacting a peptide of the formula (VI)
R6 is a peptide;
X is a direct bond or a linking group;
R7 is (C1-C6)alkyl;
with a fluorescent molecule having an electrophilic moiety that is reactive with the aminooxy groups(s) to provide the peptide conjugate.
Description The invention described herein was made with United States Government support under Grant Number MCB-9812248 awarded by the National Science Foundation, and under Grant Numbers CA58689 and GM 57464 awarded by the National Institutes of Health. The United States Government has certain rights in this invention. This is a U.S. National Stage application under 35 U.S.C. 371, from PCT/US00/26821 filed 29 Sep. 2000 and published in English on 11 Apr. 2002 as International Publication Number WO 02/28890 A1, which application and publication are incorporated herein by reference. Modified peptides and proteins are valuable biophysical tools for studying biological processes, both in vitro and in vivo. They are also useful in assays to identify new drugs and therapeutic agents. In particular, quantitative live cell imaging using fluorescent proteins and peptides is revolutionizing the study of cell biology. An exciting recent development within this field has been the construction of peptide and protein biosensors exhibiting altered fluorescence properties in response to changes in their environment, oligomeric state, conformation upon ligand binding, structure, or direct ligand binding. Appropriately labeled fluorescent biomolecules allow spatial and temporal detection of biochemical reactions inside living cells. See for example Giuliano, K. A., et al., Annu. Rev. Biophys. Biomol. Struct. 1995,24:405-434; Day, R. N. Mol. Endocrinol. 1998, 12:1410-9; Adams, S. R., et al., Nature 1991, 349:694; Miyawaski, A., et al., Nature 1997, 388:882-7; Hahn, K., et al., Nature 1992, 359:736; Hahn, K. M., et al., J. Biol. Chem. 1990,265: 20335; and Richieri, G. V., et al., Mol. Cell. Biochem. 1999, 192:87-94. Procedures for site-specific modification of polypeptides have been described, including: chemically selective labeling in solution Brinkley, M. Bioconjugate Chemistry 1992, 3:2-13) and on resin bound peptides (Hackeng, T., et al., J. Biol. Chem. Submitted); introduction of ketone amino acids through synthetic procedures (Rose, K. J. Am. Chem. Soc. 1994, 116:30-33; King, T. P., et al., Biochemistry, 1986, 25:57745779; Rose, K., et al., Bioconjugate Chem. 1996, 7:552-556; Marcaurelle, L. A., Bertozzi, C. R. Tett. Lett. 1998, 39:7279-7282; and Wahl, F., Mutter, M. Tett. Lett. 1996, 37:6861-6864); and molecular biology techniques (Cornish, V. W., et al., J. Am. Chem. Soc. 1996, 118:8150). While each of these methods has utility for producing a particular class of biosensor or labeled polypeptide, all have limitations that restrict their general use. Labeling of natural amino acid sidechains in solution is often impractical because of the existence of many other competing nucleophiles. Additionally, the use of unnatural amino acids, such as those bearing ketones for selective labeling, requires the synthesis of dye constructs or amino acids that are difficult to make and not available commercially. Currently, the major obstacles to the development of fluorescent biosensors remain: (1) The difficulty in site-specific placement of the dye in the polypeptide and (2) determining exactly which site is optimal for dye placement (Giuliano, K. A., et al., Annu. Rev. Biophys. Biomol. Struct. 1995, 24:405-434). Solvent-sensitive dyes and other biophysical probes must be placed precisely for optimal response to changes in protein structure without interference with biological activity. Also, the need for site-specific incorporation of two dyes without impairment of biological activity has proven a serious limitation for utilization of fluorescence resonance energy transfer (FRET) within a single protein. Total chemical synthesis of proteins provides a potential solution to these problems (Wilken, J., Kent, S. B. H. Curr. Op. Biotechnology. 1998, 9:412; Kent, S. B. H. Ann. Rev. Biochem. 1988, 57, 957-989; Dawson, P. E., et al., Science 1994, 266:776-779; Muir, T. W., et al., Proc. Natl. Acad. Sci. 1998, 95:6705-6710; and Cotton, G. J., et al., J. Ann. Chem. Soc. 1999, 121:1100-1101). However, many biophysical probes suitable for fluorescent biosensors or other purposes are not stable to the various conditions used for peptide synthesis, and site-specific incorporation after synthesis is difficult to achieve. Thus, there is currently a need for peptide synthons having protected functional groups that can be selectively modified to incorporate one or more functional molecules (e.g. a biophysical probe such as a fluorescent label) following peptide synthesis. A highly efficient method for the site-specific attachment of biophysical probes or other molecules to unprotected peptides following chemical synthesis has been developed. The methodology utilizes amino acids having one or more protected aminooxy groups, which can be incorporated during solid-phase peptide synthesis or which can be combined with recombinant peptides through post expression steps. It has been discovered that the protected aminooxy group can be unmasked following peptide synthesis, and reacted with an electrophilic reagent to provide a modified (e.g. a labeled) peptide. The aminooxy group reacts selectively with electrophiles (e.g. an activated carboxylic ester such as an N-hydroxysuccinimide ester) in the presence of other nucleophilic groups including cysteine, lysine and amino groups. Thus, selective peptide modification (e.g. labeling) can be accomplished after synthesis using commercially available and/or chemically sensitive molecules (e.g. probes). The methodology is compatible with the synthesis of C-thioester containing peptides and amide-forming ligations, required steps for the synthesis of proteins by either total chemical synthesis or expressed protein ligation. An aminooxy containing amino acid can be introduced into different sites by parallel peptide synthesis to generate a polypeptide analogue family with each member possessing a single specifically-labeled site. The parallel synthesis enables the development of optimized biosensors or other modified polypeptides through combinatorial screening of different attachment sites for maximal response and minimal perturbation of desired biological activity. Thus, a simple and efficient methodology for site-specific modification (e.g. labeling) of peptides after synthesis has been developed that provides high yield, selectivity, and compatibility with both solid-phase peptide synthesis and Cα-thioester peptide recombinant synthesis. Accordingly, the invention provides a synthetic intermediate (i.e. a synthon) useful for preparing modified peptides, which is a compound of formula (I):
Peptides including one or more aminooxy groups are also useful synthetic intermediates that can be modified to provide related peptides having altered biological, chemical, or physical properties, such as, for example, a peptide linked to a fluorescent label. Accordingly, the invention also provides a peptide having one or more (e.g. 1, 2, 3, or 4) aminooxy groups; provided the peptide is not glutathione. The invention also provides a peptide having one or more (e.g. 1, 2, 3, or 4) secondary aminooxy groups. The invention generally provides intermediates and methods that allow for site-specific modification of peptides after synthesis. Accordingly, functional molecules can be selectively linked to a peptide to provide a peptide conjugate having altered biological, chemical, or physical properties. For example, functional molecules (e.g. biophysical probes, peptides, polynucleotides, and therapeutic agents) can be linked to a peptide to provide a peptide conjugate having differing and useful properties. Thus, the invention also provides a compound of formula (III): R6 is a peptide; X is a direct bond or a linking group; R7 is hydrogen, (C1-C6)alyl, an amino protecting group, or a radical comprising one or more aminooxy groups; Y is a direct bond or a linking group; and D is a functional molecule. Preferably the functional molecule is a biophysical probe, such as a fluorescent group that can be used for FRET studies or other studies involving fluorescent signals, such as excimer pair formation. Processes for preparing synthons of the invention as well as the protein conjugates of the invention are provided as further embodiments of the invention and are illustrated by the procedures in the Examples below. Thus, the invention also provides a method for preparing a peptide conjugate comprising a peptide and a functional molecule, comprising reacting a peptide having one or more aminooxy groups with a corresponding functional molecule having an electrophylic moiety, to provide the peptide conjugate. The following definitions are used, unless otherwise described: alkylene, alkenylene, alkynylene, etc. denote both straight and branched groups; but reference to an individual radical such as �propylene� embraces only the straight chain radical, a branched chain isomer such as �isopropylene� being specifically referred to. Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. The term �amino acid,� includes the residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g. phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, α-methylalanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). The term also includes natural and unnatural amino acids bearing a conventional amino protecting group (e.g. acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g. as a (C1-C6)alkyl, phenyl or benzyl ester or amide). Other suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, T. W. Greene, Protecting Groups In Organic Sythesis; Wiley: New York, 1981, and references cited therein). The term �peptide� includes any sequence of 2 or more amino acids. The sequence may be linear or cyclic. For example, a cyclic peptide can be prepared or may result from the formation of disulfide bridges between two cysteine residues in a sequence. Thus, the term includes proteins, enzymes, antibodies, oligopeptides, and polypeptides. Peptide sequences specifically recited herein are written with the amino terminus on the left and the carboxy terminus on the right. An �aminooxy group� is a group having the following formula The term �functional molecule� includes any compound that can be linked to a peptide to provide a peptide conjugate having useful properties. Such conjugates may be useful for studying the structure or function of the peptide. Such conjugates may also be useful for drug screening, as pharmacological tools, as research tools, or as therapeutic agents. For example, the term functional molecule includes biophysical probes, peptides, polynucleotides, therapeutic agents, cross-linking groups (chemical or photochemical), a compound that modifies the biological activity of the peptide, or a caged molecule (e.g. a reporting molecule or a biologically active agent that is masked and that can be unmasked by photoactivation or chemical means). The term �biophysical probe� includes any group that can be detected in vitro or in vivo, such as, for example, a fluorescent group, a phosphorescent group, a nucleic acid indicator, an ESR probe, another reporting group, a moiety, or a dye that is sensitive to pH change, ligand binding, or other enviornmental aspects. Aminoacids and peptides that include one or more aminooxy groups are useful intermediates for preparing peptide conjugates. The aminooxy group(s) can typically be positioned at any suitable position on the aminoacid or peptide. For example, the aminooxy group(s) can conveniently be incorporated into the side chain of the amino acid or into one or more side chains of the peptide. Thus, as used herein with respect to the amino acids and peptides of the invention, the term �a radical comprising one or more aminooxy groups� includes any organic group that can be attached to the amino acid or peptide that includes one or more aminooxy groups. For example, the term includes a carbon chain having two to ten carbon atoms; which is optionally partially unsaturated (i.e. contains one or more double or triple bonds); which chain is optionally interrupted by one or more (e.g. 1, 2, or 3) �NH�, �O�, or �S�; which chain is optionally substituted on carbon with one or more (e.g. 1, 2, or 3) oxo (═O) groups; and which chain is optionally substituted with one or more (e.g. 1, 2, or 3) aminooxy groups. Preferably, the aminooxy group(s) are secondary aminooxy groups. The term �cross-linking group� refers to any functionality that can form a bond with another functionality, such as photoaffinity label or a chemical crosslinking agent. The term �caged molecule� includes a molecule or reporter group that is masked such that it can be activated (i.e. unmasked) at a given time or location of choice, for example using light or a chemical agent. Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents. Specifically, (C1-C6)alkylene can be methylene, ethylene, propylene, isopropylene, butylene, iso-butylene, sec-butylene, pentylene, or hexylene; (C3-C8)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl; (C2-C6)alkenylene can be vinylene, allylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 3-butenylene, 1,-pentenylene, 2-pentenylene, 3-pentenylene, 4-pentenylene, 1-hexenylene, 2-hexenylene, 3-hexenylene, 4-hexenylene, or 5-hexenylene; (C2-C6)alkynylene can be ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 3-butynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 4-pentynylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 4-hexynylene, or 5-hexynylene; and aryl can be phenyl, indenyl, or naphthyl; A specific value for R is a radical of formula (V):
A specific value for R1 is hydrogen or benzyloxycarbonyl. A specific value for R2 is hydrogen. A specific value for R3 is methyl. A specific value for R4 is hydrogen, 2-chlorobenzyloxycarbonyl, or benzyloxycarbonyl. A specific value for R5 is hydrogen. A specific value for R6 is an antibody. A specific value for R6 is a peptide that includes about 2 to about 1000 amino acids. A more specific value for R6 is a peptide that includes about 5 to about 500 amino acids. An even more specific value for R6 is a peptide that includes about 10 to about 100 amino acids. Specifically X is a linking group that is about 5 angstroms to about 100 angstroms in length. More specifically, X is a linking group of about 5 angstroms to about 25 angstroms in length. Specifically X is �Ra�C(═O)�NH�Rb� wherein each of Ra and Rb is independently (C1-C6)alkylene. Preferably, each of Ra and Rb-is methylene (�CH2�). A preferred value for R6 is KKKEKERPEISLPSDFEHTIHVGF DACTGEFTGMPEQWARLLQT (SEQ ID NO: 1) A specific value for R7 is hydrogen. Another specific value for R7 is (C1-C6)alkyl. A preferred value for R7 is methyl. Specificaly Y is a linking group that is about 5 angstroms to about 100 angstroms in length. More specifically, Y is a linking group of about 5 angstroms to about 25 angstroms in length. A specific value for Y is (C1-C6)alkylene. A preferred value for Y is methylene (�CH2�). Preferably, the biophysical probe can be an Alexa dye, a solvatochromic dye, an electrochromatic dye, or a dye that is sensitive to pH change, ligand binding, or other enviornmental aspects. Preferred fluorescent groups include molecules that are capable of absorbing radiation at one wavelength and emitting radiation at a longer wavelength, such as, for example, Alexa-532, Hydroxycoumarin, Aminocoumarin, Methoxycoumarin, Amino methylcoumarin, Cascade Blue, Lucifer Yellow, NBD, P-Phycoerythrin, R-Phycoerthrin, (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, Fluorescein, BODIPY-FL, BODIPY TR, BODIPY TMR, Cy3, TRITC, X-Rhodamine, Lissamine Rhodamine B, PerCP, Texas Red, Cy5, Cy7, Allophycocyanin (APC), TruRed, APCy7 conjugates, Oregon Green, Tetramethylrhodamine, Dansyl, Indo-1, Fura-2, FM 143, DilC18(3), Carboxy-SNARF-1, NBD, lndo-1, Fluo-3, DCFH, DHR, SNARF, Monochlorobimane, and Calcein. More preferred fluorescent groups include rhodamine and Alexa-532. Preferred nucleic acid indicators include intercolating agents and oligonucleotide strands, such as, for example, YOYO-1, Propidium Iodide, Hoechst 33342, DAPI, Hoerchst 33258, SYTOX Blue, Chromomycin A3, Mithramycin, SYTOX Green, SYTX Orange, Etkhidium Bromide, 7-AAD, Acridine Orange, TOTO-1, TO-PRO-1, Thiazole Orange, Propidium Iodide, TOTO-3, TO-PRO-3, LDS 751. The synthetic intermediates (i.e. synthons) of the invention that include one or more aminooxy groups can be incorporated into peptides using a variety of techniques that are known in the art. For example, as discussed below, the synthons can be incorporated into a peptide using solid-phase peptide synthesis, solution-phase peptide synthesis, native chemical ligation, intein-mediated protein ligation, and chemical ligation. Peptides may be prepared using solid-phase peptide synthesis (SPPS). For example, according to the SPPS technique, protected amino acids in organic solvents can be added one at a time to a resin-bound peptide chain, resulting in the assembly of a target peptide having a specific sequence in fully-protected, resin-bound form. The product peptide can then be released by deprotection and cleavage from the resin support (Wade, L. G., JR., Organic Chemistry 4th Ed. (1999)). As illustrated in Example 2 below, amino acids containing an aminooxy functional group can be incorporated into peptides using SPPS. Use of this methodology allows an amino acid containing an aminooxy functional group to be positioned at a desired location within a synthesized peptide chain. Amino acids containing an aminooxy group can also be incorporated into a peptide using solution-phase peptide synthesis (Wade, L. G., JR. Organic Chemistry 4th Ed. (1999)). Solution-phase peptide synthesis involves protecting the amino-terminus of a peptide chain followed by activation of the carboxyl-terminus allowing the addition of an amino acid or a peptide chain to the carboxy-terminus (Wade, L. G., JR. Organic Chemistry 4th Ed. (1999)). Native chemical ligation is a procedure that can be used to join two peptides together thereby producing a single peptide having a native backbone structure. Native chemical ligation is typically carried out by mixing a first peptide with a carboxy-terminal α-thioester and a second polypeptide with an amino-terminal cysteine (Dawson, P. E., et al., (1994), Science 266:776-779; Cotton, G. J., et al., (1999), J. Am. Chem. Soc. 121:1100-1101). The thioester of the first peptide undergoes nucleophilic attack by the side chain of the cysteine residue at the amino terminus of the second peptide. The initial thioester ligation product then undergoes a rapid intramolecular reaction because of the favorable geometric arrangement of the alpha-amino group of the second peptide. This yields a product with a native peptide bond at the ligation site. A polypeptide beginning with cysteine can be chemically synthesized or generated by intein vectors, proteolysis, or cellular processing of the initiating methionine. This method allows mixing and matching of chemically synthesized polypeptide segments. The synthons of the invention are particularly useful in combination with native chemical ligation, because native chemical ligation allows a synthetic peptide having a specifically positioned amino acid (e.g. a synthon of the invention) to be selectively ligated to another peptide. The ability to specifically incorporate aminooxy modified amino acids into a peptide chain allows useful moieties to be linked at any position within a peptide. Examples of such moieties that can be incorporated into a peptide using this method include, but are not limited to, phosphorylated or glycosylated amino acids, unnatural amino acids, tags, labels, crosslinking reagents, biosensors, reactive groups, and fluorophores. Another advantage of native chemical ligation is that it allows incorporation of peptides into a peptide that are unable to be added by ribosomal biosynthesis. Intein-mediated protein ligation may also be used to selectively place amino acids containing aminooxy functional groups into peptides. Inteins are intervening sequences that are excised from precursor proteins by a self-catalytic mechanism and thereby expose reactive ends of a peptide. Intein vectors have been developed that not only allow single-step purification of proteins, but also yield polypeptides with reactive ends necessary for intein-mediated protein ligation (IPL) (also called expressed protein ligation)(EPL) (Perler, F. R. and Adam, E., (2000) Curr. Opin. Biotechnol. 11(4):377-83; and Evans, T. C., et al., (1998) Protein Sci 7:2256-2264). This method allows a peptide having a selectively placed amino acid containing an aminooxy functional group to be readily ligated to any peptide with reactive ends generated by intein excision. Two peptides may also be linked through use of chemical ligation. Chemical ligation occurs when two peptide segments are each linked to functional groups that react with each other to form a covalent bond producing a non-peptide bond at the ligation site (Wilken, J. and Kent, S. B. H., (1998) Curr. Opin Biotechnol. 9:412-426). This method can be used to ligate a peptide having a specifically positioned aminooxy functional group to another peptide to produce a desired peptide that may be later linked to a detectable group. A functional molecule (�D�) can be attached to a peptide comprising an aminooxy group through a direct linkage (e.g. an amide bond O�N�C(═O)�D) or through a linking group. The structure of the linking group is not crucial, provided it does not interfere with the use of the resulting labeled peptide. Preferred linking groups include linkers that separate the aminooxy nitrogen and the detectable group by about 5 angstroms to about 100 angstroms. Other preferred linking groups separate the aminooxy nitrogen and the detectable group by about 5 angstroms to about 25 angstroms. For example, the linking group can conveniently be linked to the detectable group through an: 1) amide (�N(H)C(═O)�, �C(═O)N(H)�), 2) ester (�OC(═O)�, �C(═O)O�), 3) ether (�O�), 4) thioether (�S�), 5) sulfinyl (�S(O)�), or 6) sulfonyl (�S(O)2) linkage. Such a linkage can be formed from suitably functionalised starting materials using synthetic procedures that are known in the art. The linking group can conveniently be linked to the nitrogen of the aminooxy group to form an amide (�O�N(H)C(═O)� or a thiourea linkage (�O�N�C(═S)�N�) linkage, using reagents and conditions that are known in the art. The aminooxy group can be attached to a peptide through a direct bond (e.g. a carbon-oxygen bond) between the aminooxy oxygen and a side chain of the peptide, or the aminooxy group can be attached to the peptide through a linking group. The structure of the linking group is not crucial, provided it does not interfere with the use of the resulting labeled peptide. Preferred linking groups include linking groups that separate the aminooxy oxygen and the side chain of the peptide by about 5 angstroms to about 100 angstroms. Other preferred linking groups separate the aminooxy oxygen and the side chain of the peptide by about 5 angstroms to about 25 angstroms. A specific linking group (e.g. X or Y) can be a divalent (C1-C6)alkylene, (C2-C6)alkenylene, or (C2-C6)alkynylene chain, or a divalent (C3-C8)cycloalkyl, or aryl ring. Thus, a simple and efficient synthetic methodology for site-specific labeling of peptides after synthesis has been developed that provides high yield, selectivity and compatibility with both solid-phase peptide synthesis and Cα-thioester peptides. The approach and primary advantages can be summarized as follows: (1) A protected aminooxy amino acid which can be incorporated into peptides has been synthesized; (2) Procedures have been optimized to yield highly efficient and specific modification of the aminooxy nitrogen in the presence of unprotected competing nucleophiles, including cysteine, lysine and amino groups; (3) One preferred electrophile that can be used for labeling, an activated carboxylic ester, is readily available in the majority of commercially available fluorescent dyes and labels; (4) Labeling of the aminooxy group occurs after synthesis and purification, thus enabling the use of chemically sensitive fluorophores and labels that would otherwise not survive earlier synthetic procedures; (5) The synthetic methodology is compatible with the steps required for the synthesis of proteins by total chemical synthesis or expressed protein ligation, namely synthesis of Cα-peptide thioesters and amide-forming ligations; and (6) combinatorial screening of both the functional molecule and its placement will enable the rapid synthesis of optimally labeled polypeptide-based biosensors. The invention will now be illustrated by the following non-limiting Examples. Under controlled pH conditions, the low pKa and enhanced nucleophilicity of an aminooxy group relative to other nucleophilic side chains found in peptides suggested the possibility of site-specific reaction with standard electrophiles such as succimidyl esters ( A test peptide containing both a secondary aminooxy group and nucleophilic amino acids that were most likely to interfere with selective labeling at the aminooxy nitrogen (lysine, cysteine, and the amino-terminus) was prepared. As illustrated in Conditions for selectively labeling the secondary aminooxy group were determined by varying the reaction pH and dye stoichiometry. Labeling with the succinimide ester of tetramethylrhodamine (TMR-OSu) was determined to be optimal in a solvent system consisting of 50% oDMSO/50% aqueous acetate buffer at pH of 4.7 with 2 equivalents of dye per mole of peptide (Table 1). The crude reaction products were separated from unreacted dye, and characterized by RP-HPLC and ESI-MS. Under these conditions, a single molecule of dye was incorporated on the aminooxy group with a 78% yield, based on HPLC quantification. However, side reaction products were also isolated and determined to be either SA-test peptide labeled with two dye molecules (�13%) or acetylated peptide products (�5%). The selectivity, as defined by the ratio of the peak areas of desired single-labeled product over double-labeled products, was 6/1 (Table 1).
It was determined that many of the side reactions occurred during size exclusion chromatography in the ammonium bicarbonate solvent used to separate reaction products. Using an acidic solvent system, 0.1% TFA, virtually eliminated multiply labeled side products leading to considerable improvements in both selectivity and yield. Including a mild reducing agent, tris(2-carboxyethyl)phosphine CTCEP), in the reaction buffer also significantly curtailed several minor side reactions revealed by HPLC, especially disulfide formation. Labeling and gel filtration under these optimized conditions produced a 70% recovered yield of labeled SA-test peptide (90% yield based on HPLC quantitation), 90% of which was labeled with only a single dye at the aminooxy amine. The labeling selectivity was increased to 22/1 (Table 1). The purity of the product was confirmed using RP-HPLC, mass spectroscopy, further chemical reaction, and isolation of purposefully overlabeled products. The labeled peptide eluted as a single peak under all HPLC conditions tested with a mass consistent with that predicted for singly-labeled SA-test peptide (Mass=1970). To determine that the labeling site was indeed at the N-methylaminooxy group, a selective zinc/acetic acid reduction procedure was used to cleave the N�O bond ( To eliminate the unlikely possibility that the HPLC peak containing isolated single-labeled SA-test peptide product was a mixture of two labeled species, SA-test peptide was reacted under higher pH conditions (pH 9.0) to label all reactive sites. SA-test peptide contained 3 nucleophilic labeling sites which would be irreversibly labeled: aminooxy, lysine, and N-terminal amine. Dye labeling at high pH generated a mixture of peptides labeled at all possible combinations of sites with 1, 2 or 3 dyes. HPLC analysis of this reaction mixture showed 8 peaks, indicated by ESI-MS to correspond to unreacted SA-test peptide, SA-test peptide single-labeled at the aminooxy nitrogen, and six additional peaks corresponding to two single-labeled peptides, three peptides bearing two dyes and a single triply-labeled peptide species. This experiment revealed the HPLC retention times of all these products, none of which coeluted with the peak identified as SA-test peptide labeled with a single dye on the secondary aminooxy nitrogen. Site-specific labeling of the aminooxy group could even be achieved at basic pH (Table 1). Using pH 9.0 carbonate buffer in our solvent systern, addition of 0.5 equivalents of dye produced, after 3 hours, �50% conversion of the starting SA-test peptide to a single peak with the elution time of the desired singly-labeled product. After addition of another. 0.5 equivalents of TMR-OSu and an additional 3 hours of reaction time, HPLC showed �85% conversion to a peak with the retention time of the desired product Two minor peaks (�2-3% of total peak area), were also apparent and corresponded to the two other single-labeled SA-test peptide species identified in the multiple labeling experiment above. The N-hydroxysuccinimide ester of rhodamine clearly showed selective reactivity with the aminooxy group. The selectivity observed at higher pH cannot be explained by the nucleophicity of the aminooxy group alone. In fact, others have shown that at, in an an uncatalyzed reaction with phenyl acetate at high pH, amines are more reactive than O-alkylaminooxy groups. Therefore we suggest that kinetic factors are contributing to the selective reactivity of the N-methylaminooxy group, even when competing groups are not protonated. Possible reasons for this include: (1) the aminooxy oxygen localizes the nitrogen near the activated ester via formation of a hydrogen bonded �bridged� intermediate (2) a base catalyzed reaction pathway under the conditions of our reaction. This exceptional reactivity has important practical implications, as it can allow the selective labeling of acid-labile polypeptides and synthetic proteins under physiological or basic conditions. Preparation of Proteins by Total Chemical Synthesis Often Requires the ligation of large polypeptides prepared by solid-phase peptide synthesis on thioester-linker resins. The most generally applicable methods available for ligations are native chemical ligation and expressed protein ligation. These processes utilize the same basic chemistry to join two peptides, one with an N-terminal cysteine and the other with a C-terminal thioester, through a regiospecific and site-specific reaction to generate a larger polypeptide. The application of aminooxy-labeling chemistry to the synthesis of large polypeptides and proteins requires compatibility with these solid-phase peptide synthesis and ligation chemistries. The optimal approach for utilizing aminooxy-labeling chemistry in the chemical synthesis of proteins is direct incorporation of the aminooxy group as part of an amino acid used during standard solid-phase peptide synthesis. For this purpose, we generated a suitably protected N-methylaminooxy amino acid, α-Boc-β�[N�(2 Chlorobenzyloxycarbonyl)-N-Methylaminooxy Acetyl]-α,β-Diaminopropionic Acid [Boc-2-Cl-Z(SA)Dapa-OH] (4), as shown in Ligation of the LY-(SAOD)-AG-MPAL (SEQ ID NO: 3) thioester peptide with the peptide CRANK-NH2 (SEQ ID NO: 4), was tested using standard procedures employing phosphate buffer with 6M guanidine hydrochloride at neutral pH in the presence of 2-3% thiophenol by volume. The ligation proceeded over 24 hours and generated the desired ligation product, LY-(SAOD)-AGCRANK-NH2 (SEQ ID NO: 5), at �85% yield. The major side product was attributable to modification of unligated CRANK-NH2 peptide under the ligation conditions (mass=714.5, data not shown), and was not related to the presence of the aminooxy group. There was also a single time-dependent side reaction, which generated a product of 14 mass units lower than the desired. Using high concentrations of reacting peptides and isolating the ligation product after 24 hours reduced this side reaction to acceptable levels (<5%). The ligation of a peptide containing multiple potentially reactive functional groups, including a hexahistidine tag useful for affinity chromatography was also tested. Coupling CEYRIDRVRLFVDKLDNIAQVPRVGAA-HHHHHH (SEQ ID NO: 6) to LY-(SAOD)-AG-MPAL thioester proceeded to completion in 5 hours with minimal side reactions. In both ligation reactions, there was less than 1% LY-(SAOD)-AG-MPAL self condensation product, indicating that the aminooxy group and thioester do not appreciably react with one another under the ligation conditions. These results demonstrate that the inclusion of an unprotected aminooxy group in the peptide chain is compatible with native chemical ligation. Labeling of the two ligation products using tetramethylrhodamine succinimide ester proceeded with selectivity similar to that for the SA-test peptide. HPLC integration indicated that the product of the LY-(SAOD)-AG-MPAL ligation with CRANK-NH2 was labeled with greater that 95% efficiency and with a selectivity of 34:1. Mass spectral analysis and zinc reduction demonstrated labeling at only the aminooxy group. For the longer hexahistidine-containing polypeptide ligation product, selectivity for the aminooxy group was greater than 10:1, but it was difficult to achieve high yields. The histidines could potentially have been affecting yield and selectivity by catalyzing nucleophilic attack on the succinimide ester of the reactive dye. Inclusion of guanidine hydrochloride in the reaction solvent increased the yield to approximately 50%, indicating that folding or poor solubility of the peptide was a factor in preventing access of the reactive dye to the aminooxy group. Selectivity was also improved, presumably because of the availability of the reactive secondary aminooxy group. Single-site labeling at the aminooxy group was proven by mass spectral analysis of trypsin and α-chymotrypsin digests of the labeled polypeptide product. As a control to establish the selectivity of labeling for aminooxy amino acid, the labeling of native β-Lactoglobulin with tetramethylrhodamine N-hydroxysuccinimide ester was attempted. It was found that non-specific labeling of this 162 aa protein containing 15 lysines and 4 cysteines was minimal (<1%) even after 6 hours. Finally, the GTPase binding domain of p21 activated kinase (45 aa, 4 lys, 1 cys) with a secondary aminooxy amino acid incorporated at the amino-terminus (SAOD-PBD) was prepared. Previous experiments have demonstrated that PBD domains labeled with fluorescent reporter dyes at this terminus could be used as biosensors of GTPase activation. Labeling of PBD using the new methodology would enable the production of sufficient quantities to apply the biosensors in vivo and in pharmaceutical screening applications, and would allow incorporation of sensitive detectable groups enabling applications within living cells. SAOD-PBD was readily labeled with Alexa-532 N-hydroxy-succinimide ester by titration addition of dye at pH 4.7 over 72 hours. The labeling efficiency was commensurate with that reported for the longest model peptide (�50% yield by HPLC quantitiation) and there was no indication of multiple labeling. In this case, isolation of labeled SAOD-PBD by RP-HPLC proved difficull Separation to baseline resolution was not achieved, but small quantities of unlabelled PBD in the labeled product do not preclude the use of the labeled material in biosensor applications. Previous reports indicate that separation of labeled product from starting polypeptide is highly dependent on the specific peptide and the attached dye. These results demonstrate that the optimized site-specific labeling chemistry reported here is compatible with the steps required for the preparation of proteins by total chemical synthesis. General: For column chromatography, silica gel (230-400 mesh) was used in standard glass columns with gravity or air pressure. Reversed-phase high performance liquid chromatography (RP-HPLC) was performed on a Waters HPLC system with UV detection at 214 nm using either a Vydac C-18 analytical column (5 μm, 0.46�25 cm), a Waters RCM 8�10 module equipped with a semipreperative Delta Pack C-18 Radial Pack cartridge column (15 μm, 8�100 mm) from Millipore, or a Vydac C-18 preparative scale column (15 μm, 1.0�25 cm). Linear gradients of solvent B (0.09% TFA in 90% acetonitrile/10% water) in solvent A (0.1% TFA in water) were used for all HPLC chromatographic separations. Mass spectra of peptides were obtained either with a Sciex API-I electrospray ionization (ESI) triple-quadruple mass spectrometer (PE Biosystems, Foster City), or matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) instruments from Thermo Bioanalysis (Ihermo Bioanalysis, LTD., UK) or Kratos Analytical (Chestnut Ridge, N.Y.). For ESI-MS, the observed masses reported were derived from the experimental m/z states for all observed charge states of a molecularspecies using the program MacSpec (Sciex, version 2.4.1) for electrospray mass spectrometry. MALDI-MS observed masses were relative to internal calibration using α-cyano-hydroxycinnammic acid or sinipinic acid matrices. Calculated masses reported were derived from either MacProMass (Terry Lee and Sunil Vemuri, Beckman Research Institute, Duarte, Calif.) or PAWS (Version 8.1.1, ProteoMetrics) and reflect the average isotope composition of the singly-charged molecular ion. Proton nuclear magnetic resonance spectrometry was recorded on a Bruker AC-250 mass spectrometer and data was analyzed using WinNMR (Bruker Instruments). Ultraviolet-Visible spectroscopy was performed on a Hewlett-Packard photodiode-array spectrophotometer. Boc-L-amino acids were purchased form Novabiochem (La Jolla, Calif.) or Bachem Bioscience, Inc. (King of Prussia, Pa.). [[4 (Hydroxymethyl)phenyl]-acetamido]methyl (�OCH2-Pam) Resin was purchased from PE Biosystems (Foster City, Calif.) and methylbenzhydrylamine (MBHA) resin was purchased from Peninsula Laboratories, Inc. (San Carlos, Calif.). Solvents were Synthesis grade or better and were purchased from Fisher Scientific (Tustin, Calif.). Trifluoroacetic acid (TFA) and anhydrous hydrogen fluoride were purchased from Halocarbon (New Jersey) and Matheson Gas (Rancho Cucamonga, Calif.). Dyes were obtained from Molecular Probes (Eugene, Oreg.). All other reagents were analytical grade or better and were purchased from Aldrich (Milwaukee, Wis.), Lancaster (Windham, N.H.), Peptides International (Louisville, Ky.) or Richelieu Biotechnologies (Montreal, Canada). Peptide Segment Synthesis. Synthesis of peptides was carried out manually using optimized stepwise solid-phase synthesis methods with in situ neutralization and HBTU activation procedures for Boc chemistry on either �OCH2-Parn, MBHA, or Trt-protected mercaptopropionyl-Leu (TAMPAL) resin (Hojo, H., et al., BulL Chem. Soc. Jpn. 1993, 66:2700-2706; Hackeng, T. M.et al., Proc. Natl. Acad Sci. USA. In press; and Schnolzer, M., et al., Int. J. Peptide Protein Res. 1992, 40:180-193). Standard Boc protecting group strategies were employed. Coupling was monitored by quantitative ninhydrin assay after 15 minute coupling cycles. After chain assembly, standard deprotection and cleavage from the resin support was carried out by treatment at 0� C. for 1 hour with anhydrous HF containing either 10% p-cresol or anisole as scavenger. Purification was performed using RP-HPLC. Synthesis of TAMPAL Resin (Hojo, H., et al., Bull. Chem. Soc. Jpn. 1993, 66:2700-2706). 2.5 grams of MBHA resin (0.865 mmol/g, 2.16 mmol of amine) was swelled in DMF. Boc-Leu-OH (1.1 grams, 4.4 mmol) was activated with HBTU (8 ml, 0.5M solution) and DIEA (2 ml), then coupled to the MBHA resin until complete reaction by ninhydrin assay. The Nα-Boc group of the linked leucine was removed with neat TFA, then S-Trt-β-mercaptopropionic acid (1.5 grams, 4.3 mmol), activated in the same manner as Boc-Leu-OH, was added to the deprotected Leu-MBHA resin and allowed to couple until complete reaction. The S-Trt-β-mercaptopropionyl-Leu-MBHA resin was washed extensively with DMF, then DCMIMeOH (1/1), and finally dried in vacuo to yield 3.39 grams of thioester resin. Substitution calculated by weight gain yielded 0.549 mmol/gram. Deprotection of TAMPAL Resin: S-trityl protection was removed by two 5 minute treatments with 95% TFA/5% triisopropylsilane. The deprotected resin was extensively with DMF before coupling the first amino acid, activated using optimized in situ neutralization protocols.
Selective Labeling of LY-(SAOD)-AGCRANK-NH2 and LY-(SAOD)-AGCEYRIDRVR-LFVDKLDNIAQVPRVGAA-HHHHHH with Tetramethylrhodamine N-hydroxy-succinimidyl Ester. A sample of 20 μL of LY-(SAOD)-AGCRANK-NH2 (2.5 μg/μl, 2.18 mM) in 200 mM citrate buffer, pH=4.7, 5 mM TCEP was labeled using 4.3 equivalents of dye in DMSO, purified and analyzed. Expected Mass=1580, Observed Mass=1580. A 20 μL sample of LY-(SAOD)-AGCEYRIDRVRLFVDKLDNIAQVPRVGAA-HHHHH (SEQ ID>NO: 12) (9.7 μg/μl, 2.12 mM) in 200 mM citrate buffer, pH=4.7, containing 5 mM TCEP and 3M guanidine hydrochloride was labeled using a modified procedure. 18 μl of a solution of tetramethylrhodamine N-hydroxysuccinimide (10 μg/μl) in DMSO was added in 6 μl aliquots over 15 minutes with rapid mixing. The reaction was incubated at room temperature for 5 hours prior to gel filtration/RP-HPLC purification and mass spectral analysis. Expected Mass=4930, Observed Mass=4929. Labeling of β-Lactoglobulin with Tetramethylrhodamine N-hydroxysuccinimide ester: 10 μL of a 10 mg/ml solution of tetramethylrhodamine N-hydroxysuccinimide ester in DMSO (9.5 equivalents compared to protein), was added to a solution containing 10 μL of DMSO and 20 μL of a solution of β-Lactoglobulin (20.3 mg/ml or 1.1 mM) in 2.8 M guanidine hydrochloride with 5 mM TCEP (pH 4.7). After 3 hours, protein was separated from unreacted dye by gel filtration, and labeling was determined by analysis of the dye-to-protein ratio (protein concentration was determined by the method of Waddel and ε for tetramethylrhodamine in phosphate buffer, pH=8.0 of 81,000).
All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. Patent Citations
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