MICROWAVE-ASSISTED METHOD FOR SYNTHESIS OF OLIGO- AND POLYSACCHARIDES ON SOLID PHASE

Abstract

The present invention relates to a method for synthesizing oligo- and polysaccharides using microwave radiation, in particular to a method and a device for automated synthesis of oligo- and polysaccharides.

Claims

1. A method for synthesizing oligo- and polysaccharides, comprising the following steps: a) providing a solid support with at least one immobilized saccharide; b) adding a further saccharide bearing at least one protecting group and a glycosylation reagent to 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 under microwave irradiation.

2. The method according to claim 1, wherein step c) reads as follows: c) increasing the temperature after the coupling reaction of step b) by means of microwave irradiation and performing removal of the at least one protecting group of the further saccharide under microwave irradiation.

3. The method according to claim 1 or 2, wherein the saccharide immobilized on the solid support is a glycosyl acceptor comprising 1 to 20 monosaccharide units.

4. The method according to claim 1, wherein the further saccharide is a glycosyl donor comprising a glycal, epoxide or orthoester group or having a leaving group at the reducing end selected from halogen, —O—C(═NH)—CCl.sub.3, —O—C(═NPh)-CF.sub.3, —OAc, —SR.sup.5, —SO-Ph, —SO.sub.2-Ph, —O—(CH.sub.2).sub.3—CH═CH.sub.2, —O—P(OR.sup.5).sub.2, —O—PO(OR.sup.5).sub.2, —O—CO—OR.sup.5, —O—CO—SR.sup.5, —O—CS—SR.sup.5, —O—CS—OR.sup.5, ##STR00019## wherein R.sup.5 represents an alkyl or aryl group.

5. The method according to claim 1, wherein the at least one protecting group of the further saccharide is a temporary protecting group selected from allyl, p-methoxybenzyl, 2-naphthylmethyl, tri-isopropylsilyl, tert-butyldimethylsilyl, tert-butylmethoxyphenylsilyl, triethylsilyl, trimethylsilyl, 2-trimethylsilylethoxymethyl, 9-fluorenylmethoxycarbonyl and levulinoyl.

6. The method according to claim 1, wherein the immobilized saccharide comprises at least one permanent protecting group and/or the further saccharide comprises at least one permanent protecting group, in addition to the at least one protecting group, selected from acetyl, phenyl, benzyl, isopropylidene, benzylidene, benzoyl, p-methoxybenzyl, p-methoxybenzylidene, p-methoxyphenyl, p-bromobenzylidene, p-nitrophenyl, allyl, allyloxycarbonyl, monochloroacetyl, isopropyl, p-bromobenzyl, dimethoxytrityl, trityl, 2-naphthylmethyl, pivaloyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, tert-butylmethoxyphenylsilyl, triethylsilyl, trimethylsilyl, 2-trimethylsilylethoxymethyl, 9-fluorenylmethoxycarbonyl, tert-butyloxycarbonyl, benzyloxymethyl, methyloxymethyl, tert-butyloxymethyl, methoxyethyloxymethyl, and levulinoyl.

7. The method according to claim 1, wherein the solid support obtained in step b) is treated with a capping reagent, optionally under microwave irradiation.

8. The method according to claim 1, wherein the glycosylation reagent is a Lewis acid selected from: AgOTf, BF.sub.3.OEt.sub.2, trimethylsilyl trifluoromethanesulfonate, trifluoromethanesulfonic acid, trifluoromethanesulfonic anhydride, lanthanoid(III) triflates, NIS/AgOTf, NIS/TfOH or dimethyl(methylthio)sulfonium trifluoromethanesulfonate.

9. The method according to claim 1, wherein the removal of the at least one protecting group is performed at a reaction temperature of at least 40° C.

10. The method according to claim 1, wherein step b) is performed at a reaction temperature below 0° C. and optionally under microwave irradiation.

11. The method according to claim 1, wherein in step b) the glycosylation reagent is cooled to a temperature of at least 0° C. before addition to the further saccharide and the solid support.

12. The method according to claim 1 further comprising step a′) between step a) and step b): a′) washing the solid support of step a) with an acidic solution.

13. The method according to claim 1, wherein step b) is performed without microwave irradiation.

14. The method according to claim 1, wherein in step b) the coupling reaction is complete within 30 minutes reaction time.

15. The method according to claim 1 further comprising the steps: d) cleaving the saccharide obtained in step c) from the solid support; e) purifying the saccharide obtained in step d).

16. A synthesizer for microwave-assisted automated multistep synthesis of oligo- and polysaccharides on a solid support comprising: (a) a microwave transparent reaction vessel equipped with a temperature sensor, (b) a microwave generator component, (c) a reagent storing component, (d) a reagent delivery system, (e) a cooling device OW for cooling the microwave transparent reaction vessel, (f) a thermal controller for controlling the temperature inside the microwave transparent reaction vessel, wherein the thermal controller is connected to the cooling device OW and the temperature sensor and the microwave generator component, and is configured to control and adjust the temperature inside the microwave transparent reaction vessel by operating the cooling device OW and the microwave generator component.

17. The synthesizer comprising the components (a)-(e) as defined in claim 16 and comprising further: (f) a pre-cooling device for pre-cooling the reagents to be supplied to the microwave transparent reaction vessel, (g) a thermal controller for controlling the temperature inside the microwave transparent reaction vessel, wherein the thermal controller is connected to the cooling device and the temperature sensor and the microwave generator component and the pre-cooling device, and is configured to control and adjust the temperature inside the microwave transparent reaction vessel by operating the cooling device and the pre-cooling device and the microwave generator component.

Description

DESCRIPTION OF THE FIGURES

[0965] FIG. 1 provides examples of interconnecting molecules for immobilizing saccharides to solid support.

[0966] FIG. 2 provides examples of commercially available anchoring groups.

[0967] FIG. 3 shows the structures of exemplarily saccharide building blocks which can be used in the present invention.

[0968] FIG. 4 shows the structures of further exemplarily saccharide building blocks which can be used in the present invention.

[0969] FIG. 5A 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 (400). The Fmoc deprotection period is indicated.

[0970] FIG. 5B shows analytical results by MALDI from Example 11. No product was observed.

[0971] FIG. 6 shows the temperature readings inside the reaction vessel (400), taken during a synthesis cycle of the inventive method, in the presence and absence of pre-cooling and without microwave irradiation/heating.

[0972] FIG. 7 shows the thermal profile during a blank experiment. The reaction vessel (400) is made of PFA. The solid black line shows the temperature inside the reaction vessel (400); the solid gray line is the temperature at the wall of the jacket adjusted by an active cooling device (350). At the beginning, 3 mL of DCM were delivered in the reactor vessel. The temperature is adjusted by the active cooling device (350) from room temperature to −12° C. The increased to −2° C. by the active cooling device (350). The reaction vessel (400) was discharged and the temperature of the jacket was adjusted again to cool down to −12° C.; while Fmoc deprotection module is performed using microwave heating against the cooling effect of the cooling device (350). The microwave radiation was set with a maximum power of 50 W and regulated to maintain a temperature around 45° C. or at least below 60° C.

[0973] FIG. 8A 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 (400). The coupling and deprotection steps are indicated. The reaction was completed in 6 h and 30 min FIG. 8B shows analytical results by MALDI (the mass of the product is mark plus sodium ion) and HPLC of experiments from synthesis Example 12.

[0974] FIG. 9A shows charts comparing temperature readings inside the glass reaction vessel (400) and the chiller side, taken during a synthesis cycle via phosphate-glycosylation, with the post coupling reactions assisted by microwave irradiation.

[0975] FIG. 9B shows charts comparing temperature readings inside the glass reaction vessel (400), taken during a synthesis cycle via phosphate-glycosylation, with and without the microwave irradiation during the post coupling reactions.

[0976] FIG. 9C shows analytical results by MALDI and HPLC of experiments from FIG. 9A.

[0977] FIG. 10A shows charts comparing temperature readings inside the glass reaction vessel (400) and the chiller side, taken during a synthesis cycle via phosphate-glycosylation, with temperature adjustment during the coupling and post coupling reactions via microwave irradiation.

[0978] FIG. 10B shows analytical results by MALDI and HPLC of experiments from FIG. 10A.

[0979] FIG. 11A shows temperature charts comparing fast coupling (10 min, example 5) and regular coupling (25 min, example 4). The temperature inside the reaction 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 was, coupling/glycosylation, capping, deprotection. The fast coupling allows a complete cycle in less than 45 min, while regular coupling take above 1 h.

[0980] FIG. 11B shows analytical results by MALDI and HPLC of experiments from fast coupling synthesis example 5.

[0981] FIG. 12A 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 (400). The NAP deprotection period is indicated.

[0982] FIG. 12B shows analytical results by MALDI (the mass of the product is mark plus sodium ion) and HPLC of experiments from synthesis EXAMPLE 6.

[0983] FIG. 13A 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 (400). The Lev deprotection period is indicated.

[0984] FIG. 13B shows analytical results by MALDI (the mass of the product is mark plus sodium ion) and HPLC of experiments from synthesis Example 7.

[0985] FIG. 14A 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 (400). The Fmoc deprotection period is indicated.

[0986] FIG. 14B shows analytical results by MALDI (the mass of the product is mark plus sodium ion) and HPLC of experiments from synthesis Example 8.

[0987] FIG. 15A 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 (400). The chloroacetyl deprotection period is indicated.

[0988] FIG. 15B shows analytical results by MALDI (the mass of the product is mark plus sodium ion) and HPLC of experiments from synthesis Example 9.

[0989] FIG. 16A shows temperature charts of the first cycle during the synthesis of protected 5-(benzyl(benzyloxycarbonyl)amino)-pentyl 3-(α-D-mannopyranosyl)-4-(α-D-mannopyranosyl)-6-(α-D-mannopyranosyl)-α-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 (400). The coupling and deprotection periods are indicated.

[0990] FIG. 16B shows analytical results by MALDI (the mass of the product is mark plus sodium ion) and HPLC of experiments from synthesis Example 10.

[0991] FIG. 17A shows a block diagram illustrating the automated synthesizer (100) using the inventive methods.

[0992] FIG. 17B shows a block diagram illustrating the automated synthesizer (100) with a pre-cooling device (300) using the inventive methods.

[0993] FIG. 18A shows a block diagram illustrating the automated synthesizer (100) with a gas valve manifold using the inventive methods.

[0994] FIG. 18B shows in detail the manifold implemented in the inventive methods.

[0995] FIG. 18C shows the technical drawing of the manifold mounting layer (861) implemented in the inventive methods.

[0996] FIG. 18D shows the technical drawing of the manifold substrate layer (862) implemented in the inventive methods.

[0997] FIG. 18E shows the technical drawing of the manifold side element layer (863) implemented in the inventive methods.

LIST OF REFERENCE SIGNS

[0998] 100 synthesizer [0999] 200 computing device, processor [1000] 300 pre-cooling device [1001] 350 cooling device for reaction vessel [1002] 400 reaction vessel [1003] 450 temperature sensor [1004] 500 microwave generator component [1005] 600 reagent delivery system, top delivery system [1006] 630-632 reagent containers [1007] 660, 760 reagent storing component [1008] 700 reagent delivery system, bottom delivery system [1009] 704 waste container [1010] 800 inert gas delivery system [1011] 801 gas container [1012] 802 manifold [1013] 803-809, 848 pressure (regulator) valves [1014] 810 flow control valve [1015] 820, 820a-820h tubing connectors [1016] 823-829 pressure sensors [1017] 833-839 check valves [1018] 840 manifold line [1019] 841a-841n mounting feet [1020] 842a-f, 843 substrate channels [1021] 844a-844o substrates (elements) [1022] 845a-845g end substrate connectors [1023] 846a-846g substrate-to-manifold connectors [1024] 847a-847n lockdown bars [1025] 849a-849g pressure indicators [1026] 850a-850g two-port check valves [1027] 851 two ports metering valve with knurled handle [1028] 852 manifold output lines [1029] 861 first (mounting) layer [1030] 862 second (substrate) layer [1031] 863 third (side) layer [1032] 900 thermal controller

EXAMPLES

[1033] 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.

[1034] 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.

Abbreviations

[1035] Ac.sub.2O=Acetic anhydride [1036] ClAc=Chloroacetyl [1037] DBU=1,8-Diazabicyclo[5.4.0]undec-7-ene [1038] DDQ=2,3-Dichloro-5,6-dicyano-1,4-benzoquinone [1039] EGME=2-Methoxyethanol [1040] Fmoc=Fluorenylmethyloxycarbonyl [1041] HFIP=Hexafluoroisopropanol [1042] Lev=Levulinic ester [1043] MsOH=Methanesulfonic acid [1044] NAP=2-Naphthylmethyl ether [1045] NDM=1-Dodecanethiol [1046] TIS=Triisopropylsilane [1047] DCM=Dichloromethane [1048] DMF=N—N-Dimethylformamide [1049] DCE=1,2-Dichloroethane [1050] THF=Tetrahydrofuran

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

[1051] The improvement in conditions and reaction time for the capping and deprotection steps with microwave radiation was investigated.

[1052] 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 (500) 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.

TABLE-US-00001 TABLE 1 Without Microwave With Microwave Radiation Module Conditions Time Conditions Time Capping 2% MsOH in 25 min 2% MsOH in 5 min CH.sub.2Cl.sub.2/Ac.sub.2O CH.sub.2Cl.sub.2/Ac.sub.2O (5:1), 25° C. (5:1), 25° C. Deprotection: Lev 7% N.sub.2H.sub.4•HOAc in 90 min 7% N.sub.2H.sub.4•HOAc in 9 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), 35° C. Deprotection: NAP 2% DDQ in 240 min  2% DDQ in 100 min  DCE/MeOH/H.sub.2O DCE/MeOH (64:16:1), 40° C. (4:1), 45° C. Deprotection: Fmoc 20% Piperidine in  5 min 20% Piperidine in 1 min DMF, 25° C. DMF, 60° C. Deprotection: ClAc 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

[1053] The temperature development inside of the reaction vessel (400) during a synthesis cycle in the presence and absence of pre-cooling device (300) was investigated. FIG. 6 shows the results charts comparing temperature readings inside the reaction vessel (400), taken during a synthesis cycle, in the presence and absence of a pre-cooling device (300).

[1054] The three thermal stages are shown. Temperature spikes appear when a liquid is dispensed in the reaction vessel (400). 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.

Example 3: Synthesis of Mannose Tetramer Via Phosphate Donor with Auxiliary Reactions (Post-Coupling) Accelerated by Microwave Heating

[1055] ##STR00010##

[1056] Automated Synthesis Working Modules

[1057] The timing and quantity of solvents/reagents transferred to the reaction vessel (400) in each step is controlled by the software. The reagent delivery system (600) 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.

[1058] Module 1: Acidic Washing: The resin loaded into the reaction vessel (400) 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 (400) was adjusted in the range of −22° C. to −20° C. by cooling device (350) (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 (400) via the pre-cooling device (300) 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 (400).

[1059] Module 2: Glycosylation: 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 (400) 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 (400) 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 (400) via the pre-cooling device (300) which cools the solution of phosphate building block (when it is programmed to do so) to a temperature of −18° C. After the set temperature in the range of −22° C. to −20° 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 (400) from the respective activator storing component via the pre-cooling device (300) 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., linearly ramped to 0° C. (when it is programmed 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).

[1060] Module 3: Capping: While the temperature of the active cooling element is adjusted to range of −22° C. to −20° C. preparing for the next coupling cycle. The temperature of the reactor vessel is adjusted between 70 and 20° C. by microwave irradiation of the washing solvents 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) was delivered and the resin is incubated for one minute. Up to this point the microwave irradiation power is adjust at 50 W. The microwave irradiation power is then adjusted 150-180 W to proceed with 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 one minute; then 1 mL of DCM in added to dilute the solution and the incubation continues for another one minute. The solution is drained from the reactor vessel and the resin is washed 3 time with DCM (2 mL for 15 s).

[1061] Module 3: Fmoc Deprotection: The temperature of the reactor vessel is adjusted between 70 and 20° C. by microwave irradiation of the washing solvents and reagents adjusting the irradiation power (40 W). 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 (400) is adjusted between 70-20° 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 (400). 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.

[1062] Resin functionalized with a photo-cleavable linker (64 mg; loading 0.392 mmol/g; 25.1 μmol) was loaded into the reaction vessel (400) of the synthesizer (100) and swollen in 2 mL DCM. The sequence of reaction steps for the formation of tetramannose was as follows:

[1063] 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.

[1064] 2. Module 2 was performed with 5 equiv Building Block and 2% TMSOTf in DCM solution.

[1065] 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.

[1066] 4. Module 4 was carried out with 20% piperidine in DMF.

[1067] 5. After Module 1 took place the Module 2 repeated twice (for the first coupling). The Modules 3 and 4 were then performed.

[1068] 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.

[1069] 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 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.

Example 4: Synthesis of Mannose Tetramer Via Phosphate Donor with Temperature Regulation Via Microwave Heating

[1070] ##STR00011##

[1071] Automated Synthesis Working Modules

[1072] The timing and quantity of solvents/reagents transferred to the reaction vessel (400) in each step is controlled by the software. The reagent delivery system (600) 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.

[1073] Module 1: Acidic Washing: The resin loaded into the reaction vessel (400) 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 (400) was adjusted in the range of −22° C. to −20° C. by cooling device (350) (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 (400) via the pre-cooling device (300) 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 (400).

[1074] Module 2: Glycosylation: 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 (400) 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 (400) 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 (400) via the pre-cooling device (300) which cools the solution of phosphate building block (when it is programmed to do so) to a temperature of −18° C. After the set temperature in the range of −22° C. to −20° 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 (400) from the respective activator storing component via the pre-cooling device (300) 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 10 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. (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 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).

[1075] Module 3: Capping: 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 adjusted between 70 and 20° C. by microwave irradiation of the washing solvents 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) was delivered and the resin is incubated for one minute. Up to this point the microwave irradiation power is adjust at 50 W. The microwave irradiation power is then adjusted 150-180 W to proceed with 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 1 min; 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).

[1076] Module 3: Fmoc Deprotection: The temperature of the reactor vessel is adjusted between 70 and 20° C. by microwave irradiation of the washing solvents and reagents adjusting the irradiation power (40 W). 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 (400) is adjusted between 70-20° 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 (400). 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.

[1077] Resin functionalized with a photo-cleavable linker (64 mg; loading 0.392 mmol/g; 25.1 μmol) was loaded into the reaction vessel (400) of the synthesizer (100) and swollen in 2 mL DCM. The sequence of reaction steps for the formation of tetramannose was as follows:

[1078] 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.

[1079] 2. Module 2 was performed with 5 equiv Building Block and 2% TMSOTf in DCM solution.

[1080] 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.

[1081] 4. Module 4 was carried out with 20% Piperidine in DMF.

[1082] 5. After Module 1 took place the Module 2 repeated twice (for the first coupling). The Modules 3 and 4 were then performed.

[1083] 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.

[1084] 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 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.

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

[1085] 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.

##STR00012##

[1086] Automated Synthesis Working Modules

[1087] The timing and quantity of solvents/reagents transferred to the reaction vessel (400) in each step is controlled by software. The reagent delivery system (600) 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 (400).

[1088] Module 1: Acidic Washing: The resin loaded into the reaction vessel (400) 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 (400) was adjusted in the range of −22° C. to −20° C. by cooling device (350) (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 (400) via the pre-cooling device (300) 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 (400).

[1089] Module 2: Glycosylation: 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 (400) 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 (400) 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 (400) via the pre-cooling device (300) 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 (400) from the respective activator storing component via the pre-cooling device (300) 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 2 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 an additional 3 min. Once incubation time is finished, the reaction mixture is drained and the resin is washed with DCE (once, 2 mL for 5 s).

[1090] Module 3: Capping: 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.

[1091] 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).

[1092] Module 4: Fmoc Deprotection: 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 (400). 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.

[1093] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 1) was loaded into the reaction vessel (400) of the synthesizer (100) and swollen in 2 mL DCM.

[1094] The sequence of reaction steps for the formation of tetramannose 9 was as follows:

[1095] 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.

[1096] 2. Module 2 was performed with 5 equiv Building Block and 2% TMSOTf in DCM solution.

[1097] 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.

[1098] 4. Module 4 was carried out with 20% piperidine in DMF.

[1099] 6. Subsequently, modules 1-4 were repeated four times in order to obtain a tetramer.

[1100] 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-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 photoreactor with additional 18 mL DCM (flow rate: 700 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 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 photocleavage process was evaporated in vacuo and the crude material was analyzed by MALDI-TOF, and HPLC.

[1101] After weighing, the recovered crude was 27 mg, which correspond to a 65% yield. This experiment demonstrates that high yields were obtained by combining 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 the Presence of Fmoc-, Lev-, ClAc- Temporal Protecting Groups

[1102] ##STR00013##

[1103] Automated Synthesis Working Modules

[1104] The timing and quantity of solvents/reagents transferred to the reaction vessel (400) in each step is controlled by software. The reagent delivery system (600) 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.

[1105] Module 1: Acidic Washing: The same acidic washing module was applied as in Example 5.

[1106] Module 2: Glycosylation: 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 (400) 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 (400) 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 (400) via the pre-cooling device (300), 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 (400) from the respective activator storing component via the pre-cooling device (300), 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).

[1107] Module 3: NAP Deprotection (ca. 60 min): 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 (400). 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.

[1108] Then, the resin is washed (3 times) with the following solvent sequence DMF, THF and DCM (3 mL for 120 s each). After this module, the resin is ready for the next glycosylation cycle.

[1109] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 2) was loaded into the reaction vessel (400) of the synthesizer (100) and swollen in 2 mL DCM.

[1110] The sequence of reaction steps for the formation of tetramannose was as follows:

[1111] 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.

[1112] 2. Module 2 was performed twice with 5 equiv Building Block and 2% TMSOTf in DCM solution.

[1113] 3. Module 3 was carried out with 2% DDQ and 13% methanol in DCE.

[1114] 4. After Module 1 took place, the Module 2 repeated twice (for the first and last coupling). Then Module 3 was performed.

[1115] 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.

[1116] After buildup of the tetramer on the resin the oligosaccharide was cleaved from solid support in a photoreactor as described in Example 5. The desired product was obtained in 10% yield estimated by HPLC analysis. (see FIG. 12B)

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-, ClAc- Temporal Protecting Groups

[1117] ##STR00014##

[1118] Automated Synthesis Working Modules

[1119] The timing and quantity of solvents/reagents transferred to the reaction vessel (400) in each step is controlled by software. The reagent delivery system (600) 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.

[1120] Module 1: Acidic Washing: The same acidic washing module was applied as in Example 5.

[1121] Module 2: Glycosylation: 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 (400) 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 (400) 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 (400) via the pre-cooling device (300) 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 (400) from the respective activator storing component via the pre-cooling device (300) 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).

[1122] Module 3: Lev Deprotection (ca. 5 min): 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 (400). 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, THF and DCM (3 mL for 15 s each). After this module the resin is ready for the next glycosylation cycle.

[1123] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 3) was loaded into the reaction vessel (400) of the synthesizer (100) and swollen in 2 mL DCM.

[1124] The sequence of reaction steps for the formation of 5-Amino-pentyl α-(1.fwdarw.3)-D-tetramannopyranoside was as follows:

[1125] 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.

[1126] 2. Module 2 was performed twice with 5 equiv Building Block and 2% TMSOTf in DCM solution.

[1127] 3. Module 3 was carried out with 1% hydrazine acetate and 21% acetic acid in pyridine.

[1128] 4. After Module 1 took place the Module 2 repeated twice (for the first coupling). Then Module 3 was performed.

[1129] 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.

[1130] 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-, ClAc- Temporal Protecting Groups

[1131] ##STR00015##

[1132] Automated Synthesis Working Modules

[1133] The timing and quantity of solvents/reagents transferred to the reaction vessel (400) in each step is controlled by software. The reagent delivery system (600) 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.

[1134] Module 1: Acidic Washing: The same acidic washing module was applied as in Example 5.

[1135] Module 2: Glycosylation: The same glycosylation module was used as in Example 6.

[1136] Module 3: Fmoc Deprotection (ca. 5 min): 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 (400). 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.

[1137] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 4) was loaded into the reaction vessel (400) of the synthesizer (100) 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:

[1138] 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.

[1139] 2. Module 2 was performed twice with 5 equiv Building Block and 2% TMSOTf in DCM solution.

[1140] 3. Module 3 was carried out with 20% triethylamine in DMF.

[1141] 4. After Module 1 took place the Module 2 repeated twice (for the first coupling). Then Module 3 was performed.

[1142] 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.

[1143] 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.

[1144] 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 ClAc Temporal Protecting Group in the Presence of Fmoc-, Lev-, NAP- Temporal Protecting Group

[1145] ##STR00016##

[1146] Automated Synthesis Working Modules

[1147] The timing and quantity of solvents/reagents transferred to the reaction vessel (400) in each step is controlled by software. The reagent delivery system (600) 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.

[1148] Module 1: Acidic Washing: The resin loaded into the reaction vessel (400) 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 (400) was adjusted in the range of −22° C. to −20° C. by cooling device (350) (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 (400) via the pre-cooling device (300) 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 (400).

[1149] Module 2: Glycosylation: 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 (400) 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 (400) 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 (400) via the pre-cooling device (300) 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 (400) from the respective activator storing component via the pre-cooling device (300), 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).

[1150] Module 3: ClAc (ca. 5 min): The resin is washed with DMF (three times with 2 mL for 15 s). For ClAc deprotection 2 mL of a solution of 4% thiourea and 9% of pyridine in 2-methoxyethanol was delivered to the reaction vessel (400). 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 ClAc 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.

[1151] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 5) was loaded into the reaction vessel (400) of the synthesizer (100) and swollen in 2 mL DCM.

[1152] The sequence of reaction steps for the formation of 5-Amino-pentyl α-(1.fwdarw.6)-D-tetramannopyranoside was as follows:

[1153] 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.

[1154] 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.

[1155] 3. Module 3 was carried out with 4% thiourea and 9% of pyridine in 2-methoxyethanol.

[1156] 4. After Module 1 took place the Module 2 repeated twice (for the first coupling). Then Module 3 was performed.

[1157] 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.

[1158] After buildup of the tetramer on the resin, the oligosaccharide was cleaved from solid support in a photoreactor as described in Example 8. 22 mg of crude product were obtained, which correspond to a yield of 58%.

Example 10: Synthesis of a Protected 5-(benzyl(benzyloxycarbonyl)amino)-pentyl 3-(α-D-mannopyranosyl)-4-(α-D-mannopyranosyl)-6-(α-D-mannopyranosyl)-α-D-mannopyranoside Via Phosphate Glycosylation and Selective Deprotection of a Donor Bearing Four Temporal Protecting Groups

[1159] ##STR00017##

[1160] Automated Synthesis Working Modules

[1161] The timing and quantity of solvents/reagents transferred to the reaction vessel (400) in each step is controlled by software. The reagent delivery system (600) 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.

[1162] Module 1: Acidic Washing: The same acidic washing module was applied as in Example 5.

[1163] Module 2: Glycosylation: The same glycosylation module was used as in Example 7.

[1164] Module 3: ClAc (ca. 5 min): The same chloroacetyl deprotection module was used as in Example 9.

[1165] Module 4: Fmoc Deprotection (ca. 5 min): The same Fmoc deprotection module was used as in Example 8.

[1166] Module 5: Lev Deprotection (ca. 5 min): The same Lev deprotection modules was used as in Example 7.

[1167] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 6) was loaded into the reaction vessel (400) of the synthesizer (100) and swollen in 2 mL DCM.

[1168] The sequence of reaction steps for the formation of 3-(α-D-mannopyranosyl)-4-(α-D-mannopyranosyl)-6-(α-D-mannopyranosyl)-α-D-mannopyranoside was as follows:

[1169] 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.

[1170] 2. Module 2 was performed twice with 5 equiv Building Block and 2% TMSOTf in DCM solution.

[1171] 3. Module 3 was carried out with 4% thiourea and 9% of pyridine in 2-methoxyethanol.

[1172] 4. Module 4 was carried out with 20% triethylamine in DMF.

[1173] 5. Module 5 was carried out with 1% hydrazine acetate and 21% acetic acid in pyridine.

[1174] 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.

[1175] 5. Subsequently, Module 2 repeated three times in order to obtain a tetramer 17.

[1176] 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

[1177] 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.

[1178] Automated Synthesis Working Modules

[1179] The timing and quantity of solvents/reagents transferred to the reaction vessel (400) in each step is controlled by software. The reagent delivery system (600) 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 (400). All solvents were pre-cooled before they are delivered inside the reaction vessel (400).

[1180] Module 1: Glycosylation: The same glycosylation module was used as in Example 5.

[1181] Module 2: Fmoc Deprotection: The same Fmoc deprotection module was used as in Example 5.

[1182] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 1) was loaded into the reaction vessel (400) of the synthesizer (100) and swollen in 2 mL DCM.

[1183] The sequence of reaction steps for the formation of tetramannose 9 was as follows:

[1184] 1. Module 1 was performed with 5 equivalent Building Block and 2% TMSOTf in DCM solution.

[1185] 2. Module 2 was carried out with 20% piperidine in DMF.

[1186] 3. Subsequently, Modules 1 and 2 were repeated four times in order to obtain a tetramer.

[1187] 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. 5B).

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

[1188] ##STR00018##

[1189] Automated Synthesis Working Modules

[1190] The timing and quantity of solvents/reagents transferred to the reaction vessel (400) in each step is controlled by software. The reagent delivery system (600) 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 (400).

[1191] Module 1: Acidic Washing: The resin loaded into the reaction vessel (400) 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 (400) was adjusted in the range of −20° C. to −16° C. by cooling device (350) (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 (400) via the pre-cooling device (300), 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 (400).

[1192] Module 2: Glycosylation: 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 (400) 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 (400) 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 (400) via the pre-cooling device (300), 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 (400) from the respective activator storing component via the pre-cooling device (300), 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).

[1193] Module 3: Fmoc Deprotection: 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 (400). 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.

[1194] Module 4: Acidic Washing: The resin loaded into the reaction vessel (400) 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 (400) was adjusted in the range of −30° C. to −26° C. by cooling device (350) (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 (400) via the pre-cooling device (300), 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 (400).

[1195] Module 5: Glycosylation: 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 (400) 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 (400) 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 (400) via the pre-cooling device (300), 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 (400) from the respective activator storing component via the pre-cooling device (300), 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).

[1196] Module 6: Lev Deprotection: 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 (400). 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.

[1197] Module 7: Glycosylation: 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 (400) 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 (400) 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 (400) via the pre-cooling device (300), 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 (400) from the respective activator storing component via the pre-cooling device (300), 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).

[1198] Module 8: Glycosylation: 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 (400) 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 (400) 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 (400) via the pre-cooling device (300) 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 (400) from the respective activator storing component via the pre-cooling device (300), 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).

[1199] The resin functionalized with a photo-cleavable linker (45 mg; loading 0.30 mmol/g) (see Scheme 7) was loaded into the reaction vessel (400) of the synthesizer (100) and swollen in 2 mL DCM. The sequence of reaction steps for the formation of protected Lewis antigen 21 was as follows:

[1200] 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.

[1201] 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.

[1202] 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).

[1203] 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.

[1204] 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.

[1205] 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).

[1206] 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.

[1207] 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).

[1208] 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.

[1209] 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.

[1210] 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).

[1211] After buildup 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%.