METHOD FOR PREPARING MULTIPLE ANTIGEN GLYCOPEPTIDE CARBOHYDRATE CONJUGATES
20170283466 · 2017-10-05
Assignee
Inventors
Cpc classification
A61K47/645
HUMAN NECESSITIES
A61K47/55
HUMAN NECESSITIES
C07K9/00
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to a method for preparing carbohydrate T cell epitope conjugates of formula (I): M(T-B).sub.n (I) wherein M, T, B and n ore as defined in claim 1.
Claims
1-2. (canceled)
3-24. (canceled)
25. A method for preparing a carbohydrate T cell epitope conjugate of formula (I): ##STR00005## wherein T is a peptide QYIKANSKFIGITEL (SEQ ID NO: 1); and R.sub.1 is: ##STR00006## said method comprising the step of removing the Pr protecting groups from a compound of formula (II) ##STR00007## wherein R.sub.1 is ##STR00008## and Pr is a protecting group selected from the group consisting of: benzyl and acetyl.
26. A method for preparing a carbohydrate T cell epitope conjugate immobilized on a solid support, Z, via a β-Ala-residue, said T cell epitope conjugate having formula (III): ##STR00009## wherein T is a peptide QYIKANSKFIGITEL (SEQ ID NO: 1); R.sub.1 is: ##STR00010## and Pr is a protecting group, said protecting group selected from the group consisting of: benzyl and acetyl; R.sub.2 is Fmoc or H; said method comprising the step of coupling compounds of formula (IV): ##STR00011## to a compound immobilized on a solid support via a β-Ala-residue, said compound immobilized on a solid support, Z, having formula (V): ##STR00012## wherein T is a peptide QYIKANSKFIGITEL (SEQ ID NO: 1) and R.sub.1 is ##STR00013##
27. A carbohydrate T cell epitope conjugate of the formula (VI): ##STR00014## wherein T is a peptide QYIKANSKFIGITEL (SEQ ID NO: 1); R.sub.1 is: ##STR00015## and wherein R.sub.2 is Fmoc or H.
28. The method of claim 25, wherein the Pr protecting groups are benzyl.
29. The method of claim 28, wherein the benzyl groups are removed in the presence of TfOH or H.sub.2.
30. The method of claim 25, wherein the Pr protecting groups are acetyl.
31. The method of claim 30, wherein the acetyl groups are removed in the presence of hydrazine or MeONa.
32. The method of claim 26, wherein the Pr protecting groups are benzyl.
33. The method of claim 26, wherein the Pr protecting groups are acetyl.
34. The method of claim 32, wherein R.sub.2 is Fmoc.
35. The method of claim 32, wherein R.sub.2 is H.
36. The method of claim 33, wherein R.sub.2 is Fmoc.
37. The method of claim 33, wherein R.sub.2 is H.
38. (New The carbohydrate T cell epitope conjugate of claim 27, wherein the Pr protecting groups are benzyl.
39. The carbohydrate T cell epitope conjugate of claim 27, wherein the Pr protecting groups are acetyl.
40. The carbohydrate T cell epitope conjugate of claim 38, wherein R.sub.2 is Fmoc.
41. The carbohydrate T cell epitope conjugate of claim 38, wherein R.sub.2 is H.
42. The carbohydrate T cell epitope conjugate of claim 39, wherein R.sub.2 is Fmoc.
43. The carbohydrate T cell epitope conjugate of claim 39, wherein R.sub.2 is H.
Description
FIGURES
[0165]
[0166]
BBREVIATIONS
[0167] AA amino acid
[0168] Ac acetyl
[0169] AcOH acetic acid
[0170] Bn benzyl
[0171] Boc tert-butoxycarbonyl
[0172] tBu tert-butyl
[0173] DIC N,N′-diisopropylearbodiimide
[0174] DIPEA diisopropylethylamine
[0175] DIPE diisopropyl ether
[0176] DMAP 4-dimethylaminopyridine
[0177] DMF dimethylformamide
[0178] DMS dimethylsulfide
[0179] DVB divinylbenzene
[0180] EtOH ethanol
[0181] FA formic acid
[0182] Fmoc 9-fluorenylmethoxycarbonyl
[0183] HATU 2-(1H-9-azabenzotriazole-l-yl)-1,1,3,3 -tetramethyluronium hexafluorophosphate
[0184] HOBt N-hydroxybenzotriazole
[0185] HPLC/MS high performance liquid chromatography/mass spectroscopy
[0186] MAG multiple antigenic glycoppeptide
[0187] MeOH methanol
[0188] MeONa sodium methylate
[0189] ESMS electrospray mass spectrometry
[0190] MW molecular weight
[0191] NMP N-methylpyrrolidone
[0192] NMR nuclear magnetic resonance
[0193] PyBOP benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
[0194] RP-HPLC reverse phase high-performance liquid chromatography
[0195] RT room temperature
[0196] SELDI-TOF MS surface-enhanced laser desorption/ionization time-of-flight mass spectrometry
[0197] TBS tert-butyldimethylsilyl
[0198] TFA trifluoroacetic acid
[0199] TfOH trifluoromethanesulfonic acid
[0200] THF tetrahydrofuran
[0201] TIS triisopropylsilane
[0202] TMSBr trimethylsilyl bromide
[0203] Trt trityl
EXAMPLES
Example 1
Preparation of MAG-Tn3 via Protocol A and B
[0204] General Methods
[0205] The synthesis of 6 was performed stepwise on solid-phase using Fmoc chemistry. Amino acid side chain protective groups used were Trt on Gln and Asn, Boc on the Lys.sup.7 and Lys.sup.11, tBu on Tyr, Ser and Thr, OtBu on Glu. For the Lys.sup.19 and Lys.sup.20, the protective groups were Fmoc.
[0206] The net peptide contents were determined by nitrogen analysis or quantitative amino acid analysis using a Beckman 6300 analyser after hydrolysis of the compounds with 6N HCl at 110° C. for 20 h.
[0207] The HPLC/MS analyses were performed on an Alliance 2695 system coupled to a UV detector 2487 (220 nm) and to a Q-Tofinicro™ spectrometer (Micromass) with an electrospray ionisation (positive mode) source (Waters). The samples were cooled to 4° C. on the autosampler. The linear gradient was performed with acetonitrile+0.025% FA (A)/water+0.04% TFA+0.05% FA (B) over 20 min. The column was a Zorbax 300SB C18(3.5μ, 3×150 mm) (Agilent) (gradient 13-53% A) or a XBridge™ BEH130 C18(3.5μ, 2.1×150 mm) (Waters) (gradient 15-40% A). The temperature of the source was maintained at 120° C. and the desolvation temperature at 400° C. The cone voltage was 40V.
[0208] The ESMS analyses were recorded in the positive mode by direct infusion in the same mass spectrometer. The samples were dissolved at ˜5 μM concentration in water/acetonitrile (1/1) with 0.1% formic acid.
[0209] The SELDI-TOF analyses were performed on a PCS 4000 mass spectrometer (Bio-Rad Labs). H4 ProteinChip array surfaces were activated with 1 μL CH.sub.3CN. Spots were incubated with the reaction mixture (2.5 μL, 1 mg/mL) in a box at RT for 20 min. They were then washed with the reaction buffer (3×1 min) and H.sub.2O (3×1 min). The matrix (2×0.6μL of sinapinic acid saturated in 50% CH.sub.3CN/0.5% TFA) was applied on each spot and allowed to air-dry. Spectra were generated from each array spot with a laser setting ˜3 μJ. The instrument was externally calibrated with bovine ubiquitin, bovine cytochrome C, β-lactoglobulin with the matrix and the settings as described above.
[0210] The purity of 6 was analyzed by RP-HPLC using an Agilent 1200 pump system with a UV detector at 220 nm. The column was a Zorbax 300SB C18 (3.5μ, 3×150 mm) (Agilent) and the gradient was performed with acetonitrile+0.1% TFA (A)/water+0.1% TFA (B) over 40 min, from 13 to 53% A (0.8 mL/min, retention time 20.5 min) .
[0211] The molar equivalents of all reagents are indicated relative to amino groups. The molar amounts of the protected intermediates 4 and 5 arc calculated based on the starting Fmoc-β-Ala-resin 1 substitution. The overall yields (Table) include all the synthetic steps from 1. They were calculated on the net peptide content of the final product 6 from the Fmoc-β-Ala-resin 1 substitution.
[0212] Protocol A (
[0213] Fmoe-β-Ala-Resin (Low-Substituted Resin) 1
[0214] 2 g of p-benzyloxybenzyl alcohol resin (Wang resin, 0.91 mmol/g , 100-200 mesh, polymer matrix: copoly(styrene-1% DVB), Novabiochem) were swelled in DMF (Applied Biosystems) for lh in a dry round-bottomed flask. 311 mg (1 mmol) of dry Fmoc-β-Ala-OH (Novabiochem, Merck Chemicals Ltd) were dissolved in 8 mL of anhydrous CH.sub.2Cl.sub.2 (Acros). Four drops of DMF were added to complete the dissolution. After the addition of 77 μL (0.5 mmol) of DIC (Fluka), the reaction mixture was stirred for 20 min under argon, at room temperature. The reaction mixture was evaporated to dryness and the rotary evaporator opened under argon. The residue was dissolved in the minimum volume of DMF (6 mL) and the solution was added to the resin. 6 mg (0.05 mmol) of DMAP (Acros) dissolved in 0.5 mL of DMF were added and the suspension was stirred gently for 2 h at room temperature.
[0215] The resin substitution rate was measured by UV analysis of a resin sample according to the following procedure. 2 to 6 mg of resin were transferred with a Pasteur pipette to a small sintered glass furmel. The resin was washed with DMF, CH.sub.2Cl.sub.2 (Carlo Erba) and dried. The resin was transferred in an UV cell, precisely weighted and then added with 2.8 mL of 20% piperidine (Aldrich) in DMF. The suspension was agitated with the aid of a Pasteur pipette for 2 min. The absorbance was read at 300.5 nm (ε=7800) with the reference cell containing 20% piperidine in DMF. The extent of loading was found to be 0.1 mmol/g.
[0216] The resin was washed three times with DMF. The residual hydroxyl groups were capped using the following protocol. The resin was resuspended in 13 mL of DMF. 1.55 mL (16.4 mmol) of Ac.sub.2O (Sigma) in 1 mL of DMF, and then 660 mg (5.41 mmol) of DMAP in 1 mL of DMF were added. After gently stirring for 30 min at room temperature, the suspension was filtered in a sintered glass funnel, successively washed three times by DMF, three times by CH.sub.2Cl.sub.2 and then was dried overnight in a desiccator. The resin 1 was stored at 4° C.
TABLE-US-00001 [QYIKANSKFIGITEL].sub.4-K.sub.2-K-β-Ala-Resin (protected peptide) 2
[0217] The tetravalent peptide was synthesized from 250 mg (25 μmol) of Fmoc-β-Ala-resin 1 on an Applied Biosystems peptide synthesizer 433A using Fmoc chemistry. The Applied standard synthesis protocol was followed except for an additional washing step after each coupling step. Briefly, the Fmoc groups were removed with 22% piperidine in NMP (Applied Biosystems) and the deprotection was monitored by conductivity. The lysine core was constructed by successively coupling two levels of Fmoc-Lys(Fmoc)-OH (Applied Biosystems) (1.sup.st cycle: 40eq, 2.sup.nd cycle: 20eq) using HATU (Applied Biosystems) (1.sup.st cycle: 40eq, 2.sup.nd cycle 20eq)/DIPEA (Applied Biosystems) (1.sup.st cycle 80eq, 2.sup.nd cycle: 40eq) as the coupling reagents and NMP as solvent (Note: the use of this very large excess of reagents should be not necessary for the efficiency of the reaction and is only due to the fact that the prepacked cartridges are filled with lmmol of amino acid). The stepwise introduction of the subsequent Fmoc-protected amino acids (Applied Biosystems, /10eq/amine) carrying standard side-chain protective groups was performed with HATU (10eq/amine) I DIPEA (20eq/amine) in NMP. The AA in positions 15-16 and 9-10 were incorporated as, respectively, Fmoc-Ile-Thr(Ψ.sup.Me,Me pro)-OH and F/inoc-Asn(Trt)-Ser(Ψ.sup.Me,Mepro)-OH (10eq/amine) (Novabiochem, Merck Chemicals Ltd) with HATU (10eq/amine) and DIPEA (20eq/amine).
TABLE-US-00002 [S(α-D-GalNAc(OBn).sub.3)-T(α-D-GalNAc(OBn).sub.3)-T(α-D- GalNAc(OBn).sub.3)-QYIKANSKFIGITEL].sub.4-K.sub.2-K-β-Ala 5
[0218] Starting from 2 (25 μmol), the glycosylated building blocks were incorporated manually: Fmoc-T(α-D-GalNAc(OBn).sub.3)-OH (Ficher Chemicals AG) (1.sup.st cycle), Fmoc-T(α-D-GalNAc(OBn).sub.3)-OH (2.sup.nd cycle) and Fmoc-S(α-D-GalNAc(OBn).sub.3)-OH (Ficher Chemicals AG) (3.sup.rd cycle). Briefly, the dry building block (0.3 mmol, 3eq/free amino group) was dissolved in the minimum amount of DMF (˜mL). A solution of 55 mg (0.145 mmmol) of HATU (Novabiochem) in 0.5 mL DMF was added and the resulting mixture was added to the resin. After adding 52 μL (0.3 mmol ) of DIPEA (Aldrich), the suspension was mechanically stirred. The three coupling steps were monitored by the Kaiser test [1] and were completed, respectively, in 1 h, 1 h and 1 h. After each coupling steps, the resin was washed with DMF (four times). All Fmoc cleavages were carried out by treatment of the resin with 20% piperidine in DMF. Following each deprotection, the resin was successively washed by DMF (six times), CH.sub.2Cl.sub.2 (six times), and DMF (six times). At the end of the synthesis, the resin was extensively washed with DMF and CH.sub.2Cl.sub.2, and dried in a desiccator. 10 mL of TFA (Applied Biosystems)/water/TIS (Acros) (95/2.5/2.5 v/v/v) were added to the resin at 4° C. and the mixture was stirred for 1 h30 at room temperature. After filtration of the resin, the solution was concentrated and the crude product precipitated with diethyl ether. After centrifugation, the pellet was dissolved in water and lyophilized to yield 229 mg of the crude glycopeptide 5.
TABLE-US-00003 [S(α-D-GalNAc)-T(α-D-GalNAc)-T(α-D-GalNAc)- QYIKANSKFIGITEL].sub.4-K.sub.2-K-β-Ala or MAG-Tn3 6
[0219] From 5, [0220] With TfOH [2-4]
[0221] 200 mg (0.014 mmol) of 5 were dissolved in 2.96 mL of TFA, 1.78 mL of DMS (Sigma-Aldrich) and 587 μL of metacresol (Sigma-Aldrich) at RT. The solution was cooled to −10° C. and 587 μL, of TfOH (Fluka) was added and the mixture was stirred 1 h15 at −10° C. (TfOH/TFA/DMS/m-cresol 1/5/3/1 v//v/v/v). The product was precipitated with diethyl ether and, after centrifugation, the pellet was dissolved in water and lyophilized to yield 372 mg of the crude glycopeptide. The product was dissolved in 7.7 mL of 0.05 M ammonium acetate buffer and the pH adjusted to 7 with 1 M ammonia. After 1 h at room temperature, the solution was lyophilized to yield to 412 mg of the crude product. The product was purified by RP-HPLC using an Agilent 1200 pump system with a UV detector at 230 nm. The column was a Zorbax C18 (5μ, 300 Å, 9.4×250 mm) (Agilent) and the gradient was performed with water (0.1% TFA)/acetonitrile over 20 min, from 73/27 to 60/40. The purification gave 3.9 mg (net peptide content) of 6 in 95.90% purity. The overall yield is 1.6%.
[0222] Protocol B (
TABLE-US-00004 [QYIKANSKFIGITEL].sub.4-K.sub.2-K-β-Ala-Resin (protected peptide) 2
[0223] Until the incorporation of Tyr.sup.5, the tetravalent peptide was synthesized from 36.9 g (4.8 mmol) of Fmoc-β-Ala-Tentagel R Trt resin 1 (0.13 mmol/g) (Rapp Polymere) on a manual peptide synthesizer equipped with a Schmizo reactor. Before the elongation process, the resin was swelled in DMF for 2 to 3 hours and was washed with 240 mL of DMF (three times, 2 min/cycle). Following each coupling, the Fmoc groups were removed with 20% piperidine in 240 mL of DMF (three steps, 20 min each). In the case of Glu.sup.17, Asn.sup.9 and GIn.sup.4, 2% HOBt was added to the deprotection solution. Following each deprotection, the resin was successively washed by 240 mL of DMF (4 times, 2 min/cycle), 240 mL of 2% HOBt in DMF (twice, 5 min/cycle), and 240 mL of DMF (twice, 2 min/cycle).
[0224] The amino acid couplings (1.5 to 2eq/amine) were performed in DMF (111 mL) at room temperature with DIC/HOBT (1.5 to 2eq each/amine) (see details below). The AA in positions 15-16 and 9-10 were incorporated as, respectively, Fmoc-Ile-Thr(Ψ.sup.Me,Mepro)-OH and Fmoc-Asn(Trt)-Ser(Ψ.sup.Me,Me pro)-OH. After 30 min, a fresh portion of DIC (1.5 to 2eq) was added to the reaction mixture. The coupling steps were monitored by the Kaiser test [1]. From Leu.sup.18 to Ser.sup.1, after 1 h coupling with DIC/HOBT (in equal amount), PyBOP reagent was added (see details below) and the pH was adjusted to 7 by dropwise addition of DIPEA. After 30 min, the resin was washed with 240 mL of DMF (5 times, 2 min/cycle) and an acetylation step was carried out from Leu.sup.18 to Thr.sup.2. The acetylation was performed at room temperature with acetic anhydride (1eq/amine) in the presence of pyridine (1 eq/amine) in 111 mL of DMF. After 20 min, the resin was washed with 240 mL of DMF (6 times, 2 min/cycle). After the incorporation of Tyr.sup.5, the resin was extensively washed with 240 mL of DMF (8 times, 2 min/cycle) and 240 mL of CH.sub.2Cl.sub.2 (8 times, 2 min/cycle), before drying.
[0225] After the incorporation of Tyr.sup.5, the assembly was pursued on a 0.15 mmol scale or a 4.65 mmo1 scale using the same protocole and afforded the peptide-resin 2 for, respectively, 4 and 5.
TABLE-US-00005 DIC/HOBt: 1/1 Coupling PyBOP Amino acids (eq/amine, reaction (eq/ (eq/amine, mmol) mmol) (min) amine) 20. Fmoc-Lys(Fmoc)-OH 1.75, 2.1 60 0.5 (1.75, 2.1) 19. Fmoc-Lys(Fmoc)-OH 2, 4.8 105 1 (2, 4.8) 18. Fmoc-Leu-OH (2, 9.6) 2, 9.6 60 1 17. Fmoc-Glu(OtBu) (2, 9.6) 2, 9.6 60 1 16-15. Fmoc-Ile- 1.5, 7.2 60 0.5 Thr(Ψ.sup.Me-Mepro)-OH (1.5, 7.2) 14. Fmoc-Gly-OH (2, 9.6) 2, 9.6 60 1 13. Fmoc-Ile-OH (2, 9.6) 2, 9.6 60 1 12. Fmoc-Phe-OH (2, 9.6) 2, 9.6 60 1 11. Fmoc-Lys(Boc)-OH 2, 9.6 60 1 (2 eq, 9.6 mmol) 10-9. Fmoc-Asn(Trt)- 1.5, 7.2 60 0.5 Ser(Ψ.sup.Me-Mepro)-OH (1.5, 7.2) 8. Fmoc-Ala-OH (2, 9.6) 2, 9.6 60 1 7. Fmoc-Lys(Boc)OH 2, 9.6 60 1 (2, 9.6) 6. Fmoc-Ile-OH (2, 9.6) 2, 9.6 60 1 5. Fmoc-Tyr(tBu)OH 2, 9.6 60 1 (2, 9.6) 4. Fmoc-Gln(Trt)-OH (2, 1.2) or 60 1 (2, 1.2 for 4) or (2, 37.2) (2, 37.2 for 5)
TABLE-US-00006 [S(α-D-GalNAc(OAc).sub.3)-T(α-D-GalNAc(OAc).sub.3)-T(α-D- GalNAc(OAc).sub.3)-QYIKANSKFIGITEL].sub.4-K.sub.2-K-β-Ala 4
[0226] The synthesis was performed from 2 (0./15 mmol) as previously described for 2. The coupling steps were performed with the following AA building blocks [5] and reagents.
TABLE-US-00007 DIC/HOBt: 1/1 Coupling PyBOP Amino acids (eq/amine, reaction (eq/ (eq/amine, mmol) mmol) (min) amine) 3. Fmoc-Thr(α-D- 1.5, 0.9 60 0.5 GalNAc(OAc)3)-OH (1.5, 0,9) 2. Fmoc-Thr(α-D- 1.5, 0.9 60 0.5 GalNAc(OAc)3)-OH (1.5, 0.9) 1. Fmoc-Ser(α-D- 1.5, 0.9 60 0.5 GalNAc(OAc)3)-OH (1.5, 0.9)
[0227] At the end of the synthesis, the glycopeptide-resin (0.15 mmol) was suspended in a TFA/TIS/H.sub.2O(95/2.5/2.5 v/v/v) (10 mL/g of glycopeptide-resin) and stirred for 1 h at 20° C.=2° C. After filtration, the resin was washed twice with the same TFA mixture (2 mL/g of glycopeptide-resin per wash). The filtrates and the washes were gathered and stirred for additional 30 min at 20° C. ±2° C. After concentration (bath temperature ≦35° C.), the crude product was precipitated with DIPE (˜35 mL/g of glycopeptide-resin). After filtration and washing with DIPE, the solid was dried (t°≦30° C.) and gave 750 mg of crude 4.
[0228] ESMS: 12409.589 (C.sub.553 H.sub.855 N.sub.107 O.sub.213 calcd 12410,465)
TABLE-US-00008 [S(α-D-GalNAc(OBn).sub.3)-T(α-D-GalNAc(OBn).sub.3)-T(α-D- GalNAc(OBn).sub.3)-QYIKANSKFIGITEL].sub.4-K.sub.2-K-β-Ala 5
[0229] The synthesis was performed from 2 (4.65 m/mol) as previously described for 2. The coupling steps were performed with the following AA building blocks (Ficher Chemicals AG) and reagents. At the end of the synthesis, 84.87 g of glycopeptide-resin were obtained.
TABLE-US-00009 DIC/HOBt: 1/1 Coupling PyBOP Amino acids (eq/amine, reaction (eq/ (eq/amine, mmol) mmol) (min) amine) 3. Fmoc-Thr(α-D- 1.5, 27.9 60 0.5 GalNAc(OBn)3)-OH (1.5, 27.9) 2. Fmoc-Thr(α-D- 1.5, 27.9 60 0.5 GalNAc(OBn)3)-OH (1.5, 27.9) 1. Fmoc-Ser(α-D- 1.5, 27.9 60 0.5 GalNAc(OBn)3)-OH (1.5, 27.9)
[0230] The glycopeptide-resin (20 g, 1.096 mmo1) was treated as previously described for 4 and afforded 9.95 g of crude 5.
[0231] ESMS: 14141.433 (C.sub.733H.sub.999N.sub.107 calcd 14141,610)
[0232] Note: This protocol gave a comparable crude compound as that obtained according to protocol A, i.e. starting from Fmoc-β-Ala-p-benzyloxybenzyl alcohol resin (Wang resin) (see above).
TABLE-US-00010 [S(α-D-GalNAc(OH).sub.3)-T(α-D-GalNAc(OH).sub.3)-T(α-D- GalNAc(OH).sub.3)-QYIKANSKFIGITEL].sub.4-K.sub.2-K-β-Ala or MAG-Tn3 6
[0233] From4
[0234] With Hydrazine [7]
[0235] 100 mg (20 μmol) of 4 were dissolved in 3.2 mL of MeOH. 567 μL (11.3 mmol) of hydrazine monohydrate were added and the solution was stirred at room temperature. After 2 h30, the solution is cooled to 0° C. and 3.2 mL of acetone were added. After 1 h, the solution was concentrated and co-distilled five times with acetone. The crude glycopcptide was lyophilized to yield 117 mg. The product was purified by RP-HPLC using an Agilent 1200 pump system with a UV detector at 230 nm. The column was a Zorbax C18 (5 μ, 300 Å, 9.4×250 mm) (Agilent) and the gradient was performed with water (0.1% TFA)/acetonitrile over 20 min, from 72/28 to 62/38. The purification gave 5.8 mg (net peptide content) of 6 in 96.4% purity. The overall yield is 2.7%.
[0236] With MeONa 24 mg (4.8 μmol) of 4 were dissolved in 3.2 mL of MeOH. The pH was adjusted to 14 (pH meter, moist pH paper ˜10.5) with 1% MeONa in MeOH and the solution stirred at RT. The reaction was monitored by RP-HPLC. After 2 h, the reaction is neutralized with dry ice and evaporated to dryness. The crude peptide was dissolved in 1% aqueous TFA and lyophilized. The product was purified by RP-HPLC using an Agilent 1200 pump system with a UV detector at 230 nm. The column was a Kromasil C4 (5 μ, 100 Å, 10×250 mm) (AIT) and the gradient was performed with 0.1% aqueous TFA (VWR)/acetonitrile (Carlo Erba) over 30 min, from 73/27 to 62/38. The purification gave 754 μg (net peptide content) of 6 in 91.4% purity. The overall yield is 1.4%.
[0237] From 5,
[0238] With H.sub.2
[0239] 10 g (1.1 mmol) of 5 were dissolved in 800 mL of NMP/H.sub.2O 87.5112.5. After addition of 4 g of 10% Pd/C type 39 (Johnson Matthey), the reaction was stirred at 37° C. and 5 bar for 170 h. Two additional amounts of catalyst were added portionwise after 72 h (2 g) and 120 h (2 g). At the end of the reaction, the catalyst was filtrated on celite and washed with NMP/H.sub.2O 87.5112.5. The resulting filtrate was gathered with other filtrates issued from similar reaction (1.35 mmo1 in total). After dilution with H.sub.2O (until NMP/H.sub.2O 10/90), the filtrates were purified by RP-HPLC in two steps. The primary purification was carried out on a Vydac C18 column (300 Å, 10-15 μm, 50 mL/mn) with TFA/H.sub.2O/CH.sub.3CN 0.1/94.9/5.0 v/v/v (A) and with TFA/H.sub.2O/CH.sub.3CN 0.1/49.9/50.0 v/v/v (B). The gradient was 0% B over 15 min, 0-40% B over 5 min, 40-80% B over 60 min. The secondary purification was carried out on a Vydac C18 column (300 Å, 10-15 μm, 50 mL/mn) with AcOH/H.sub.2O/CH.sub.3CN 0.5194.5/5.0 v/v/v (A) and with AcOH/H.sub.2O/CH.sub.3CN 0.5/49.5/50.0 v/v/v (B). The gradient was 0% B over 15 min, 0-20% B over 5 min, 20-60% B over 60 min. After concentration by RP-HPLC on a Daisogel SP-300-10-ODS-AP column (20 mL/mn, isocratic TFA/H.sub.2O/CH.sub.3CN 0.1/49.9//50.0 v/v/v), the solution was evaporated on rotary evaporator and lyophilized to afford 225 mg (net peptide content) of 6 in 95.3% purity. The overall yield is 1.5%.
[0240] ESMS: 10897.387 (C.sub.481H.sub.783N.sub.107O.sub.177 calcd 10897,123)
[0241] Conclusion
[0242] The obtained results are summarized in the following table:
TABLE-US-00011 Initial process I.sup.1 New process II Carbohydrate None (H) TBS Ac Bn protective group (R) Deprotection — TFA NH.sub.2—NH.sub.2 TfOH H.sub.2 method Overall yield.sup.2 1-10 mg scale — 2.7% (96.4%) 1.6% (95.9%) 1.5% (95.3%) (Purity).sup.3 3% (94.5%) >10 mg scale <1% (<95%) Comment Impurities and No expected Scale ~5 mg Compromise Scale 225 mg Scale.sup.4 reproducibility compound (partial between complete issues during deprotection) deprotection and scale-up => new Impurities++ degradation. synthesis route Scale ~5 mg with carbohydrate protection .sup.1ref 5 and 9, (Ref 11 of WO 9843677 Multiple antigen glycopeptide carbohydrate, vaccine comprising the same and use thereof) .sup.2calculated on the net peptide content from the Fmoc-βAla-resin substitution (includes all the synthesis steps from 1). .sup.3as determined by RP-HPLC: Column Zorbax 300SB C18 (3.5μ, 3 × 150 mm) (Agilent), 0.8 mL/min, A: acetonitrile + 0.1% TFA, B: water + 0.1% TFA, gradient 13% to 53% of A over 40 min, detection at 220 nm. .sup.4Refers to the final product (net peptide content)
[0243] Compared to the initial synthesis using unprotected carbohydrate synthons (
[0247] Among the tested protocols in the new process (Table,
[0248] The Bn/TfOH method afforded the MAG-Tn3 with an overall yield of 1.6%. This method relies on a compromise between complete deprotection and degradation. Alternatively, the Bn/H.sub.2 method afforded the MAG-Tn3 with similar yield (1.5%) and, most importantly, the process has been validated on a 225 mg scale. Finally the Ac/hydrazine method gave the highest overall yield (2.7%, compared to 1.6% and 1/.5%).
[0249] A MAG-Tn3 based on another peptide (PV=KLFAVWKITYKDT) (SEQ ID No 4) has also been prepared according to the method of invention (Ac/Hydrazine, Bn/TfOH).)
Example 2
Influence of the Resin Substitution Ratio and of the Nature of the Stationary Phase used for the Purification of MAG-Tn3
[0250] MAG-Tn3 was prepared according to protocol B from a polystyrene resin functionalized with Fmoc-β-Ala (sold under the trade name Fmoc-β-Ala-TentaGel R Trt), with two different substitution ratios: 0.13 or 0.1 mmol/g (namely the number of Fmoc-β-Ala grafted groups relative to the weight of the non grafted resin).
[0251] The purification of the crude MAG-Tn3 was then performed by RP-HPLC on three distinct stationary phases (reversed phases) based on a silica gel grafted by octadecyl groups, namely Vydac®, Jupiter® and Daisogel®. The primary purification was carried out with TFA/H.sub.2O/CH.sub.3CN 0.1194.9/5.0 v/v/v (A) and with TFA/H.sub.2O/CH.sub.3CN 0.1/49.9/50.0 v/v/v (B). The gradient was 0% B over 15 min, 0-40% B over 5 min, 40-80% B over 60 min. The secondary purification was carried out with AcOH/H.sub.2O/CH.sub.3CN 0.5/94.5/5.0 v/v/v (A) and with AcOH/H.sub.2O/CH.sub.3CN 0.5/49.5/50.0 v/v/v (B). The gradient was 0%B over 15 min, 0-20% B over 5 min, 20-60% B over 60 min.
[0252] The results are reported in the following table.
TABLE-US-00012 Resin Purification Overall MAG-Tn3 substitution stationary Quantity Purity yield batch (mmol/g) phase (g).sup.a (%).sup.b (%).sup.c 1 0.13 Vydac ® 0.275 95.3 2-4 2 0.13 Jupiter ® 3.63 96.6 6 3 0.1 Daisogel ® 4.65 99.2 11 4 0.1 Daisogel ® 4.72 99.0 11 .sup.aPowder weight .sup.bAnalysis by RP-HPLC: Zorbax 300SB C18 (3.5μ, 3 × 150 mm, Agilent), A: acetonitrile + 0.1% TFA, B: H.sub.2O + 0.1% TFA, 15-53% A (40 min). .sup.cThe yield includes all the synthetic steps from Fmoc-β-Ala-resin and was calculated on the net peptide content of the final product. Vydac ® C18, 300 Å, 10-15 μm (Grace), ref 218MSB1015 or 218TPB1015 or 238TPB1015 Jupiter ® C18, 300 Å, 10 μm (Phenomenex), ref 04G-4055 Daisogel ® C18, 300 Å, 10 μm (Daiso), ref SP-300-10-ODS-RPS
[0253] These results demonstrate that both the yields of the process of preparation of the conjugates according to the invention, and the purity of the obtained conjugates can be highly improved by reducing the substitution ratio of the resin and/or by using an appropriate stationary phase.
REFERENCES
[0254] 1. Kaiser, E., R. L. Colescott, C. D. Bossinger, and P. I. Cook, Anal Biochem (1970). 34: 595-8.
[0255] 2. Maemura, M., A. Ohgaki, Y. Nakahara, H. Hojo, and Y. Nakahara, Bioscience Biotechnology and Biochemistry (2005). 69: 1575-1583.
[0256] 3. Tam, J. P., W. F. Heath, and R. B. Merrifield, J Am Chem Soc (1986). 108: 5242-5251.
[0257] 4. Tanaka, E., Y. Nakahara, Y. Kuroda, Y. Takano, N. Kojima, H. Hojo, and Y. Nakahara, Bioscience Biotechnology and Biochemistry (2006). 70: 2515-2522.
[0258] 5. Bay, S., R. Lo-Man, E. Osinaga, H. Nakada, C. Leclerc, and D. Cantacuzène, J. Peptide Res. (1997). 49: 620-625;
[0259] 6.: Fmoc solid phase peptide synthesis, A practical approach, Edited by W. C. Chan and P. D. White, Oxford University Press.
[0260] 7. Sander, J. and II. Waldmann, Chem Eur J (2000). 6: 1564-1577.
[0261] 8. Lo-Man, R., S. Vichier-Guerre, S. Bay, E. Dériaud, D. Cantacuzène, and C. Leclerc, J. Immunol. (2001). 166: 2849-2854.
[0262] 9. La-Man, R., S. Vichier-Guerre, R. Perraut, E. Dériaud, V. Huteau, L. BenMohamed, O. M. Diop, P. O. Livingston, S. Bay, and C. Leclerc, Cancer Res (2004). 64: 4987-4994
[0263] 10. Babino, A., D. Tello, A. Rojas, S. Bay, E. Osinaga, and P. M. Alzari, FEBS Lett. (2003). 536: 106-110.
[0264] 11. WO9843677—Multiple antigen glycopeptide carbohydrate, vaccine comprising the same and use thereof