The Breaking Beads Approach for Photocleavage from Solid Support
Photocleavage from polystyrene beads is a pivotal reaction for solid phase synthesis that relies on photolabile linkers. The photocleavage, usually performed by batch irradiation, suffers from incomplete and slow cleavage. To overcome these issues, continuous flow and high-energy lamps are frequently used, but these setups are hazardous, technically challenging, and expensive. We developed a photocleavage approach that relies on a benchtop LED lamp and magnetic stirring. In this approach, we crush the beads instead of keeping their integrity to increase the yield of photocleavage. This approach proved very efficient for photocleavage of protected oligosaccharides.
Introduction<!>Results and Discussion<!>Time-dependent photocleavage efficiency.<!>Conclusions<!>Conflicts of interest
<p>Photolabile groups provide an additional level of orthogonality to other common protecting groups. [1][2][3] They are used as handles to connect a solid support to a synthesized molecule in solid phase synthesis (SPS). 4 These linkers are very attractive for SPS because they are stable to many reaction conditions and can be liberated under very mild conditions. [5][6][7][8][9][10][11][12][13][14][15][16] Photolabile linkers are especially valuable for oligosaccharides and glycoconjugates synthesis because they proved to be orthogonal and stable to the multiple synthetic manipulations required. [17][18][19][20][21][22][23] Photochemical reactions on solid support are not as straightforward as in solution. Polystyrene beads are insoluble, not transparent and porous. The efficiency of irradiation depends on the ability of the light not only to cover the entire surface of the beads but also to penetrate into the pores that contain the synthesized molecules anchored to photolabile linkers. Therefore, batch irradiation of solid support tends to suffer from heterogeneity problems, and photocleavage is inefficient. 24 Irradiation by mercury lamp in continuous flow reactors provides more efficient photocleavage because the beads are exposed to the light for a longer time and from a close distance. [25][26][27] Flow-based irradiation reactor is a technical improvement that contributed dramatically to expedite automated synthesis of even extremely large oligosaccharides and made photolabile linkers the preferred choice for these transformations. [28][29][30] The major drawbacks of continuous flow irradiation reactors are that they are expensive, they rely on the use of a hazardous and difficult to handle mercury lamp, and they cannot be paused during irradiation to check the progress. 6,[14][15][16] It is also difficult to reach high cleavage efficiency probably because light cannot penetrate to the inner part of the beads. 24 As a common solid phase practice, the integrity of the beads is preserved in these photocleavage processes. 31 We figured that since the photocleavage is the last step performed on the solid support, the integrity of the beads is not important anymore. A recent report suggested that after stirring polystyrene beads with a magnetic bar, the crushed beads are much smaller than the original mesh size but are still big enough to be separated from the cleaved product by a simple filtration of the solution. 32,33 We thought that crushing the beads during the irradiation will increase the surface area exposed to light and might improve cleavage efficiency while it will not hamper the simple filtration separation process. Herein, we present a simple benchtop light-emitting diode (LED) batch system for photocleavage from solid support. The system takes advantage of magnetic stirring to crush the beads during irradiation in order to expose more surface area to the light source. We evaluated the effect of stirring and the size of the beads on the irradiation efficiency and on the ability to cleave protected oligosaccharides.</p><!><p>Preparation and labeling of photolabile linkers.</p><p>Two different photocleavage handles 1 and 2 were prepared in a solution following previously reported procedure. 20,25,26 A Merrifield resin was treated with 1 under basic conditions to provide hydroxyl functionalized photolabile linker HP-Linker (Scheme 1). The HP-Linker was reacted with Fmoc-Cl to provide FHP-Linker that can be used for loading quantification and cleavage efficiency evaluation. In our irradiation setup, 25-100 mg of solid support was inserted into a quartz cuvette with a magnetic bar and 2 ml of dichloromethane (DCM) were added. This cuvette was placed on a stirring plate (Figure 1A). A high Power 365 nm LED lamp was positioned in a 2 cm distance from the cuvette and the entire setup was covered prior to commencing irradiation (Figure 1A). After irradiation, the beads were transferred and collected in a fritted filter Sep-Pak. The cleavage efficiency was determined either by Fmoc quantification of the remaining uncleaved product (FHP-Linker) or by fluorescent microscopy (FlAP-Linker) (Figure 1). The DCM filtrate from the initial washing was used to quantify the amount of linker that was cleaved by irradiation as will be demonstrated later.  The fluorescence intensity of the beads decreased as a factor of irradiation time showing that N-Cbz-Nfluorescein-hexanediamine 4 was cleaved from the solid support. After four hours of irradiation, there was still significant fluorescence signal of the beads (Figure 2B). Full cleavage was not observed even after eight hours of irradiation. This proves that without stirring it was not possible to achieve complete removal of the product from the solid support. The images also showed that the integrity of the beads is preserved during irradiation without stirring.</p><p>The effect of stirring rate on photocleavage efficiency.</p><p>Four irradiation experiments using FHP-Linker were performed at increasing stirring rates. Each irradiation was performed for four hours and after each irradiation experiment, the cleavage efficiency was determined using a standard Fmoc quantification analysis (Figure 3). 33,34 This analysis showed that increasing the stirring rate improves cleavage efficiency. Stirring at 1060 rpm for four hours provided a cleavage of over 80%. The integrity of the beads was visualized by microscopy and showed that increasing the stirring rate enhanced the deformation of the beads (Figure 3A).  It is possible that elevated stirring rates improve the exposure of the particles to the UV light that leads to an increase in cleavage efficiency. Another option is that there is a correlation between the shape and size of the beads to the photocleavage efficiency. Smaller beads, which are formed by compressive stress from stirring, have more surface area. The improved cleavage efficiency can be explained by the amount of linker exposed to the light during the irradiation.</p><!><p>We checked what will be the time required to achieve optimal photocleavage. We irradiated FHP-Linker at 1060 rpm for one up to six hours (Figure 4). Cleavage efficiency was determined by Fmoc quantification of the beads after irradiation. The cleavage after six hours reached over 80%. As an orthogonal analysis, the amount of cleaved N-Cbz-O-Fmoc-hexanolamine 3 in the filtrate was quantified by UV spectroscopy. To achieve this, 3 was synthesized in solution and diluted to provide standard solutions at different concentrations. These solutions were used to prepare a calibration curve to correlate between the concentration of 3 and absorbance at 301 nm.</p><p>Measuring the UV absorbance of the filtrate solution after irradiation and fitting it to the calibration curve enabled us to determine the cleavage efficiency by different methods (Figure 4 and SI). There was a clear correlation between the cleavage efficiency determined by the amount of 3 in the filtrate to the one calculated by Fmoc quantification for the beads (Figure 4). The slightly lower cleavage yields calculated for the filtrate are probably because we applied only two DCM washes without shaking and some of the product remains inside the beads. The accurate quantitative method to evaluate the amount of cleaved material in the filtrate relies on the use of known standards and precise analytical protocol. It can be used to evaluate the efficiency of photocleavage setups or photolabile linkers. Considering the ongoing debate regarding the accuracy of Fmoc quantification protocols, 35 we feel that our analytical evaluation of the cleavage is a very good alternative.</p><p>To prove the applicability of the method to larger amount of solid support, we irradiated 100 mg of FHP-Linker for four hours, evaporated the filtrate solvent and took proton and carbon NMR of the 3 that was cleaved off directly and without purification (SI). The NMR analysis showed a spectrum that was very similar to 3 that was synthesized in solution. HRMS and NMR proved that the new cleavage process can provide clean products. This also confirms that the process will afford enough material for biology studies.</p><p>The contribution of crushing the beads prior to photocleavage.</p><p>Four to six hours of irradiation time is relatively short compared with other deprotection strategies (e.g. hydrogenolysis using atmospheric hydrogen for 8-16 h) but is a bit longer compared to other cleavage procedures (e.g. TFA 1-4 h, flow photocleavage). This might limit the number of irradiation experiments that can be performed in a typical workday. We decided to crush the beads prior to the irradiation in order to check the influence of beads size on cleavage efficiency. We stirred FHP-Linker beads for four hours at 1060 rpm without irradiation. The crushed FHP-Linker was then irradiated only for one hour using 1060 rpm stirring. As a control, we irradiated FHP-Linker beads that were not pre-crushed under the same conditions. Fmoc quantification after irradiation of the pre-crushed beads showed that over 80% of the linker was cleaved off while in the control experiment only 40% was cleaved (Figure 5). Microscope analysis after irradiation showed that the pre-crushed contained beads had much smaller particles in comparison with the control. Fluorescence microscopy images of crushed FlAP-Linker beads mesh before and after irradiation further demonstrate the difference that results from the cleavage protocol (SI). Since irradiation time and stirring rate were identical in both experiments, it proved that the increased surface area contributed to the great cleavage efficiency, and not only the stirring rate itself. Achieving over 80% cleavage in only one hour of irradiation suggests that pre-crushing can be used as a routine to expedite the process and allow for multiple experiments in a typical workday.</p><p>Figure 5. Irradiation of FHP-Linker with and without pre-crushing of the beads. This led to increase in cleavage efficiency. Crushing the beads four hours prior to one hour of irradiation significantly increased cleavage efficiency compared to the irradiation of intact beads.</p><p>Synthesis of α-1-6-Mannose disaccharide and its removal from the support using the breaking beads approach.</p><p>The fully protected α-1-6-dimannose 5 was synthesized on the FHP-Linker using automated synthesizer glyconeer 2.1 (Scheme 2). HP linker was placed in the glyconeer 2.1 reaction vessel. The rest of the process was performed using typical automated glycan assembly (AGA) protocols. 19 Glycosylations of Fmoc protected thiomannoside 6 was performed using NIS/TfOH as activators at low temperature after which Fmoc was removed and another glycosylation cycle was performed using the exact conditions as the first one. We intentionally kept the Fmoc on the disaccharide to enable us to determine the efficiency of cleavage using spectroscopic analytical methods. After the second glycosylation, the solid support was removed from the reaction vessel and the quantity of Fmoc was determined. The calculated loading after AGA was 0.392 mmol/g because 30 % of the beads mass were attributed to the protected saccharide. Disaccharide 5 was cleaved off from the same amount of beads using two irradiation methods. Beads were irradiated either for one hour without stirring or while stirring at 1060 rpm after four hours of pre-crushing. After each cleavage, the amount of Fmoc that remains on the solid support was determined. The calculated Fmoc loading after irradiation without stirring was 0.376 mmol/g. This implies that only 4 % of the disaccharide was cleaved. The calculated Fmoc loading determined following irradiation with stirring after pre-crushing the beads was 0.09 mmol/g. This shows that using the new cleavage protocol, 77 % of the disaccharide was removed within one hour of irradiation.</p><p>After irradiation of the disaccharide using the breaking beads protocol, the filtrate was collected and analyzed by HPLC, NMR, and MS of the crude showed a purity of over 95% of the disaccharide. This Disaccharide 5 contains eight aromatic moieties and is a good model for a typical oligosaccharide synthesized on such linkers. 17,29,36 The analysis of the cleavage of disaccharide 5 confirms that the new strategy is applicable also for complex compounds. Our study further highlights the difference between the two cleavage protocols and shows that the advantage observed for simple molecules is also relevant for complex molecules. We also tried to calculate the cleavage yield by mass. There was a dramatic difference between the measures mass after irradiation with stirring and crushing of the beads and without. After irradiation of the crushed beads we had enough material for NMR, HPLC, and MS but this analysis proved to be inaccurate for such low amounts of material to be able to provide quantitative data. Scheme 2: Synthesis of disaccharide by AGA. The disaccharide was synthesized on HP-Linker in glyconeer 2.1 from two monomers by performing two cycles of deprotection coupling. Afterwards, the saccharide cleaved by UV irradiation with and without crushing the beads prior to irradiation. In all our studies, browning of the resin was observed during irradiation. Browning of polystyrene beads is a known phenomenon that probably results from the formation of nitrosoaldehyde during photocleavage. 37 Such moiety, which absorbs UV light, might decrease the efficiency of photocleavage overtime. 37 However, by crushing the beads prior to irradiation we make sure that the surface area is large enough to minimize the effect of browning on photocleavage. The accuracy of our calculated cleavage efficiency using three orthogonal methods proves that the browning does not play a crucial role when our method is used. Although the nature of the Merrifield polystyrene resin itself might also contribute to this phenomenon, replacing it with another solid support might hamper its applicability for peptide and oligosaccharide synthesis. Using smaller beads with higher surface area throughout the synthesis process might be an interesting approach but will probably result in clogging of the filters during solid phase synthetic manipulations.</p><p>As a note, the exact irradiation efficiency might be setup-dependent. Although there might be variability when the cleavage will be performed by similar setup in other labs, the differences between irradiation of beads with and without crushing were reproduced multiple times proving the validity of the observation. It is logical to think that the presence of UV active moieties on peptides or saccharides might decrease the efficiency of irradiation. However, photocleavage of biopolymers have been demonstrated even with up to hundreds of UV active moieties. 28,29 The effect of crushing the beads will not have any negative effect on the efficiency. In contrast, as demonstrated for disaccharide 5, the enhanced exposure will make sure that the process still surpasses the common strategies and might provide an advantage when many UV absorbing groups are present. Many reports in the past failed to reach efficient cleavage yields. Most of these methods rely on very powerful mercury lamp setups which also have brood irradiation wavelengths. 6,[14][15][16] In order to control the irradiation, many of these strategies apply filters which might also absorb much of the light. 19,25 Unlike other protocols, we rely on a LED lamp with a specific wavelength and do not use any filters. This might result in the improved cleavage observed in this study.</p><!><p>In summary, a new approach to cleave compounds from photolabile linkers on solid support was described. A simple combination of a benchtop LED irradiation and magnetic stirring provides efficient photocleavage. By using a number of complementary analytical techniques, we showed that increasing the surface area of the polystyrene beads by crushing them expedite photocleavage. The strategy was demonstrated for a fully protected oligosaccharide model, which proves that it is straightforward, applicable for bio relevant targets and can be done in any standard laboratory. This can make the use of photolabile linkers more accessible to the community. The method can be very valuable for cleaving complex molecules from photolabile linkers in high efficiency.</p><!><p>There are no conflicts to declare.</p>