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Spyder Technology

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. http://pubs.acs.org/journals/jcchff/index.html).

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.

Figure 1
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 Figure 1.

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.

Figure 2
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 Figure 2. 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

Figure 3
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.)

Figure 4

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

Figure 5
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%.

Figure 6

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

Figure 7
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 , overflow 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

Figure 9
shows HPLC traces of products synthesized in adjacent wells and Figure 7 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

Figure 10
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 ) connected 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. Figure 10

Figure 11
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 inert atmosphere.

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

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 (Figure 13 ). Centrifuge can be driven

Figure 13
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 14 ). Software is capable of performing individual steps (Figure 15 ), or the whole process can be programmed (Figure 16 ). Every 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

Figure 14
well content. After stopping the rotation, microtiterplate is placed (rotor is turned) under the multichannel (96 channel) liquid delivery

Figure 15
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

Figure 16
is repeated as many times as many washes are required. The plates are

Figure 17
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

Figure 19
used in the synthesis of 768 tetrahydroisoquinolinones, and Figure 19 shows HPLC traces of all

Figure 18
products from one microtiterplate. In all cases the main peak corresponded to the expected product. Peaks marked by the dot contain diastereomeric molecule.

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 21. Figure 20 shows the results from the synthesis of tetrapeptides containing arginine. 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.

Figure 20

Figure 21


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.


  1. Leblova Z, Lebl M. Compilation of papers in molecular diversity field. 2001. INTERNET World Wide Web address: http://www.5z.com/divinfo/.
  2. Merrifield RB. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J.Amer.Chem.Soc. 1963;85:2149-54.
  3. Cargill JF, Lebl M. New methods in combinatorial chemistry: Robotics and parallel synthesis. Curr.Opin.Chem.Biol. 1997;1(1):67-71.
  4. 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.
  5. 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.

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Copyright (c) 1999 Spyder Instruments, Inc.