DEVICE FOR AUTOMATED SYNTHESIS OF OLIGO- AND POLYSACCHARIDES

Abstract

The present invention generally relates to automated synthesis technology, and more particularly, to a device and method for automated synthesis of oligo- and polysaccharides on a solid support. In particular the present invention relates to a device for automated synthesis of oligo- and polysaccharides on a solid support comprising a reaction vessel, a reagent storing component, a reagent delivery system, a cooling device for cooling reaction vessel, and a pre-cooling device for pre-cooling the reagents to be supplied.

Claims

1. A device for automated synthesis of oligo- and polysaccharides on a solid support, the device comprising: (a) a reaction vessel; (b) a reagent storing component; (c) a reagent delivery system; (d) a cooling device for cooling the reaction vessel, and (e) a pre-cooling device for pre-cooling the reagents to be supplied; wherein the reagent delivery system connects the reagent storing component with the reaction vessel; wherein the pre-cooling device is interposed between the reaction vessel and the reagent delivery system; and wherein the pre-cooling device is in thermal communication with the reagent delivery system.

2. The device according to claim 1 further comprising a computing device comprising at least one processor configured to control one or more components of the device.

3. The device according to claim 1, the device being adapted for reactions under anhydrous and inert atmosphere.

4. The device according to claim 1, further comprising a microwave generator component having a chamber in which the reaction vessel is located, and wherein the reaction vessel is microwave transparent.

5. The device according to claim 1, wherein the pre-cooling device is configured to cool the reagents to be supplied to the reaction vessel a temperature which is not higher than 3° C. below the temperature of the reaction mixture in the reaction vessel.

6. The device according to claim 1, wherein the pre-cooling device is configured to cool the reagents to be supplied to the reaction vessel to a temperature in the range of −40° C. to −9° C.

7. The device according to claim 1, wherein the cooling device comprises a cooling jacket.

8. The device according to claim 1, further comprising an inert gas delivery system.

9. The device according to claim 1, wherein the pre-cooling device is upstream to the cooling device.

10. The device according to claim 1, wherein the reaction vessel is interchangeable.

11. The device according to claim 1, wherein the reaction vessel is made of a fluoropolymer such as perfluoroalkoxy alkanes (PFA) or glass.

12. The device according to claim 1, wherein the reaction vessel comprises one or more inlets at the top of the reaction vessel and one or more inlets at the bottom of the reaction vessel.

13. The device according to a claim 1, wherein the reagent delivery system connects the reagent storing component with the reaction vessel via the pre-cooling device.

14. The device according to claim 1, wherein the pre-cooling device is a part of the cooling device using the same cooling circuit together.

15. The device according to claim 1, the reagent delivery system being in fluid communication with the reaction vessel through one or more reagent delivery lines; and the pre-cooling device being in thermal communication with the one or more reagent delivery lines.

16. The device according to claim 1, wherein the reaction vessel, the pre-cooling device, the reagent delivery system and the reagent storing component are successively connected in the following sequence: reagent storing component—reagent delivery system—pre-cooling device—reaction vessel.

17. The device according to claim 1, the reagent delivery system being in fluid communication with the reagent storing component and being further in fluid communication with the reaction vessel.

18. The device according to claim 1, wherein the reagent storing component and the pre-cooling device are connected through the reagent delivery system.

19. The device according to claim 1, wherein at least one liquid line between the reagent delivery system and the reaction vessel is pre-cooled by the pre-cooling device located between the reagent delivery system and the reaction vessel.

20. The device according to claim 1, wherein the pre-cooling device is a thermoelectric cooler.

21. The device according to claim 1, wherein the pre-cooling device is positioned between the reaction vessel and the reagent delivery system so that the reagents along their way from the pre-cooling device to the reaction vessel do not increase their temperature for more than 0.5° C.

22. The device according to claim 1, wherein the pre-cooling device is positioned downstream to the reagent delivery system and upstream to reaction vessel.

23. A method for synthesizing oligo- and polysaccharides with a device according to claim 1, the method comprising the following steps: a) providing a solid support with at least one immobilized saccharide in a reaction vessel; b) adding a further saccharide bearing at least one protecting group and a glycosylation reagent to the reaction vessel containing the solid support in order to initiate a coupling reaction of the further saccharide to the saccharide immobilized on the solid support; c) performing removal of the at least one protecting group of the further saccharide; wherein in step b) at least the glycosylation reagent is pre-cooled to a temperature of at least 40° C. to −9° C. by a pre-cooling device during delivery to and before addition to the reaction vessel.

24. The method according to claim 23, wherein in step b) the glycosylation reagent and the further saccharide are pre-cooled to a temperature of at least 40° C. to −9° C. by the pre-cooling device during delivery to and before addition to the reaction vessel.

25. The method according to claim 23, wherein in step a) the reaction vessel is cooled to a temperature of at least 40° C. to −9° C. by a cooling device.

Description

DESCRIPTION OF THE FIGURES

[1110] FIG. 1A is a block diagram illustrating the inventive device for automated oligo- and polysaccharide synthesis on solid support.

[1111] FIG. 1B shows a diagram of an example of the device comprising two reagent delivery systems (600, 700) in accordance with one or more aspects of the invention.

[1112] FIG. 1C shows a diagram of the device according to the invention further comprising a microwave generator component (500).

[1113] FIG. 2 is a flow diagram illustrating components of FIG. 1A-C in more details, and more particularly, the inert gas and liquid distribution, in accordance with one or more aspects of the invention.

[1114] FIG. 3A is a conceptual diagram illustrating a side view of an example implementation of the pre-cooling device (300), in accordance with one or more aspects of the invention;

[1115] FIG. 3B shows a compact embodiment of the pre-cooling device (300) and cooling device (350) comprising a cooling jacket (307) forming together a single component of the inventive device;

[1116] FIG. 3C shows a compact embodiment of the pre-cooling device (300) and cooling device (350) comprising a cooling coil (307) forming together a single component of the inventive device.

[1117] FIG. 4A is a first conceptual diagram illustrating a cross-section view of an example implementation of a reaction vessel, in accordance with one or more aspects of the invention;

[1118] FIG. 4B is a second conceptual diagram illustrating a cross-section view of an example implementation of a reaction vessel, in accordance with one or more aspects of the invention.

[1119] FIG. 5 is a conceptual diagram illustrating an example implementation of the liquid top delivery system, in accordance with one or more aspects of the invention.

[1120] FIG. 6 is a conceptual diagram illustrating an example implementation of the liquid bottom delivery system, in accordance with one or more aspects of the invention.

[1121] FIG. 7 is a conceptual diagram illustrating an example implementation of the inert gas delivery system, in accordance with one or more aspects of the invention.

[1122] FIG. 8 is a block diagram illustrating a process of controlling components of the device through a computer program, in accordance with one or more aspects of the invention.

[1123] FIG. 9 shows the thermal profile during the synthesis of a mannose-α-1,6-tetramer “standard test” in the presence and absence of pre-cooling. The reaction vessel is made of glass, using thioglycoside donor as building block, and active cooling is ON in both experiments. By using a precooling device, the thermal perturbation due to the delivery of reagents is suppressed (solid line), specifically the delivery of activator reagents and donor solution. The washing solvent was not pre-cooled to highlight the stark contrast in the thermal spike.

[1124] FIG. 10 shows the analytical results of the experiments described in FIG. 9 and mass spectrometry (MALDI) and HPLC chromatograms.

[1125] FIG. 11 shows the thermal profile during the synthesis of a mannose-α-1,6-tetramer “standard test” in the presence and absence of pre-cooling. The reaction vessel is made of glass, using glycosyl phosphate donor as building block, and active cooling is ON in both experiments. By using a pre-cooling device, the thermal perturbation due to the delivery of reagents is suppressed (solid line), specifically the delivery of activator reagents and donor solution. The washing solvent was not pre-cooled to highlight the stark contrast in the thermal spike.

[1126] FIG. 12 shows the analytical results of the experiments described in FIG. 11 and mass spectrometry (MALDI) and HPLC chromatogram. The reaction without the pre-cooling shows a HPLC chromatogram with more side product traces (peaks at 2.3 min and 3.6 min).

[1127] FIG. 13 shows the thermal profile during the synthesis of a mannose-α-1,6-tetramer “standard test” in the presence and absence of precooling. The reaction vessel is made of PFA, using glycosyl phosphate donor as building block, and active cooling is OFF in both experiments. By using a pre-cooling device, there is a brief drop in temperature when the pre-cooled reagents/building blocks are delivered (solid line). In both cases the temperature oscillates around 20° C.

[1128] FIG. 14 shows the analytical results of the experiments described in FIG. 13 and HPLC chromatograms. In both experiments no desired product was observed. Only side products are visible in HPLC chromatogram (peaks at 2.3 min and 3.4 min).

[1129] FIG. 15 shows the temperature differential (maximum temperate reached minus the actual reactor temperature) within a glass reaction vessel when 1 mL of dichloromethane (DCM) is delivered at different flow rates to a reactor vessel at −18° C. The white circle series refers to the solvent delivered without precooling; the black triangle series refers to precooled solvent. By using the precooling, the reagent can be delivered 3 times faster than the current state of the art with a thermal perturbation lesser than ±3° C. (highlighted in box).

[1130] FIG. 16 shows the thermal perturbation in the reaction vessel when different volumes of Dichloromethane are delivered at 1.05 mL/min in a reaction vessel at −18° C. in the presence or absence of pre-cooling. The solid line refers to the thermal perturbation with precooling. The dash line refers to the thermal perturbation without pre-cooling. The pre-cooling reduces the thermal perturbation even when 4 mL are delivered which is 4 times the current scale of the current state of the art synthesizer.

[1131] FIG. 17 depicts certain commonly used Merrifield resins 1101, 1102, 1103, 1104, 1105 functionalized with organic linkers, which were developed for the automated synthesis of oligo- and polysaccharides on a solid support.

[1132] FIG. 18 shows a set of common building blocks (glycosyl donors) used for automated synthesis in the inventive device.

[1133] FIG. 19A shows temperature charts comparing fast coupling (10 min, example 5) and regular coupling (25 min, example 4). The temperature inside the reaction vessel/reaction mixture was taken during a synthesis cycle via phosphate glycosylation; with constant cooling and temperature adjustment during the coupling and post coupling reactions via microwave irradiation. Each chart shows the sequence of four chemical steps: Acidic washing, coupling/glycosylation, capping and deprotection. The fast coupling allows a complete cycle in less than 45 min, while regular coupling take above 1 h.

[1134] FIG. 19B shows analytical results by MALDI and HPLC of experiments from fast coupling synthesis example 5.

[1135] FIG. 20A shows temperature charts of the first cycle during the synthesis of 5-amino-pentyl α-(1.fwdarw.2)-D-tetramannopyranoside (example 6). The solid line follows the temperature inside the reaction. The dash line shows the temperature at the cooling jacket side. The grey areas highlight the microwave irradiation periods allowing the adjustment of the temperature inside the reaction vessel. The NAP deprotection period is indicated.

[1136] FIG. 20B shows analytical results by MALDI (the mass of the product is mark plus sodium ion) and HPLC of experiments from synthesis EXAMPLE 6.

[1137] FIG. 21A shows temperature charts of the first cycle during the synthesis of 5-amino-pentyl α-(1.fwdarw.3)-D-tetramannopyranoside (Example 7). The solid line follows the temperature inside the reaction. The dash line shows the temperature at the cooling jacket side. The grey areas highlight the microwave irradiation periods allowing the adjustment of the temperature inside the reaction vessel. The Lev deprotection period is indicated.

[1138] FIG. 21B shows analytical results by MALDI (the mass of the product is mark plus sodium ion) and HPLC of experiments from synthesis Example 7.

[1139] FIG. 22A shows temperature charts of the first cycle during the synthesis of 5-Amino-pentyl α-(1.fwdarw.4)-D-tetramannopyranoside (Example 8). The solid line follows the temperature inside the reaction. The dash line shows the temperature at the cooling jacket side. The grey areas highlight the microwave irradiation periods allowing the adjustment of the temperature inside the reaction vessel. The Fmoc deprotection period is indicated.

[1140] FIG. 22B shows analytical results by MALDI (the mass of the product is mark plus sodium ion) and HPLC of experiments from synthesis Example 8.

[1141] FIG. 23A shows temperature charts of the first cycle during the synthesis of 5-Amino-pentyl α-(1.fwdarw.6)-D-tetramannopyranoside (Example 9). The solid line follows the temperature inside the reaction. The dash line shows the temperature at the cooling jacket side. The grey areas highlight the microwave irradiation periods allowing the adjustment of the temperature inside the reaction vessel. The chloroacetyl deprotection period is indicated.

[1142] FIG. 23B shows analytical results by MALDI (the mass of the product is mark plus sodium ion) and HPLC of experiments from synthesis Example 9.

[1143] FIG. 24A shows temperature charts of the first cycle during the synthesis of α-(1-.fwdarw.3)-α-(1-.fwdarw.4)-α-(1.fwdarw.6)-D-mannopyranoside (Example 10). The solid line follows the temperature inside the reaction. The dash line shows the temperature at the cooling jacket side. The grey areas highlight the microwave irradiation periods allowing the adjustment of the temperature inside the reaction vessel. The coupling and deprotection periods are indicated.

[1144] FIG. 24B shows analytical results by MALDI (the mass of the product is mark plus sodium ion) and HPLC of experiments from synthesis Example 10.

[1145] FIG. 25A shows temperature charts of the first cycle during the synthesis of 1-6 mannose tetramer (Example 11) without the acidic wash step between the deprotection step and the next glycosylation. The solid line follows the temperature inside the reaction. The dash line shows the temperature at the cooling jacket side. The grey areas highlight the microwave irradiation periods allowing the adjustment of the temperature inside the reaction vessel. The Fmoc deprotection period is indicated.

[1146] FIG. 25B shows analytical results by MALDI from Example 11. No product was observed.

[1147] FIG. 26A shows temperature chart during the synthesis of protected Lewis antigen tetramer (Example 12). The solid line follows the temperature inside the reaction. The dash line shows the temperature at the cooling jacket side. The grey areas highlight the microwave irradiation periods allowing the adjustment of the temperature inside the reaction vessel. The coupling and deprotection steps are indicated. The reaction was completed in 6 h and 30 min

[1148] FIG. 26B shows analytical results by MALDI (the mass of the product is mark plus sodium ion) and HPLC of experiments from synthesis Example 12.

[1149] FIG. 27 compares the thermal profile during the synthesis of a Lewis antigen fragment Example 13 in the presence and absence of pre-cooling. Active cooling for the reaction vessel is provided identically. By using a pre-cooling element (ON), the thermal perturbation due to the delivery of reagents is suppressed (solid line), specifically the delivery of activator reagents and donor solution. The washing solvent was pre-cooled. In dash line the temperature profile inside of the reaction vessel when the precooling was turned OFF.

[1150] FIG. 28A the analytical results of the experiments as shown in FIG. 27 are shown. Mass spectrometry (MALDI) and HPLC chromatogram are provided. For the reaction with the precooling device ON.

[1151] FIG. 28A the analytical results of the experiments as shown in FIG. 27 are shown. Mass spectrometry (MALDI) and HPLC chromatogram are provided. For the reaction with the precooling device ON. Mainly the product is observed FIG. 28B the analytical results of the experiments as shown in FIG. 27 are shown. Mass spectrometry (MALDI) and HPLC chromatogram are provided. For the reaction with the precooling device OFF. The HPLC chromatogram shows peaks for the product and significant deletion sequence.

[1152] FIG. 29 shows a schematic drawing of an embodiment of the gas valve manifold of the inventive synthesizer comprising a pressure regulator valve, a pressure sensor and a check valve at each output line.

[1153] FIG. 30A shows the technical drawing of the manifold first (mounting) layer.

[1154] FIG. 30B shows the technical drawing of the manifold second (substrate) layer.

[1155] FIG. 30C shows the technical drawing of the manifold third (side element) layer.

[1156] FIG. 31 is a block diagram illustrating the inventive device for automated oligo- and polysaccharide synthesis on solid support further comprising a thermal controller (900).

[1157] FIG. 32 is a further block diagram illustrating the inventive device for automated oligo- and polysaccharide synthesis on solid support.

ABBREVIATIONS

[1158] Ac.sub.2O=Acetic anhydride [1159] CIAc=Chloroacetyl [1160] DBU=1,8-Diazabicyclo[5.4.0]undec-7-ene [1161] DDQ=2,3-Dichloro-5,6-dicyano-1,4-benzoquinone [1162] EGME=2-Methoxyethanol [1163] Fmoc=Fluorenylmethyloxycarbonyl [1164] HFIP=Hexafluoroisopropanol [1165] Lev=Levulinic ester [1166] MsOH=Methanesulfonic acid [1167] NAP=2-Naphthylmethyl ether [1168] NDM=1-Dodecanethiol [1169] TIS=Triisopropylsilane [1170] DCM=Dichloromethane [1171] DMF=N-N-Dimethylformamide [1172] DCE=1,2-Dichloroethane [1173] THF=Tetrahydrofuran

LIST OF REFERENCE SIGNS

[1174] 100 device [1175] 200 computing device, processor [1176] 201-203 connection line/communication line [1177] 204 signal processor [1178] 205-213 communication lines [1179] 300 pre-cooling device/pre-cooling component [1180] 303 metal cooling surface [1181] 304 coolant fluid reservoir/coolant reservoir [1182] 305 Peltier cooler/contact-cooling device [1183] 306 cooling circuit pump [1184] 307 cooling coil/cooling jacket [1185] 308, 309 lines [1186] 310 cooling line [1187] 311 cooling means [1188] 350 cooling device for reaction vessel [1189] 400 reaction vessel [1190] 401 effective loading space [1191] 402 bottom inlet/bottom exit [1192] 403 bottom compartment [1193] 404 frit [1194] 405 temperature sensor/sensor [1195] 406 channel [1196] 407 building block line [1197] 408 activator solution line [1198] 409 cap [1199] 410 isolation cover [1200] 500 microwave generator component/microwave generator [1201] 501 microwave radiation [1202] 502 chamber/microwaves chamber [1203] 600 reagent delivery system, top delivery system [1204] 601 building block delivery line [1205] 602 activator solution delivery line [1206] 603 syringe pump [1207] 605 valve [1208] 606, 607 rotary valve/rotary valve distributor [1209] 608-611 lines [1210] 612-615 containers [1211] 616, 617 lines [1212] 618 splitter [1213] 619-624 lines [1214] 625, 626 loop lines [1215] 627-629 lines [1216] 630-632 containers [1217] 633 splitter [1218] 634-636 lines [1219] 637 multiple ways valve/four way magnetic valve [1220] 638-641 solvent containers [1221] 642-645 delivery lines [1222] 646-649 lines [1223] 650 splitter [1224] 651 washing solvent distribution component/washing solvents component [1225] 652 activator distribution component [1226] 653 building blocks distribution component [1227] 654 cooling means [1228] 655 exhaust gases exit [1229] 656 waste delivery line [1230] 657 single valve [1231] 660, 760 reagent storing component [1232] 661 washing solvent storing component [1233] 662 activator storing component [1234] 663 building block storing component [1235] 700 reagent delivery system, bottom delivery system [1236] 701 loop line/line [1237] 702, 703 lines [1238] 704 waste container/waste collector [1239] 705,706 lines [1240] 707 multi-port valve/multiple ways magnetic valv [1241] 708-711 containers [1242] 712, 713 splitter [1243] 714-718 lines [1244] 719 deprotection distribution component [1245] 720 capping distribution component [1246] 761 deprotection storing component [1247] 762 capping storing component [1248] 800 inert gas delivery system [1249] 801 gas container [1250] 802 manifold [1251] 803-809 seven valves [1252] 810 valve [1253] 811 solvent line [1254] 812 pressure line [1255] 813-815 gas lines [1256] 816 capping line [1257] 817 deprotection line [1258] 818 gas line [1259] 820 tubing connector [1260] 820a-820h—tubing connector [1261] 823-829 seven pressure sensors [1262] 833-839 seven checks or one way valves [1263] 840 manifold channel [1264] 841a-841n mounting feet [1265] 842a-842f substrate channels [1266] 843 substrate channels [1267] 844a-844o substrate connectors [1268] 845a-845g substrate connectors [1269] 846a-846g substrate-to-manifold connector [1270] 847a-847n lockdown bars [1271] 848a-848g pressure regulator [1272] 849a-849g pressure indicators [1273] 850a-850g two-port check valves [1274] 851 two ports metering valve with knurled handle [1275] 852a-852g manifold output lines [1276] 861 first layer of gas valve manifold [1277] 862 second layer of gas valve manifold [1278] 863 third layer of gas valve manifold [1279] 900 thermal controller [1280] 1101-1105 resins EXAMPLES

[1281] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[1282] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

Example 1: Investigation of Microwave-Assisted Capping and Deprotection Steps

[1283] The improvement in conditions and reaction time for the capping and deprotection steps with microwave radiation was investigated. During a typical AGA cycle there is the glycosylation coupling step as well as several auxiliary steps (acetyl capping and temporary group deprotection). These auxiliary steps increase the overall yield of the final oligo- or polysaccharide (glycan) by terminating unglycosylated nucleophiles as well as they remove temporary protecting groups that allows the next coupling to occur. These auxiliary steps have been a bottleneck in the overall time required for one AGA cycle. The investigation of the overall time required for one AGA cycle by using microwave radiation during these auxiliary steps has shown that microwave assisted deprotection and capping steps drastically reduce the reaction time. The results shown in Table 1 demonstrate that the utilization of a microwave generator component is not only instrumental for hastening the cooling to heating process, microwave-assisted synthesis also drastically reduces chemical reaction time. Under these rapid microwave-assisted conditions, the steps remained orthogonal and few side reactions were observed. With shortened reaction times for these auxiliary steps the overall duration of a standard AGA cycle was successfully reduced from 100 minutes to below 60 minutes and even to 45 minutes. First results have also shown that the glycosylation reaction benefits from the use of microwave radiation.

TABLE-US-00001 TABLE 1 Conditions and time for performing capping and deprotection steps without microwave radiation and with microwave radiation. Without Microwave With Microwave Radiation Module Conditions Time Conditions Time Capping 2% MsOH in  25 min 2% MsOH in  1 min CH.sub.2Cl.sub.2/Ac.sub.2O CH.sub.2Cl.sub.2/Ac.sub.2O (5:1), 25° C. (5:1), 45° C. Deprotection: Lev 7% N,H, HOAc in  90 min 7% N.sub.2H.sub.4 HOAc in  1 min CH.sub.2Cl.sub.2/Pyr/HOAc/H.sub.2O CH.sub.2Cl.sub.2/Pyr/HOAc/H.sub.2O (20:16:4:1), 25° C. (20:16:4:1), 40° C. Deprotection: NAP 2% DDQ in 120 min 0.5% MsOH in  1 min DCE/MeOH/H.sub.2O CH.sub.2Cl.sub.2/HFIP/TIS (64:16:1), 40° C. (20:2:1), 40° C. Deprotection: Fmoc 20% Piperidine in  5 min 20% Piperidine in  1 min DMF, 25° C. DMF, 40° C. Deprotection: CI Ac Thiourea in EGME, 360 min Thiourea in EGME, 45 min 80° C. 80° C.

Example 2: Investigation of Temperature Fluctuations of the Reaction Mixture by Addition of Pre-Cooled Building Block and Activator Solutions

[1284] The temperature development inside of the reaction vessel during a synthesis cycle in the presence and absence of pre-cooling device was investigated. FIG. 9 shows the results charts comparing temperature readings inside the reaction vessel, taken during a synthesis cycle, in the presence and absence of a pre-cooling device.

[1285] The three thermal stages are shown. Temperature spikes appear when a liquid is dispensed in the reaction vessel. The dashed curve shows the temperature profile for the device without pre-cooling of the reagents. The thermal spikes are remarkable at the pre-coupling regime (subzero temperatures). The solid line depicts the temperature profile with active pre-cooling. The incoming solution of reagents is pre-cooled and this suppresses the temperature spikes. Since there are three thermal stages during a glycosylation cycle: (1) The pre-coupling regime (−40° C. to −10° C.). The building block and activator are allowed to impregnate the resin and for diffusion through the porous solid. The low temperature prevents the early decomposition of the intermediate before the actual coupling; (2) the coupling regime (around 0° C.). The increase in the temperature allows for initiating the coupling reaction by promoting the formation of the intermediates; and (3) the post-coupling regime (room temperate or above). The capping and deprotection reactions take place at higher temperature. These reactions close out the glycosylation cycle. Then, the next coupling or process termination takes place.

[1286] In FIG. 9 the thermal profile during the synthesis of a mannose-α-1,6-tetramer “standard test” in the presence and absence of pre-cooling is shown. Here, the reactor is made of glass and a thioglycoside donor is used as building block, and active cooling of the reaction vessel by a cooling device is ON in both experiments. By using a pre-cooling device according to the present invention, the thermal perturbation due to the delivery of reagents is suppressed (solid line), specifically the delivery of activator reagents and donor solution. In this example, the washing solvent was not pre-cooled to highlight the strong contrast in the thermal spike.

[1287] In FIG. 10 the analytical results of the experiments as described in FIG. 9 are shown. Mass spectrometry (MALDI) and HPLC chromatogram are provided. The experiment performed in presence of a pre-cooling device shows superior results: only the desired product was obtained. An overall yield of 60% was achieved. The reaction without the pre-cooling delivers a mixture of tetra-mannose and some partially deprotected tetra-mannose. This further complicates the purification process and results in lower overall yield.

[1288] In FIG. 11 the thermal profile during the synthesis of a mannose-α-1,6-tetramer “standard test” in the presence and absence of pre-cooling is demonstrated. The reaction vessel is made of glass, and a glycosyl phosphate donor is used as building block, and active cooling of the reaction vessel by a cooling device is ON in both experiments. By using a pre-cooling element, the thermal perturbation due to the delivery of reagents is suppressed (solid line), specifically the delivery of activator reagents and donor solution. The washing solvent was not pre-cooled to highlight the stark contrast in the thermal spike.

[1289] In FIG. 12 the analytical results of the experiments as shown in FIG. 11 are shown. Mass spectrometry (MALDI) and HPLC chromatogram are provided. The experiment performed in presence of a pre-cooling device shows superior results: only the desired product was obtained. An overall yield of 60% was achieved. The reaction without the pre-cooling shows a HPLC chromatogram with more side product traces (peaks at 2.3 min and 3.6 min).

[1290] In FIG. 13 the thermal profile during the synthesis of a mannose-α-1,6-tetramer “standard test” in the presence and absence of pre-cooling is demonstrated. The reaction vessel is made of PFA, and a glycosyl phosphate donor is used as building block, and active cooling of the reaction vessel by a cooling device is OFF in both experiments. By using a pre-cooling element, there is a brief drop in temperature when the pre-cooled reagents/building blocks are delivered (solid line). In both cases the temperature oscillates around 20° C. In FIG. 14 the analytical results of the experiments as described in FIG. 13 are shown. HPLC chromatogram are provided. In both experiments no desired product was observed. Only side products are visible in HPLC chromatogram (peaks at 2.3 min and 3.4 min). Precise control of the temperature is instrumental during the automated synthesis of oligosaccharides.

[1291] In FIG. 15 the temperature differential (maximum temperate reached minus the actual reaction vessel temperature) within a glass the reaction vessel when 1 mL of Dichloromethane is delivered at different flow rates to a reactor vessel at −18° C. is shown. The white circle series refer to the solvent delivered without pre-cooling; the black triangle series refers to pre-cooled solvent. By using the pre-cooling device, the reagent can be delivered 3 times faster than the current state of the art with a thermal perturbation lesser than ±3° C. (highlighted in box).

[1292] In FIG. 16 the thermal perturbation in the reaction vessel when different volumes of Dichloromethane are delivered at 1.05 mL/min in a reactor vessel at −18° C. in the presence or absence of pre-cooling is shown. The solid line refers to the thermal perturbation with pre-cooling. The dash line refers to the thermal perturbation without pre-cooling. The pre-cooling reduces the thermal perturbation even when 4 mL are delivered which is 4 times the current scale of the current state of the art synthesizer. This presents a major advantage of the present invention in the up-scaling the synthesis of oligosaccharides.

[1293] The advantages and improvement of the automated glycosylation via pre-cooling of the reagents are most apparent in FIG. 10 and FIG. 12. Furthermore, it has been found that by pre-cooling of the reagents, the overall duration of a synthesis cycle could be reduced as the pre-cooling device allows rapid delivery of pre-cooled reagents. By using the pre-cooling device of the present invention, the donor could be delivered 3 times faster than the current state of the art devices; this was achieved without a thermal perturbation larger than 3° C. compared to the actual reaction vessel temperatures. In addition, the precooling reduces the thermal perturbation even when 4 mL are delivered which is 4 times the current scale of the current state of the art synthesizers. This represents a major advantage of the present invention in the up-scaling the synthesis of oligosaccharides.

Example 3: Synthesis of Mannose Tetramer Via Thioglycoside Donor

[1294] ##STR00003##

Automated Synthesis Working Modules

[1295] The current example was performed with and without pre-cooling of the activators solution and building block/donor solution; within a glass reaction vessel.

[1296] The timing and quantity of solvents/reagents transferred to the reaction vessel in each step is controlled by the software. The reagent delivery system is based on valve-pressured control in which the entire platform is constantly pressurized so that the specific solvent/reagent is transferred from the respective storage components by timing the opening and closing of the appropriate valves. Some of the reagents are delivered by the withdrawing and/or dispensing action of a syringe pump. Before start the modules, the program initializes all the components (valves, active cooling, pre-cooling, pump, sensors and actuators); renews the driving solvent in the loops 625 and 626; the program empties and flushes the reagents delivery lines.

Module 1: Acidic Wash:

[1297] The resin loaded into the reaction vessel is washed with DMF, THF, DCM (six times each with 3 mL for 15 s). The resin is swollen in 2 mL DCM, and the temperature of the reaction vessel was adjusted in the range of −18° C. to −17° C. For acidic washing, 1 mL of the solution of TMSOTf is delivered to the reaction vessel via the pre-cooling device which reaches a temperature of −15° C. After three minutes, the solution is drained. Finally, 3 mL DCM is added to the reaction vessel, incubated for 15 s, and drained out.

Module 2: Glycosylation:

[1298] Thioglycoside building block is dissolved in the proper solvent mixture (4 mL for four glycosylation cycles) in the designated building block storing component. The reaction vessel is set to reach the initial glycosylation temperature. During the adjustment of the temperature, the DCM in the reaction vessel is drained and 1 mL of thioglycoside building block (6.5 eq. in 1.0 mL DCM) is delivered from the building block storing component to the reaction vessel via the pre-cooling device which cools the solution of thioglycoside building block to a temperature of −15° C. After the temperature reached the range of −18° C. to −17° C. ° C., 1.0 mL NIS and TfOH solution in DCM and dioxane (v/v, 2:1) is delivered to the reaction vessel from the respective activator storing component via the pre-cooling device which cools the activator solution down to a temperature of −15° C. The glycosylation mixture is incubated for 5 min at the temperature range of −18° C. to −17° C., linearly ramped to 0° C., and after reaching 0° C. the reaction mixture is incubated for an additional 20 min. Once incubation time is finished, the reaction mixture is drained and the resin is washed with DCM (once, 2 mL for 15 s). Then the resin is washed with 2 mL of Dioxane. Finally, the resin is washed twice with DCM (2 mL for 15 s).

Module 3: Capping:

[1299] The temperature of the reaction vessel is adjusted to 25° C. The resin is washed twice with DMF (3 mL for 15 s). 2 mL of Pyridine solution (10% in volume in DMF) was delivered and the resin is incubated for one minute. The resin is washed three times with DCM (2 mL for 15 s). The capping of the unreacted acceptor groups is done by delivering 4 mL of methanesulfonic acid (2% in volume) and acetic anhydride (10% in volume) in DCM. The resin and the reagents are incubated for 10 min; then 1 mL of DCM in added to dilute the solution and the incubation continues for another 10 min. The solution is drained from the reaction vessel and the resin is washed 3 times with DCM (2 mL for 15 s).

Module 4: Fmoc Deprotection:

[1300] The resin is washed with DMF (three times with 3 mL for 15 s), swollen in 2 mL DMF and the temperature of the reaction vessel is adjusted to 25° C. For Fmoc-deprotection the DMF is drained and 2 mL of a solution of 20% Piperidine in DMF was delivered to the reaction vessel. After 5 min the reaction solution is drained from the reactor vessel. Then, the resin is washed with DMF (three times with 3 mL for 15 s) and DCM (five times with 3 mL). After this module the resin is ready for the next glycosylation cycle.

[1301] Resin 1103 (see FIG. 17) functionalized with a photo-cleavable linker (40 mg; loading 0.33 mmol/g; 25.1 μmol) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM. The sequence of reaction steps for the formation of mannose tetramer was as follows: [1302] 1. Module 1 was performed with 1 mL TMSOTf solution at −20° C. for 3 min. [1303] 2. Module 2 was performed with 5 equiv. building block 1 (see FIG. 18) and 5 equiv. NIS solution. [1304] 3. Module 3 was carried out in two steps; first 2 mL of Pyridine solution (10% in volume in DMF); then in a second step with 4 mL of Methanesulfonic acid (2% in volume) and Acetic anhydride (10% in volume) in DCM. [1305] 4. Module 4 was carried out with 20% Piperidine in DMF. [1306] 5. Subsequently, modules 1-4 were repeated four times in the same manner as described in steps 1-4 in order to obtain a tetramer.

[1307] After buildup of the tetramer on the resin the oligosaccharide was cleaved from solid support in a photo-reactor: A mercury lamp is turned on 30 min prior to the first cleavage event. The fluorinated-ethylene-propylene (FEP) tubing was washed with 20 mL DCM at a flow rate of 5 mL/min before cleavage. The solid support was pre-swelled in the dark in DCM for 30 min at least before being taken up with a 20 mL disposable syringe. The suspension of solid support in DCM was slowly injected from the disposable syringe (20 mL) into the FEP tubing using a syringe pump. The suspension was pushed through the FEP tubing into the photo-reactor with additional 18 mL DCM (flow rate: 700 μL/min). The photo-cleavage took place inside the reactor while solid support travelled toward the exit point of the reactor. The suspension leaving the reactor was directed into a syringe equipped with polyethylene filter frit where the resin was filtered off and the solution containing the cleaved oligosaccharide is collected in a separate glass vial. The tubing as washed with 20 mL DCM (flow rate: 2 mL/min) until any remaining resin exited the reactor and the remaining oligosaccharide solution is collected. The tubing was re-equilibrated with 20 mL DCM using a flow rate of 5 mL/min and the entire cleavage procedure was repeated. The combined solution that was collected in the photo-cleavage process was evaporated in vacuo and the crude material was analyzed by MALDI-TOF, and HPLC.

Example 4: Synthesis of Mannose Tetramer Via Phosphate Donor

[1308] ##STR00004##

Automated Synthesis Working Modules

[1309] The current example was performed within a PFA/glass reactor, in a factorial experimental design with two factors: active cooling of the reaction vessel and pre-cooling action of the activators solution and building block/donor solution, each factor had two levels with and without active temperature control (on and off stage of the corresponding device).

[1310] The timing and quantity of solvents/reagents transferred to the reaction vessel in each step is controlled by the software. The reagent delivery system is based on valve-pressured control in which the entire platform is constantly pressurized so that the specific solvent/reagent is transferred from the respective storage components by timing the opening and closing of the appropriate valves. Some of the reagents are delivered by the withdrawing and/or dispensing action of a syringe pump. Before start the modules, the program initializes all the components (valves, active cooling, precooling, pump, sensors and actuators); renews the driving solvent in the loops 625 and 626; the program empties and flushes the reagents delivery lines.

Module 1: Acidic Washing:

[1311] The resin loaded into the reaction vessel is washed with DMF, THF, DCM (six times each with 3 mL for 15 s). The resin is swollen in 2 mL DCM, and the temperature of the reaction vessel was adjusted in the range of −18° C. to −17° C. by cooling device (when it is programed to do so). For acidic washing 1 mL of the solution of 2% TMSOTf in DCM is delivered to the reaction vessel via the pre-cooling device which cools the solution to a temperature of −15° C. (when it is programed to do so). After three minutes, the solution is drained. Finally, 3 mL DCM is added to the reaction vessel.

Module 2: Glycosylation:

[1312] Phosphate building block is dissolved in the proper solvent mixture e.g. DCM (5 mL for one initial double cycle for the first coupling and three more single glycosylation cycles to build up the tetramer) in the designated building block storing component. The reaction vessel is set to reach the initial glycosylation temperature. During the adjustment of the temperature in the reactor vessel (when it is programed to do so), the DCM in the reaction vessel is drained and 1 mL of phosphate building block 2 (5.0 eq. in 1.0 mL DCM) is delivered from the building block storing component to the reaction vessel via the pre-cooling device which cools the solution of phosphate building block (when it is programed to do so) to a temperature of −15° C. After the set temperature in the range of −18° C. to −17° C. is reached the resin is incubated in the solution of phosphate building block for 10 min. Then 1.0 mL of the solution of 2% TMSOTf in DCM is delivered to the reaction vessel from the respective activator storing component via the pre-cooling device which cools the activator solution down to a temperature of −15° C. (when it is programed to do so). The glycosylation mixture is incubated for 30 min in the temperature range of −18° C. to −17° C., linearly ramped to 0° C. (when it is programed to do so), and after reaching 0° C. the reaction mixture is incubated for an additional 10 min. Once incubation time is finished, the reaction mixture is drained and the resin is washed with DCE (once, 2 mL for 5 s).

Module 3: Capping:

[1313] The temperature of the reactor vessel is adjusted to 25° C. The resin is washed twice with DMF (3 mL for 15 s). 2 mL of Pyridine solution (10% in volume in DMF) was delivered and the resin is incubated for one minute. The resin is washed three times with DCM (2 mL for 15). The capping of the unreacted acceptor groups is done by delivering 4 mL of Methanesulfonic acid (2% in volume) and Acetic anhydride (10% in volume) in DCM. The resin and the reagents are incubated for 10 min; then 1 mL of DCM in added to dilute the solution and the incubation continues for another 10 min. The solution is drained from the reactor vessel and the resin is washed 3 time with DCM (2 mL for 15 s).

Module 4: Fmoc Deprotection:

[1314] The resin is washed with DMF (three times with 3 mL for 15 s), swollen in 2 mL DMF and the temperature of the reaction vessel is adjusted to 25° C. For Fmoc deprotection the DMF is drained and 2 mL of a solution of 20% Piperidine in DMF was delivered to the reaction vessel. After 5 min the reaction solution is drained from the reactor vessel. Then, the resin is washed with DMF (three times with 3 mL for 15 s) and DCM (five times with 3 mL). After this module the resin is ready for the next glycosylation cycle.

[1315] Resin 1103 (see FIG. 17) functionalized with a photo-cleavable linker (40 mg; loading 0.33 mmol/g; 25.1 μmol) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM. The sequence of reaction steps for the formation of mannose tetramer was as follows: [1316] 1. Module 1 was performed with 1 mL TMSOTf solution at the temperature range of −18° C. to −17° C. (when it is programed to do so) for 3 min. [1317] 2. Module 2 was performed with 5 equiv building block 2 (see FIG. 18) and 2% TMSOTf in DCM solution. [1318] 3. Module 3 was carried out in two steps; first 2 mL of Pyridine solution (10% in volume in DMF); then in a second step with 4 mL of Methanesulfonic acid (2% in volume) and Acetic anhydride (10% in volume) in DCM. [1319] 4. Module 4 was carried out with 20% Piperidine in DMF. [1320] 5. After Module 1 took place the Module 2 repeated twice (for the first coupling). The Modules 3 and 4 were then performed. [1321] 6. Subsequently, modules 1-4 were repeated three times in the same manner as described in steps 1-4 in order to obtain a tetramer.

[1322] After buildup of the tetramer on the resin the oligosaccharide was cleaved from solid support in a photoreactor: A mercury lamp is turned on 30 min prior to the first cleavage event. The fluorinated-ethylene-propylene (FEP) tubing was washed with 20 mL DCM at a flow rate of 5 mL/min before cleavage. The solid support was pre-swelled in the dark in DCM for 30 min at least before being taken up with a 20 mL disposable syringe. The suspension of solid support in DCM was slowly injected from the disposable syringe (20 mL) into the FEP tubing using a syringe pump. The suspension was pushed through the FEP tubing into the photoreactor with additional 18 mL DCM (flow rate: 700 μL/min). The photo-cleavage took place inside the reactor while solid support travelled toward the exit point of the reactor. The suspension leaving the reactor was directed into a syringe equipped with polyethylene filter frit where the resin was filtered off and the solution containing the cleaved oligosaccharide is collected in a separate glass vial. The tubing as washed with 20 mL DCM (flow rate: 2 mL/min) until any remaining resin exited the reactor and the remaining oligosaccharide solution is collected. The tubing was re-equilibrated with 20 mL DCM using a flow rate of 5 mL/min and the entire cleavage procedure was repeated. The combined solution that was collected in the photo-cleavage process was evaporated in vacuo and the crude material was analyzed by MALDI-TOF, and HPLC.

Example 5: Fast Synthesis of Mannose Tetramer Via Phosphate Donor with Temperature Regulation Via Microwave Heating

[1323] The automated synthesis of tetramannose as shown in Scheme 1 was conducted by combining constant cooling with microwave radiation to adjust and control the temperature of the reagents during the glycosylation cycle.

##STR00005##

Automated Synthesis Working Modules

[1324] The timing and quantity of solvents/reagents transferred to the reaction vessel in each step is controlled by software. The reagent delivery system utilizes pressure control valves, which constantly pressurize the entire platform, so that the specific solvent/reagent is transferred from the respective storage components by timing the opening and closing of the appropriate valves. All the solvents are pre-cooled before they are delivered inside the reaction vessel.

Module 1: Acidic Washing:

[1325] The resin loaded into the reaction vessel is washed with DMF, THF, DCM (six times each with 3 mL for 15 s). The resin is swollen in 2 mL DCM, and the temperature of the reaction vessel was adjusted in the range of −22° C. to −20° C. by cooling device (when it is programmed to do so). For acidic washing, 1 mL of the solution of 2% TMSOTf in DCM is delivered to the reaction vessel via the pre-cooling device which cools the solution to a temperature of −20° C. (when it is programmed to do so). After three minutes, the solution is drained. Finally, 3 mL DCM is added to the reaction vessel.

Module 2: Glycosylation:

[1326] Phosphate building block is dissolved in the proper solvent (6.5 eq. in 1.0 mL DCM) and loaded in the designated building block storing component. The reaction vessel is set to reach the initial glycosylation temperature. During the adjustment of the temperature, the DCM in the reaction vessel is drained and 1 mL of phosphate building block (6.5 eq. in 1.0 mL DCM) is delivered from the building block storing component to the reaction vessel via the pre-cooling device, which cools the solution of phosphate building block to a temperature of −15° C. After the temperature reached the range of −18° C. to −17° C., 1.0 mL NIS and TfOH solution in DCM and dioxane (v/v, 2:1) is delivered to the reaction vessel from the respective activator storing component via the pre-cooling device, which cools the activator solution down to a temperature of −15° C. The glycosylation mixture is incubated for 5 min at the temperature range of −18° C. to −17° C., linearly ramped to 0° C., and after reaching 0° C. the reaction mixture is incubated for an additional 30 minutes. Once incubation time is finished, the reaction mixture is drained and the resin is washed with DCM (once, 2 mL for 15 s). Then the resin is washed with 2 mL of Dioxane. Finally the resin is washed twice with DCM (2 mL for 15 s).

Module 3: Capping:

[1327] While the temperature of the active cooling element is kept in the range of −22° C. to −20° C. preparing for the next coupling cycle. The temperature of the reactor vessel is kept between 70° C. and 20° C. by microwave irradiation of the washing solution and reagents, adjusting the irradiation power. The resin is washed twice with DMF (3 mL for 15 s). 2 mL of pyridine solution (10% in volume in DMF) were delivered and the microwave irradiation power is then adjusted to 40-50 W. The resin is incubated for one minute between 70° C. and 20° C. The resin is washed three times with DCM (2 mL for 15). The microwave irradiation power is then set to 150-180 W to proceed the capping of the unreacted acceptor groups. The capping is done by delivering 4 mL of methanesulfonic acid (2% in volume) and acetic anhydride (10% in volume) in DCM. The resin and the reagents are incubated for 1 min. The temperature of the reactor vessel is adjusted between 70° C. and 20° C. by microwave irradiation; then 1 mL of DCM in added to dilute the solution and the incubation continues for another 1 min. The solution is drained from the reactor vessel and the resin is washed 3 times with DCM (2 mL for 15 s).

Module 4: Fmoc Deprotection:

[1328] The resin is washed with DMF (three times with 3 mL for 15 s), swollen in 2 mL DMF. For Fmoc deprotection, 2 mL of a solution of 20% piperidine in DMF were delivered to the reaction vessel. The temperature of the reagents inside the reactor vessel is adjusted between 70° C. and 20° C. by microwave irradiation (40 W). After 1 min, the reaction solution is drained from the reactor vessel. Then, the resin is washed with DMF (three times with 3 mL for 15 s) and DCM (five times with 3 mL). After this module, the resin is ready for the next glycosylation cycle.

[1329] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 1) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM.

[1330] The reaction sequence for the formation of tetramannose 9 was as follows:

[1331] 1. Module 1 was performed with 1 mL TMSOTf solution at the temperature range of −22° C. to −20° C. (when it is programmed to do so) for 3 min.

[1332] 2. Module 2 was performed with 5 equiv Building Block and 2% TMSOTf in DCM solution.

[1333] 3. Module 3 was carried out in two steps; first 2 mL of pyridine solution (10% in volume in DMF); then in a second step with 4 mL of methanesulfonic acid (2% in volume) and acetic anhydride (10% in volume) in DCM.

[1334] 4. Module 4 was carried out with 20% piperidine in DMF.

[1335] 6. Subsequently, modules 1-4 were repeated four times in order to obtain a tetramer.

[1336] After build-up of the tetramer on the resin, the oligosaccharide was cleaved from solid support in a photoreactor: A mercury lamp is turned on 30 min prior to the first cleavage event. The fluorinated-ethylene-propylene (FEP) tubing was washed with 20 mL DCM at a flow rate of 5 mL/min before cleavage. The solid support was pre-swollen in the dark in DCM for 30 min at least before being taken up with a 20 mL disposable syringe. The suspension of solid support in DCM was slowly injected from the disposable syringe (20 mL) into the FEP tubing using a syringe pump. The suspension was pushed through the FEP tubing into the photo-reactor with additional 18 mL DCM (flow rate: 700 μL/min). The photo-cleavage took place inside the reactor while solid support travelled toward the exit point of the reactor. The suspension leaving the reactor was directed into a syringe equipped with polyethylene filter frit where the resin was filtered off and the solution containing the cleaved oligosaccharide is collected in a separate glass vial. The tubing was washed with 20 mL DCM (flow rate: 2 mL/min) until any remaining resin exited the reactor and the remaining oligosaccharide solution is collected. The tubing was re-equilibrated with 20 mL DCM using a flow rate of 5 mL/min and the entire cleavage procedure was repeated. The combined solution that was collected in the photo-cleavage process was evaporated in vacuo and the crude material was analyzed by MALDI-TOF, and HPLC.

[1337] After weighing, the recovered crude was 27 mg, which correspond to a 65% yield. This experiment demonstrates that high yields were obtained by combining pre-cooling of the reagents and constant cooling with microwave radiation even with short coupling times of 30 minutes.

Example 6: Synthesis of 5-Amino-Pentyl α-(1→2)-D-Tetramannopyranoside Via Phosphate Glycosylation and Selective Deprotection of NAP Temporal Protecting Group in Presence of Fmoc-, Lev-, CIAc-Temporal Protecting Groups

[1338] ##STR00006##

Automated Synthesis Working Modules

[1339] The timing and quantity of solvents/reagents transferred to the reaction vessel in each step is controlled by software. The reagent delivery system is utilizes a pressure control syringe pump system, which constantly pressurize the entire platform, so that the specific solvent/reagent is transferred from the respective storage components by timing the opening and closing of the appropriate valves, or withdrawing and dispensing with the motorized syringe in connection with a rotary valve.

Module 1: Acidic Washing: The same acidic washing module was applied as in Example 5.

Module 2: Glycosylation:

[1340] Phosphate building block is dissolved in the proper solvent mixture, e.g. DCM (5 mL for one initial double cycle for the first coupling and three more single glycosylation cycles to build up the tetramer) in the designated building block storing component. The reaction vessel is set to reach the initial glycosylation temperature. During the adjustment of the temperature in the reactor vessel (when it is programmed to do so), DCM is drained in the reaction vessel and 1 mL of phosphate building block (5.0 eq. in 1.0 mL DCM) is delivered from the building block storing component to the reaction vessel via the pre-cooling device, which cools the solution of phosphate building block (when it is programmed to do so) to a temperature of −18° C. Then 1.0 mL of a solution of 2% TMSOTf in DCM is delivered to the reaction vessel from the respective activator storing component via the pre-cooling device, which cools the activator solution down to a temperature of −18° C. (when it is programmed to do so). The glycosylation mixture is incubated for 20 min in the temperature range of −22° C. to −20° C. Keeping constant the active cooling action by microwave transparent coolant flowing in the jacket, the linearly ramped to 0° C. in 5 min (when it is programmed to do so) by the microwave radiation, adjusting the maximum radiation power to 180 W, and after reaching 0° C. the reaction mixture is incubated for additional 10 minutes. Once incubation time is finished, the reaction mixture is drained and the resin is washed with DCE (once, 2 mL for 5 s).

Module 3: NAP Deprotection (ca. 60 min):

[1341] The resin is washed with DCM (three times with 2 mL for 15 s). For NAP deprotection, 2 mL of a solution of 2% DDQ and 13% methanol in DCE was delivered to the reaction vessel. The temperature of the reagents inside the reactor vessel is adjusted between 60° C. and 20° C. by microwave irradiation (180 W). After 30 min, the reaction solution is drained from the reactor vessel. The resin is washed with DCM (three times with 2 mL for 15 s); the incubation in NAP deprotection solution between 60° C. and 20° C. by microwave irradiation (180 W) and the DCM washes were repeated twice more. Then, the resin is washed (3 times) with the following solvent sequence DMF, THE and DCM (3 mL for 120 s each). After this module, the resin is ready for the next glycosylation cycle.

[1342] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 2) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM.

[1343] The sequence of reaction steps for the formation of tetramannose was as follows:

[1344] 1. Module 1 was performed with 1 mL TMSOTf solution at the temperature range of −22° C. to −20° C. (when it is programmed to do so) for 3 min.

[1345] 2. Module 2 was performed twice with 5 equiv Building Block and 2% TMSOTf in DCM solution.

[1346] 3. Module 3 was carried out with 2% DDQ and 13% methanol in DCE.

[1347] 4. After Module 1 took place, the Module 2 repeated twice (for the first and last coupling). Then Module 3 was performed.

[1348] 5. Subsequently, modules 1-3 were repeated three times in the same manner as described in steps 1-3 in order to obtain a tetramer 11.

[1349] After buildup of the tetramer on the resin, the oligosaccharide was cleaved from solid support in a photoreactor as described in Example 5.

Example 7: Synthesis of 5-Amino-Pentyl α-(1→3)-D-Tetramannopyranoside Via Phosphate Glycosylation and Selective Deprotection of Lev Temporal Protecting Group in the Presence of Fmoc-, NAP-, CIAc-Temporal Protecting Groups

[1350] ##STR00007##

Automated Synthesis Working Modules

[1351] The timing and quantity of solvents/reagents transferred to the reaction vessel in each step is controlled by software. The reagent delivery system is utilizes a pressure control syringe pump system, which constantly pressurize the entire platform, so that the specific solvent/reagent is transferred from the respective storage components by timing the opening and closing of the appropriate valves, or withdrawing and dispensing with the motorized syringe in connection with a rotary valve.

Module 1: Acidic Washing:

[1352] The same acidic washing module was applied as in Example 5.

Module 2: Glycosylation:

[1353] Phosphate building block is dissolved in the proper solvent mixture e.g. DCM (5 mL for one initial double cycle for the first coupling and three more single glycosylation cycles to build up the tetramer) in the designated building block storing component. The reaction vessel is set to reach the initial glycosylation temperature. During the adjustment of the temperature in the reactor vessel (when it is programmed to do so), the DCM in the reaction vessel is drained and 1 mL of phosphate building block (5.0 eq. in 1.0 mL DCM) is delivered from the building block storing component to the reaction vessel via the pre-cooling device which cools the solution of phosphate building block (when it is programmed to do so) to a temperature of −18° C. Then 1.0 mL of the solution of 2% TMSOTf in DCM is delivered to the reaction vessel from the respective activator storing component via the pre-cooling device which cools the activator solution down to a temperature of −18° C. (when it is programmed to do so). The glycosylation mixture is incubated for 30 min in the temperature range of −22° C. to −20° C. Keeping constant the active cooling action by microwave transparent coolant flowing in the jacket, the linearly ramped to 0° C. in 5 min (when it is programmed to do so) by the microwave radiation, adjusting the maximum radiation power to 180 W, and after reaching 0° C. the reaction mixture is incubated for additional 10 min. Once incubation time is finished, the reaction mixture is drained and the resin is washed with DCE (once, 2 mL for 5 s).

Module 3: Lev Deprotection (ca. 5 min):

[1354] The resin is washed with DCM (three times with 2 mL for 15 s). For Lev deprotection, 2 mL of a solution of 1% hydrazine acetate and 21% acetic acid in pyridine was delivered to the reaction vessel. The temperature of the reagents inside the reactor vessel is adjusted between 40° C. and 20° C. by microwave irradiation (180 W). After 1 min, the reaction solution is drained from the reactor vessel. The resin is washed with DCM (three times with 2 mL for 15 s); the incubation in Lev deprotection solution between 40° C. and 20° C. by microwave irradiation (180 W) and the DCM washes were repeated twice more. Then, the resin is washed (3 times) with the following solvent sequence DMF, THE and DCM (3 mL for 15 s each). After this module the resin is ready for the next glycosylation cycle.

[1355] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 3) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM.

[1356] The sequence of reaction steps for the formation of 5-Amino-pentyl α-(1.fwdarw.3)-D-tetramannopyranoside was as follows:

[1357] 1. Module 1 was performed with 1 mL TMSOTf solution at the temperature range of −22° C. to −20° C. (when it is programed to do so) for 3 min.

[1358] 2. Module 2 was performed twice with 5 equiv Building Block and 2% TMSOTf in DCM solution.

[1359] 3. Module 3 was carried out with 1% hydrazine acetate and 21% acetic acid in pyridine.

[1360] 4. After Module 1 took place the Module 2 repeated twice (for the first coupling). Then Module 3 was performed.

[1361] 5. Subsequently, modules 1-3 were repeated three times in the same manner as described in steps 1-3 in order to obtain a tetramer 12.

[1362] After buildup of the tetramer on the resin the oligosaccharide was cleaved from solid support in a photoreactor as described in Example 5. 13 mg of the crude product were obtained, which correspond to a yield of 36%.

Example 8: Synthesis of 5-Amino-Pentyl α-(1→4)-D-Tetramannopyranoside Via Phosphate Glycosylation and Selective Deprotection of Fmoc Temporal Protecting Group in the Presence of NAP-, Lev-, CIAc-Temporal Protecting Groups

[1363] ##STR00008##

Automated Synthesis Working Modules

[1364] The timing and quantity of solvents/reagents transferred to the reaction vessel in each step is controlled by software. The reagent delivery system is utilizes a pressure control syringe pump system, which constantly pressurize the entire platform, so that the specific solvent/reagent is transferred from the respective storage components by timing the opening and closing of the appropriate valves, or withdrawing and dispensing with the motorized syringe in connection with a rotary valve.

Module 1: Acidic Washing:

[1365] The same acidic washing module was applied as in Example 5.

Module 2: Glycosylation:

[1366] The same glycosylation module was used as in Example 6.

Module 3: Fmoc Deprotection (ca. 5 min):

[1367] The resin is washed with DMF (three times with 2 mL for 15 s). For Fmoc deprotection 2 mL of a solution of 20% triethylamine in DMF was delivered to the reaction vessel. The temperature of the reagents inside the reactor vessel is adjusted between 70 and 20° C. by microwave irradiation (50 W). After 1 min the reaction solution is drained from the reactor vessel. The resin is washed with DMF (three times with 2 mL for 15 s); the incubation in Fmoc deprotection solution between 70 and 20° C. by microwave irradiation (40 W) and the DMF washes were repeated twice more. Then, the resin is washed with the following solvent sequence DMF (3 times) and DCM (3 times) 3 mL for 15 s each time. After this module the resin is ready for the next glycosylation cycle.

[1368] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 4) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM. The sequence of reaction steps for the formation 5-Amino-pentyl α-(1.fwdarw.4)-D-tetramannopyranoside was as follows:

[1369] 1. Module 1 was performed with 1 mL TMSOTf solution at the temperature range of −22° C. to −20° C. (when it is programmed to do so) for 3 min.

[1370] 2. Module 2 was performed twice with 5 equiv Building Block and 2% TMSOTf in DCM solution.

[1371] 3. Module 3 was carried out with 20% triethylamine in DMF.

[1372] 4. After Module 1 took place the Module 2 repeated twice (for the first coupling). Then Module 3 was performed.

[1373] 5. Subsequently, modules 1-3 were repeated three times in the same manner as described in steps 1-4 in order to obtain a tetramer 13.

[1374] After buildup of the tetramer on the resin, the oligosaccharide was cleaved from solid support in a photoreactor: A mercury lamp is turned on 30 min prior to the first cleavage event. The fluorinated-ethylene-propylene (FEP) tubing was washed with 20 mL DCM at a flow rate of 5 mL/min before cleavage. The solid support was pre-swelled in the dark in DCM for 30 min at least before being taken up with a 20 mL disposable syringe. The suspension of solid support in DCM was slowly injected from the disposable syringe (20 mL) into the FEP tubing using a syringe pump. The suspension was pushed through the FEP tubing into the photoreactor with additional 18 mL DCM (flow rate: 800 μL/min). The photocleavage took place inside the reactor while solid support travelled toward the exit point of the reactor. The suspension leaving the reactor was directed into a syringe equipped with polyethylene filter frit where the resin was filtered off and the solution containing the cleaved oligosaccharide is collected in a separate glass vial. The tubing as washed with 20 mL DCM (flow rate: 2 mL/min) until any remaining resin exited the reactor and the remaining oligosaccharide solution is collected. The tubing was re-equilibrated with 20 mL DCM using a flow rate of 5 mL/min and the entire cleavage procedure was repeated. The combined solution that was collected in the photocleavage process was evaporated in vacuo and the crude material was analyzed by MALDI-TOF, and HPLC.

[1375] 15 mg of the crude product were recovered, which correspond to a yield of 51%.

Example 9: Synthesis of 5-Amino-Pentyl α-(1→6)-D-Tetramannopyranoside Via Thioglycosylation and Selective Deprotection of CIAc Temporal Protecting Group in the Presence of Fmoc-, Lev-, NAP-Temporal Protecting Group

[1376] ##STR00009##

Automated Synthesis Working Modules

[1377] The timing and quantity of solvents/reagents transferred to the reaction vessel in each step is controlled by software. The reagent delivery system is utilizes a pressure control syringe pump system, which constantly pressurize the entire platform, so that the specific solvent/reagent is transferred from the respective storage components by timing the opening and closing of the appropriate valves, or withdrawing and dispensing with the motorized syringe in connection with a rotary valve.

Module 1: Acidic Washing:

[1378] The resin loaded into the reaction vessel is washed with DMF, THF, DCM (six times each with 3 mL for 15 s). The resin is swollen in 2 mL DCM, and the temperature of the reaction vessel was adjusted in the range of −22° C. to −20° C. by cooling device (when it is programmed to do so). For acidic washing, 1 mL of the solution of 1% TMSOTf in DCM is delivered to the reaction vessel via the pre-cooling device which, cools the solution to a temperature of −20° C. (when it is programmed to do so). After three minutes, the solution is drained. Finally, 3 mL DCM is added to the reaction vessel.

Module 2: Glycosylation:

[1379] Thioglycoside building block is dissolved in the proper solvent mixture e.g. DCM (6 mL for two double cycles for the first and last coupling and two more single glycosylation cycles couplings between to build up the tetramer) in the designated building block storing component. The reaction vessel is set to reach the initial glycosylation temperature. During the adjustment of the temperature in the reactor vessel (when it is programmed to do so), the DCM in the reaction vessel is drained and 1 mL of thioglycoside building block (6.5 eq. in 1.0 mL DCM) is delivered from the building block storing component to the reaction vessel via the pre-cooling device which cools the solution of phosphate building block (when it is programmed to do so) to a temperature of −15° C. to −18° C. Then 1.0 mL NIS and TfOH solution in DCM and dioxane (v/v, 2:1) are delivered to the reaction vessel from the respective activator storing component via the pre-cooling device, which cools the activator solution down to a temperature of −15° C. to −18° C. (when it is programmed to do so). The glycosylation mixture is incubated for 5 min in the temperature range of −15° C. to −22° C. Keeping constant the active cooling action by microwave transparent coolant flowing in the jacket, the temperature linearly ramped to 0° C. in 5 min (when it is programmed to do so) by the microwave radiation, adjusting the maximum radiation power to 180 W, and after reaching 0° C. the reaction mixture is incubated for additional 20 min. Once incubation time is finished, the reaction mixture is drained and the resin is washed with mixture of DCM and dioxane (v/v, 2:1) (once, 2 mL for 5 s).

Module 3: CIAc (ca. 5 min):

[1380] The resin is washed with DMF (three times with 2 mL for 15 s). For CIAc deprotection 2 mL of a solution of 4% thiourea and 9% of pyridine in 2-methoxyethanol was delivered to the reaction vessel. The temperature of the reagents inside the reactor vessel is adjusted between 90° C. and 20° C. by microwave irradiation (180 W). After 22 min the reaction solution is drained from the reactor vessel. The resin is washed with DMF (three times with 2 mL for 15 s); the incubation in CIAc deprotection solution between 90 and 20° C. by microwave irradiation (180 W) and the DMF washes were repeated once more. Then, the resin is washed with the following solvent sequence DMF (3 times) and DCM (5 times) 3 mL for 15 s each time. After this module the resin is ready for the next glycosylation cycle.

[1381] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 5) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM.

[1382] The sequence of reaction steps for the formation of 5-Amino-pentyl α-(1.fwdarw.6)-D-tetramannopyranoside was as follows:

[1383] 1. Module 1 was performed with 1 mL TMSOTf solution at the temperature range of −22° C. to −20° C. (when it is programmed to do so) for 3 min.

[1384] 2. Module 2 was performed twice with 6.5 equiv Building Block and NIS and TfOH solution in DCM and dioxane (v/v, 2:1) solution.

[1385] 3. Module 3 was carried out with 4% thiourea and 9% of pyridine in 2-methoxyethanol.

[1386] 4. After Module 1 took place the Module 2 repeated twice (for the first coupling). Then Module 3 was performed.

[1387] 5. Subsequently, modules 1-3 were repeated three times in the same manner as described in steps 1-4 in order to obtain a tetramer 13.

[1388] After buildup of the tetramer on the resin, the oligosaccharide was cleaved from solid support in a photo-reactor as described in Example 8. 22 mg of crude product were obtained, which correspond to a yield of 58%.

Example 10: Synthesis of α-(1→3)-α-(1→4)-α-(1→6)-D-Mannopyranoside Via Phosphate Glycosylation and Selective Deprotection of a Donor Bearing Four Temporal Protecting Groups

[1389] ##STR00010##

Automated Synthesis Working Modules

[1390] The timing and quantity of solvents/reagents transferred to the reaction vessel in each step is controlled by software. The reagent delivery system is utilizes a pressure control syringe pump system, which constantly pressurize the entire platform, so that the specific solvent/reagent is transferred from the respective storage components by timing the opening and closing of the appropriate valves, or withdrawing and dispensing with the motorized syringe in connection with a rotary valve.

Module 1: Acidic Washing:

[1391] The same acidic washing module was applied as in Example 5.

Module 2: Glycosylation:

[1392] The same glycosylation module was used as in Example 7.

Module 3: CIAc (ca. 5 min):

[1393] The same chloroacetyl deprotection module was used as in Example 9.

Module 4: Fmoc Deprotection (ca. 5 min):

[1394] The same Fmoc deprotection module was used as in Example 8.

Module 5: Lev Deprotection (ca. 5 min):

[1395] The same Lev deprotection modules was used as in Example 7.

[1396] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 6) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM.

[1397] The sequence of reaction steps for the formation α-(1.fwdarw.3)-α-(1.fwdarw.4)-α-(1.fwdarw.6)-D-mannopyranoside was as follows: 1. Module 1 was performed with 1 mL TMSOTf solution at the temperature range of −22° C. to −20° C. (when it is programmed to do so) for 3 min.

[1398] 2. Module 2 was performed twice with 5 equiv Building Block and 2% TMSOTf in DCM solution.

[1399] 3. Module 3 was carried out with 4% thiourea and 9% of pyridine in 2-methoxyethanol.

[1400] 4. Module 4 was carried out with 20% triethylamine in DMF.

[1401] 5. Module 5 was carried out with 1% hydrazine acetate and 21% acetic acid in pyridine.

[1402] 4. After Module 1 took place, Module 2 was repeated twice (for the first coupling). Then Modules 3-5 were performed. Then Module 1 was performed.

[1403] 5. Subsequently, Module 2 repeated three times in order to obtain a tetramer 17.

[1404] After buildup of the tetramer on the resin the oligosaccharide was cleaved from solid support in a photoreactor as described in the previous Example: 15 mg of the crude product were obtained, which correspond to yield of 51%.

Example 11: Attempted Tetramannose Synthesis without Acidic Washing Module Between Deprotection and Glycosylation

[1405] The synthesis referred on the scheme 1 was attempted without the acidic was step between the deprotection and the following glycosylation. In the preceding examples, at least the eleven washing steps with solvent were performed between the deprotection and glycosylation module.

Automated Synthesis Working Modules

[1406] The timing and quantity of solvents/reagents transferred to the reaction vessel in each step is controlled by software. The reagent delivery system utilizes pressure control valves, which constantly pressurize the entire platform, so that the specific solvent/reagent is transferred from the respective storage components by timing the opening and closing of the appropriate valves. All the solvents are pre-cooled before they are delivered inside the reaction vessel.

[1407] All solvents were pre-cooled before they are delivered inside the reaction vessel.

Module 1: Glycosylation:

[1408] The same glycosylation module was used as in Example 5.

Module 2: Fmoc Deprotection:

[1409] The same Fmoc deprotection module was used as in Example 5.

[1410] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 1) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM.

[1411] The sequence of reaction steps for the formation of tetramannose 9 was as follows:

[1412] 1. Module 1 was performed with 5 equivalent Building Block and 2% TMSOTf in DCM solution.

[1413] 2. Module 2 was carried out with 20% piperidine in DMF.

[1414] 3. Subsequently, Modules 1 and 2 were repeated four times in order to obtain a tetramer.

[1415] After buildup of the tetramer on the resin, the oligosaccharide was cleaved from solid support in a photoreactor as described in Example 5. MALDI analysis revealed that no title compound was formed (see FIG. 19B).

Example 12: Total Synthesis of a Lewis Antigen Tetramer with Temperature Regulation Via Microwave Heating

[1416] ##STR00011##

Automated Synthesis Working Modules

[1417] The timing and quantity of solvents/reagents transferred to the reaction vessel in each step is controlled by software. The reagent delivery system utilizes pressure control valves, which constantly pressurize the entire platform, so that the specific solvent/reagent is transferred from the respective storage components by timing the opening and closing of the appropriate valves. All the solvents are pre-cooled before they are delivered inside the reaction vessel.

Module 1: Acidic Washing:

[1418] The resin loaded into the reaction vessel is washed with DMF, THF, DCM (six times each with 3 mL for 15 s). The resin is swollen in 2 mL DCM, and the temperature of the reaction vessel was adjusted in the range of −20° C. to −16° C. by cooling device (when it is programmed to do so). For acidic washing, 1 mL of the solution of 2% TMSOTf in DCM is delivered to the reaction vessel via the pre-cooling device, which cools the solution to a temperature of −15° C. (when it is programmed to do so). After three minutes, the solution is drained. Finally, 3 mL DCM is added to the reaction vessel.

Module 2: Glycosylation:

[1419] Thioglycoside building block (18) is dissolved in the proper solvent (6.5 eq. in 1.0 mL DCM) and loaded in the designated building block storing component. The reaction vessel is set to reach the initial glycosylation temperature. After the temperature reached the range of −18° C. to −15° C. the DCM in the reaction vessel is drained and 1 mL of thioglycoside building block (6.5 eq. in 1.0 mL DCM) is delivered from the building block storing component to the reaction vessel via the pre-cooling device, which cools the solution of thioglycoside building block to a temperature of −15° C. Then, 1.0 mL NIS and TfOH solution in DCM and dioxane (v/v, 2:1) is delivered to the reaction vessel from the respective activator storing component via the pre-cooling device, which cools the activator solution down to a temperature of −15° C. The glycosylation mixture is incubated for 5 min at the temperature range of −18° C. to −17° C., the temperature linearly ramped during 5 min to 0° C. by microwave radiation, and after reaching 0° C., the reaction mixture is incubated for additional 20 min. Once incubation time is finished, the reaction mixture is drained and the resin is washed with DCM (once, 2 mL for 15 s). Then the resin is washed with 2 mL of DCM:dioxane 2:1 volume ratio. Finally, the resin is washed twice with DCM (2 mL for 15 s).

Module 3: Fmoc Deprotection:

[1420] The resin is washed with DMF (three times with 3 mL for 15 s), swollen in 2 mL DMF. For Fmoc deprotection 2 mL of a solution of 20% piperidine in DMF was delivered to the reaction vessel. The temperature of the reagents inside the reactor vessel is adjusted between 70° C. and 20° C. by microwave irradiation (50 W). After 1 min the reaction solution is drained from the reactor vessel. Then, the resin is washed with DMF (three times with 3 mL for 15 s) and DCM (five times with 3 mL). After this module the resin is ready for the next glycosylation cycle.

Module 4: Acidic Washing:

[1421] The resin loaded into the reaction vessel is washed with DMF, THF, DCM (six times each with 3 mL for 15 s). The resin is swollen in 2 mL DCM, and the temperature of the reaction vessel was adjusted in the range of −30° C. to −26° C. by cooling device (when it is programmed to do so). For acidic washing, 1 mL of the solution of 2% TMSOTf in DCM is delivered to the reaction vessel via the pre-cooling device, which cools the solution to a temperature of −15° C. (when it is programmed to do so). After three minutes, the solution is drained. Finally, 3 mL DCM is added to the reaction vessel.

Module 5: Glycosylation:

[1422] Thioglycoside building block (19) is dissolved in the proper solvent (6.5 eq. in 1.0 mL DCM) and loaded in the designated building block storing component. The reaction vessel is set to reach the initial glycosylation temperature. After the temperature reached the range of −30° C. to −26° C., the DCM in the reaction vessel is drained and 1 mL of thioglycoside building block 19 (6.5 eq. in 1.0 mL DCM) is delivered from the building block storing component to the reaction vessel via the pre-cooling device, which cools the solution of thioglycoside building block to a temperature of −15° C. Then, 1.0 mL NIS and TfOH solution in DCM and dioxane (v/v, 2:1) is delivered to the reaction vessel from the respective activator storing component via the pre-cooling device, which cools the activator solution down to a temperature of −15° C. The glycosylation mixture is incubated for 10 min at the temperature range of −30° C. to −26° C., the temperature linearly ramped during 5 min to 0° C. by microwave radiation, and after reaching 0° C. the reaction mixture is incubated for additional 30 min. Once incubation time is finished, the reaction mixture is drained and the resin is washed with DCM (once, 2 mL for 15 s). Then the resin is washed with 2 mL of DCM:dioxane 2:1 volume ratio. Finally, the resin is washed twice with DCM (2 mL for 15 s).

Module 6: Lev Deprotection:

[1423] The resin is washed with DCM (three times with 2 mL for 15 s). For Lev deprotection 2 mL of a solution of 1% hydrazine acetate and 21% acetic acid in pyridine was delivered to the reaction vessel. The temperature of the reagents inside the reactor vessel is adjusted between 40° C. and 20° C. by microwave irradiation (180 W). After 3 min, the reaction solution is drained from the reactor vessel. The resin is washed with DCM (three times with 2 mL for 15 s); the incubation in Lev deprotection solution between 40° C. and 20° C. by microwave irradiation (180 W) and the DCM washes were repeated twice more. Then, the resin is washed (3 times) with the following solvent sequence DMF, THE and DCM (3 mL for 15 s each). After this module the resin is ready for the next glycosylation cycle.

Module 7: Glycosylation:

[1424] Thioglycoside building block (20) is dissolved in the proper solvent (6.5 eq. in 1.0 mL DCM) and loaded in the designated building block storing component. The reaction vessel is set to reach the initial glycosylation temperature. After the temperature reached the range of −35° C. to −26° C., the DCM in the reaction vessel is drained and 1 mL of thioglycoside building block 20 (6.5 eq. in 1.0 mL DCM) is delivered from the building block storing component to the reaction vessel via the pre-cooling device, which cools the solution of thioglycoside building block to a temperature of −15° C. Then, 1.0 mL NIS and TfOH solution in DCM and dioxane (v/v, 2:1) is delivered to the reaction vessel from the respective activator storing component via the pre-cooling device, which cools the activator solution down to a temperature of −15° C. The glycosylation mixture is incubated for 10 min at the temperature range of −35° C. to −26° C., the temperature linearly ramped during 5 min to 0° C. by microwave radiation, and after reaching 0° C. the reaction mixture is incubated for additional 30 min. Once incubation time is finished, the reaction mixture is drained and the resin is washed with DCM (once, 2 mL for 15 s). Then the resin is washed with 2 mL of DCM:dioxane 2:1 volume ratio. Finally, the resin is washed twice with DCM (2 mL for 15 s).

Module 8: Glycosylation:

[1425] Thioglycoside building block (18) is dissolved in the proper solvent (6.5 eq. in 1.0 mL DCM) and loaded in the designated building block storing component. The reaction vessel is set to reach the initial glycosylation temperature. After the temperature reached the range of −30° C. to −26° C., the DCM in the reaction vessel is drained and 1 mL of thioglycoside building block 18 (6.5 eq. in 1.0 mL DCM) is delivered from the building block storing component to the reaction vessel via the pre-cooling device which cools the solution of thioglycoside building block to a temperature of −15° C. Then, 1.0 mL NIS and TfOH solution in DCM and dioxane (v/v, 2:1) is delivered to the reaction vessel from the respective activator storing component via the pre-cooling device, which cools the activator solution down to a temperature of −15° C. The glycosylation mixture is incubated for 5 min at the temperature range of −30° C. to −26° C., the temperature linearly ramped during 5 min to 0° C. by microwave radiation, and after reaching 0° C. the reaction mixture is incubated for additional 20 min. Once incubation time is finished, the reaction mixture is drained and the resin is washed with DCM (once, 2 mL for 15 s). Then the resin is washed with 2 mL of DCM:dioxane 2:1 volume ratio. Finally, the resin is washed twice with DCM (2 mL for 15 s).

[1426] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 7) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM. The sequence of reaction steps for the formation of protected Lewis antigen 21 was as follows:

[1427] 1. Module 1 was performed with 1 mL TMSOTf solution at the temperature range of −22° C. to −16° C. (when it is programmed to do so) for 3 min.

[1428] 2. Module 2 was performed with 6.5 equiv Building Block 18 and 2% TMSOTf in DCM solution. In the temperature range of −22° C. to 0° C.

[1429] 3. Module 3 was carried out with 20% piperidine in DMF at the temperature range of 25° C. to 60° C. (when it is programmed to do so).

[1430] 4. Module 4 was performed with 1 mL TMSOTf solution at the temperature range of −30° C. to −26° C. (when it is programmed to do so) for 3 min.

[1431] 5. Module 5 was performed with 6.5 equiv Building Block 19 and 2% TMSOTf in DCM solution. In the temperature range of −35° C. to −10° C.

[1432] 6. Module 6 was carried out with 1% hydrazine acetate and 21% acetic acid in pyridine at the temperature range of 25° C. to 60° C. (when it is programmed to do so).

[1433] 7. Module 7 was performed with 6.5 equiv Building Block 20 and 2% TMSOTf in DCM solution in the temperature range of −35° C. to −10° C.

[1434] 8. Module 3 was carried out with 20% Piperidine in DMF at the temperature range of 25° C. to 60° C. (when it is programmed to do so).

[1435] 9. Module 4 was performed with 1 mL TMSOTf solution at the temperature range of −30° C. to −26° C. (when it is programmed to do so) for 3 min.

[1436] 10. Module 8 was performed with 6.5 equiv Building Block 18 and 2% TMSOTf in DCM solution in the temperature range of −35° C. to −10° C.

[1437] 11. Module 3 was carried out with 20% piperidine in DMF at the temperature range of 25° C. to 60° C. (when it is programmed to do so).

[1438] After build-up of the tetramer on the resin, the oligosaccharide was cleaved from solid support in a photoreactor as described in Example 5. The combined solution that was collected in the photocleavage process was evaporated in vacuo and the crude material was analyzed by MALDI-TOF, and HPLC. 14 mg of crude product were obtained, which correspond to a yield of 47%.

Example 13: Investigation of Pre-Cooled and Non-Precooled Building Block and Activator Solutions Addition for Highly Sensitive Building Blocks

[1439] ##STR00012##

[1440] In FIG. 27 the thermal profile during the synthesis of a Lewis antigen fragment in the presence and absence of pre-cooling is demonstrated. The reaction vessel is made of PFA, and a thioglycoside donor is used as building block, and active cooling of the reaction vessel by a cooling device is ON in both experiments. By using a pre-cooling device, the thermal perturbation due to the delivery of reagents is suppressed (solid line), specifically the delivery of activator reagents and donor solution. The washing solvent was not pre-cooled to highlight the stark contrast in the thermal spike.

[1441] In FIG. 28 the analytical results of the experiments as shown in FIG. 27 are shown. Mass spectrometry (MALDI) and HPLC chromatogram are provided. The experiment performed in presence of a pre-cooling device shows superior results: only the desired product was obtained (FIG. 28A). An overall yield of 68% respect to the loading was achieved recovering 8.7 mg of the product. The reaction without the pre-cooling (FIG. 28B) shows a HPLC chromatogram with deletion sequences (peaks at 33 min corresponding to the deletion sequence compound 24, the mass of such highly polar compound barely shows up on the MALDI spectra because the TCA (trichloroacetic acid) group).

Automated Synthesis Working Modules

[1442] The timing and quantity of solvents/reagents transferred to the reaction vessel in each step is controlled by software. The reagent delivery system utilizes pressure control valves, which constantly pressurize the entire platform, so that the specific solvent/reagent is transferred from the respective storage components by timing the opening and closing of the appropriate valves. All the solvents are pre-cooled before they are delivered inside the reaction vessel.

Module 1: Acidic Washing:

[1443] The resin loaded into the reaction vessel is washed with DMF, THF, DCM (six times each with 3 mL for 15 s). The resin is swollen in 2 mL DCM, and the temperature of the reaction vessel was adjusted in the range of −30° C. to −26° C. by cooling device (when it is programmed to do so). For acidic washing, 1 mL of the solution of 2% TMSOTf in DCM is delivered to the reaction vessel via the pre-cooling device, which cools the solution to a temperature of −15° C. (when it is programmed to do so). After three minutes, the solution is drained. Finally, 3 mL DCM is added to the reaction vessel.

Module 2: Glycosylation:

[1444] Thioglycoside building block (20) is dissolved in the proper solvent (6.5 eq. in 1.0 mL DCM) and loaded in the designated building block storing component. The reaction vessel is set to reach the initial glycosylation temperature. After the temperature reached the range of −30° C. to −26° C., the DCM in the reaction vessel is drained and 1 mL of thioglycoside building block 20 (6.5 eq. in 1.0 mL DCM) is delivered from the building block storing component to the reaction vessel via the pre-cooling device, which cools the solution of thioglycoside building block to a temperature of −15° C. Then, 1.0 mL NIS and TfOH solution in DCM and dioxane (v/v, 2:1) is delivered to the reaction vessel from the respective activator storing component via the pre-cooling device, which cools the activator solution down to a temperature of −15° C. The glycosylation mixture is incubated for 10 min at the temperature range of −30° C. to −26° C., the temperature linearly ramped during 5 min to 0° C. by microwave radiation, and after reaching 0° C. the reaction mixture is incubated for additional 30 min. Once incubation time is finished, the reaction mixture is drained and the resin is washed with DCM (once, 2 mL for 15 s). Then the resin is washed with 2 mL of DCM:dioxane 2:1 volume ratio. Finally, the resin is washed twice with DCM (2 mL for 15 s).

Module 3: Lev Deprotection:

[1445] The resin is washed with DCM (three times with 2 mL for 15 s). For Lev deprotection 2 mL of a solution of 1% hydrazine acetate and 21% acetic acid in pyridine was delivered to the reaction vessel. The temperature of the reagents inside the reactor vessel is adjusted between 40° C. and 20° C. by microwave irradiation (180 W). After 3 min, the reaction solution is drained from the reactor vessel. The resin is washed with DCM (three times with 2 mL for 15 s); the incubation in Lev deprotection solution between 40° C. and 20° C. by microwave irradiation (180 W) and the DCM washes were repeated twice more. Then, the resin is washed (3 times) with the following solvent sequence DMF, THF and DCM (3 mL for 15 s each). After this module the resin is ready for the next glycosylation cycle.

Module 4: Glycosylation:

[1446] Thioglycoside building block (21) is dissolved in the proper solvent (6.5 eq. in 1.0 mL DCM) and loaded in the designated building block storing component. The reaction vessel is set to reach the initial glycosylation temperature. After the temperature reached the range of −35° C. to −26° C., the DCM in the reaction vessel is drained and 1 mL of thioglycoside building block 21 (6.5 eq. in 1.0 mL DCM) is delivered from the building block storing component to the reaction vessel via the pre-cooling device, which cools the solution of thioglycoside building block to a temperature of −15° C. Then, 1.0 mL NIS and TfOH solution in DCM and dioxane (v/v, 2:1) is delivered to the reaction vessel from the respective activator storing component via the pre-cooling device, which cools the activator solution down to a temperature of −15° C. The glycosylation mixture is incubated for 10 min at the temperature range of −35° C. to −26° C., the temperature linearly ramped during 5 min to 0° C. by microwave radiation, and after reaching 0° C. the reaction mixture is incubated for additional 30 min. Once incubation time is finished, the reaction mixture is drained and the resin is washed with DCM (once, 2 mL for 15 s). Then the resin is washed with 2 mL of DCM:dioxane 2:1 volume ratio. Finally, the resin is washed twice with DCM (2 mL for 15 s).

Module 5: Fmoc Deprotection:

[1447] The resin is washed with DMF (three times with 3 mL for 15 s), swollen in 2 mL DMF. For Fmoc deprotection 2 mL of a solution of 20% piperidine in DMF was delivered to the reaction vessel. The temperature of the reagents inside the reactor vessel is adjusted between 70° C. and 20° C. by microwave irradiation (50 W). After 1 min the reaction solution is drained from the reactor vessel. Then, the resin is washed with DMF (three times with 3 mL for 15 s) and DCM (five times with 3 mL). After this module the resin is ready for the next glycosylation cycle.

[1448] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 9) was loaded into the reaction vessel of the synthesizer and swollen in 2 mL DCM. The sequence of reaction steps for the formation of protected Lewis antigen 23 was as follows:

[1449] 1. Module 1 was performed with 1 mL TMSOTf solution at the temperature range of −30° C. to −26° C. (when it is programmed to do so) for 3 min.

[1450] 2. Module 2 was performed with 6.5 equiv Building Block 20 and 2% TMSOTf in DCM solution in the temperature range of −35° C. to −10° C.

[1451] 3. Module 3 was carried out with 1% hydrazine acetate and 21% acetic acid in pyridine at the temperature range of 25° C. to 60° C. (when it is programmed to do so).

[1452] 4. Module 4 was performed with 6.5 equiv Building Block 21 and 2% TMSOTf in DCM solution in the temperature range of −35° C. to −10° C.

[1453] 5. Module 5 was carried out with 20% piperidine in DMF at the temperature range of 25° C. to 60° C. (when it is programmed to do so).

[1454] After build-up of the tetramer on the resin, the oligosaccharide was cleaved from solid support in a photoreactor as described in previous examples. The combined solution that was collected in the photo-cleavage process was evaporated in vacuo and the crude material was analyzed by MALDI-TOF, and HPLC. By peak integration of the chromatograph a 90% yield is observed from the reaction with pre-cooling 14 mg of crude product were obtained, which correspond to a yield of 47% respect to the resin loading. By peak integration of the chromatograph 50% yield is observed from the reaction without pre-cooling.