Innovations
`
`Lead compound,
`phone home
`IRORI
`
`Chemistry & Biology February 1998,
`5:R39–R40
`
`© Current Biology Ltd ISSN 1074-5521
`
`Before the advent of combinatorial
`chemistry a chemist needed only a
`felt-tipped pen to keep track of her
`compounds. But with the synthesis
`of huge libraries of compounds now
`common, a system for labeling newly
`synthesized compounds is enough to
`form the basis of a company.
`One such company is IRORI (La
`Jolla, California), which sells capsules
`of solid-phase synthesis resin with
`embedded radiofrequency tags, and
`sorting machines that can read the
`code stored on the tags (Figure 1).
`Synthesis software directs the sorting
`of the capsules, and keeps track of
`which synthesis history is associated
`with which code.
`
`A new use for an old component
`Radiofrequency (rf) tags are far from
`new. They are found in everything
`from building-entrance keys to
`injectable pet ID tags, and the
`IRORI tags are not spectacularly
`different in terms of design or
`communications capability. What was
`new in 1995, when Michael Nova
`and Andrew Senyei of IRORI and
`K.C. Nicolaou (of Scripps Research
`Institute, La Jolla, California, and an
`Editor of this journal) reported the
`construction of their first rf tags, was
`the application of rf technology to
`combinatorial chemistry.
`In combinatorial chemistry, one of
`a large number of possible subunits is
`added to each of several variable sites
`on a molecule. Conceptually, the
`simplest way to make these
`compounds is by parallel synthesis,
`using a large matrix of individual
`
`Figure 1
`
`Rectifier
`
`Antenna
`
`R39
`
`Transmitter/receiver(cid:13)
`circuitry
`
`Control logic
`
`Glass housing
`
`Synthesis manager data base
`1 2 3
`
`1-naphthoyl
`nicotinoyl
`1-bromonicotinoyl
`
`reactions. But if every
`possible chemical group is
`to be combined with every
`other, a lot of time can be
`saved by using the
`split/pool method. For
`example, the three reaction
`products A, B and C can be
`combined, mixed, and then
`split into four mixtures to
`be reacted with components
`D, E, F and G to produce
`12 different compounds.
`Further rounds of splitting
`and pooling give X · Y · Z
`products from only
`X + Y + Z reactions. A series
`of four reactions, each using
`ten different components,
`should result in 10,000
`different products.
`The trick with
`split/pool is to keep track of
`all those compounds. The
`resin beads used for solid-
`phase synthesis solve one problem:
`they keep many molecules of the
`same compound in one place. But
`the identity of the compound on the
`bead must still be decoded. Methods
`for directly determining the structure
`of compounds on a single bead are
`still in their infancy, so a tag or code
`that details the synthesis history of
`the bead is the next best option.
`
`The IRORI radiofrequency tag. An antenna receives energy
`to power up the device, with the rectifier converting the
`radiofrequency pulse to direct circuit current. The chip code
`is then read, and communicated to the external reader using
`a radiofrequency pulse generated by the antenna. This code
`tells the synthesis manager software where to send the
`capsule for the next synthesis step. At the end of the
`synthesis, the code is read one last time. The identity of the
`code is compared to a database to derive the synthesis
`history of the capsule, and thus the expected structure of
`the synthesized compound, which is on the matrix
`surrounding the tag. Image courtesy of IRORI.
`
`‘directed sorting’ used by IRORI,
`one microreactor per compound is
`enough. This means less chemicals,
`less resin, and less decoding at the
`end. Rf tags are re-usable, but
`keeping their numbers down in any
`one experiment is also important, as
`they go for $8.50 a pop.
`The software to direct the
`synthesis, and the sorter to
`redistribute up to 10,000
`microreactors in 10 hours, are also
`not cheap, selling for a total of
`~$100,000. Is all this technology
`really necessary? “I remember when
`I first saw [the rf tag system] I
`thought it was cute, but that it
`looked like a two dollar solution to a
`two cent problem,” admits Tony
`Czarnik, now Vice-President for
`Chemistry at IRORI. “But it is
`actually a very elegant solution to a
`problem that there aren’t obvious
`alternatives to.” According to Matt
`Francis of Harvard University
`(Cambridge, Massachusetts): “It lets
`you make something with the
`benefits of a parallel library, with
`discrete compounds made in
`significant amounts, but using the
`efficiency of split/pool synthesis.”
`
`The IRORI system
`In their original paper in Angewandte
`Chemie, the IRORI chemists wrote a
`code to each microreactor (containing
`resin and rf tag) whenever a
`synthesis step occurred. The more
`recent commercial version assigns a
`code at the outset to a proposed final
`compound. A sorter reads the code at
`the conclusion of every step and
`sends the microreactor to the
`appropriate reaction chamber.
`Although the new system sounds
`more cumbersome, it results in huge
`savings in the numbers of reactions.
`To ensure that most compounds will
`be made in a given split/pool reaction
`sequence, the chemist must include
`at least ten times more beads than
`prospective products. But with the
`
`Luminex Ex. 1011
`Luminex/Irori - Page 1
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`R40 Chemistry & Biology 1998, Vol 5 No 2
`
`The unique tag is encased in
`glass, so it is “inert to all the slings
`and arrows that a synthetic chemist
`can throw at it,” says Czarnik. “And
`the code,” he says, “can be read with
`a very reliable, very fast method that
`doesn’t depend on orientation.”
`The tags are enclosed in one of
`two containers. In a MicroKan™
`reactor, the tag is placed in the
`center of the vessel and any solid-
`phase resin can be added to the
`surrounding chamber. This offers a
`familiar substrate for synthesis, but
`involves some labor in setting up the
`microreactors. The MicroTube™
`reactors are ready to go, with
`synthesis occurring on a polymer
`tube that surrounds the tag.
`Chemistry happens on the surface of
`the polymer, whereas reactions with
`resin occur primarily inside the resin
`bead. As a result, some synthesis
`condition need to be modified for
`the MicroTube™ reactor. In both
`cases, the amount of product is in the
`tens of milligrams: a large amount in
`the world of combinatorial chemistry.
`The sorter can scan and
`distribute up to 10,000 microreactors
`in one 10-hour run. This speed, and
`the cost of the reactors, constrain the
`size of the libraries. “It’s the
`medium-sized libraries we think the
`IRORI system will be best at,” says
`Francis. Libraries of less than one
`hundred can be done by manual
`parallel synthesis, while libraries of
`more than ~20,000 may be too
`expensive and take too long to make
`with the current technology. “To get
`to libraries of more than 20,000,” says
`Czarnik, “you probably need to
`make less compound” by reducing
`the size of the reactors. And, he adds,
`“the sorting has to get faster.”
`IRORI is working on a faster
`sorter, but reducing the size of the rf
`tag may be difficult. “It’s not clear
`that there is a direct way to make the
`tags smaller, as the antenna [which
`takes up ~90% of the volume of the
`tag] has to be big enough to power
`itself up.” Solutions to both the size
`and cost problems are under
`development at IRORI.
`
`According to some chemists, the
`big libraries that demand smaller and
`cheaper tags may not even be
`desirable. “There is as much range of
`opinion [on library size] as there are
`people,” says Czarnik. “There are
`devout believers in spending as
`much time thinking about what you
`should make as possible, and then
`never making more than a few
`hundred compounds. Then there are
`those who say, ‘I would like to know
`exactly what to make, but I would
`also like to know the face of God, so
`perhaps we should make lots of
`compounds.’”
`
`The many alternatives
`Almost coincident with the IRORI
`paper was a report in the Journal of
`the American Chemical Society from
`researchers at Ontogen Corporation
`(Carlsbad, California). They
`proposed rf tags that were essentially
`the same as the IRORI tags, while
`their synthetic strategy used directed
`sorting. More recently, however,
`Ontogen has concentrated on
`building large automated machines
`for parallel solid-phase synthesis.
`A research group at Pfizer, Inc.
`(Groton, Connecticut) led by Eric
`Roskamp has reported a simple
`solution to the tagging problem:
`numbering the vessels. Roskamp
`used a heated printing press and
`graphite ink to imprint a number on
`his plastic microreactors. A video
`camera and optical character
`recognition software could scan
`through ~1000 microreactors in a day.
`“In the worst case, if you really rub
`on it, the black will wear off,” he
`says. “But you can still read [the
`imprint] by eye.” He will not give
`details of the new version of this
`proprietary machine, except to say
`that it “does the same things, but
`faster and more efficiently.”
`An additional tagging system for
`IRORI scientists may incorporate the
`2D bar codes they described late last
`year in Angewandte Chemie. The
`patterns of dark and light squares are
`laser-etched onto a ceramic surface,
`and then read by a camera.
`
`The predecessors of these high-
`tech solutions include oligo-
`nucleotides and peptides, with a
`single base or amino acid added at
`each step of synthesis. The
`chemistry of these tags is not,
`however, compatible with many
`reactions used in small molecule
`synthesis. This led Clark Still of
`Columbia University to develop sets
`of small, halogen-substituted
`aromatics that could be used as tags
`(technology now licensed to
`Pharmacopeia, Inc. (Princeton, New
`Jersey)), while researchers at
`Affymax (Palo Alto, California)
`devised secondary amine tags.
`Some methods bypass tags
`altogether. Deconvolution identifies
`a hit compound by deducing the
`preferred component at each position
`in turn. But this method relies on
`both extensive resynthesis and the
`testing of mixtures of compounds,
`and it identifies only one hit
`compound.
`Perhaps the most widespread
`method, even in solid-phase
`synthesis, is to keep each reaction
`separate by spatial coding. Steps like
`washing and rinsing can be pooled,
`but many of the time-saving
`advantages of split/pool are lost. The
`method remains popular because it is
`not a proprietary technology.
`Solid-phase synthesis allows a
`chemist to drive reactions to
`completion with excess reagents, and
`to isolate products free from
`contaminants. Chemists are working
`on automated purification schemes
`for the solution phase, but even
`without these methods the more
`familiar techniques of solution-phase
`synthesis remain popular. “The
`biggest barrier is a cultural one,” says
`Czarnik, but he is confident that
`barrier will come down. “We are
`placing a sizable bet that split/pool is
`an irresistible force,” says Czarnik,
`“and that people will see this as the
`direction to move in.”
`
`William A. Wells, Biotext Ltd
`1095 Market Street #516, San Francisco,
`CA 94103-1628, USA; wells@biotext.com.
`
`Luminex Ex. 1011
`Luminex/Irori - Page 2
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`Innovations IRORI R41
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`Luminex Ex. 1011
`Luminex/Irori - Page 3
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