Small-scale solid-phaseO-glycopeptide synthesis of linear and cyclized hexapeptides from blood-clotting factor IX containingO-(alpha;-D-Xyl-1rarr;3-alpha;-D-Xyl-1rarr;3-beta;-D-Glc)-L-ser

摘要

925J. CHEM. soc. PERKIN TRANS. I 1993 Small-scale Solid-phase 0-Glycopeptide Synthesis of Linear and Cyclized Hexapeptides from Blood-clotting Factor IX containing O-(a-~-Xyl-l+3-a-D-Xyl-1 -+S-p-~-Glc)-~-Ser Kerry B. ReirneC Morten Meldal,**a Shoichi Kusumoto,b Koichi Fukaseb and Klaus Bocka a Carisberg Laboratory, Department of Chemistry, Gamle Carlsberg, Vej 10 DK-2500 Valby, Copenhagen,Denmark Department of Chemistry, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan Glycopeptide sequences corresponding to residues 51 -56 of the EG F-like domains of human and bovine blood-clotting factor IX have been synthesized using the Fmoc/Dhbt strategy. Building blocks consisting of Fmoc-Ser(R)-OPfp, where R = P-D-GIc or a-D-Xyl-(l-3)-~t-~-xyl-(1-3)-P-~-Gk, have been synthesized.The building blocks were prepared by treatment of the unprotected glycosylated serine compounds with N-(fluoren-9-ylmethoxycarbonyl)succinimide in 1,4-dioxane, followed by treatment with pentafIuorop henol and dicyclo hexylcarbodi imide in tetra hydrof u ran. The glycosylated building blocks were then used in a solid-phase peptide synthesis to give the corresponding glycopeptides. Cyclic glycopeptides were prepared from the acetamidomethyl-protected linear glycopeptides by treatment with thallium(iii) trifluoroacetate in trifluoroacetic acid. The cyclic glycopeptides were fully characterized by NMR spectroscopy and mass spectrometry. Many 0-glycosylated proteins carry very complex oligosac- OH charides which are available in only minute amounts from the natural protein and are often heterogeneous when isolated.These oligosaccharides linked to amino acids may be obtained by synthesis, which, depending on its complexity, is often tedious and time consuming, leading to small quantities of 1 glycosyl amino acid to be further manipulated for the incorporation into a target glycopeptide with a well defined ology for the protection and incorporation of the completely composition. We have recently described a method in which unprotected 0-glycosyl amino acid 1into a solid-phase peptide oligosaccharides are treated with N"-fluoren-9-ylmethoxy-synthesis. Such methodology will be valuable in the future when carbonyl (Fmoc) and 2,3,4,5,6-pentafluorophenyl(Pfp) ester it may be expected that such 0-linked building blocks for solid- protected hydroxyamino acids to yield larger amounts of fully phase glycopeptide syntheses become available from over-protected building blocks for solid-phase synthesis. ',2 Tech-expression of glycoproteins or from other natural sources.niques for the conversion and incorporation of very small amounts of complex synthetic or isolated glycosyl amino acid into glycopeptides by solid-phase peptide synthesis has not yet Results and Discussion been reported. Synthetic compound 1 was available only in a small quantity Blood-clotting factors VII and IX are plasma glycoproteins (-20 mg), therefore the glycopeptide with the more readily (zymogens of serine proteases) which are involved in the blood available glucose conjugate of serine was first prepared.This coagulation ca~cade.~?~ These proteins are synthesized in the allowed the investigation of the chemistry of protection and liver and are glycosylated prior to secretion. Recently it has peptide synthesis, as well as affording the glycopeptide with a been shown that the first epidermal growth factor (EGF)-like truncated oligosaccharide for comparison in biological testing. domains of human and bovine blood-clotting factors VII and Glycosylation of W-Fmoc-Ser-OPfp with the donor 3,2910 IX, as well as human and bovine Protein Zs and bovine 2,3,4,6-tetra-0-benzoyl-a-~-glucopyranosy1bromide '2, under thrombospondin,6 contain unique 0-linked sugar chains silver trifluoromethanesulfonate promotion in dichloromethane consisting of glucose and xylose.The above proteins all contain (-30 "C), afforded the fully protected building block 4 in 72 a conserved sequence of amino acids consisting of a Cys-X-Ser- yield (Scheme 1). The product could be isolated directly from X-Pro-Cys sequence. The sequence for the factor IX blood- the crude syrup by trituration from an ethyl acetate-diethyl clotting proteins is Cys-Glu-Ser-Asn-Pro-Cys, corresponding ether-hexane mixture. The glycosylated serine derivative 4 was to residues 51-56. In these proteins the serine residue bears then used in an automated continuous-flow solid-phase peptide either a disaccharide (a-D-xyl-l-+3-P-~-Glc) or a trisaccharide synthesis of a hexapeptide portion (residues 51-56) of the (a-D-Xyl-1 -+3-a-D-Xyl-l-+3-P-~-Glc)chain.7 The biological human blood-clotting factor IX, HBF-IX (51-56) (see the function of these sugar chains remains unknown. The chemical Experimental section for details of the peptide synthesis).The synthesis of the disaccharide and trisaccharide chains benzoyl groups of glycopeptide 5 could be removed by sodium conjugated to serine have been carried out. methoxide-catalysed transesterification in methanol. The In order to investigate the biological significance of the sample was dissolved in methanol and made basic (pH -12 oligosaccharide portions of these proteins a sample of the by moist pH paper) by the dropwise addition of 1 mol dm-3 trisaccharide conjugated to serine, compound 1, was incorpor- sodium methoxide.The progress of the reaction was ated into a peptide sequence to give a glycopeptide corre- followed by analytical reversed-phase HPLC, and after 4 h sponding to residues 51-56 of the human and bovine blood- was found to be complete. When the reaction mixture was clotting factor IX. allowed to become too basic, p-elimination of the glucose This glycopeptide synthesis required development of method- residue occurred; therefore the reaction conditions were 926 J. CHEM. SOC. PERKIN TRANS. I 1993 OBz NHFmoc I OBz NHFmoc Br 0 2 3 4 Solid-phaseH peptide synthesis (Fmoc-amino acid-ODhbt) S S 79II I Cys-Glu-Ser-Asn-Pro-Cys -iv Cys(Acm)-Glu-Ser-Asn-Pro4ys(Acm) 0 AI83 iii 71 SR=BZ .40ROR OH7 c6R=H 0FmoC= rsquo;yo Am = -CH2NHCOMe / Scheme 1 Reagents and conditions: i, AgO,SCF,, CH,CI,, -30 OC, 2 h; then recrystallization from Et,O-EtOAc-hexane; ii, 4 (3.8 mol equiv.relative to the amount of peptide); iii, NaOMe, MeOH; iv, Tl(OCOCF,),, PhOMe, TFA lsquo;OBZ II bH 00 4 8 6mhr 1 OH NHFmoc NHFmoc - V H O W - H 0HO - yOH 0 6996 HO OH 0 10 9 I Cys(Acm)-Glu-+r-Asn-I0 Pro-C ys( Am) OHvl ?amp;OH 6 Scheme 2 Reagents and conditions: i, HOBt, aq. DMF; ii, morpholine; iii, NaOMe, MeOH; iv, Fmoc-Su, 1,4-dioxane, sat. aq. NaHCO,; v, Pfp-OH, DCCI, THF; vi, 10 (I .5 mol equiv. relative to amount of peptide) monitored carefully. The partially deprotected glycopeptide 6 and therefore more suitable for conformational analysis by was isolated after reversed-phase HPLC in 71 yield.The NMR spectroscopy studies. glycopeptide was characterized with the cysteine residues The cyclic peptide was prepared by an intramolecular protected as their acetamidomethyl (Acm) derivatives, in oxidation procedure, using thallium(1n) trifluoroacetate in order to protect them from oxidation. trifluoroacetic acid (TFA). Preparative HPLC afforded glyco- It is not known if the natural protein sequence contains a peptide 7 in 83 yield. The cyclic nature of the peptide was disulfide bridge between residues 51 and 56; such a bridge is confirmed by amino acid analysis which gave an analysis for possible since there is a proline in position 55 facilitating the half-cystine very close to the theoretical value, whereas the formation of such a structure.In light of this it was decided to preceding linear peptide 6 gave a much reduced amount, which prepare the cyclic glycopeptide 7 as well. Additionally, a cyclic is commonly observed for Cys(Acm) and Cys not involved in structure would likely be conformationally more well defined a disulfide bond. J. CHEM. SOC. PERKIN TRANS. I 1993 An analogue of compound 1 was prepared by removal of all the blocking groups of the ester 4 to give the fully deprotected glycosylated serine derivative 8 (Scheme 2). The deprotec- tion was achieved by hydrolysis of the Pfp-ester, catalysed by 1-hydroxybenzotriazole (HOBt) in aq. dimethylformamide (DMF),followed by the removal of the Fmoc group using 50 morpholine in DMF, and finally removal of the benzoyl esters by sodium methoxide-catalysed transestefication as before.The deprotection reactions were carried out without purifi- cation of the intermediate products, and the only work-up involved evaporation of the reaction mixtures between steps. The final deprotected compound 8 was obtained in 65 overall yield after purification. Compound 8 was then used as a model compound for the more complex compound 1. The first step was protection of the free!amino group of the serine moiety with the Fmoc group. The reaction was carried out by a procedure described previously,' and the Fmoc-protected derivative 9 was obtained in 68 yield. It was decided to prepare the Pfp-ester to serve as both a protection and a source of activation of the carboxyl terminus.This had the advantage of giving a compound which was stable enough to be purified by reversed-phase HPLC, as well as being reactive enough to serve directly as a building block for glycopeptide synthesis.' The semiprotected serine derivative 10 was obtained in 69 yield. It was decided to leave the hydroxy groups of the sugar residue unprotected to limit the number of chemical steps in the overall synthesis of the target glyco- peptides. There are examples in the literature of couplings of glycosylated amino acids where the hydroxy groups of the sugar residues have been left unprotected. 14v1 The building block 10 was then used in a solid-phase peptide synthesis using only 1.5 mol equiv.of compound 10 relative to the amount of functionalized resin used; the other amino acid building blocks were added in 6-fold excess. The yield of the glycopeptide 6 was 60 after purification. This result compared favourably to the results obtained earlier using the fully blocked glycosylated building block 4. The overall yield of product 6 including the peptide synthesis and the deprotection step was approximately 56, whereas the same compound 6 was prepared in 60 yield when using a partially protected building block. Based on these results it was decided to use a partially protected derivative of compound 1 rather than a fully protected building block. A sequence of protection, analogous to that carried out for compound 8,was performed using trisaccharide 1as the starting material.The amino group of the serine portion of the molecule was protected with the Fmoc group, to give protected acid 11in 76 yield (Scheme 3). The next reaction, protection of the carboxylic acid group as its Pfp-ester, did not proceed as smoothly as with the analogous glucose derivative. The starting material did not dissolve completely in tetrahydrofuran (THF) and some DMF was added to bring the compound completely into solution for the activation with N,N'-dicyclohexyl-carbodiimide (DCCI) and Pfp-OH. Addition of DMF to increase solubility has frequently been reported in the formation of symmetrical anhydrides although is also well documented that activation of carboxylic acids by DCCI is favoured in apolar media.' Following purification by HPLC, compound 12 was obtained in only 42 yield.This yield may be improved by the reversed-addition procedure" or by the use of fully protected intermediates. This partially protected building block was then used directly in a solid-phase peptide synthesis protocol. Normally, during a solid-phase synthesis, the acylating reagent (the activated amino acid) is added in excess, usually 2-3 or more mol. equiv. In this particular case the acylating reagent, compound 12, was available in only a very small amount so it was decided to make it the limiting reagent, using only 0.37 mol equiv. to afford a greater incorporation of 11 12 iii Solld-phase peptidesynthesis 24 8 8I IhrCys-GloSer-Asn-Pro-Cys 7Cys(Acm)Gl u-Ser-Asn-Pro-Cys(Acm) I I OR OR 14 13 =R Scheme 3 Reagents and conditions: i, Fmoc-Su, 1,4-dioxane, sat.aq. Na,CO,; ii, Pfp-OH, DCCI, THF-DMF; iii, 12 (0.37 mol. equiv. relative to amount of peptide); iv, TI(OCOCF,),, PhOMe, TFA glycosylated amino acid with less being wasted. With this approach the desired product is not the major product. In order to avoid acetylation of hydroxy groups, it was decided not to cap the unchanged amino groups after incorporation of compound 12 but instead to continue directly with the peptide synthesis. This led to a large amount of an impurity comprising a pentapeptide sequence lacking the glycosylated serine residue. It was assumed that separation of this unglycosylated peptide impurity from the desired glycosylated product would be a simple matter due to the presence of the oligosaccharide structure on the target peptide.Unfortunately this was not the case, but the desired compound could be purified by using a combination of gel filtration chromatography and repeated reversed-phase HPLC. After Chromatography a pure sample of compound 13 was obtained in 24 yield (based on the amount of building block 12 consumed). The incorporation of the building block 12 into glycopeptide product was lower than with the corresponding unit 10. This may be due to the greater effect of the unprotected hydroxy groups with the trisaccharide relative to a monosaccharide.The effect of having unprotected uersus protected hydroxy groups on larger oligosaccharides both in the protection scheme and in the peptide synthesis is currently being investigated with the analogous maltotriose conjugates of serine. Thus the acylation conditions are being investigated with derivatives of Fmoc-protected serine bearing either a maltotriose or a glucose unit with the hydroxy groups either protected or unprotected. Compound 13 was finally cyclized, in a similar manner to its analogue 6, to give the cyclic glycopeptide 14 in 75 yield. Studies are currently being performed using the molecular modelling force-field l8 and NMR techniques to assess the conformational preferences of the glycopeptides, synthesized in the present work.Experimental General Methods.-Analytical TLC was performed on Merck silica gel 60 F254aluminium plates, with detection by UV light and/or charring with sulfuric acid. Medium-pressure chromato- graphy was performed on Merck Kieselgel60 (0.0154.040 mm) with a Biichi B-680 chromatographic system. Solvents were purchased from Labscan, were distilled before use, and were dried, as required, by literature procedures. W(Fluoro-9- ylmethoxycarbony1)succinimide (Fmoc-Su) and Fmoc-amino acid derivatives were purchased from Bachem or Novabiochem, while DCCI, Pfp-OH and 3,4-dihydr0-3-hydroxy-4-0~0-1,2,3- benzotriazine (Dhbt-OH) were from Fluka. Extracts were evaporated under reduced pressure, and below 40 "C. Semi- preparative HPLC separations were performed on a Waters- 600E HPLC system using a DELTA PAK C-18 column, except where noted (300 A; 25 mm x 10 cm), with a flow rate of 10 cm3 min-' with detection at 200, 215, 235 and 270 nm using a photodiode array detector (Waters-991); solvents systems A: 0.1 TFA in water, B: 0.1 TFA in 90 acetonitrile-10 water.Gel exclusion chromatography was carried out by using a column of Biogel P-2 (fine) (58 cm x 1.5 cm) with a flow rate of 21 cm3 h-' with water as the eluent, and UV detection at 235 nm. Elemental analyses were carried out at LEO Pharma- ceutical Products Denmark. Mass spectra were kindly per- formed at the Department of Chemistry, Royal Veterinary and Agricultural University of Denmark. Mass spectra of com- pounds in a matrix of o-nitrobenzyl alcohol were recorded by liquid secondary-ion mass spectrometry (LSIMS) on a VEGA 70SE instrument with a Cs ion source calibrated with caesium iodide.Quantitative amino acid analyses were per- formed on a Pharmacia LKB Alpha Plus amino acid analyser following hydrolysis with 6 mol dm-, HCl at 110 "C for 24 h. Asn was determined as Asp, and Cys was determined as half- cystines. Nomenclature is according to the IUPAC recom- mendations. Optical rotations were measured with a Perkin- Elmer 241 polarimeter and are given in units of lo-' deg cm2 g-'. 'H and I3C NMR spectra were recorded with a Bruker AM-500 or AMX-600 spectrometer at 500.13 or 600.13 MHz for proton, and 125.76 or 150.91 for carbon, respectively.All spectra were recorded at 300deg;C. Chemical shifts are given in ppm with the solvent peaks for CDCl,, D20, (CD3),S0 and CD30D set toamp; 7.26,4.75,2.50 and 3.35, respectively. The pD of D20solutions was adjusted by using 0.2 mol dm-, Na,CO, in D20and then reading the value on a standard pH instrument. The coupling constants for the amide protons were measured in 90 water-lO D,O. The assignment of the proton chemical shifts was made by examination of the 2D homonuclear chemical-shift correlation (COSY) spectra; in addition, phase-sensitive two-dimensional total correlation (TOCSY) spectra were obtained for compounds 5, 6 and 13, using the pulse sequence d1-9O0-amp;MLEV spinlock-FID, with a solvent presaturation pulse of 1 s during dl.For the 2D spectra an initial data set of 51 2 2K experiments was acquired, and zero-filled once in the Fl-direction with no zero filling in F2, to give a final data set of 1K x 1K real data points. The spin lock (45 ps 90") was applied for a period of 200 ms. 'H NMR chemical shifts of overlapping signals were obtained from the centre of the cross-peaks in either the COSY or TOCSY spectra. Solid-phase Peptide Synthesis, General Procedure.-Synthesis of the glycopeptides was carried out on a Kieselguhr-supported poly(dimethylacry1amide) resin (Macrosorb SPR-250 pur-chased from Sterling Organics) which was converted into the amine form with ethylenediamine. The linker, 4-(hydroxy- methy1)phenoxyacetic acid, was attached using 4-(hydroxy- methy1)phenoxyacetic acid 3,4-dihydro-4-oxo- 1,2,3-benzotri- azin-3-yl (Dhbt) ester.The first amino acid was attached to the resin-bound linker (2.5 g of resin) by esterification of Na-Fmoc- Cys(Acm)-OH (0.52 g 1.25 mmol) promoted by l-mesityl- sulfonyl-3-nitro-l,2,4-triazole(MSNT) (0.37 g, 1.25 mmol) in dichloromethane with the presence of N-methylimidazole 2o (0.075 cm3, 0.94 mmol). The esterification procedure was repeated using the above amounts of reagents to ensure quantitative functionalization of the linker. A sample of the resin with the first amino acid attached was deblocked and acylated with Na-Fmoc-Ala-Dhbt by using a double coupling procedure to ensure complete acylation, and the level of incorporation of the first amino acid (Cys) was estimated based on the level of incorporation of Ala, which was in turn J.CHEM. SOC. PERKIN TRANS. 1 1993 determined by quantitative amino acid analysis (0.165 mmol g-' resin). The glycopeptides were synthesized in DMF, with the amino acids being added as their Na-Fmoc-protected Dhbt- esters, where the side chains of Cys and Glu were protected with Acm and tert-butyl groups, respectively. The glycosylated amino acids were added as their Pfp-esters with the addition of Dhbt-OH as an acylation catalyst as well as an indicator of the end-point of the acylation. Deprotection of the Fmoc groups was achieved with a solution of 50 morpholine in DMF for 30 min. The syntheses were carried out using either a custom- made, fully automatic, continuous-flow peptide synthesizer, with monitoring of the acylation reactions using a solid-phase spectrophotometer operating at 440 nm (Procedure A); or manually using a sintered glass funnel, and monitoring of the acylation times visually by the decrease in the yellow colour of the resin as the acylation proceeded (Procedure B).The latter procedure was better suited to small-scale syntheses. After the final removal of the Fmoc-group the resin was washed suc- cessively with DMF and diethyl ether and lyophilized. The peptide-resin was then treated 2 h with 95 aq. TFA (30 cm3 g-' resin) for the simultaneous removal of tert-butyl protection and cleavage of the peptide from the resin. The TFA was removed under reduced pressure and the residue was triturated with diethyl ether.The crude peptides were purified by HPLC as described below . Nu-(Fluoren-9-ylmethoxycarbonyl)-O-(2,3,4,6-tetra-O-ben-zoyl-P-D-glucopyranosy1)-L-serinePentafluorophenyl Ester 4.-A sample of N"-Frnoc-L-Ser-OPfp 3 (0.95 g, 1.9 mmol), together with silver trifluoromethanesulfonate (0.82 g, 3.2 mmol) and 3 A molecular sieves, was dissolved in dry dichloromethane (23 cm3). The solution was cooled to -30 "C, and a solution of 2,3,4,6-tetra-O-benzoyl-~t-~-glucopyranosylbromide '2 (1.8 g, 2.7 mmol) in dichloromethane (20 cm3) was added. The reaction mixture was stirred in the dark under argon and was allowed to warm to room temperature. After 2 h TLC hexane-ethyl acetate (2: l) indicated that the reaction was complete.The reaction mixture was then filtered, and the filtrate was washed with 5 aq. sodium hydrogen carbonate, dried (MgSO,), and concencentrated to a syrup. The syrup was triturated with a mixture of ethyl acetate-diethyl ether-hexane ( -1:1:1) to give title compound 4 as a powder (1.14 g, 1.05 mmol). The mother liquor was chromatographed hexane+thyl acetate (2 :1) as eluent and crystallized as above to give a second batch (0.32 g, 0.30 mmol) of compound to give an overall yield of 72, a;' +0.01 (c 1.27, CHC13); 'H and 13C NMR data are presented in Tables 1 and 5, respectively (Found: C, 65.0; H, 4.1; N, 1.2. C58H42F5N014 requires C, 64.99; H, 3.95; N, 1.31). Bis-5-acetamidomethyl-Cys',Cy~~~-0-(2,3,4,5-tetra-O-benzoyZ-~-~-glucopyranosyl)-Ser~~-HBF-IX(5 1-56) 5.-The solid-phase peptide synthesis (procedure A), and the coupling of the first amino acid to the resin, was carried out as described above.The loading of the first amino acid onto the resin was estimated by quantitative amino acid analysis to be 0.165 mmol g-'. The peptide synthesis was carried out using resin (0.464 g, equivalent to 0.0765 mmol of peptide). In the acylation step with the glycosylated serine 4 an excess of 3.76 mol equiv. (0.309 g, 0.288 mmol) was used. The acylation times varied from 30 min (Pro55) to 13 h (Am5,) with the acylation time for substrate 4 being 25 h. After cleavage of the protected glycopeptide 5 from the resin it was purified by semipreparative HPLC using isocratic solvent A for 5 min, followed by a linear gradient of amp;20 solvent B over a period of 40 min, followed by a linear gradient of 20-100 solvent B over a period of 40 min (retention time of 72 min).The yield of glycopeptide 5 was 82.4 mg, 78.5; 'H NMR data are presented in Table 2. J. CHEM. SOC. PERKIN TRANS. I 1993 Table 1 'H NMR data"for compounds 4 and 8-10 O-(p-D-Ghcopyranosyl)-L-serine8.-A sample of the fully protected serine derivative 4 (1.43 g, 1.33 mmol) was dissolved 8 9 10 in DMF (20 an3).To the stirred solution were added HOBt I-H 4.88 4.48 4.29 4.35 2-H (8.0) 5.52 (8.0) 3.3 1 (8-5) 3.21 (8.5) 3.24 3-H (8.0,9.2) 5.95 (8.0,9.0) 3.48 (18.5)b 3.37 (8.5,9.0) 3.39 4-H (19.5)b 5.68 (1 8.5) 3.37 (1 8.5) 3.28 (18.5)'' 3.28 5-H (19.5)b 4.18 (I 8.5) 3.34 3.27 3.30 6-H 4.66 3.89 3.87 3.88 6-H' (3.0, 12.0) 4.45 (2.0, 12.5) 3.70 (2.0, 12.0) 3.67 (2.0, 12.0) 3.65 Ser-a-H (5.0, 12.0) 4.87 (6.0, 12.5) 3.97 (5.5, 12.0) 4.44 (6.0, 12.0) 4.90 Ser-P-H 4.50 4.28 4.38 3.90 (3.5, 10.0) 4.03 (5.5, 11.5) 4.04 3.79 (3.5, 10.5) 3.65 Ser NH (3.5, 10.0) 5.71 (3.5, 11.5) (5.5, 10.5) Fmoc CH (8.0)4.I4 4.25 4.25 Fmoc CH, ( 14.0)b 4.40 (14.0) 4.40 (13.5)* 4.46 (7.5, 10.5) 4.27 (7.0, 10.5) 4.36 (6.8, 10.5) 4.41 ArH (7.5, 10.5) 8.1-7.2 (28 H) (7.0, 10.5) 7.80 (2 H) 7.69 (2 H) 7.39 (2 H) 7.32 (2 H) (6.8, 10.5) 7.79 (2H) 7.68 (2H) 7.38 (2H) 7.30(2 H) " Compound 4 in CDCl,; compound 8 in D,O, pD 7.1; compounds 9 and 10 in CD,OD.The numbers in parentheses denote coupling constants in Hz. The values are the sums of the individual coupling constants, J,, + JBx. (67.8 mg, 0.502 mmol) and water (2.0 cm3). The solution was stirred at room temperature for 20 h and then morpholine (10 cm3) was added. After a further 20 min the solution was evaporated to dryness, and the residue was taken up in methanol and the solution made basic (pH 12) towards moist pH paper by the addition of 1 mol dm-3 sodium methoxide. The solution was kept at room temperature for 18 h, and then neutralized by the addition of solid CO,. The solution was then evaporated to dryness and the residue was purified by silica gel chromatography using ethyl acetate-methanol-water-acetic acid (6:2: 1:1) as the eluent to afford the title compound (231 mg, 64.8).The 'H NMR data are given in Table 1 and are in accord with previously published data., ' Nu-(Fluoren-9-ylmethoxycarbonyl)-O-(~-D-glucopyranosyl)-L-serine 9.-A sample of the fully deblocked serine derivative 8 (26.1 mg, 0.0977 mmol) was dissolved in saturated aq. sodium hydrogen carbonate (5.0 cm3). The solution was cooled to 0 "C and a solution of Fmoc-Su (1 36 mg, 0.403 mmol) in 1,4-dioxane (5.0 cm3) was added dropwise. The solution was stirred for 2 h at 0 "C, and for 1 h further at room temperature. To the reaction mixture was added water (50 an3) and the solution was acidified to pH 4 by the addition of 1 mol dmP3 hydrochloric acid.The solution was then extracted with ethyl acetate (3 x 30 cm3) and the aqueous layer was lyophilized to give a residue, which was purified by semipreparative HPLC using isocratic solvent A for 20 min, followed by a linear gradient of amp;90 solvent B over a period of 30 rnin (fR 40 rnin). Compound 9 (32.2 mg, 67.8) was obtained, after lyophilization, as a solid; +0.01 (c 1.06, MeOH); 'H and I3C NMR data are presented in Tables 1 and 5, respectively; m/z 490 (M + H)+ and 512 (M + Na)' (C2,H,7N0,, requires M, 489.1). Bis-5-acetamidomethyl-CysS',Cy~~~-O-(fJ-~-glucopyrano-syl)-Ser53HBF-IX (51-56) 6.-Compound 5 was debenzoyl- ated as follows. A sample (26.0 mg, 18.9 mmol) was dissolved in methanol (3.0 cm3) and to this solution was added 1 mol dmP3 sodium methoxide until a wetted pH paper indicated pH 12.After 4 h the pH of the solution was adjusted by the addition of solid C02. The solvent was removed by evaporation and the resulting residue was purified by semipreparative HPLC using a linear gradient of 0-20 solvent B over a period of 40 min (tR 21 min). The HPLC fractions were lyophilized to give com- pound 6 as a solid (12.9 mg, 71.4); 'H and 13C NMR data are presented in Tables 2 and 6, respectively. Amino acid analyses (theoretical values in parentheses): Asn 1.10 (l), Ser 0.95 (l), Glu 1.05 (l), Cys 0.74 (2) and Pro 0.89 (1); m/z 956 (M + H)+ and 978 (M + Na)+ (C3,H,,N90,,S, requires M, 955.3). Cyclic O-( P-~-Ghcopyranosyamp;Ser~3HBF-IX (51-56) 7.-A sample of compound 6 (4.4 mg, 0.0046 mmol) was dissolved in TFA (2.4 cm3), together with anisole (10 mm3, 0.092 mmol).The solution was cooled to O"C, and a solution of thallium(I1r) trifluoroacetateinTFA(0.8cm3;7.5 ~moldm-~) wasadded. After 75 rnin the reaction mixture was evaporated to dryness and diethyl ether (2 cm3) was added to the residue. After the mixture had settled for 30 min, the ether was decanted off, and the residue was purified by semipreparative HPLC using isocratic solvent A (tR 11 min). Compound 7 was obtained, after lyophilization, as solid (3.1 mg, 83); 'H and 13C NMR data are presented in Tables 2 and 6, respectively. Amino acid analyses (theoretical values in parentheses): Asn 1.07 (l), Ser 0.95 (I), Glu 1.03 (l), Cys 1.99 (2) and Pro 0.95 (1); m/z 812 (M + H)' and 834 (M + Na)' (C,gH4sN701,S, requires M, 81 1.2).Nu-(Fluoren-9-ylmethoxycarbonyl)-O-(fJ-~-glucopyrano-sy1)-L-serine PentaJIuorophenyl Ester 10.-A sample of com- pound 9 (32.2 mg, 0.657 mmol) was dissolved in dry THF (4.0 cm3). To this solution was added DCCI (17.1 mg, 0.0828 mmol). The solution was stirred at room temperature and a solution of Pfp-OH (22.0 mg, 0.119 mmol) in THF (2.0 cm3) was added. The reaction mixture was stirred for 20 h and was then evaporated to dryness, the residue was taken up in ethyl acetate, and the solution was filtered. The filtrate was concentrated and the residue was purified by semipreparative HPLC using a linear gradient of 35-55 solvent B over a period of 40 min (tR 34 min) to give compound 10 (0.0297 g, 68.9); -0.16 (c 0.90, MeOH); 'H and I3C NMR data are presented in Tables 1 and 5, respectively; m/z 656 (M + H)+ and 678 (M + Na)+ (C3,H,,FsN0,, requires M, 655.1).O-(P-~-Ghcopyranosyl)-Ser3HBX-IX (51-56) 6.-The solid-phase peptide synthesis was carried out by method B as described above, using resin (1 13 mg, equivalent to 0.0186 mmol of peptide). The loading of the first amino acid onto the resin was estimated by quantitative amino acid analysis to be 0.165 mmol g-'. The activated amino acids were added in six-fold excess, except for the glycosylated serine derivative 10 which was added in 1-5-fold excess. The acylation times, as judged by the decrease in the yellow colour of the resin during the acylation, varied from 73 rnin (Pros5) to 4 h (Ams4), with the acylation time for compound 10 being 16 h.Following cleavage from the resin the glycopeptide 6 was purified by semiprepara- tive HPLC using the same solvent system as for compound 6 from the tetrabenzoyl precursor 5 to give title compound 6 as a solid (1 0.6 mg, 59.6). The NMR data of the purified product were identical with those obtained above. 930 J. CHEM. SOC. PERKIN TRANS. 1 1993 Table 2 'H NMR data" for compounds 5-7 NH a-H P-H, Y-H2 6-H2 Cys5' 5 4.09 3.02(4,5, 14.5),2.81 (9.0, 14.5) 6 4.30 3.17 (5.5, 14.5), 3.03 (8.0, 14.5)7 4.40 (5.5, 7.4) 3.48, 3.37 (7.4, 14.5) GIu5' 5 8.43 (9.0) 4.36 1.90 2.26 6 8.97 (7.0) 4.46 2.00, 2.12 2.48 ( 15.0) 7 4.34 (6.5, 8.5) 2.16,2.07 2.47 Ser53 5 8.08 (10.5) 4.46 3.89 (8.0, 10.0), 3.82 (5.0, 10.0) 6 8.64(7.0) 4.60 4.13 (6.5, 10.5), 3.88 (5.0, 10.5) 7 4.74 4.00 (6.5, 10.5), 3.88 (7.0, 10.5) Asn" 5 8.28 (8.0) 4.71 2.62 (7.5, 16.5), 2.35 (6.0, 16.5) 6 8.48 (7.5) 5.01 2.83 (6.5, 15.5), 2.67 (8.0, 15.5)7 4.76 2.72 (5.5, 15.9, 2.62 (8.5, 15.0) pro55 5 4.37 1.95 1.85 3.57 6 4.46 2.22 1.99 3.75 7 4.7 1 2.36, 2.20 1.96 3.64, 3.49 CYS~~ 5 8.07 (11.0) 4.35 2.98 (4.5, 14.0), 2.86 (9.0, 14.0) 6 8.34(7.5) 4.55 3.13 (4.5, 14.0), 2.96 (8.5, 14.0) 7 4.60 (4.5, 9.0) 3.37 (4.5, 14.0), 3.03 (9.0, 14.0) Acm 5 6 8.57, 8.46 8.59, 8.57 4.28,4.16 (2 H, 4.12) 4.38,4.29,4.26,4.25 1.86 (3 H), 1.84 (3 H) 2.00 (3 H), 1.95 (3 H) 1-H 2-H 3-H 4-H 5-H 6-H2 pGlc-( I-3)-Ser 5 5.24 (8.0) 6 4.45 (8.0)7 4.46 (8.0) 5.33 (8.0, 9.5) 3.25 (8.0,9.0) 3.32 (8.0,9.5) 5.95 (19.0) 3.48 (18.5)* 3.50 5.60 (19.0)b 3.35 (19.0)b 3.39 (19.0)b 4.484.55' 3.43 3.47 4.484.55' 3.90 (2.5, 12.5), 3.68 (6.0, 12.5) 3.94 (2.5, 12.5), 3.74 (6.0, 12.5) " Compound 5 in (CD,),SO; compound 6 in 90 water-lO D,O, pH 2.8; compound 7in D,O, pD -6.0.The numbers in parentheses denote coupling constants in Hz. The values are the sums of the individual coupling constants, JAx+ JBx.'The signal is contained within the indicated range. Table 3 'H NMR data" for compounds 11 and 12 1 -H 2-H 3-H 4-H 5-H 6-H2 ~~ P-Glc--(I-3)-Ser 11 4.32 (8.0) 3.32 3.50 (1 3.57 3.19 3.85 (2.0, 12.0), 3.67 12 4.39(8.0) 3.35 3.52 3.54 3.32 3.87,3.66 (6.0, 12.0) ~-Xyl-(l-3)-GIc11 5.19 (2.5) 3.54 3.73 3.70 3.91 (21 .5),b3.56 12 5.21 (3.5) 3.54 3.74 (18.0)b 3.71 3.88 (21 .O),b 3.56 (21 .5)b ~-Xyl-(l-3)-Xyl 11 5.17 (2.5) 3.42 (2.5, 9.5) 3.63 (18.5)b 3.47 3.86 (21 .5),b 3.55 12 5.17 (3.5) 3.42 (3.5,9.5) 3.63 (18.5)b 3.48 3.86, 3.55 Ser a-H Ser P-H2 FmocCH FmocCH, ArH ~~~~~~ Other signals 11 4.42 4.38, 3.81 4.25 (14.0)b 4.38,4.35 7.80, 7.69, 7.39, 7.32 12 4.90 4.55, 3.93 4.25 (13.5)b 4.46,4.41 7.80, 7.68, 7.38, 7.31 " Solvent CD30D.The numbers in parentheses denote coupling constants in Hz.The values are the sums of the individual coupling constants, JAX + JBX. Nu-(Fluoren-9-ylmethoxycarbonyl)-O-a-~-xylopyranosyl-( solvent B over a period of 50 rnin (fR 54 min). Following 1-3)-(a-~-xylopyranosyl-( 11.-lyophilization, compound 11 was obtained as a solid (22.0 mg, 1-3)-P-~-glucopyranosyl-~-serine A sample of 0-a-D-xylopyranosyl-( 1 -3)-a-u-xylopyranosy1- 76.0); 01h'.~ + 1.06 (c 0.15, MeOH); 'H and I3C NMR (1-3)-P-~-glucopyranosyl-~-serine (20.4 mg, 0.0384 mmol) data are presented in Tables 3 and 5, respectively; m/z 7541 was dissolved in a mixture of 1,4-dioxane (4.0 cm3) and 10 aq.(M + H)+ and 776 (M + Na)+ (C34H43N018 requires M, sodium carbonate (3.0 cm3). The solution was cooled to 0 OC, 75 3.2). and a solution of Fmoc-Su (19.4 mg, 0.0575 mmol) in 1,4- a-~-xylopyranosyl-dioxane (1 .O cm3) was added dropwise during 25 min. After 30 Nu-(Fluoren-9-ylmefhoxycarbonyl)-O-1-3)-P-~-glucupyranosyl-~-serinemin the flask was removed from the ice-bath and the mixture (1-3)-cr-~-xylupyranosyl-( was stirred for an additional 90 min before being transferred to a Penfa-uorophenyl Ester 12.-Compound 11 (20.5 mg, 0.0272 separatory funnel by using portions of water and ethyl acetate.mmol) was dissolved in THF (3.0 cm3), with the addition of The ethyl acetate layer was extracted with portions of water and DMF (0.5 cm3) to bring the sample into solution. To the stirred the combined aqueous fractions were lyophilized. The resulting solution, at room temperature, was added DCCI (9.5 mg, 0.046 residue was purified by semipreparative HPLC, using isocratic mmol). The solution was stirred at room temperature and a solvent A for 15 min followed by a linear gradient of 0-50 solution of Pfp-OH (10 mg, 0.054 mmol) in THF (0.2 cm3) was J.CHEM. SOC. PERKIN TRANS. I 1993 93 1 Table 4 'H NMR data" for compounds 13 and 14 ~~ 1-H 2-H 3-H 4-H 5-H 6-H2 P-G/c-( 1-3)-Ser 13 4.47 (8.0) 3.37 3.60 3.63 3.45 3.90, 3.71 14 4.46 3.41 3.60 3.61 3.46 3.91, 3.72 ~-Xyl-(-3I-Glc 13 5.27 (4.0) 3.65 3.78 3.82 3.88, 3.62 I 14 5.29 (3.8) 3.64 3.80 3.79 3.92, 3.61 x-Xyf-(1-3)-Xyl 13 5.30 (4.0) 3.53 (4.0, 10.0) 3.66 3.59 3.82, 3.64 14 5.31 (3.8) 3.53 (4.0, 9.5) 3.66 3.58 3.84, 3.64 NH a-H P-H2 Y-H2 6-H, ~~ Cyssl 13 4.29 3.18 (6.0, 15.0) 3.04 (8.0, 15.0) 4.38 3.45, 3.33 14 GIu5' 13 8.77 (7.0) 4.45 2.12,2.01 2.44 (15.0)b (2 H) 14 4.29 2.11, 1.99 2.36 (2 H) Ser53 13 8.58(8.0) 4.6 1 4.14 (6.0, 1 l.O), 3.91 14 4.72 3.99 (6.5, 10.5), 3.85 AS^^^ 13 8.45 (8.0) 5.02 2.84 (7.0, 15.0), 2.68 (8.0, 15.0) 14 4.74 2.70 (6.5, 15.0), 2.60 (8.0, 15.0) Pross 13 S.OS(8.0) 4.46 2.25 (2 H) 2.03 (2 H) 3.76 (2 H) 14 4.69 2.32, 2.18 I .93 (2 H) 3.62, 3.47 CYS~~ 4.42 3.11 (5.0, 14.0),13 2.97 (8.0, 14.0) 14 4.56 3.34, 2.99 (9.0, 14.0) ____~~~~ ~ Acm 13 8.55,8.54 4.42 (2 H), 2.01 (3 H), 1.98 (3 H) 4.30 (2 H) " Compound 13 in 90 water-lO D20, pH 3.6; compound 14 in D20,pD 4.2.The numbers in parentheses denote coupling constants in Hz. The value is the sum of the individual coupling constants, JAx + JBX. Table 5 13CNMR data" for compounds 4 and 9-12 ~~~~ ~~~~~ c-1 c-2 c-3 c-4 c-5 C-6 P-Gfc-C1-3)-Ser 4 101.1 71.8 72.5 69.4 72.5 62.8 9 104.7 75.0 77.9 71.5 78.1 62.6 10 104.2 75.1 78.0 71.7 78.2 62.9 11 104.9 73.7 86.0 71.4 77.6 62.4 12 104.5 73.8 86.0 71.5 77.8 62.6 a-Xy/-(l-3)-Glc 11 101.9 72.5 83.2 71.5 63.4 12 101.9 72.5 83.1 71.6 63.4 a-Xyl-(l-3)-Xyl 11 101.6 74.1 75.1 71.5 63.6 12 101.6 74.1 75.1 71.6 63.6 Fmoc Fmoc Ser C-a Ser C-P CH CH, Fmoc aromatic C Pfp c C0,Pfp OCONH Other signal 4 54.2 68.6 47.1 67.3 143.7, 143.6, 141.3, 127.8, 127.1, 140.8(250), 139.7 155.8 125.1, 120.0 (250), 137.7 (245) 9 55.7 70.8 48.4 68.2 145.3, 142.6, 128.8, 128.2, 126.3, 120.9 10 55.8 69.8 48.4 68.2 145.2, 145.1, 142.6, 128.7, 128.2, 142.5 (253), 139.4 168.2 158.6 126.2, 120.9 (197)11 55.9 71.0 48.4 68.1 145.3, 142.6, 128.8, 128.2, 126.3, 168.3 120.9 12 55.9 70.0 48.4 68.2 145.2, 145.1, 142.6, 128.8, 128.2, 142.4(259), 139.3 158.6 126.2, 120.9 (244) a Compound 4 in CDCI,, compounds 9-12 in CD,OD.These values are the one-bond 19F-13C coupling constants J(19F-' ,C) in Hz. added. After 24 h the mixture was evaporated to dryness, and {Bis-5-acetamidomethyl-Cys Cys46-0-a-xylopyranosyl-the residue was purified by semipreparative HPLC using a (1-3)-a-D-xylopyranosyl-(I -3)-P-D-gluCOpyranOSyl-~er53}-linear gradient of 30-60 solvent B over a period of 60 min (fR HBF-IX (51-56) 13.-The solid-phase peptide synthesis was 40 min). Compound 12 was obtained as a solid (10.6mg, 42.4); carried out using method B as described above, using resin (1 78 'H and 13C NMR data are presented in Tables 3 and 5, mg, equivalent to 0.0294 mmol of peptide).The loading of the respectively; m/z 920 (M + H)+ and 942 (M + Na)+ first amino acid onto the resin was estimated by quantitative (C40H42F5N018requires M, 919.2). amino acid analysis to be 0.165 mmol g-'. The activated amino Table 6 3CNMR data' for compounds 6 and 7 C-1 c-P c-y c-6 CyP 6 7 52.9 52.8 31.8 39.4 GW2 6 53.9 27.0 30.7 7 54.3 26.7 32.5 Ser5 6 54.3 69.2 7 55.8 69.6 6 49.4 37.1 7 49.6 37.5 pro55 6 61.6 30.2 25.2 48.7 7 60.9 31.1 22.8 48.5 CyP 6 53.7 32.8 7 52.8 39.6 Acm 6 41.9141.5 22.9122.9 c-1 C-2 C-3 C-4 C-5 C-6 ~~ P-GIc-(I-3)-Ser 6 7 103.1 103.3 73.9 73.9 76.4 76.3 70.5 70.5 76.7 76.7 61.7 61.6 a Compound 6 in 90 water-lO D,O, pH 2.8; compound 7 in D20, pD -6.0.acids were added in 3.5-fold excess, except for the glycosylated serine derivative 12 which was added in only 0.37 mol equiv. (10.1 mg, 0.110 mmol). The end-points of the acylations were estimated by the decrease in the yellow colour of the resin during the acylation. The end-point of the acylation for the glycosylated serine derivative 12 could not be determined by the decrease in the yellow colour of the resin (compound 12 was added in only 0.37 mol equiv.), therefore the reaction mixture was left for 43 h to ensure consumption of substrate 12. Purification of compound 13 was complicated by the presence of the deletion peptide which was a result of the expected incomplete acylation during the coupling of the glycosylated serine derivative.This impurity was expected to have a much different HPLC mobility than the glycosylated peptide; however, the chromatographic mobilities of the two products were almost identical on C-18 and C-8 reversed-phase columns as well as on a Biogel P-2 column. The desired compound was finally purified by repeated HPLC on a semipreparative C-8 column using a linear gradient of amp;20 solvent B over a period of 80 rnin (tR 31 min, with the deletion peptide eluting earlier at 27 min). Compound 13was finally obtained as a solid (3.2 mg, 24 yield based on the amount of compound 12 incorporated into peptide product). 'H NMR data are presented in Table 4; m/z 1220 (M + H)' (C45H,3N,02,S, requires M, 1219.4).Cyclic (O-a-D-Xylopyranosyl-( 1 -3)-a-~-xylopyranosyl-1-3)-P-D-glucopyranosyl-Sers3)HBF-IX (51-56) 14.-A sample of compound 13 (3.0 mg, 0.0024 mmol) was dissolved in TFA acid (3.0 cm3) containing anisole (10 mm3, 0.092 mmol). The solution was cooled to 0 OC, and a solution of thallium(r1I) trifluoroacetate in TFA (0.25 cm3; 15.5 pmol dm-3) was added. After 105 rnin the reaction mixture was evaporated to dryness and diethyl ether (2 cm3) was added to the residue. After the mixture had been stirred for 40 rnin the ether was decanted off, J. CHEM. SOC. PERKIN TRANS. i 1993 and the residue was purified by semipreparative HPLC using a linear gradient of amp;20 solvent B over a period of 80 rnin (rR 18 min). The product was further purified by passage through a Biogel P-2 column with water as the eluent.The cyclic glycopeptide 14 was obtained, after lyophilization, as a solid (2.0 mg, 75); 'H NMR data are presented in Table 4. Amino acid analyses (theoretical values are in parentheses); Asn 1.04 (I), Ser 0.91 (l), Glu 1.08 (I), Cys 1.97 (2) and Pro 1.OO (1); m/z 1076 (M + H)' and 1098 (M + Na)' (C3,H,,N,02,S2 requires M, 1075.3). Acknowledgements This work was supported by a Natural Sciences and Engineering Research Council of Canada Post-Doctoral Fellowship to K. B. R. We also thank Carl Erik Olsen, The Royal Veterinary and Agricultural University, Denmark for record of the mass spectra. References 1 A. M. Jansson, M. Meldal and K. Bock, J. Chem. SOC., Perkin Trans.I, 1992, 1699. 2 M. Meldal and K. J. Jensen, J. Chem. Soc., Chem. Commun., 1990,483. 3 C. M. Jackson and Y. Nemerson, Annu. Rev. Biochem., 1980,49,765. 4 K. G. Mann, R. J. Jenny and S. Krishnaswamy, Annu. Rev. Biochem., 1988,57, 915. 5 H. Nishimura, S.-I. Kawabata, W. Kisiel, S. Hase, T. Ikenaka, T. Takao, Y. Shimonishi and S. Iwanaga, J. Biol. Chem., 1989, 264, 20320. 6 H. Nishimura, S. Yamashita, Z. Zeng, D. A. Walz and S. Iwanaga, J. Biochem. (Tokyo), 1992, I 11,460. 7 S. Hase, H. Nishimura, S.-I. Kawabata, S. Iwanaga and T. Ikenaka, J. Biol. Chem., 1990,265, 1858. 8 B. Luning, T. Norberg and J. Tejbrant, J. Carbohydr. Chem., 1992, 11,933. 9 K. Fukase, S. Hase, T. Ikenaka and S. Kusumoto, Bull. Chem. Soc. Jpn., 1992,65,436. 10 L. Kisfaludy and I. Schon, Synthesis, 1983, 325. 11 R. K. Ness, H. G. Fletcher, Jr. and C. S. Hudson, J.Am. Chem. Soc., 1950,72,2200. 12 N. Fujii, A. Otaka, S. Funakoshi, K. Bessho and H. Yajima, J. Chem. Soc., Chem. Commun., 1987, 163. 13 L. A. Carpino and N. Y. Han, J. Org. Chem., 1972, 37, 3404; A. Paquet, Can. J. Chem., 1982,60,976. 14 F. Filira, L. Biondi, F. Cavaggion, B. Scolaroand R.Rocchi, Peptides 1989, Chemistry, Structure and Biology, Proceedings of the American Peptide Symposium, Ilth, La Jolla, 1990, p. 804. 15 L. Otvos, L. Urge, M. Hollosi, K. Wroblewski, G. Graczyk, G. C. Fasman and J. Thurin, Tetrahedron Lett., 1990,31, 5889. 16 B. J. Balcom and N. 0.Petersen, J. Org. Chem., I989,54, 1922. 17 M. Meldal, Acfa Chem. Scand., Sect. B, 1986,40,242; J. Kovacs, L. Kisfaludy and M. Q. Ceprini, J.Am. Chem. Soc., 1967,89, 183. 18 R. Stuike-Prill and B. Meyer, Eur. J. Biochem., 1990, 194,903. 19 R. Arshady, E. Atherton, D. L. J. Clive and R. C. Sheppard, J. Chem. Soc., Perkin Trans. I, 1981, 529. 20 B. Blankemeyer-Menge, M. Nimitz and R. Frank, Tetrahedron Lett., 1990,31, 1701. 21 J. M. Lacombe, A. A. Pavia and J. M. Rocheville, Can. J. Chem., 198 1,59,482. Paper 2/06856G Received 24th December 1992 Accepted 26th January 1993