Combinatorial Chemistry & High Throughput Screening, 2005, 8, 499-512 499 1386-2073/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd. High Throughput Sonication: Evaluation for Compound Solubilization Kevin Oldenburg 1 , Douglas Pooler 1 , Kurt Scudder 1 , Christopher Lipinski 2 and Michele Kelly 2, * 1 MatriCal, Inc., Spokane, WA, 2 Pfizer Global Research and Development, Groton, CT, USA Abstract: Dissolution of organic compounds in DMSO in HTS plate or tube format is a difficult problem as users move to higher compression plate formats. Precipitation of compounds from DMSO screening stocks is a recognized problem in the HTS materials management process. The adverse effect of freeze thaw cycles on DMSO stock solutions stored in plate format as a result of cherry picking operations has led to the gradual replacement of plate-based storage with tube-based storage so as to minimize the number of freeze thaw cycles. Compound solubility in DMSO is markedly decreased by uptake of small quantities of water. We attribute this effect to the non ideal properties of DMSO water mixtures such that cavity formation in solvent, a necessary step in dissolution, is more difficult in wet DMSO than in dry DMSO or in pure water. We report here that efficient compound dissolution is possible even in 384 well format by the use of in-well plate-based sonication. Surprisingly, compounds precipitated from DMSO stocks either by water uptake or repeated freeze thaw cycles can be re-dissolved by low energy sonication. Finally, we demonstrate that precipitation of compound from DMSO stock solutions is synergistically enhanced by water uptake into DMSO compound stock solutions as well as by increasing the number of freeze thaw cycles. Keywords: Sonication, compound solubility, freeze-thaw cycle, water in DMSO, precipitation, solubilization. INTRODUCTION Sonication has been used in basic science research for over 40 years for such things as degassing of solutions [1], lysing of mammalian [2], fungal [3], or bacterial cells [4], shearing of DNA [5-6], and sonochemistry [7]. Sonication has not been used in the high throughput arenas of pharmaceutical chemistry and screening due primarily to the fact that previously, sonicators could not accommodate the number of samples that needed to be prepared at one time. Anecdotally, a diverse compound collection may have up to 20% of the compounds precipitated or degraded to at least some extent. The proportion of insoluble compounds could potentially be higher within a closely related chemical library or structural class. As a consequence, when a high throughput screening (HTS) campaign is performed against a compound collection, a large number of samples are either not in solution to the extent expected or are not in solution at all, resulting in an inordinate number of “false negative” results. In a worst-case scenario, the loss of this data can lead to misinterpretation of the screening results and consequently, directing chemistry down the wrong path or missing potential lead compounds. More recently, systematic investigations have been undertaken to better understand the extent of this problem [8-10]. Compound solubility is governed by two basic laws: kinetics and thermodynamics. Sonication can address one of these, kinetics, but is not likely to affect the other, thermodynamics. If a compound is soluble at a given concentration, then sonication can be used to accelerate that compounds solubility. That is, if the compound would “normally” take hours or days to dissolve, sonication can be used to drive that compound into solution in a matter of *Address correspondence to this author at the Pfizer Global R&D; Groton, CT 06340, USA; Tel: (860) 715-5125; Fax: (860) 715-9684; E-mail: michele.a.kelly@pfizer.com seconds. However, sonication, in most cases, can do nothing about the thermodynamic portion of the equation. That is, if a compound is only soluble to a concentration of 10 mM in its lowest energy crystalline form, then sonication cannot be used to drive it into solution at a higher concentration. It will, however, insure that the compound is driven into solution to the maximum extent allowed by thermodynamics. The caveat to this statement is that because sonication inputs energy into the sample, it may, in a local environment convert a thermodynamically more stable less soluble crystal form into a higher energy more soluble form, perhaps even to an amorphous form. This is one possible reason why sonication is effective in redissolving precipitated materials. Conversely, high energy sonication has been applied to induce crystallization of supersaturated conditions [11-12]. In this paper, the degree of compound precipitation is measured as a function of multiple freeze thaw cycles and of water contamination present in a sample. 16 commercial compounds chosen specifically to be representative of the chemistry found in typical drugs were tested along with a small Pfizer compound subset. Compound precipitation was induced through a combination of water contamination to the DMSO (dimethyl sulfoxide) dissolving the samples and by multiple freeze thaw cycles. The ability of a multi-well high throughput sonication system was then tested to determine its ability to drive these precipitated compounds back into solution. Optimal sonication conditions were determined along with heating profiles during sonication, possible contamination of samples with metal ions from the sonication device, and effects of sonication on compound stability were determined. In general, sonication was capable of driving many of the precipitated compounds back into solution and had no effect on compound stability. In addition, compounds that initially went into solution with difficulty (>5 hours