Sonication to purify solid product- the crude product just disappeared

I am forming Fmoc-valine-citrulline dipeptide, and below is the literature protocol I am following (from Bioconjugate Chem 2002, 13 (4), 855-869):

Formation of Fmoc-Val-Cit

Fmoc-Val-OSuc (succinimidyl ester) ($$\pu{14.91 mmol}$$) in DME ($$\pu{40 mL}$$) was added to a solution of Cit ($$\pu{2.743 g}$$, $$\pu{1.05 equiv.}$$) and NaHCO3 ($$\pu{1.315 g}$$, $$\pu{1.05 equiv.}$$) in water ($$\pu{40 mL}$$). THF ($$\pu{20 mL}$$) was added to aid solubility, and the mixture was stirred at room temperature for 16 h. Aqueous citric acid (15%, $$\pu{75 mL}$$) was added, and the mixture was extracted with 10% 2-propanol/ethyl acetate ($$\pu{2 \times 100 mL}$$). The solid product began to precipitate but remained in the organic layer. The suspension was washed with water ($$\pu{2 \times 150 mL}$$), and the solvents were evaporated. The resulting white solid was dried in vacuo for 5 h and then treated with ether ($$\pu{80 mL}$$). After brief sonication and trituration, the white solid product was collected by filtration

I confirmed the formation of the crude product through HRMS and NMR. I then proceeded to carry out sonication to purify. I thought the purpose of sonication was to initiate crystallization. However, when I sonicated the crude product (~20 mins), I observed that the product was well-dissolved in ether, rather than forming a crystal. I used a Büchner funnel to collect the product but there was nothing left on the filter paper. Would pouring more slowly over the filter paper solve the problem? And more importantly, I want to understand the purpose of sonication step.

• Did you do the trituration? Nov 28 '20 at 7:29
• @Waylander No...do you think trituration is critical? Nov 28 '20 at 8:17
• @chemrese I think so. The product is clearly soluble in neat ether, so trituration with something less polar (hexane?) will be needed to crystallise it. Nov 28 '20 at 9:32
• I would also note that the procedure calls for "brief" sonication, 20 mins is way too much Nov 28 '20 at 10:17
• I think you got it: the most likely hypothesis from what is described here is that the solid you treated with ether was not soluble in ether (it's not even very soluble in EtOAc!), but it still contained too much iPrOH (and maybe water?), and that caused its dissolution. I mean, it would be silly to advise to triturate and sonicate a product in a whopping 80 mL ether if it were so soluble in it. You would not normally dry a solid for 5 h before trituration, so drying was meant to remove a solvent; one must check that it has actually happened, before continuing. Nov 28 '20 at 18:14

2 Answers

So I think we have established the problem (thank you @user6376297) . The material still contained enough iPrOH to cause it to dissolve in ether rather than give a filtrable solid because it was not adequately dried. What you need to do with your ethereal solution of product is remove the solvent under reduced pressure then dry the resulting solid under vacuum for at least 5hrs and preferably overnight. Then briefly (~1 min) sonicate the dried solid with ether and collect by filtration.

There is a lesson here: if you have a literature procedure that delivers your desired product in good yield then you follow it - without shortcuts.

• I think the problem arose also because I ignored the scale of work-up solvents. I did 0.2 mmol scale to test out the reaction, but didn't properly calculate the amount of work-up solvents needed relative to the scale I was using. I only cared about the reaction solvent amount (DME, THF..). I realised I shouldn't have done like that Nov 29 '20 at 1:49
• I did hard vac it overnight, actually, but maybe there was too much of the IPA and water. Nov 29 '20 at 11:24
• I followed everything in the second time but still, the product all disappeared... maybe my hand is just bad. Dec 3 '20 at 14:42
• Then your product is in the ether solution. Concentrate that down, add toluene and vac that off whic hwill remove residual iPrOH. You should get solid product at that point that you can recrystallise. Dec 3 '20 at 15:57

There is some confusion on the purpose of sonication here, which is understandable, as to quote, for example, from Wikipedia on Sonication:

Sonication is the act of applying sound energy to agitate particles in a sample, for various purposes such as the extraction of multiple compounds from plants, microalgae and seaweeds.[1]

which relates to physical separation techniques. However, a key further comment:

Sonication has numerous effects, both chemical and physical. The chemical effects of ultrasound are concerned with understanding the effect of sonic waves on chemical systems, this is called sonochemistry.

Related to this question, assuming possible sonochemistry (also referred to as sonolysis) occurring, to quote a source: Free radical formation from sonolysis of water in the presence of different gases:

Free radical formation by sonolysis of water has been considered to be generated by cavitation especially when ultrasound was irradiated with low frequency (from a few dozens to several hundred kHz).(8–10) Cavitation is a phenomenon which refers to the formation, growth, and collapse of small bubbles formed in liquids. During the process of cavitation, the extremely high temperature (several thousand degrees K) and high pressure (hundreds of atmospheres) of imploding cavitation bubbles lead to the thermal dissociation of water molecule, which in turn results in generation of •OH and •H.(11–13)

And importantly, further comments:

Only •OH was detected in the O2-dissolved water sample, and the amount of the radical was much greater than that determined in any of other gas-dissolved water samples. One of the possible reasons to explain why the •H radical was not detected in the O2-dissolved water is that the •H reacts with O2 to form •OOH. In the H2-dissolved water, only •H was detected, suggesting that H2 reduces or neutralizes •OH. In the N2-dissolved water, both •OH and •H were detected at comparable level. In the water samples dissolved with rare gases (Ar, Ne, and He), the amount of •H was almost double as compared with that of •OH, and both •OH and •H yields increased in the order Ar > Ne > He.

The above implies that application of a sonication treatment other than 'brief' in the presence of water could have crossed a boundary into sonochemistry.

However, as to what particular impact a radical presence could have produced, it is not entirely clear, as product chemistry varies distinctly, for example, between the •OH and •H radical.

However, expecting some possible active radical chemistry products also is likely reasonable with a '20-minute' session.

• Here US seems to replace some trituration. It has to do with removing impurities, it is basically trituration. This is what I get from the posted procedure. Anyway informative... Nov 29 '20 at 9:55