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Bioconjugate Chem. 1998, 9, 126−131
TECHNICAL NOTES A New Method for the Synthesis of Neoglycopeptides† Paul R. Hansen,* Carl E. Olsen, and Arne Holm Research Center for Medical Biotechnology, Chemistry Department, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. Received May 21, 1997; Revised Manuscript Received September 30, 1997X
A new method for conjugation of small carbohydrates to peptides based on maleimido-thiol chemistry is described. The reducing carbohydrate is reacted with S-trityl-2-mercaptoethylamine in methanol. The resulting Schiff base is then stabilized by acetylation with acetic anhydride to give an S-tritylprotected glycosylamide. Activation of the carbohydrate is effected by brief treatment with trifluoroacetic acid/triisopropylsilane, followed by precipitation in ether. The thiol-functionalized carbohydrate is then reacted with a maleimido-modified peptide to give a neoglycopeptide. The potential of this method was investigated using maltose as the model compound. Furthermore, we prepared five maleimido-modified model peptides. In contrast to some literature reports, we found that the maleimido group is stable to treatment trifluoroacetic acid/triisopropylsilane which was used to cleave the modified peptides from the resin.
INTRODUCTION
Scheme 1
Glycopeptides containing an unnatural type of linkage between the saccharide moiety and peptide (e.g. a spacer) are termed neoglycopeptides. For a review of the area, see Magnusson (2) and references cited therein. Neoglycopeptides have been used for various purposes, including the determination of the influence of carbohydrate molecules on biological activity, conformation, and stability of the parent peptides (3). Neoglycopeptides have been prepared by reacting an amino group in the carbohydrate with a carboxyl group in the peptide (C-terminal, Asp,1 Glu) (3, 4) or by reacting 3-mercaptopropionic acid spacer glycosides with the N terminus of a peptide (5). Furthermore, reductive amination (6), use of a protected valine Amadori compound in peptide synthesis (7), and † This work was presented in part at the 24th European Peptide Symposium, Edinburgh, Scotland, September 8-13, 1996, and will be published in the proceedings (1). * Author to whom correspondence should be addressed. Telephone: +45-3528-2573. Fax: +45-3528-2398. E-mail: prh@ pepsyn.chem.kvl.dk. X Abstract published in Advance ACS Abstracts, December 15, 1997. 1Abbreviations used for amino acids are according to the IUPAC-IUB Commission on Biochemical Nomenclature [(1985) J. Biol. Chem. 260, 14-42]. Additional abbreviations: BME, β-mercaptoethanol; CCK, cholecystokinin; DCM, dichloromethane; DIC, N,N′-diisopropylcarbodiimide; DIEA, N,N-diethylisopropylamine; DMF, dimethylformamide; FAB-MS, fast atom bombardment mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbonyl; HOBt, 1-hydroxybenzotriazole; MALDI-TOF-MS, matrixassisted laser desorption/ionization time-of-flight mass spectrometry; Mca, 6-maleimidocaproyl; RP-HPLC, reversed-phase highperformance liquid chromatography; SPDP, N-succinimidyl-3(2-pyridyldithio)propionate; SPPS, solid-phase peptide synthesis; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TLC, thinlayer chromatography; Trt, triphenylmethyl.
periodate oxidation followed by reductive amination (8) have been described. Boas et al. (9) reported the coupling of an unprotected sugar to the N terminus of a protected resin-bound peptide, following the method described by Blomberg et al. (10) for immobilization of reducing sugars to matrices. The formed glycosylamine was stabilized by N-acetylation and the product cleaved from the resin. However, the yield was low, and many byproducts were observed. Schneller and Geiger (11) described a method in which a sulfhydryl group is introduced into the carbohydrate by reductive amination with cysteamine. The resulting product is then coupled to a SPDP-modified protein to form a disulfide bridge. In this paper, we describe a new strategy for conjugation of small carbohydrates to peptides based on maleimido-thiol chemistry. Maleimido moieties react fast, selectively, and irreversibly with sulfhydryl groups to form a thioether as shown in Scheme 1. This approach has been used for a variety of applications, including conjugation of enzymes to insulin (12), peptides to proteins (13), phosphorothioate nucleotides to proteins (14), and lipids to peptides (15), radioiodolabeling of hormones (16), and immonoassays (17). However, to the best of our knowledge, this chemistry has not been utilized for synthesis of neoglycopeptides. RESULTS AND DISCUSSION
The principle of our approach is shown in Scheme 2 using maltose and a 6-maleimidocaproic acid-modified peptide as an example.
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Technical Notes
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Scheme 2
Figure 1. Analytical HPLC of 6 following purification by preparative HPLC.
First, the protected thiol spacer S-trityl-2-mercaptoethylamine 1 is reacted with the reducing carbohydrate 2 in methanol. The resulting imine 3, which is in equilibrium with the glycosylamine 4, is then stabilized by N-acetylation with acetic anhydride. This principle was first described by Blomberg et al. (10) for immobilization of sugars to matrices. N-Acetyl-S-trityl-2mercaptoethylamine 5 is generated as a coproduct in the reaction. When using a small carbohydrate such as 2, the resulting spacer saccharide 6 may be purified by preparative RP-HPLC. Alternatively, the byproduct 5 may be removed from the crude reaction mixture by extraction. The S-trityl group, which is a commonly used protecting group for cysteine in Fmoc solid-phase peptide synthesis (18), is cleaved by brief treatment with trifluoroacetic acid/triisopropylsilane. Most glycosidic linkages and carbohydrate moieties are stable to treatment with TFA for 2 h, even with unprotected hydroxyl groups (19). Triisopropylsilane (20) was chosen as the scavenger for the trityl group, since water or thiol scavengers cannot be present during deprotection. Following precipitation in ether, the thiol-functionalized carbohydrate is then reacted with a maleimido-modified peptide2 or protein to 2For obvious reasons, a cysteine residue with an unprotected thiol group cannot be present in the maleimido-modified peptide.
give a neoglycopeptide or neoglycoprotein. When small carbohydrates are conjugated to peptides, the neoglycopeptide may be purified by preparative RP-HPLC. When small carbohydrates are conjugated to proteins, workup may be done by gel filtration. Synthesis of a Spacer-Modified Carbohydrate Model Compound. Using the above-described procedure with maltose, we prepared the model spacer saccharide N-acetyl-[2-[S-(triphenylmethyl)mercapto]ethyl][R-D-glucopyranosyl(1f4)]-β-D-glucopyranosylamine (6). The course of the reaction was easily monitored by RPHPLC. After overnight reaction, two peaks were observed. The imine 3 and starting material 1 eluted approximately after 30 and 40 min, respectively. After acetylation with a large excess of acetic anhydride and overnight reaction, the aforementioned peaks had disappeared; two new peaks appeared at approximately 37 min (6) and 47 min (5). HPLC analysis of the crude reaction mixture before workup always revealed some accompanying O-acetylated carbohydrate, with a tR of ∼39 min, usually less than 5%. Attempts to recover unreacted maltose by collecting the elute from the first 10 min only resulted in minute amounts of sugar. Compound 6 was obtained in 62% yield following preparative RP-HPLC (Figure 1). An alternative purification procedure for 6 involved washing the crude reaction mixture extensively with DCM. Using this approach, 6 could be obtained in 43.5% yield. The crude product may be used without further purification since unreacted maltose will not interfere with the subsequent conjugation reaction. When using 5 equiv of S-trityl-2-mercaptoethylamine, the reaction
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product was completely soluble in DCM. In this case, crude 6 was obtained by washing the lyophilized mixture extensively with water. As described by Blomberg et al. (10), we observed two methyl signals in the 1H NMR spectrum of 6 (at 1.82 and 2.10 ppm), indicating the presence of two rotamers in the ratio 1:3.4. The J1,2 values for the two rotamers (9.4 Hz, 5.24 ppm, H-1a; and 8.9 Hz, 4.65 ppm, H-1b) show that the glycosylamide 6 has the β-configuration. For R-linked N-glycopeptides, the J1,2 value is typically 5-6 Hz (21). The 13C NMR spectrum of 6 revealed 22 signals stemming from the major rotamer. All the expected peaks were identified. As with the 1H NMR spectrum, minor peaks indicated the presence of the other rotamer. Synthesis of a Maleimido-Modified Model Peptide. Keller and Rudinger (22), Marburg et al. (23), and Wu¨nsch et al. (24) have reported that the maleimido group is sufficiently stable toward TFA to allow its incorporation into a peptide chain. However, Papini et al. (25) reacted several histidine-containing porcine CCKrelated peptides with N-hydroxy succinimido maleoyl-βalaninate in DMF. They found that during workup under neutral conditions the Nim-imino function of the side chain of histidine reacts nearly quantitatively with the maleimido function. Furthermore, Papini and coworkers found this intramolecular cyclization reaction to proceed at remarkably higher rates than the bimolecular alkylation of histidine derivatives with N-ethylmaleimide. Jensen and Jakobsen (26) reported the direct solidphase peptide synthesis of N-maleoyl-β-alanyl-Leu-AlaGly-Val-OH using Fmoc chemistry. The product was cleaved from the resin with neat TFA, TFA/H2O, or TFA/ triethylsilane and isolated in good purity after gel filtration. Contrary to the above, Rusiecki et al. (27) claim that the maleimido group is degraded in TFA/CH2Cl2. In order to investigate the stability of the maleimido group to cleavage conditions of Fmoc SPPS, the model peptide Mca-WHRQDGKL-OH was synthesized. The peptide was designed so that it would contain the most commonly used side chain protecting groups (Boc, tBu, trityl, and Pmc) in Fmoc SPPS. Furthermore, we chose 6-maleimidocaproic acid (Mca) as the spacer, since this compound would be less prone to intramolecular cyclization compared to N-hydroxy succinimido maleoyl-βalaninate. After each coupling step, starting with Asp5, 50 mg of resin was removed and derivatized with 6-maleimidocaproic acid. The product was then cleaved from the resin with TFA/TIS (95:5) and subjected to analytical HPLC. In each case, the desired maleimido-modified peptide was obtained in >80% purity and showed the correct mass by FAB-MS. Analytical HPLC of crude McaWHRQDGKL-OH is shown in Figure 2. In order to establish that the maleimido function was available for conjugation, we reacted both Mca-HRQDGKL-OH and Mca-WHRQDGKL-OH with β-mercaptoethanol (BME) in 0.1 M NaOAc buffer at pH 4. After 18 h, the mixture was analyzed by HPLC and FAB-MS. In both cases, FAB-MS showed that BME had reacted with the maleimido group. Furthermore, the analytical HPLC showed no sign of starting material, but one new peak eluting 0.5 min earlier than the maleimido-modified peptide. Conjugation of a Spacer-Modified Carbohydrate to Maleimido-Modified Peptides. Encouraged by these results, we then synthesized two maltose-containing model neoglycopeptides, R-D-Glcp(1f4)-β-D-Glcp-1N-(Ac-(CH2)2-S-Mca-DRVYIHPF (9a) and R-D-Glcp(1f4)β-D-Glcp-1-N-Ac-(CH2)2-S-Mca-YETPTRMLVAGIAA (9b)
Hansen et al.
Figure 2. Analytical HPLC of crude WHRQDGKL-OH modified with 6-maleimidocaproic acid.
in a small scale using the approach outlined in Scheme 2. The crude maleimido-modified peptides Mca-DRVYIHPF and Mca-YETPTRMLVAGIAA were obtained in greater than 80% purity and used without further purification. The conjugation reaction was carried out in 0.1 M NaOAc at pH 4. The progress of the reaction was followed by analytical RP-HPLC and found to be complete after 18 h. Following purification by RP-HPLC, the neoglycopeptides were isolated in 71.2 and 55.4% yield, based on 6. Both 9a (Figure 3) and 9b (Figure 4) were isolated in high purity. The products were analyzed by MALDI-TOF-MS, and in both cases, the expected mass was obtained. In conclusion, we have investigated a new strategy for conjugation of reducing carbohydrates to peptides based on maleimido-thiol chemistry. We were able to show that this method works on a small scale for short carbohydrates by conjugating the maltose spacer saccharide N-acetyl-N-[2-[S-(triphenylmethyl)mercapto]ethyl][R-D-glucopyranosyl(1f4)]-β-D-glucopyranosylamine (6) to some maleimido-modified peptides. In both cases, very pure products were obtained, upon purification by RPHPLC. EXPERIMENTAL PROCEDURES
General. Analytical HPLC was performed using a Waters C18 reversed-phase column (Delta-Pak, 100 Å, 15 µm, Millipore) on a Waters 600E system equipped with Millennium software. Samples were chromatographed at a flow rate of 1.5 mL/min starting with 0.1% aqueous TFA (buffer A) for 10 min and increasing over 45 min to 0.1% TFA in CH3CN/H2O (9:1) (buffer B), with detection at 220 nm. For preparative HPLC, a Waters C18 reversedphase column (RCM-module, Millipore) was used.
Technical Notes
Figure 3. Analytical HPLC of 9a following purification by preparative HPLC.
NMR spectra were recorded on a Bruker AC 250P instrument. Amino acid analyses were performed on a Waters Pico-Tag analyzer after samples were hydrolyzed with 6 N aqueous HCl/0.1% phenol at 110 °C for 18 h. MALDI-TOF-MS was carried out on a Fisons TofSpec E instrument in the linear mode. FAB-MS was carried out on a JEOL JMS AX505W instrument. TLC was carried out on Kiesel Gel 60 F254 Plates (Merck, Germany). Spots were visualized by UV or spraying with a dilute solution of sulfuric acid followed by heating. Melting points were determined on a Gallenkamp melting point apparatus. Chemicals. Protected amino acids and Pepsyn KA resin were purchased from Milligen (Bedford, MA). Triisopropylsilane, DIC, MBS, and HOBt were obtained from Aldrich. 6-Maleimidocaproic acid was obtained from Fluka. DMF and DCM were from Riedel de Hae¨n (Seelze, Germany). Peptide Synthesis. Peptide synthesis was accomplished manually in a syringe equipped with a filter by a stepwise solid-phase peptide synthesis procedure starting with a Pepsyn K resin (0.200 g, 0.1 mequiv/g) using the Fmoc/tBu protection scheme (18). The following side chain-protected amino acid derivatives were used: Arg(Pmc), Asp(tBu), His(Boc), Tyr(tBu), Lys(Boc), Thr(tBu), and Trp(Boc). The amino acids were coupled in 3-fold excess using DIC (3 equiv) and HOBt (1.5 equiv) in DMF for 2 h. Fmoc removal was accomplished by treatment for 2 × 10 min with 20% piperidine in DMF. Following peptide synthesis, 6-maleimidocaproic acid (10 equiv) was coupled overnight to the peptidyl resin using DIC (10 equiv) and HOBt (1.5 equiv) in DMF. The product was cleaved from the support with TFA/TIS (90: 5, v:v) (2 mL) for 2 h and the resin washed with TFA.
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Figure 4. Analytical HPLC of 9b following purification by preparative HPLC.
The combined TFA washes (3 × 5 mL) were evaporated in vacuo, and the product was precipitated in dry ether (5 mL). After centrifugation, the product was washed three times with ether (3 × 5 mL) and dried. In order to investigate the stability of the 6-maleimidocaproic acid (Mca) moiety to cleavage conditions of Fmoc SPPS, a 6-maleimidocaproic acid-modified model peptide, McaWHRQDGKL-OH, was synthesized. After each coupling step, starting with Asp5, 50 mg of resin was removed and derivatized with 6-maleimidocaproic acid as described above and subjected to analytical HPLC. Mca-DGKL-OH HPLC purity: >95%. FAB-MS: calcd for [M + H+] (C28H44N6O10) 624, found 625. McaQDGKL-OH HPLC purity: >95%. Mca-RQDGKL-OH HPLC purity: >95%. Mca-HRQDGKL-OH HPLC purity: >80%. FAB-MS: calcd for [M + H+] (C45H71N15O14) 1046, found 1068 [M + Na]+. Amino acid analysis (found/ theory): Asp (1.0/1), Glu (1.0/1), Gly (1.1/1), His (0.9/1), Arg (1.0/1), Leu (1.1/1), Lys (1.1/1). Mca-WHRQDGKLOH HPLC purity: >80%. MALDI-TOF-MS: calcd for [M + H+] (C56H82N17O15) 1233, found 1233. In order to establish that the maleimido function was available for conjugation, Mca-HRQDGKL-OH (1 mg, 1.0 × 10-3 mmol) was dissolved in 0.1 M NaOAc buffer at pH 4 (1 mL), to which was added BME (10 µL, 1.2 × 10-1 mmol). After 18 h, the reaction mixture was analyzed by HPLC which showed no sign of starting material, but one new peak eluting 0.5 min earlier as compared to the maleimido-modified peptide. FAB-MS: calcd for [M + H+] (C47H76N15O15S) 1123, found 1124. A similar experiment was carried out with McaWHRQDGKL-OH. MALDI-TOF-MS: calcd for [M + H+] (C58H87N17O16S) 1310, found 1308. Mca-DRVYIHPF amino acid analysis (found/theory): Asp (0.9/1), His (1.0/
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1), Arg (0.8/1), Pro (0.8/1), Tyr (0.9/1), Val (1.0/1), Ile (0.9/ 1), Phe (1.0/1). MALDI-TOF-MS: calcd for [M + H+] (C60H83N14O15) 1240, found 1241. HPLC showed one major peak with a tR of 34 min (>85% purity). McaYEVTPTRMLVAGIAA amino acid analysis (found/ theory): Glu (0.8/1), Gly (1.0/1), Arg (0.8/1), Ala (3.0/3), Thr (1.5/2), Pro (0.9/1), Tyr (0.8/1), Val (1.7/2), Met (1.0/ 1), Ile (0.9/1), Leu (1.0/1). MALDI-TOF-MS: calcd for [M + H+] (C81H130N119O24S) 1786, found 1786. HPLC showed one major peak with a tR of min 37 (>80% purity). Synthesis of S-Trityl-2-mercaptoethylamine (1). The title compound was prepared as described by Herman et al. (28). Synthesis of N-Acetyl-N-[2-[S-(triphenylmethyl)mercapto]ethyl][R-D-glucopyranose(1f4)]-β-D-glucopyranosylamine (6). Method A. Maltose monohydrate (18 mg, 0.05 mmol) and S-trityl-2-mercaptoethylamine (32 mg, 0.1 mmol) in MeOH were refluxed for 18 h. Next, Ac2O (500 µL, 5.0 mmol) was added at 0 °C, and the mixture was left stirring for 18 h. The solvent was then removed by evaporation and the residue dissolved in H2O/CH3CN (4: 1) and lyophilized. The resulting white powder was dissolved in methanol (2 mL) and purified by preparative HPLC to give 21.2 mg (61.8%) of the title compound. FAB-MS: calcd for [M + H+] (C35H44O11NS) 686, found 686. Selected 1H NMR data (a ) minor rotamer, b ) major rotamer; 250 MHz, CH3OD): δ 7.3 (m, 15H, aromatic H), 5.24 (J1,2 ) 9.39 Hz, β-D-Glcp, H-1a), 5.18 (J1,2 ) 3.9 Hz, R-D-Glcp, H-1), 4.65 (J1,2 ) 8.9 Hz, β-D-Glcp, H-1b), 3.10 (t, 2.2H, CH2N), 2.5 (m, 2H, CH2S), 2.10, (s, 2.3H, methyl), 1.82 (s, 0.7H, methyl). 13C NMR (62 MHz, CH3OD): δ 174.7 (CON), 146.5 (C-1 aromatic ring), 130.9, 129.1, 129.0, 128.1, 128.0 (aromatic ring C), 103.0 (C-1, R-D-Glcp), 88.6 (C-1, β-D-Glcp), 81.1 (C-4, β-D-Glcp), 79.2, 78.8 (C-5, R-D-Glcp and β-D-Glcp), 75.1, 74.9 (C-3, R-DGlcp and β-D-Glcp), 74.2 (C-4, R-D-Glcp), 71.5, 71.3 (C-2, R-D-Glcp and β-D-Glcp), 68.0 [SC(Ph)3], 62.7 (C-6, R-DGlcp and β-D-Glcp), 45.3 (CH2N, a), 42.1 (CH2N, b), 32.7 (CH2S, a), 31.5 (CH2S, b), 22.0 (CH3). The carbohydrate carbon atoms were assigned on the basis of literature values (29). Anal. Calcd for C35H43O11NS: C, 61.3; H, 6.32; N, 2.04; S, 4.67. Found C, 59.9; H, 5.8; N, 1.9; S, 4.2. Method B. Maltose monohydrate (36 mg, 0.1 mmol) and S-trityl-2-mercaptoethylamine (64 mg, 0.2 mmol) were reacted in MeOH as described above. Following lyophilization, the reaction mixture was washed with DCM (10 × 2 mL) to remove excess 1 and byproduct 5. The product was then lyophilized to give 29.8 mg of crude 6 (43.5%). Method C. Maltose monohydrate (36 mg, 0.1 mmol) and S-trityl-2-mercaptoethylamine (160 mg, 0.5 mmol) were reacted in MeOH as described above, except that 2 mL of acetic anhydride was used. Following lyophilization, the product was extracted by washing with H2O (250 mL) to give 32.1 mg of product (46.9% crude yield). Synthesis of R-D-Glcp-(1f4)-β-D-Glcp-1-N-Ac-(CH2)2-SMca-DRVYIHPF (9a). 6 (1 mg, 1.5 × 10-3 mmol) was deprotected by treatment with TFA/TIS (95:5, 1 mL) for 5 min. The solution turned yellow upon adding the deprotection mixture. However, the color disappeared after a few seconds, indicating a very fast reaction. Following evaporation, the product was precipitated in ether and dried. In a separate experiment, HPLC analysis of the product showed no trace of the starting material 6. The thiol-functionalized carbohydrate was then dissolved in 500 µL of 0.1 M NaOAc buffer at pH 4, transferred to a test tube containing conjugate Mca-
Hansen et al.
DRVYIHPF (3.5 mg, 2.8 × 10-3 mmol), and diluted with buffer to a final volume of 1 mL. The reaction mixture was left on a rocking table overnight, lyophilized, and purified by preparative HPLC. The yield was 1.8 mg (71.2% based on 6). Analytical HPLC is shown in Figure 3. MALDI-TOF-MS: calcd for [M + H+] (C76H113N15O25S) 1684.9, found 1685.0. Synthesis of R-D-Glcp(1f4)-β-D-Glcp-1-N-Ac-(CH2)2-SMca-YETPTRMLVAGIAA (9b). 6 (1.5 mg, 2.2 × 10-3 mmol) was conjugated to Mca-YEVTPTRMLVAGIAA (5 mg, 2.8 × 10-3 mmol) as described above. The product was purified using preparative HPLC (Figure 4). The yield was 2.7 mg. (55.4% based on 6). MALDI-TOFMS: calcd for [M + H+] (C97H160N20O35S2) 2230.6, found 2229.3. LITERATURE CITED (1) Hansen, P. R., Heron, I., Olsen, C. E., Jakobsen, M. H., and Holm, A. (1997) A new strategy for the synthesis of neoglycopeptides based on maleimido-thiol chemistry. In Peptides 1996: Proceedings of the 24th European Peptide Symposium (R. Epton, Ed.) ESCOM, Leiden, The Netherlands ( in press). (2) Magnusson, G., Chernak, A. Y., Kihlberg, J., and Kononov, L. O. (1994) Synthesis of neoglycoconjugates. In Neoglycoconjugates: Preparation and Applications (Y. C. Lee and R. T. Lee, Eds.) pp 53-143, Academic Press, San Diego. (3) Varga-Defterdarovic, L., Horvat, S., Chung, N. N., and Shiller, P. W. (1992) Glycoconjugates of opioid peptides. Int. J. Pept. Protein Res. 39, 12-17. (4) Christiansen-Brams, I., Meldal, M., and Bock, K. (1993) Solid-phase synthesis of of a di-N-glycosylated RGD analog. In Peptides 1992. Proceedings of the Twenty-Second European Peptide Symposium (C. H. Schneider and A. N. Eberle, Eds.) pp 365-366, ESCOM, Leiden, The Netherlands. (5) Elofsson, M., Roy, S., Walse, B., and Kihlberg, J. (1993) Solid-phase synthesis and conformational studies of glycosylated derivatives of helper-T-cell immunogenic peptides from hen-egg lysozyme. Carbohydr. Chem. 240, 89-103. (6) Morehead, H., McKay, P., and Wetzel, R. (1982) Highperformance liquid chromatography analysis in the synthesis, characterization, and reactions of neoglycopeptides. Anal. Chem. 126, 29-36. (7) Forrow, N. J., and Batchelor, M. J. (1990) Synthesis of the N-glycopeptide partial sequence A1-A12 of the b-Chain og glycosylated haemoglobin HbA1c. A new approach to Amadori N-glycopeptides. Tetrahedron Lett. 31, 3493-3495. (8) Sasaki-Yagi, Y., Kimura, S., Ueda, H., and Imanishi, Y. (1994) Binding of enkephalin/dextran conjugates to opiod receptors. Int. J. Pept. Protein Res. 43, 219-224. (9) Boas, U., Heegaard, P., and Jakobsen, M. H. (1996) Solid phase coupling of unprotected reducing carbohydrates to the N-terminus of peptides. In Innovations and Perspectives in Solid Phase Synthesis (R. Epton, Ed.) Mayflower, Andover, England (in press). (10) Blomberg, L., Wieslander, J., and Norberg, T. (1993) Immobilization of reducing oligosacchararides to matrices by a glycosylamide linkage. J. Carbohydr. Chem. 12, 265-276. (11) Schneller, M., and Geiger, R. E. (1992) An effective method for the synthesis of neoglycoproteins and neogangliosides by use of reductively aminated sulfhydryl-containing carbohydrate conjugates. Biol. Chem. Hoppe-Seyler 373, 1095-1104. (12) Kitagawa, T., and Aikawa, T. (1976) Enzyme coupled immunoassay of insulin using a novel coupling reagent. J. Biochem. 76, 233-236. (13) Plaue´, S., Muller, S., Briand, J. P., and van Regenmortel, M. V. H. (1990) Recent advances in solid-phase peptide synthesis and preparation of antibodies to synthetic peptides. Biologicals 18, 147-157. (14) Karim, A. S., Johansson, C. S., and Weltman, J. K. (1995) Maleimide-mediated proteincojugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate. Nucleic Acids Res. 23, 2037-2040. (15) Moroder, L., Romano, R., Guba, W., Mierke, D. F., Kessler, H., Delporte, C., Winand, J., and Christophe, J. (1993) New
Technical Notes evidence for a membrane-bound pathway in hormone receptor binding. Biochemistry 32, 13551-13559. (16) Chorev, M., Caulfield, M. P., Roubini, E., McKee, R. L., Gibbons, S. W., Leu, C.T., Levy, J. J., and Rosenblatt, M. (1992) A novel mild, specific and indirect maleimido-based radiolabeling method. Radiolabeling of analogs derived from parathyroid hormone (PTH) and PTH-ralated protein (PTHrP). Int. J. Pept. Protein Res. 40, 445-455. (17) Moroder, L., Tzougraki, C., Go¨hring, W., Mourier, G., Musiol, H.-J., and Wu¨nsch, E. (1987) Studies on Immunoassays of Peptide Factors V. Synthesis of cholecystokinin-58[1-11]/Iso-1-cytochrome c conjugate. Biol. Chem. Hoppe-Seyler 368, 855-861. (18) Fields, G. B., Tian, Z., and Barany, G. (1992) Principle and practice of solid-phase peptide synthesis. In Synthetic Peptides: A User’s Guide (G. A. Grant, Ed.) pp 77-183, W. H. Freeman and Co., New York. (19) Urge, L., Otvos, L., Jr., Lang, E., Wroblewski, K., Laczko, I., and Hollosi, M. (1992) Fmoc-protected, glycosylated asparagines potentially useful as reagents in the solid-phase synthesis of N-glycopeptides. Carbohydr. Res. 235, 83-93. (20) Pearson, D. A., Blanchette, M., Baker, M. L., and Guidon, C. A. (1989) Trialkylsilanes as scavengers for the trifluoroacetic acid deblocking of protecting groups in peptide synthesis. Tetrahedron Lett. 30, 2739-2742. (21) Ratcliffe, A. J., Konradson, P., and Fraser-Reid, B. (1991) Application of n-pentenyl glycosides in the regio- and stereocontrolled synthesis of R-Linked N-glycopeptides. Carbohydr. Res. 216, 323-335. (22) Keller, O., and Rudinger, J. (1975) Preparation of some properties of maleimido acids and maleoyl derivatives of peptides. Helv. Chim. Acta 58, 531-541.
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