开题报告内容：（包括拟研究或解决的问题、采用的研究手段及文献综述，不少于2000字）
Part 1. Introduction on the project
Cystinuria is an autosomal recessive disorder characterized by a defect in the reabsorption of cystine from the renal proximal tubule, resulting in an accumulation of L-cystine crystals in urine. One possible approach to treat this disorder is to inhibit the crystallization of L-cystine. L-cystine diamides have previously been synthesized in our lab and were found to be effective in inhibiting L-cystine crystal growth. One of these inhibitors, R-S-S-R (“R”: a certain group containing alkyl group, and/or aromatic group, and/or heterocycle ring, etc.; “-S-S-”: disulfide bond) was found to suppress kidney stone formation in a genetic mouse model of cystinuria. An analog of R-S-S-R was designed through applying bioisosterism to further explore the structure-activity relationship of the L-cystine crystallization inhibitors.
Based on several related pharmacological research and reasonable speculation, L-cystine monoamides with mixed disulfide bond formed could have better activity compared with L-cystine diamides. One in particular, known as R-S-Cys with disulfide bond formed from R-SH and L-cysteine (“R”: a certain group containing alkyl group, and/or aromatic group, and/or heterocycle ring, etc.; “cys”: L-cysteine), could be potent for treating cystinuria. We are expected to work on the efficient route of synthesis and the simplest way of purification to achieve pure desired product of R-S-Cys. In the mean time we will have to take everything into consideration, including green chemistry, specific reaction condition and good yield in each step. However, as to properties our desired product and undesirable byproduct are supposed to be very polar and without UV activity, which will of course bring great challenges on purification after reaction. We cannot use TLC plate to monitor the reaction and separation when the certain compound has no UV activity, pushing us to find a way of quantitative monitoring. And it is always hard to separate two compounds that both are well dissolved in water and not in organic solvents.
Part 2. Methods that may be used for conducting this project
2.1 About reaction: Considering that disulfide bond is often formed under mild condition, we need to design the appropriate catalytic condition and introduce no metal or metal ions owing to thiol groups. We also need to adjust pH value of reaction solution, because on the one hand amide bond will break if too acidic or basic, and on the other hand reaction may not be complete if not enough.
2.2 How to monitor: We can utilize what apparatus Hu Lab. has for monitoring the reaction, mainly by LC-MS (Liquid Chromatograph Mass Spectrometer). We can know that the reaction is almost or completely finished when we can barely see any signal of starting materials or no signal at all from LC-MS. However, LC-MS has its own disadvantages and limits. For instance, the signal intensity of the compound only means the number of ions ionized, which not exactly has anything to do with the amount of compound. Therefore, we fail to know how much product we can get after reaction by LC-MS. But based on thiol group, we want to use bioassay reagent for quantitative monitoring. By calculating the thiol groups not reacted, we can have a better idea if the reaction is completed or halfway. Following common protocols from analytical chemistry, we can make standard curve (UV intensity to concentration), see if it is linear and evaluate the method simply by parameters like R2. Then if each part of method is good, we can apply immediately. After looking up some literatures we find DTNB, also known as Ellmanrsquo;s Reagent feasible to quantitatively detect the amount of thiol groups.
2.3 About purification: Our byproduct will mostly be L-cystine as predicted, which means there is no distinct difference between R-S-Cys, our desired product and L-cystine. They both have great polarity, good water solubility and no UV activity. Then traditional normal-phase/reversed-phase chromatography cannot be applied to this project for purification for two reasons----one is that UV activity is indispensable to systems like ISCO; the other is that they could all get attached to the column when using normal-phase chromatography. However, we are basically trying to separate two amino acids, so it occurs to us that ion exchange chromatography might be a solution to this problem. We need to have a better understanding towards the charge of amino acids to make this method work. It is important to calculate the pH of mobile phase condition and pI (isoelectric point) of our mixture. The complete approach using ion exchange resin includes: a. what kind of resin to perform the purification, strong or weak and anion or cation resin; b. how to treat the resin properly before we load them on column; c. parameters that we need to find, analyze and use from instruction of the certain type of resin; d. how to load resin right and how much resin, how big the column should be; e. decide the appropriate exchange ion, remember that too much hydroxide ions could break amide bond and if salt added is not volatile it will then become a tricky problem where we try to separate salt from product; f. overall conditions such as flow rate, gradient, collecting volume and times, how to detect, and the sequence about coming out, etc.
Part 3. A Short Review on cystinuria and inhibitors.
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