Publications relevant to Spyder technology:
Lebl, M. (2000) New technique for high-throughput synthesis of peptides, peptidomimetics and nonpeptide small organic molecule arrrays. In G.B. Fields, J.P. Tam & G. Barany (Eds.), Peptides for the New Millennium. (pp. 164-166). Kluwer Academic Publisher, Dordrecht.
Lebl, M., Ma, J., Pires, J., Dooley, C. & Houghten, R.A. (2000) Rapid parallel synthesis of 584 betides, peptides composed largely of beta-amino acids with side-chains not found in natural peptides. In G.B. Fields, J.P. Tam & G. Barany (Eds.), Peptides for the New Millennium. (pp. 174-175). Kluwer Academic Publisher, Dordrecht.
Lebl, M., Krchnak, V., Ibrahim, G., Pires, J., Burger, C., Ni, Y., Chen, Y., Podue, D., Mudra, P., Pokorny, V., Poncar, P., & Zenisek, K. (1999) Solid-phase synthesis of large tetrahydroisoquinolinone arrays by two different approaches. Synthesis-Stuttgart, 1971-1978.
Lebl, M., Pires, J., Poncar, P., & Pokorny, V. (1999) Evaluation of gaseous hydrogen fluoride as a convenient reagent for parallel cleavage from the solid support. Journal of Combinatorial Chemistry, 1, 474-479.
Lebl, M. (1999) New Technique for High-Throughput Synthesis. Bioorg. Med.
Chem. Letters, 9, 1305-1310.
Lebl, M. (1998) A New Approach to Automated Solid phase Synthesis Based on
Centrifugation of Tilted Plates. Journal of the Association of Laboratory
Automation, 3, 59-61.
Eichler, J., Houghten, R.A., & Lebl, M. (1996) Inclusion volume
solid-phase peptide synthesis. J. Peptide Sci., 2, 240-244.
Pokorny, V., Mudra, P., Jehnicka, J., Zenisek, K., Pavlík, M., Voburka,
Z., Rinnová, M., Stierandová, A., Lucka, A.W., Eichler, J., Houghten,
R.A. & Lebl, M. (1994) Compas 242. New type of multiple peptide
synthesizer utilizing cotton and tea bag technology. In R. Epton (Ed.),
Innovation and Perspectives in Solid Phase Synthesis. (pp. 643-648).
Mayflower Worldwide Limited, Birmingham.
Lebl, M., Stierandová, A., Eichler, J., Pátek, M., Pokorny, V., Jehnicka,
J., Mudra, P., Zenisek, K. & Kalousek, J. (1992) An automated multiple
solid phase peptide synthesizer utilizing cotton as a carrier. In
R. Epton (Ed.), Innovation and Perspectives in Solid Phase Peptide
Synthesis. (pp. 251-257). Intercept Limited, Andover.
Spyder Technology: A New Approach to Automated Solid Phase Synthesis
Based on Centrifugation of Tilted Plates
Abstract: High throughput solid phase synthesis can be performed with
application of the centrifugation based liquid removal. This technique
uses readily available standard microtiterplates and eliminates filtration
step. It is therefore applicable to simultaneous processing of unlimited
number of reaction compartments.
Combinatorial techniques (for reviews see e.g. http://www.5z.com/divinfo/)
require new methods for automation of synthetic processes. Solid
phase synthesis2 is optimal for automation, since the complicating
factor of unique behavior of different organic molecules is replaced
by predictable behavior of the solid support. Instruments available
on the market today are relatively complicated and expensive. Our
goal is to bring to the market the instrument that is rather simple,
therefore inexpensive, and allows each chemist to synthesize 100-1000
compounds in a batch. Such instrument can be used for deconvolution of
active compound from biologically active mixtures, synthesis of arrays
of compounds for general screening, or for compound optimization, so
called "lead explosion". The prototype of this instrument is shown in
Basic problem of solid phase synthesis is parallel separation of liquid
and solid phases. Commercial solid phase synthesizers utilize filtration
as the principle for separation of solid and liquid phase. Filtration can
lead to significant complications, especially in the case of multiple
synthesizers, since clogging of one vessel can result in overflowing of
this particular vessel during the next solvent addition and distribution
of the solid support from this vessel into neighboring ones.
We have found the simpler way for simultaneous processing of
hundreds of reaction vessels. We call this new technique "tilted
centrifugation". The principle of tilted centrifugation is shown in
Resin suspended in the
tilted flask placed at the perimeter of the centrifugal plate and
spun, does not remain at the bottom of the flask. As the surface
of liquid supernatant moves, the solid support layer moves as well.
If the speed of rotation is increased, the centrifugal force created
by rotation (which depends on the radius of rotation and the speed)
combines with gravitation and the resulting force causes liquid surface
to stabilize at the angle perpendicular to the resulting force vector.
At the ratio of relative centrifugal force (RCF) to G of 3, the angle
of the liquid surface is about 61 degrees. If the speed is increased
so that the ratio of these forces is more than 50, and we are getting
closer to the situation where RCF is infinity - therefore the liquid
(and resin) layer angle will be close to 90 degrees. The pocket created
by the tilt should allow only solid phase to remain in the pocket and
all of the liquid is expelled. The pocket can be created in the vessel
of basically any shape (see Figure 3
- flat bottom, U bottom, or V bottom vessel, as well as in the array of
vessels, e.g. in the commonly used microtiterplates.)
Situation of wells in microtiterplates placed on the perimeter of the
centrifuge depends on the distance of the individual well from the axis
of rotation. The volume of the "pocket" created by centrifugation in the
wells closer to the axis is bigger than the volume of the pocket created
in the wells more distant from the center of rotation. The volume of the
pocket is not as important as the ratio of volumes of pockets in different
wells of the microtiterplate. This ratio depends on the dimension of
the centrifuge rotor, speed of the rotation, and the tilt of a plate.
Wells placed on a rotor of very large diameter, or rotor spun very
fast, will have insignificant difference between forces exerted onto
"inside" and "outside" wells. We found an optimal tilt of 9 degrees,
350 rpm, and the diameter of centrifugal rotor of 48 cm. Under these
conditions the volume of the pocket in inner and outer wells differed
by an acceptable 8%.
If drilling of holes into inert material would create the array of wells,
the liquid expelled from one well would inadvertently enter another well
placed closer to the perimeter of the centrifuge. However, 96 well shallow
microtiterplate is actually composed of 96 small cylinders attached to a
flat polypropylene sheet and connected by a thin "rib", creating thus an
array of 96 round wells plus 117 interwell spaces. The liquid expelled
by centrifugal force from one well comes into the interwell space, flies
across this space and ends up on the outer wall of the adjacent well
(see Figure 4).
Then it flows along
the well until it detaches and flies across another interwell space,
eventually ending at the edge of the plate from where it flies onto the
wall of the centrifuge drum. To test the transfer of liquid and/or solid
material from one well into another we have loaded the wells with the
amount of colorized solid support (resin) which exceeded the capacity of
the pocket and observed the fate of the resin expelled from the well. As
can be observed on Figure 5
of the resin ended in the interwell space and we have not observed any
transfer of the resin beads into adjacent wells. In another experiment we
have analyzed products synthesized in all wells of the microtiterplate by
HPLC and mass spectroscopy. We have not found any traces of contamination
by liquid or solid transfer between wells in our model experiments. Figure 6
shows HPLC traces of products
synthesized in adjacent wells and Figure
shows the mass spectra of the
products from the same wells.
The first experiments using tilted plate centrifugation were performed
in the Savant centrifuge, which we have equipped with custom-built
rotor (Figure 8
). Later we have
built the dedicated centrifuge with 8 positions for microtiter
plates. This centrifuge is driven from the computer and all
centrifugation parameters can be flexibly changed. 96-channel
distributor (Figure 9
to 6 port selector valve delivers washing solvents and common
reagents. Centrifuge can be. Inclusion of the pipetting system allows
us to perform the whole synthesis in completely automatic regimen.
shows the view of the first
centrifuge prototype integrated with Packard Multiprobe 104 liquid
distribution system for the delivery of individual building blocks and
reagents. Figure 11
shows the detail
of the instrument deck. This compact system can be easily enclosed in
Since the goal of this instrument is its affordability in the laboratories
with any size budget, we decided to design the system, which can be
operated semi-manually and later upgraded to fully automated machine. Figure 12
shows the view of the intelligent
centrifuge Compas 768.2, which can be easily integrated with an array of
pipetting robots, but which can be run completely independently. Common
reagents and solvents are delivered again by multichannel distributor
). Centrifuge can be driven
and programmed from an internal computer to perform up to 20 washes by
6 different solvents in any order. More complicated operations can be
performed from the attached PC (Figure
). Software is capable of performing individual steps (Figure 15
), or the whole process can
be programmed (Figure 16
single parameter of the process can be changed in the software (Figure 17
The synthesis is performed in the following way. Microtiterplate with
slurry of solid support distributed into it is placed on the perimeter
of a rotor with a permanent tilt of 9 degrees. The rotor is rotated at
the speed required for complete removal of the liquid portion of the
well content. After stopping the rotation, microtiterplate is placed
(rotor is turned) under the multichannel (96 channel) liquid delivery
head. The solvent selector valve is turned into the appropriate position
and the washing solvent is delivered by actuating the syringe pump. This
operation is repeated until all plates are serviced. The rotor is spun at
the speed at which the liquid phase is just reaching the edge of the well,
wetting thus all solid support in the "pocket", and after reaching this
speed, rotation is stopped. The cycle of slow rotation and stopping
is repeated mixing thus the slurry of solid support in the liquid
phase. After shaking for the appropriate time, the plates are spun at
the high speed. The process of addition and removal of washing solvent
is repeated as many times as many washes are required. The plates are
then consecutively placed under the opening in the centrifuge cover and
appropriate building block solutions and coupling reagents are delivered
by pipetting (Multiprobe 104) through the opening from the stock solutions
placed on the centrifuge cover.
The best way to demonstrate the efficiency of the centrifugal
synthetic technique is
to show the results from the syntheses performed in the Compas 768.
Figure 18 shows the synthetic scheme
used in the synthesis of 768 tetrahydroisoquinolinones, and
Figure 19 shows HPLC traces of all
products from one microtiterplate. In all cases the main peak
corresponded to the expected product. Peaks marked by the dot contain
We have synthesized hundreds of peptides and evaluated their cleavage
from the resin by gaseous HF.
Results from peptide syntheses are given in
Figures 20 and
shows the results from the synthesis of tetrapeptides containing
Figure 21 shows peptides composed of
unnatural beta amino acids. Traces marked "a" contain products synthesized
on benzhydrylamine resin and cleaved by two step process - in the first
step the side chain protecting groups were removed by TFA and in the
second step the product was cleaved from the resin by gaseous HF. Traces
marked "b" contain products prepared on Knorr linker and cleaved in one
step by TFA.
We believe that tilted centrifugation is the most effective and simplest
method for liquid removal from multiplicity of vessels and polypropylene
microtiterplates ideal reaction vessels for tilted centrifugation based
synthesis. The fact that tilted centrifugation is the only way for removal
of liquids from unlimited number of reaction vessels simultaneously is
suggesting its application in ultraminiaturized synthesizers.
- Leblova Z, Lebl M. Compilation of papers in molecular
diversity field. 2001. INTERNET World Wide Web address: http://www.5z.com/divinfo/.
- Merrifield RB. Solid phase peptide synthesis. I. The synthesis of
a tetrapeptide. J.Amer.Chem.Soc. 1963;85:2149-54.
- Cargill JF, Lebl M. New methods in combinatorial chemistry: Robotics
and parallel synthesis. Curr.Opin.Chem.Biol. 1997;1(1):67-71.
- Krchnák V, Weichsel AS, Lebl M, Felder S. Automated solid-phase
organic synthesis in micro-plate wells. Synthesis of N-(alkoxy-acyl)amino
alcohols. Bioorg.Med.Chem.Lett. 1997;7(8):1013-6.
- Lebl M, Krchnák V. Techniques for massively parallel synthesis of
small organic molecules. In: Epton R, editor. Innovation and Perspectives
in Solid Phase Synthesis & Combinatorial Libraries. Birmingham: Mayflower
Scientific Limited; 1998.
For a version of this document without the embedded images
please click here.