Method and device for producing saccharides and saccharide arrays

Abstract

The present invention relates to a method and a device for producing saccharides and saccharide arrays. Said method is particularly useful for the synthesis of saccharides in parallel and of high-density saccharide arrays, such as microarrays, which are required for high-throughput screenings.

Claims

1. A method for synthesizing saccharide comprising the steps: A) providing a solid support with at least one immobilized acceptor group for reacting with a saccharide; B) delivering the saccharide onto the solid support; C) applying a vapor of a mixture of a glycosylation reagent and a solvent onto the solid support at a temperature below 20° C. in order to initiate a coupling reaction of the saccharide to the at least one immobilized acceptor group.

2. The method according to claim 1, wherein step C) is carried out at a temperature below 5° C.

3. The method according to claim 1, wherein the ratio of the solvent and the glycosylation reagent is in the range of 1:10 to 100,000:1.

4. The method according to claim 1, wherein the solvent is an aprotic organic solvent selected from: methylene chloride, chloroform, acetonitrile, diethyl ether, 1,4-dioxane, methyl tert-butyl ether, toluene and ethyl acetate.

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

6. The method according to claim 1, wherein the saccharide is a protected 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, ##STR00025## ##STR00026## wherein R.sup.5 represents an alkyl or aryl group.

7. The method according to claim 1, wherein in step B) the saccharide is a monosaccharide.

8. The method according to claim 1, further comprising step C′) between step B) and step C): C′) drying the solid support obtained in step B) under reduced pressure and/or heating.

9. The method according to claim 1, further comprising step K) K) performing removal of protecting groups from the saccharide obtained in step C).

10. The method according to claim 1, wherein the at least one immobilized acceptor group for reacting with the saccharide is located at discrete locations forming an array on the solid support.

11. The method according to claim 1, further comprising steps L) and M) L) cleaving the saccharide from the solid support; and M) optionally purifying the saccharide obtained from step L).

12. A method of detecting antibodies or glycan-binding proteins in a test sample comprising contacting the test sample with a saccharide array obtained by the method of claim 10 and observing whether one or more saccharides are bound by an antibody or glycan-binding protein in the test sample.

13. A saccharide synthesizer comprising: a substrate, the substrate having a surface and being configured to support a solid support with at least one immobilized acceptor group for reacting with a saccharide; means for delivering a saccharide to a solid support with at least one immobilized acceptor group for reacting with a saccharide supported by the substrate; a chamber comprising a process space; a vapor supply in fluid communication with the process space, the vapor supply configured to supply a vapor comprising a solvent and a glycosylation reagent to the process space; a cooling element positioned within the processing chamber configured to cool the solid support by heat transfer through the substrate; an exhaust port in the processing chamber configured in fluid communication with an isolation valve; a purge gas supply in fluid communication with the process space, the purge gas supply configured to supply a purge gas to the process space effective to displace the vapor from the process space, wherein the substrate is positioned in the process space of the chamber.

14. The synthesizer according to claim 13, wherein the means for delivering a saccharide to a solid support comprises a capillary needle which is in fluid connection with a reservoir containing a saccharide, optionally a syringe connected to said capillary needle or optionally a microactuator connected to said capillary needle; or a laser for transferring the saccharide in a polymer matrix onto the solid support.

15. The synthesizer according to claim 13, wherein the cooling element of the chamber cools the solid support below a temperature of 5° C.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 illustrates the inventive method for producing a saccharide on a cellulose membrane by spotting a solution of saccharide building block on to the cellulose membrane.

(2) FIG. 2 illustrates the inventive method for high-throughput synthesis of a high-density saccharide array.

(3) FIG. 3 provides examples of commercially available anchoring groups.

(4) FIG. 4 provides examples of interconnecting molecules for immobilizing saccharides to solid support.

(5) FIG. 5 shows the structures of exemplarily saccharide building blocks which can be used in the present invention.

(6) FIGS. 6A, B and C illustrate different embodiments of the chamber of the inventive synthesizer.

(7) FIG. 7 shows the preparation of acceptor slide for laser transfer of a glycosyl donor.

(8) FIG. 8 shows a setup of a vapor annealing coupling chamber and a vapor generator; the U-shaped vapor generator comprises a frit, a gas inlet, a gas outlet, an inlet for glycosylation reagent located above the frit, the generator is located in a water bath of 50° C.; the coupling chamber is equipped with a thermoelectric cooling element for controlling the reaction temperature, wherein the temperature can be controlled with a computer.

(9) FIG. 9 shows the reaction scheme of a vapor-triggered glycosylation on a slide.

(10) FIG. 10 shows complete disaccharide synthesis on glass slide using the inventive method.

(11) FIG. 11 shows an alternative setup of a vapor annealing coupling chamber.

(12) FIG. 12 shows MALDI-TOF-MS spectra after vapor-triggered glycosylation. On the left, disaccharide obtained by glycosylation of trichloroacetimidate 8 with functionalized cellulose membrane A; on the right, direct glycosylation of phosphate on functionalized polypropylene membrane B.

(13) FIG. 13 shows MALDI-TOF-MS spectra of compound 10.

(14) FIG. 14 Parameters for the spotting of glycosyl donor dissolved in dichloromethane. A solution of 200 mg of the building block in dichloromethane will be used. PG=protecting group, LG=leaving group. These parameters change for acetonitrile, which results in twice the radius (1 μL: r=3 mm, 2 μL: r=5 mm, 3 μL: r=7 mm, 4 μL: r=9 mm).

(15) FIG. 15 shows MALDI-TOF MS spectra: upper spectrum corresponds to the membrane area where only the glucose donor 13 was spotted; lower spectrum: corresponds to the membrane area where only the mannose donor 8 was spotted.

(16) The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled 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 skilled 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.

(17) 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.

EXAMPLES

(18) Chemicals & Reagents

(19) Unless otherwise stated, all chemicals and reagents were acquired from Sigma-Aldrich, TCI, Iris Biotech, ROTH, Acros, Merck, or Alfa Aesar and were of the highest purity available. Solid supports were obtained from PolyAn, Schott, or SurModics. Saccharide building blocks were obtained from GlycoUniverse or prepared by using a commercial saccharide synthesizer.

Example 1: Optimization of Vapor Triggered Glycosylation on a Functionalized Glass Slide

(20) 1.1 Acceptor slide preparation: Commercially available amine-functionalized glass slides from PolyAn GmbH were used as acceptor slides. The slides were functionalized with a C.sub.11-spacer (see FIG. 7) in presence of diisopropyl-carbodiimide (DIC) and hydroxybenzotriazole (HOBt) by contacting two slides with the Fmoc-protected C.sub.11-spacer 4 in between (sandwich) overnight. Unreacted free amine groups on the slide surface were capped using a mixture of Ac.sub.2O/DIPEA/DMF. Fmoc-group of the C.sub.11-spacer was removed by treating the slide with 20% piperidine in DMF. Galactose acceptor molecule 6 was coupled to the C11-acceptor in presence of DIC and HOBt by contacting two slides with the acceptor 6 in between (sandwich) overnight. Unreacted amino groups were capped using a mixture of Ac.sub.2O/DIPEA/DMF. The Fmoc group was removed treating the slide with 20% piperidine in DMF.

(21) 1.2 Donor slide preparation: All donor slides were generated by spin-coating a solution of the glycosyl donor 8 (building block) and an inert polymer matrix (SLEC PLT 7552, Sekisui Chemical GmbH, Düsseldorf/Germany) in dichloromethane on a glass slide covered with a polyimide foil (Kapton®). The inert polymer matrix forms a protective layer and shields the building block from environmental influences, while the Kapton® foil is needed for laser light absorption and heat induction. The donor composition is shown in table 1 below.

(22) TABLE-US-00001 TABLE 1 Composition of donor solution. glycosyl donor Matrix- dichloromethane entry 8 (mg) SLEC (mg) (mL) 1 50 50 1   2 25 25 0.5

(23) Chemical structures of compounds 4, 6 and 8 are shown FIG. 10.

(24) 1.3 Laser transfer: The glycosyl imidate 8 was transferred with laser induced forward transfer (cLIFT) onto the acceptor slide using the following laser parameters:

(25) 100 mW, 20 μm focus diameter, 7 ms pulse duration, 200 μm spot pitch. The donor slide was placed on top of the acceptor, and the laser light reached the Kapton® foil. The heat, which is produced via laser irradiation, deforms the donor slide, thus bringing the two layers in contact. The Kapton® foil, which is stable under short-term heat exposure, expands slightly due to the heat from the laser, transferring the desired compound from the donor to the acceptor slide.

(26) 1.4 Vapor coupling (CVAS): The acceptor slide covered with building block and matrix material was placed on a thermoelectric cooling element in a chamber for glycosylation as shown in FIG. 8. The chamber was then evacuated through the connection (connected to a common vacuum gas manifold, i.e. Schlenk line) on top of the chamber and flushed with argon for three times. Then, the temperature was reduced to −12° C. under inert atmosphere to achieve satisfactory deposition of solvent and activator inside the setup at low temperature. The chilled surface area of the chamber is 56.3 cm.sup.2 (7.5 cm×7.5 cm), where the slide is placed, while for the whole vapor chamber volume is 338 cm.sup.3 (13 cm×13 cm×2 cm).

(27) The glycosylation solution containing dichloromethane and activator was bubbled for 2.3 min under inert atmosphere (FIG. 8) until complete transfer of the solution inside the glycosylation-vapor chamber. The conditions for the glycosylation reaction are summarized in Table 2. The acceptor slide is left to react under vapor for 30 min in a closed setup. After completion, the acceptor-setup was warmed up to rt under vacuum. Then, the slide was removed from the setup and washed with dimethylformamide and dichloromethane. Deprotection of the benzoyl groups was accomplished under inert atmosphere using anhyd. methanol and K.sub.2CO.sub.3 overnight. Screening of the result was achieved after fluorescent staining with the tetravalent fluorescently red labelled (λ=633 nm) Concanavalin A lectin (binds selectively to α-mannopyranosyl residues), to verify the successful glycosylation (see FIG. 9).

(28) TABLE-US-00002 TABLE 2 Conditions of glycosylation reaction. Glycosylation conditions using setup shown in FIG. 7 Donor Bubbling Dichloro- TMSOTf T.sub.glyc T.sub.bath Reaction Entry Preparation time (min) methane (μL) (μL) (° C.) (° C.) time A Entry 1 2.3 1500 150 μL −12 55 30 min B Entry 2 1.0 400  25 μL −12 57 30 min C Entry 2 1.3 400  75 μL −12 40 30 min D Entry 1 1.3 400  75 μL −12 40 30 min

(29) In all four runs spots were observed after Concanavalin A staining, indicating the successful glycosylation of immobilized acceptor 6 with donor 8. Most prominent and homogenous spots were observed in runs at lower temperature of the glycosylation mixture, C and D; thereby leading to the conclusion that a slow and homogeneous transfer of the solution inside the setup takes place.

Example 2: Vapor Triggered Glycosylation on a Functionalized Glass Slide

(30) 2.1 Acceptor slide preparation: The vapor triggered glycosylation reaction of a glycosyl donor and a glycosyl acceptor on a functionalized glass slide (solid support) was accomplished on a commercially available 3D amine microarray slide from PolyAn GmbH (Berlin). In this case also other functionalized glass slides from other companies like carboxyl-(NHS-activated or not), epoxy-, maleimide-, thiol-, azide-, hydroxyl-, tetrazine-, aldehyde- or alkyne-surfaces may be used. Therefore the functional groups can be used on the surfaces directly for the attachment of a linker, spacer, interconnecting molecule (see FIG. 4) or saccharide building block (see FIG. 5) or convert them into another functional group, which can then be used for connection between the above mentioned species and the glass slide. Another option would be a self-synthesized microarray glass slide for this approach.

(31) To perform the glycosylation method—the glycosylation on a solid support using vapor—the 3D amine microarray slide from PolyAn GmbH (Berlin) was functionalized for this purpose. Therefore, first the commercially available (Iris Biotech GmbH, Marktredwitz) photocleavable linker 2a were attached on the slide, followed by spacer 4 and finally by modified galactose building block 6 (FIG. 10). All these attachments were done in solution by standard amide bond formation using the conditions shown below.

(32) Conditions for Photo-Linker 2a Attachment:

(33) Photo-linker 2a: 26.0 mg, 50 μmol DIC: 23.2 μl, 18.9 mg, 150 μmol HOBt: 6.76 mg, 50 μmol DMF: 250 μL Temperature: rt Time: 4-16 h
Conditions for Spacer 4 Attachment: Spacer 4: 21.2 mg, 50 μmol DIC: 23.2 μl, 18.9 mg, 150 μmol HOBt: 6.76 mg, 50 μmol DMF: 250 μL Temperature: rt Time: 4-16 h
Conditions for Galactose Building Block 6 Attachment: Galactose 6: 18.6 mg, 25 μmol DIC: 11.6 βl, 9.45 mg, 75 μmol HOBt: 3.40 mg, 25 μmol DMF: 250 μL Temperature: rt Time: 4-16 h

(34) The galactose building block was functionalized with a free carboxylic acid group to form an amide bond between the sugar moiety and the solid support. With the pre-functionalized surface 7 (FIG. 10) in hand, the vapor induced glycosylation method on a solid support was realized.

(35) 2.2 Donor slide preparation: First of all, the donor slide was prepared as followed for the laser induced forward transfer (cLIFT) of the glycosyl donor 8 (structure shown in FIG. 10).

(36) Conditions for Spin Coating Process of Glycosyl Imidate 8:

(37) Glycosyl imidate 8: 50 mg Polymer matrix (S-Lec): 50 mg DCM (dry): 1.00 mL Spin coater speed: 80 rounds per second Temperature: rt

(38) 2.3 Laser transfer: The glycosyl imidate 8 was transferred with laser induced forward transfer (cLIFT) onto the acceptor slide 7 using the following laser parameters: 100 mW, 20 μm focus diameter, 7 ms pulse duration, 200 μm spot pitch. For the detection of the molecules via mass spectrometry the whole surface area of the donor slide was transferred to the acceptor slide and for the detection of the molecules using fluorescently labeled Concanavalin A lectin (binds selectively to α-mannopyranosyl residues) a spot pattern was transferred to the acceptor slide (in this case no linker or spacer is needed). Thereby no coupling reaction is initiated, which is very important for the process. The amount of the building block which is transferred with this approach is typically in a micro to nanomolar range. Approach is typically in a micro to nanomolar range.

(39) 2.4 Vapor coupling (CVAS): The acceptor slide covered with building block and matrix material was placed in the chamber shown in FIG. 11 on a steel block. The chamber was then evacuated through the connection (connected to a common vacuum gas manifold, i.e. Schlenk line) on top of the chamber and flushed with argon for three times. The valve to the Schlenk line was closed and the whole setup was placed in the freezer at −20° C. for 1.5 h chilling the steel surface. The chamber was taken out of the freezer and was reconnected to the Schlenk line and evacuated and flushed with argon once more (the valve to the Schlenk line was kept open). Through the neck on the side of the chamber 20 mL of dry dichloromethane (DCM) were added into the chamber around the cold metal block. Additionally 400 μL of TMSOTf were added the same way. The chamber was evacuated for just as short as possible, switching the Schlenk line valve on and off by hand and then a constant flow of argon was connected to the chamber and the glycosylation was carried out for 1 h while the temperature inside the chamber increases slowly up to room temperature. To quench the reaction 1.00 mL of triethylamine were poured on the slide through the neck on the side of the chamber. The slide was taken out of the chamber, washed with different solvents and the molecule 10 was cleaved from the glass substrate via UV irradiation for the mass spectrometry approach and detected by MALDI-TOF-MS (FIG. 13). For the lectin staining approach after deprotection of the benzoyl groups (K.sub.2CO.sub.3 in MeOH) on the sugar moiety, the result was visualized by incubation of the slide with fluorescently labeled Concanavalin A in a HEPES-buffer and subsequent fluorescence scan.

Example 3: Glycosylation on Functionalized Membranes

(40) Cellulose membranes functionalized with β-alanine (A, see 12A in scheme below) were obtained from AIMS Scientific Products GmbH and polypropylene membranes (B, see 12B in scheme below) were obtained from AIMS Scientific Products GmbH (hydroxy-functionalized) and PolyAn GmbH (amino-functionalized). The membranes were functionalized with a photo cleavable linker 2a, a spacer 4 and the glycosyl imidate 6 to obtain modified cellulose and polypropylene membrane. An exemplarily modified cellulose membrane 12A is shown below:

(41) ##STR00018##

(42) Four different glycosylation reactions were tested on functionalized membranes A and B using galactose, mannose and glucose donors 8, 13, 14, and 15. The setup used for the vapor triggered glycosylation reactions is represented in FIG. 8.

(43) TABLE-US-00003 TABLE 3 Conditions and results of glycosylation reactions on membranes. Membrane Activator Reaction material & Spotting Solvent TMSOTf Temperature Bubbling time (min) functionalization conditions (μL) (μL) (° C.) time (min) Result 8 Membrane inert 1000 DCM  70 −12° C. to rt 2   30/FC Cellulose A inert 1000 DCM  70 −12° C. to rt 1.5 30/SM ambient 1000 DCM  70 −12° C. to rt 2   30/NC inert 1000 Tol  70 −12° C. to rt 4.5 30/SM inert 1000 Tol 100 −12° C. to rt 4.5 30/SM inert 900 DCM +  70 −12° C. to rt 2   30/SM 100 Tol Membrane inert 1000 DCM  75 −12° C. to rt 2   30/FC Cellulose B 0 13 Membrane inert 1000 DCM  70 −12° C. to rt 1   30/SM, P Cellulose A inert 1000 DCM  70 −12° C. to rt 1.5 30/SM, P 14 Membrane inert 1000 DCM  70 −12° C. to rt 1   30/SM, P Cellulose A 1000 DCM  70 −12° C. to rt 1.5 30/SM, P 15 Membrane inert 1000 DCM  75 −12° C. to rt 2   30/FC Poly- propylene B FC = full conversion of starting material, SM = starting material observed, SM, P = starting material observed/partial conversion, NC = no conversion

(44) Different types of building blocks have been used for this approach to examine the reactivity of the different leaving groups as well as the effect of the temporary and permanent protecting groups during glycosylation. Different reaction parameters have been tested to optimize the glycosylation reaction, such as variation of solvents, different amounts of activator, and different bubbling times of the glycosylation solutions as well as different spotting methods of the desired compounds under inert and under ambient temperature (Table 3).

(45) Suitable conditions for the vapor-triggered glycosylation on cellulose membranes with trichloroacetimidates 8, 13 and 14 and phosphate 15 have been found (see FIG. 12 for MALDI MS spectra of the products). At a constant reaction temperature below 20° C., a satisfactory deposition of the glycosylation solutions as well as the activation of the acceptor and the stereo-selective formation of the oligosaccharide was achieved.

Example 4: Glycosylation on Functionalized Cellulose Membrane

(46) A cellulose membrane which was purchased from AIMS Scientific Products GmbH was modified to membrane 12 as described in Example 3. Two different glycosylation reactions were tested on membrane 12. The first one was performed with glycosyl imidate 8 applying the conditions shown below and the second with thioglycoside 16, conditions also shown below. Both reactions were done in solution to verify that the glycosylation reaction in general is possible on the cellulose membrane. For both reactions the glycosylation product (disaccharide) was detected via MALDI-TOF-MS after cleavage of the molecule from the membrane via UV irradiation.

(47) Conditions for glycosylation of glycosyl imidate 8 and acceptor 12:

(48) TABLE-US-00004 Glycosyl imidate 8: 25 mg TMSOTF: 15 μl DCM: 5 mL NEt.sub.3 (quenching): 300 μl Temperature: rt Time: 30 min 8

(49) Conditions for glycosylation of thioglycoside 16 and acceptor 12:

(50) TABLE-US-00005 Thioglycoside 16: 60.0 mg TfOH: 1.4 μl NIS: 35.0 mg Dioxane: 0.333 mL DCM: 1.66 mL Temperature: −20° C. .fwdarw. 0° C. Time: 20 min 16

Example 5: Vapor-Triggered Saccharide Synthesis (Chilled Vapor Annealing Synthesis=CVAS) on Glass Slides or Membranes

(51) The herein described experiments are carried out in a reaction chamber shown in FIG. 11. The membrane or the glass slide covered with a certain pattern of glycosyl donors will be placed on the thermoelectric cooling element under inert gas atmosphere. For the patterning of the glass slides, laser induced forward transfer (cLIFT) is used as shown in FIG. 10. The patterning of the membranes will be done by using the SPOT synthesis, employing a spotting robot. The standard cellulose membrane esterified with Fmoc-β-alanine has a loading of 1000 nmol/cm.sup.2. By dissolving the glycosyl donors in an aprotic organic solvent (200 mg/ml) and spotting them on these membranes, we will obtain the measured parameters shown in FIG. 14. In addition, if a supplementary reagent is required for the glycosylation reaction (e.g. N-iodosuccinimide for thioglycosides), this substance could be co-spotted on the same spots or possibly brought into vapor phase via negative pressure. For large radii of the spots the glycosyl donors must be spotted multiple times to reach a saturation of the amine groups on the membrane. After patterning the glycosyl donors on the membrane or glass slide and placing them on the thermoelectric cooling element, the reaction chamber will be evacuated and flushed with an inert gas (N.sub.2, Ar etc.) to accomplish the glycosylation reactions avoiding moisture or air. Additionally, to get rid of traces of water and other volatile compounds adsorbed by the solid support (membrane or glass slide), the thermoelectric cooling element should be able to warm the surface up to +150° C. to remove these compounds under vacuum.

(52) For the CVAS process itself, the following parameters are used:

(53) Solvents: DCM, toluene, acetonitrile, ethers (1,4-dioxane, diethyl ether, MTBE)

(54) Gas-/Vapor Phase: Inert gas (N2, Ar.)

(55) Parameter: Vapor Laminar gas flow 0-2000 sccm/min (0-2 L/min) flow rate 0-100% vapor saturation/composition Temperature T.sub.gas=RT gas/vapor Vapor preparation: T.sub.bottle1 First 10-100 ml bottle (RT+ΔT.sub.bottle1)˜40° C. T.sub.bottle2 Second 10-100 ml bottle˜RT T.sub.sampleholder sample holder−50° C.-+150° C., with ΔT.sub.sampleholder>=0° C. Vapor chamber: Volume vapor chamber V.sub.chamber˜100-500 ml Atmospheric pressure for process, Δp≥0 Pa Vacuum for H.sub.2O removal (typical vacuum pump limits 10.sup.−2-10.sup.−3 bar) Process time (glycosylation time) t.sub.process˜1 min-12 h Sequential vapor exchange (gas vs. solvent/reagent vapor)
Vapor Preparation:

(56) In the setup (FIG. 11), the first bottle is heated to RT+ΔT to saturate the vapor. The second bottle is kept at RT, which results in 100% saturated vapor.

(57) Condensation on the substrate: The speed of vapor condensation on the sample surface is adjusted by the temperature difference ΔT (is |T.sub.sampleholder-T.sub.gas|). Condensation occurs at ΔT=0 (vapor vs. sample), when the vapor saturation is 100% or when ΔT>0, the vapor condensates with saturation<100%. The reaction time is between 10 minutes up to one hour.

(58) After the reaction is finished, the reactive support is quenched by adding a base (e.g. piperidine, triethylamine) within the reaction chamber. Then the substrate is removed of the chamber and washed. After the deprotection of a temporary protecting on the sugar moiety the substrate is used in the next CVAS glycosylation reaction.

Example 6: Parallel Vapor-Triggered Glycosylation on Membranes

(59) Two different acetimidates glucose 13 and mannose 8 were spotted onto the same membrane 12A (see Example 3) on different areas A (13) and B (8) under inert conditions and placed inside the chamber for glycosylation (FIG. 11). The glycosylation was performed with a glycosylation solution of 80 μL TMSOTf in 1000 μL DCM. Inert gas was passed through the glycosylation solution for 2 minutes and the membrane was left in the vapor for 30 minutes reaction time. The reaction temperature was −12° C. After the completion of the reaction, and the UV-cleavage, the MALDI-TOF showed that the desired sequences were formed (FIG. 15).