J. Org. Chem. 1998, 63, 9657-9668 9657 Novel Type of Rigid C-Linked Glycosylacetylene-Phenylalanine Building Blocks for Combinatorial Synthesis of C-linked Glycopeptides Todd Lowary,† Morten Meldal,*,† Arnim Helmboldt,† Andrea Vasella,‡ and Klaus Bock† Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark, and Institute of Organic Chemistry, ETH-Center, Universitätstrasse 16, CH-8092 Zürich, Switzerland Received March 18, 1998 C-linked 3- and 4-(glycosylacetylene)-Fmoc-phenylalanines were synthesized as rigid building blocks for synthesis of libraries of neoglycopeptide templates. Perbenzylated 1,5-galactonolactone, 1,5- gluconolactone, and 1,5-mannonolactone were reacted with cerium TMS-acetylide and the products reduced to the C-glycosylacetylene-TMS derivatives. The gluco and galacto configurations yielded exclusively the β-C-glycoside, whereas the mannonolactone gave a mixture of R-C- and β-C-anomer. The products were subjected to acetolysis and TMS cleavage. The resulting peracetylated glycosylacetylenes were cross-coupled with the (R,S)-NR-acetyl-3- and 4-iodophenylalanine O-methyl esters by Pd(0) catalysis in piperidine to give the eight possible C-linked glycosyl amino acid building blocks 33-40 as diastereomeric pairs. These were O-deacetylated. As an example of further conversion into neoglycopeptide templates the N-acetyl group of the 4-linked galactodiastereomeric pair 43 was hydrolyzed selectively by acylase 1 and separated from the (R)stereoisomer. The product was converted to 4-(β-C-galactosylacetylene)(NR-Fmoc-)-L-phenylalanine (52) by reaction with Fmoc-OSu. Acid hydrolysis of galactosyl acetylene phenyl alanine 43 at elevated temperature afforded the conversion of the acetylene to the 1′-oxo derivative which was also NR-Fmoc protected. The building blocks were used in the glycopeptide synthesis of three neoglycopeptide templates 54, 55, and 56 known to bind to murine MHC class II Ek and to present the glycan for interaction with the T-cell receptor. This template has previously been employed to elicit a carbohydrate specific T-cell response. The structural diversity of N- and O-linked oligosaccharides correlates with their role in cellular communication and regulatory functions. This has recently been highlighted by the discovery of the ubiquitous family of the galactose binding galectins that are involved in such important processes as development, metastasis, and apoptosis.1 Similarly, the mannose 6-phosphate receptor is involved in intracellular protein sorting and binds to glycopeptide templates with high affinities.2 The key role played by the recognition of carbohydrate epitopes by E-, L-, and P-selectins in the process of cell-cell adhesion and inflammation is well recognized, and the identification of the SLex tetrasaccharide as the ligand for selectin binding has generated enormous interest in the design and synthesis of carbohydrate-based inhibitors of SLexselectin binding as novel therapeutic agents. As a rule, the natural oligosaccharide ligands require a lengthy and costly synthesis and are not accessible in sufficiently large amounts. This has led to considerable efforts toward developing carbohydrate mimics. In a particularly attractive class of such mimics a large portion of the oligosaccharide is replaced by non-carbohydrate and particularly by peptide scaffolds.3 As a wide range of amino acids are commercially available, this approach offers great potential for the preparation of glycopeptide * Corresponding author. Phone: (45) 33275301. Fax: 45 33274708. E-mail: MPM@CRC.DK. † Carlsberg Laboratory. ‡ ETH-Center. (1) Pace, K. E.; Baum, L. G. Trends Glycosci. Glycotechnol. 1997, 9, 21-29. (2) Christensen, M. K.; Meldal, M.; Bock, K.; Cordes, H.; Mouritsen, S.; Elsner, H. J. Chem. Soc., Perkin Trans. 1 1994, 1299-1310. libraries. It is also attractive, because several glycopeptides mimicking the oligosaccharide have displayed relatively higher affinities to their receptors,2,4 while oligosaccharides often bind with relatively low affinities. However, while small glycopeptides are an attractive alternative to complex oligosaccharides, glycopeptides containing naturally occurring (O- or N-linked) carbohydrate-amino acid linkages could be anticipated to be poor therapeutics. They are metabolically unstable and possess a relatively short biological half-life. The delivery of such polar compounds into the cell across the lipid bilayer is generally difficult. Finally, while the flexibility associated with the linkage between sugar and peptide is not necessarily a negative attribute, decreased flexibility should improve the interaction with receptors or enzymes by reducing the entropy of binding. Indeed, considerable effort has been allocated to the synthesis of conformationally rigid oligosaccharide analogues for the investigation of carbohydrate-protein binding.5 We have envisaged the preparation of a new class of glycosyl-amino acid building blocks comprised of glycosylacetylenes C-linked to aromatic amino acids of the general type 1 (Schemes 1 and 2). Over the last years, (3) Meldal, M.; Christiansen-Brams, I.; Christensen, M. K.; Mouritsen, S.; Bock, K. In Complex carbohydrates in drug research. Structural and functional aspects; Bock, K., Clausen, H., Eds.; Munksgaard: Copenhagen: 1994; pp 153-165. (4) Uchiyama, T.; Woltering, T. J.; Wong, W.; Lin, C. C.; Kajimoto, T.; Takebayashi, M.; Weitz, S. G.; Asakura, T.; Noda, M.; Wong, C.-H. Bioorg. Med. Chem. 1997, 4, 1149-1165. (5) Franzyk, H.; Christensen, M. K.; Jørgensen, M.; Meldal, M.; Cordes, H.; Mouritsen, S.; Bock, K. Bioorg. Med. Chem. 1997, 5, 2140. 10.1021/jo980517h CCC: $15.00 © 1998 American Chemical Society Published on Web 12/08/1998 Lowary et al. 9658 J. Org. Chem., Vol. 63, No. 26, 1998 Scheme 1 Scheme 2 efficient methods for the synthesis of O- and N-linked glycopeptides have been reported.6-11 In the most successful approaches glycopeptide synthesis is preceded by the preparation of the required glycosyl-amino acid building blocks that are incorporated into the peptide chain using standard peptide synthesis protocols. Recently, one of us has published several syntheses of oligomers of C-glycosylacetylenes.12-20 The monomers possess a number of attractive features which may contribute to solve many of the above-mentioned problems associated with N- or O-linked glycopeptides. Similar to other C-glycosides, acetylenoglycopeptides should be tolerant of both the acidic and basic conditions normally used for glycopeptide synthesis, and their carbohydrate-protein linkages should be metabolically stable. The acetylene bridge between the sugar and amino acid is less polar than the normal ether or amide linkage of O- or N-glycopeptides and may confer improved membrane transport properties. This bridge is also rigid, so that acetylenoglycopeptides should present the saccharide moieties in a much less flexible fashion than naturally occurring glycopeptides. However, the most intriguing feature of these molecules is linked to the chemical versatility of the acetylene functionality and to the many ways in which 1 can be converted into a number of other building blocks (Scheme 1). There is thus enormous potential for creating molecular diversity from these monomers. Acetylenes can be hydrated to ketones (A), oxidized into R,β diketones (B), 21 reduced to either (E)- or (Z)-alkenes (C and D),22,23 converted to cyclohexadienes by Diels-Alder reaction with dienes (E),24 and transformed into R,β unsaturated (6) Nakahara, Y.; Iijima, H.; Ogawa, T. Synth. Oligosaccharides; ACS Symposium Series 560; American Chemical Society: Washington, DC, 1994; pp 249-266. (7) Meldal, M. Curr. Opin. Struct. Biol. 1994, 4, 710-718. (8) Meldal, M. In Neoglycoconjugates: preparation and application; Lee, Y. C., Lee, R. T., Eds.; Academic Press: San Diego: CA, 1994; pp 145-198. (9) Meldal, M.; Bock, K. Glycoconjugate J. 1994, 11, 59-63. (10) Meldal, M.; Hilaire, P. M. St. Curr. Opin. Biol. Chem. 1997, in press (11) Norberg, T.; Lüning, B.; Tejbrant, J. Methods Enzymol. 1995, 247, 87-106. (12) Alzeer, J.; Vasella, A. Helv. Chim. Acta 1995, 78, 177-193. (13) Alzeer, J.; Cai, C.; Vasella, A. Helv. Chim. Acta 1995, 78, 242264. (14) Cai, C.; Vasella, A. Helvetica Chimica Acta 1995, 78, 732757. (15) Alzeer, J.; Vasella, A. Helv. Chim. Acta 1995, 78, 1219-1237. (16) Cai, C.; Vasella, A. Helv. Chim. Acta 1995, 78, 2053-2064. (17) Cai, C.; Vasella, A. Helv. Chim. Acta 1996, 79, 255-268. (18) Bürli, R.; Vasella, A. Helv. Chim. Acta 1996, 79, 1159-1168. (19) Ernst, A.; Vasella, A. Helv. Chim. Acta 1996, 79, 1279-1294. (20) Xu, J.; Egger, A.; Bernet, B.; Vasella, A. Helv. Chim. Acta 1996, 79, 2004-2012. (21) Srinivasan, N. S.; Lee, D. G. J. Org. Chem. 1979, 44, 15741580. (22) McEwan, A. B.; Guttieri, M. J.; Maier, W. F.; Laine, Y.; Shvo, Y. J. Org. Chem. 1983, 48, 4436-4438. (23) Ulan, J. G.; Maier, W. F.; Smith, R. M. J. Org. Chem. 1987, 52, 3132-3142. Combinatorial Synthesis of C-Linked Glycopeptides J. Org. Chem., Vol. 63, No. 26, 1998 9659 aldehydes by rhodium-catalyzed hydroformylation (F).25 Many of these products could be further elaborated, e.g. by reductive amination of the ketones, R,β diketones, or R,β unsaturated aldehydes or by Michael addition to the R,β unsaturated aldehydes. As presented in Schemes 1 and 2 an indefinitely large number of glycopeptides can be envisaged resulting from a combination of the large number of conceivable glycosylacetylene-amino acid building blocks with the large number of subsequent synthetic manipulations. Related to this is a recent report20 on a Bergman cyclization of 1,2-bis(2′-glucosylalkynyl)benzenes to provide 2,3-diglucosylated naphthalenes. Reported here is the preparation of building blocks 3338 and 41-53 where glucosyl, galactosyl, and mannosyl residues are β-linked via the ethynediyl bridge to the meta or para position of phenylalanine (Figure 1 and Scheme 6). The preparation of the conjugates 39 and 40, where phenylalanine is linked to an R-mannosylacetylene is also described. The preparation of the building blocks involves the reductive introduction of the “anomeric” ethynyl moiety, resolution of the racemic phenylalanines, alkynyl-aryl coupling,26-28 and appropriate protection/ deprotection of the glycosyl and phenylalanyl moieties. For the incorporation of these glycosyl-amino acid building blocks into glycopeptides, we planned to activate the carboxyl group in the presence of the free hydroxyl groups using TBTU.29 We also report the synthesis of C-linked glycosylacetylene-conjugated analogues of the mouse hemoglobinderived decapeptide Hb(67-76), VITAFNEGLK substituted at position 72, binding well to the MHC Class II Ek molecule, and possibly eliciting a carbohydrate specific T-cell response. These conjugates were synthesized by multiple-column peptide synthesis (MCPS) employing the building blocks 52 and 53 (Scheme 6). Previous studies suggest that T-cells may recognize glycans that are in direct contact with the T-cell receptor when displayed on the Hb(67-76) epitope. Synthesis of the Glycosylacetylenes. The glucosylacetylenes 2 and 3 were prepared as reported.12 Similarly, we synthesized the galactosylacetylenes 4 and 5, as illustrated in Scheme 3. The known lactone 630 was converted in 91% yield to the ketose 7 by reaction with cerium TMS-acetylide. The hemiacetal was reduced by treatment with triethylsilane and boron trifluoride etherate to provide the β-linked C-glycoside 8 (71%). None of the corresponding R-anomer could be isolated; however, traces (4%, product characterized only by NMR) of the TES derivative 9 were formed by silyl exchange. The alkyne was desilylated with sodium hydroxide, yielding 99% of the benzylated alkyne 4. However, unlike the glucose case,12 where the benzyl groups could be removed within a few hours by acetolysis (acetic anhydride/O(trimethylsilyl)trifluoromethanesulfonate), debenzylation (24) Bastide, J.; Henri-Rosseau, O. In The chemistry of the triple bond; Wiley: New York, 1978; pp 447-552. (25) Johnson, J. R.; Cuny, G. D.; Buchwald, S. L. Angew. Chem., Intl. Ed. Engl. 1995, 34, 1760-1763. (26) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 16, 4467-4470. (27) Takahashi, S.; Kuroyama, Y.; Sonogashira, K.; Hagihara, N. Synthesis 1980, 627-630. (28) Sonogashira, K. In Comprehensive organic synthesis; Trost, B. M., Ed.; Pergamon: Oxford, U.K., 1991; pp 521-549. (29) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahedron Lett. 1989, 30, 1927-1930. (30) Lewis, M. D.; Cha, J. K.; Kishi, Y. J. Am. Chem. Soc. 1982, 104, 4976-4978. Figure 1. Lowary et al. 9660 J. Org. Chem., Vol. 63, No. 26, 1998 Scheme 3 of the galacto-isomer was very sluggish, and extended reaction times were required for complete conversion. Shorter reaction times resulted in large amounts of the 3-O-benzyl derivative 10. Nevertheless, after 3 days the desired tetraacetate 5 was obtained in 81% yield. Attempts to shorten the reaction by heating resulted in decomposition, leading to large amounts of tarry byproducts that complicated the purification. For the synthesis of the R- and β-mannosylacetylenes 16-29 (Scheme 3), we treated the mannopyranosyl lactone 1130 similarly to 6 to yield 93% of the ketose 12. However, whereas the reduction of the glucose and galactose acetylenes with triethylsilane and boron trifluoride etherate gave only the β-anomers, reduction of 12 Scheme 4 resulted in both the R- and β-C-mannosides 13 and 15, respectively. Chromatography provided pure 13 in 55% yield, while 15 was contaminated by a small amount of the TES derivative 14. Treatment of the pure β-anomer 13 with sodium hydroxide provided a 98% yield of 16. Similar treatment of the mixture of 14 and 15 followed by chromatographic separation afforded pure 17 in 22% yield from ketose 12. Acetolysis of the perbenzylated acetylenes 16 and 17 provided the tetraacetates 18 and 19 in 72% and 61% yields, respectively. Again, acetolysis was protracted; the byproduct being the 3-O-benzyl-2,4,6-triacetate. It appears that, at the least for glycosylacetylenes, acetolysis of the equatorial benzyloxy group of a pair of vicinal cis benzyloxy groups proceeds much more slowly. Synthesis of Aromatic Amino Acid Derivatives. Racemic mixtures of the 3-bromo-N-acetylphenylalanine methyl ester 20,31 the 4-bromo-N-acetylphenylalanine methyl ester 21,31 and the 4-O-triflate of N-acetyl-Ltyrosine methyl ester 2232 were prepared as described previously. Commercially available racemic 4-iodophenylalanine 23 was acetylated and esterified with dimethoxypropane and hydrogen chloride to yield 97% of racemic 24 (Scheme 4). The 3-iodo-N-acetylphenylalanine methyl ester was prepared by condensation of 3-nitrobenzaldehyde (25) with N-acetylglycine (26) followed by treatment of the azalactone 27 with methanol to provide the crystalline enamide 28 in an overall yield of 27%. Hydrogenation of 28 in the presence of Pd/C yielded 96% of the 3-aminophenylalanine derivative 29 that was transformed into the iodo analogue 30 by diazotization and treatment with potassium iodide (48%). (31) Burk, M. J.; Feaster, J. E.; Nugent, W. A.; Harlow, R. L. J. Am. Chem. Soc. 1993, 115, 10125-10138. Combinatorial Synthesis of C-Linked Glycopeptides Scheme 5 Cross-Coupling. Initial attempts at cross-coupling involved the reaction of the bromo derivative 20 and the benzylated glucosylacetylene 2. However, the use of standard conditions (triethylamine, copper iodide, (Ph3)2PdCl2, room temperature) resulted in an 82% yield of the known butadiyne 3112 (Scheme 5) and none of the desired cross-coupled product 32. Since yields in these crosscoupling reactions have been reported to be very sensitive to the solvent,33 a number of organic bases (triethylamine, N-ethylmorpholine, piperidine, and N,N,N′,N′-tetramethylethylenediamine) were investigated as solvents, without positive result. Similarly, replacement of the catalyst with (Ph3)4Pd gave none of the cross-coupled product, nor did increasing the reaction temperature (up to 120 °C in N,N,N′,N′-tetramethylethylenediamine). Similarly, attempted coupling of the glucosylacetylene 2 and the tyrosine triflate 22 under a variety of conditions led only to the butadiyne 31. In contrast, the reaction between 2 and the 4-iodophenylalanine 24 at room temperature in diethylamine with copper iodide and (Ph3)4Pd gave a 20% yield of the desired cross-coupled products 32 (Scheme 5). The diastereomeric pair was most conveniently separated later by enzymatic differentiation as described below. Encouraged by this result, we optimized the reaction conditions. Best results were obtained by heating the iodide 24 and the acetylene 2 at 80 °C in the presence of copper iodide and (Ph3)4Pd in piperidine. Under these conditions, we obtained 32 in a 81% yield. Piperidine has recently been reported20 to be the solvent of choice for the cross-coupling of 2 with other iodobenzene derivatives. Unfortunately, debenzylation by treatment of 32 with acetic anhydride and (32) Petrakis, K. S.; Nagabhushan, T. L. J. Am. Chem. Soc. 1987, 109, 2831-2833. (33) Alami, M.; Ferri, F.; Linstrumelle, G. Tetrahedron Lett. 1993, 34, 6403-6406. J. Org. Chem., Vol. 63, No. 26, 1998 9661 O-(trimethylsilyl)trifluoromethanesulfonate led to decomposition. However, cross coupling of the acetylated alkyne 3 with 24 proceeded equally well, the only problem being that during the course of the reaction the acetoxy groups were partially cleaved. The crude product was thus acetylated prior to purification. The acetylated glycosylacetylenes 3, 5, 18, and 19 were then coupled with the iodinated phenylalanine derivatives 24 and 30 leading to the products 33-40 (Figure 1) in yields between 69% and 91%. Deacetylation with sodium methoxide and methanol was followed by saponification of the methyl ester with sodium hydroxide to give 41-48, each as pairs of diastereoisomers, in yields of over 95%. Nr-Deacetylation of the Glycosyl Acetylene Amino Acids. Two methods for the NR-deacetylation of the free acids 41-48 were tested. Nonenzymatic deacetylation requires forcing conditions,34 but the glycosylacetylene amino acid conjugates possessing a carboxyl group may well be substrates for deacetylating enzymes that would ensure mild conditions. Also, enzymes should be selective for the configuration of the amino acid moiety and facilitate the isolation of one enantiomerically and diastereoisomerically pure amino acid. Chemical Deacetylation. Deacetylation of the galactose derived acetamide 43 was optimized (Scheme 6). Initially, 43 was hydrolyzed in 1 N HCl at 90 °C. TLC and analytical RP-HPLC showed the slow formation of two major and one minor product, all with shorter retention times on RP-HPLC. After neutralization, the products were isolated by semiprepartaive RP-HPLC and characterized by NMR and ES-MS or MALDI-TOF-MS. The expected (racemic) amine 49 was obtained in 29% yield. The faster migrating 51 was obtained in 43% yield. It showed a higher mass (by 18 units) than 49. For an unambiguous characterization, 51 was peracetylated under standard conditions. The NMR spectrum of the resulting tetraacetate showed the anomeric proton as a ddd at a low field, evidencing the formation of a ketone by regioselective hydration of the alkyne. A minor product eluting last must be the acetamido ketone corresponding to 51, as evidenced by a peak in the MALDI-TOF-MS at 412.7 and a ddd for H-3 at 3.80 ppm. The hydration/Ndeacetylation ratio was not influenced by the reaction temperature, as shown by further experiments. Extended reaction times lead to a quantitative transformation to 51. This unexpected hydration is preparatively useful and illustrates the versatility of the acetylene functionality. Enzymatic Deacetylation. The stereoisomerically pure phenylalanines were prepared in two steps by enzymatic resolution35 of the mixture R-R,S-stereoisomeric acetamides followed by Fmoc protection. The enzymatic deacetylation of the acetamide 43 was performed with the acylase I from Aspergillus melleus, available from Sigma, at 30 °C in phosphate buffer at pH 7.5 instead of pH 8 as indicated by Sigma. This enzyme has been demonstrated to be highly selectively for the L-configuration of the R-carbon atom and hydrolyze only N-acetylated amino acids with the L-configuration. The deacetylation was conveniently followed by HPLC. After 24 h the intensity of the HPLC peak for product and starting material were equal and constant. (34) Greene, T. W.; Wuts, P. G. M. In Protective groups in organic synthesis; Greene, T. W., Ed.; John Wiley & Sons Inc.: New York, 1991; pp 309-405. (35) Chenault, H. K.; Dahmer, J.; Whitesides, G. M. J. Am. Chem. Soc. 1997, 111, 6354-6364. Lowary et al. 9662 J. Org. Chem., Vol. 63, No. 26, 1998 Scheme 6 Preparative HPLC and hydrolysis of the methyl ester provided 41% of 50. Protection of the Building Block with Fmoc. The mixtures of the diastereoisomeric amino acid conjugates 49 and 51 and the diastereomerically pure amino acid conjugate 50 were transformed into the Fmoc derivatives using Fmoc-OSu under standard conditions. The acids precipitated upon acidification of the reaction mixture. Product remaining in the supernatant was isolated by preparative RP-HPLC. Peptide Synthesis. The peptide syntheses were performed on custom-made PEGA resin.36,37 The resin was derivatized by a hydroxymethyl phenoxyacetic acid (HMPA) linker. The linker was coupled to the resin by activation with O-(benzotriazol-1-yl)-N,N,N,N-tetramethyluronium tetrafluoroborate (TBTU) and 4-ethylmorpholine (NEM). The first amino acid, lysine, was coupled as Fmoc-Lys(Boc)-COOH by activation with 1-(mesitylenesulfonyl)-3-nitro-1,2,4-triazole (MSNT) and N-methylimidazol.38 Fmoc cleavages were effected by treatment with 20% piperidine in DMF. The successive (36) Meldal, M. Tetrahedron Lett. 1992, 33, 3077-3080. (37) Auzanneau, F. I.; Meldal, M.; Bock, K. J. Peptide Sci. 1995, 1, 31-44. amino acids were coupled as Pfp esters. The glycosylacetylene amino acids were coupled for 3 h after activation with TBTU. The resin was washed thoroughly with DMF after Fmoc removal and after each acylation step. Acidolytic cleavage and deprotection of the peptide with 95% aqueous TFA gave the desired crude glycosylacetylene-containing peptide 54 which was then purified by semipreparative RP-HPLC and fully characterized by ESMS and by 1D- and 2D-1H NMR spectroscopy. The ketones 53 yielded the peptides 55 and 56 which could easily be separated by HPLC and were similarly characterized. Conclusion A range of eight glycosylacetylene amino acid building blocks containing C-linked glycan with a configuration corresponding to Glc, Gal, and Man have been synthesized. The building blocks were formed by the palladium(0)-catalyzed cross-coupling reactions of glycosylacetylenes with iodophenylalanines. In the Glc- and Galcontaining building blocks the C-glycosides were β-linked (38) Blankemeyer-Menge, B.; Nimtz, M.; Frank, R. Tetrahedron Lett. 1990, 31, 1701-1704. Combinatorial Synthesis of C-Linked Glycopeptides whereas the Man was R- and β-linked. It has been demonstrated that these building blocks can be obtained in the diastereomerically pure form by enzymatic resolution of the N-acetate followed by Fmoc protection. It was possible to perform chemical modification of the acetylene by hydrolysis to the ketone. The different building blocks were compatible with the conditions of Fmoc-based peptide synthesis and have been used in the synthesis of C-linked glycopeptide T-cell antigens. The building blocks can be employed in the synthesis of C-linked, spaced, and templated glycopeptide libraries.3 Experimental Section General Methods. Optical rotations were measured at 22 ( 2 °C. Flash chromatography was performed on Silica Gel 60, 40-63 µM. Acylase I from Aspergillus melleus was purchased from Sigma as lyophilized powder. Suitable protected NR-Fmoc amino acid-OPfp esters were purchased from Bachem (Switzerland) and NovaBiochem (Switzerland). PEGA resin was custom synthesized at Carlsberg. All commercial reagents were used as supplied and HPLC grade solvents (LabScan, Dublin) were used for chromatography. 1H NMR chemical shifts were assigned on the basis of 1H-1H and 1H13 C correlation spectroscopy, and in cases of spectral overlap the shifts were derived from the center of the cross-peaks in the appropriate 2D experiment. For 1H NMR spectra recorded in CD3COOD-H2O the HOAc signal at 2.03 ppm was used as an internal reference. For the assignment of signals 1H-1HCOSY, 1H-1H double quantum filtered phase sensitive COSY and NOE in rotating frame (ROESY) spectroscopy experiments were used. MALDI-TOF MS was performed using a matrix of R-cyano-4-hydroxycinnamic acid. Unless otherwise stated, all reactions were carried out at room temperature. Organic solution were dried (Na2SO4) prior to concentration under vacuum at <40 °C (bath). Microanalyses were carried out by the microananlytical laboratory at the University of Copenhagen. Analytical, semipreparative, and preparative reverse phase HPLC separations were performed using analytical RCM (8 × 100 mm), Delta PAK (15 um, 300 Å, 25 × 200 mm), or 50 × 300 mm radial pressure C-18 columns with flow rates of 1, 10, and 20 cm3 min-1, respectively. Detection was at 215 and 280 nm. Solvent system: A, 0.1% TFA in water; B, 0.1% TFA in 90% acetonitrile-10% water. 4,5,6,8-Tetra-O-benzyl-1,1,2,2-tetradehydro-1,2-dideoxy1-C-(trimethylsilyl)-D-galactooct-3-ulopyranose (7). CeCl3‚ 7H2O (1.043 g, 2.80 mmol) was heated under vacuum at 120 °C for 1 h and then 140 °C for 1 h. The flask was cooled to 0 °C and flushed with argon, and then freshly distilled THF (10 mL) was added. The suspension was allowed to stir at room temperature for 2 h and then cooled to -78 °C. In a separate flask a solution of Li-TMS acetylene was prepared by dissolving TMS acetylene (0.5 mL, 3.54 mmol) in THF (4 mL) and then cooling the mixture to -78 °C before adding 1.6 M n-BuLi in hexane (2 mL). After being stirred for 45 min at -78 °C, the CeCl3 suspension was treated with the freshly prepared acetylene solution (5 mL). The resulting yellow slurry was allowed to stir at -78 °C for 30 min before 2,3,4,6-tetraO-benzylgalactopyranosyl lactone1 6 (758 mg, 1.40 mmol) in THF (10 mL) was added dropwise. After being stirred for 2 h while warming to room temperature the reaction was diluted with EtOAc and then washed with 0.1 HCl, water, and a saturated NaCl solution. The organic layer was then dried (Na2SO4) and evaporated to provide an oil that was chromatographed (6:1 petroleum ether/EtOAc) to give the product 7, as an anomeric mixture (810 mg, 91%) of an oil, Rf 0.16 (6:1 petroleum ether/EtOAc). 1H NMR (250 MHz, CDCl3): δ 6.97.5 (m, 20 H, Ph), 0.015, -0.005 (combined 9H, Si(CH3)3). Anal. Calcd for C39H44O6Si‚1/2H2O (645.87): C, 72.53; H, 7.02. Found: C, 72.52; H, 7.19. 3,7-Anhydro-4,5,6,8-tetra-O-benzyl-1,1,2,2-tetradehydro1,2-dideoxy-1-C-(trimethylsilyl)-D-glycero-L-mannooctitol (8). Ketose 7 (860 mg, 1.35 mmol) was dissolved in 7:3 J. Org. Chem., Vol. 63, No. 26, 1998 9663 CH3CN/CH2Cl2 (25 mL) and cooled to -10 °C. Separately, two stock solutions were prepared: solution A, Et3SiH (1.4 mL) in 2:1 CH3CN/CH2Cl2 (6 mL); solution B, BF3OEt2 (0.56 mL) in CH3CN (4 mL). To the solution of the ketose was added first solution A (4.65 mL, 5.6 mmol Et3SiH) followed by solution B (2.9 mL, 5.3 mmol BF3OEt) and the reaction allowed to stir at -10 °C for 1 h, before being diluted with EtOAc and washed with water and a saturated NaCl solution. After drying (Na2SO4) and evaporation of the solvent the residue was chromatographed (9:1 petroleum ether/EtOAc) to give the product 8 as an oil: Rf 0.52 (6:1 petroleum ether/EtOAc); [R]D -11.6° (c 0.9, CHCl3); selected 1H NMR (250 MHz, CDCl3) δ 4.10 (1 H), 3.61 (1 H), 3.57 (1 H), 4.04 (1 H), 4.10 (1 H), 3.68 (2 H); 13C NMR δ 102.55, 90.50, 70.62, 77.21, 83.26, 73.71, 79.02, 68.37. Complete and assigned NMR data are presented in the Supporting Information Tables 1 and 2. Anal. Calcd for C39H44O5Si (620.86): C, 75.45; H, 7.14. Found: C, 75.20; H, 7.09. 3,7-Anhydro-4,5,6,8-tetra-O-benzyl-1,1,2,2-tetradehydro1,2-D-glycero-L-mannooctitol (4). TMS derivative 8 (2.69 g, 4.33 mmol) was dissolved in 5:1 CH3OH/CH2Cl2 (150 mL), 1 N NaOH (8 mL) was added, and the reaction was stirred for 1 h before being neutralized by the addition of 1 N HCl (8 mL). The solvent was then evaporated and the residue partitioned between EtOAc and water. The organic layer was washed with water and a saturated solution of NaCl, before being dried (Na2SO4) and evaporated to give the product 4 (2.35 g, 99%) as an oil that solidified upon standing: Rf 0.15 (9:1 petroleum ether/EtOAc): [R]D +5.9° (c 0.8, CHCl3); selected 1H NMR (250 MHz, CDCl3) δ 2.68(1 H), 4.20 (1 H), 3.73 (1 H), 3.70 (1 H), 4.15 (1 H), 4.21(1 H), 3.77 (2 H); 13C NMR δ 73.72, 79.02, 70.00, 77.32, 83.36, 73.68, 78.78, 68.55. Complete and assigned NMR data are presented in the Supporting Information Tables 1 and 2. Anal. Calcd for C36H36O5 (548.68): C, 78.81; H, 6.61. Found: C, 78.44; H, 6.67. 3,7-Anhydro-4,5,6,8-tetra-O-acetyl-1,1,2,2-tetradehydro1,2-D-glycero-L-mannooctitol (5). Benzylated derivative 4 (2.35 g, 4.28 mmol) was dissolved in Ac2O (100 mL) and then cooled to 0 °C before TMSOTf (5.5 mL, 28.5 mmol) was added. The reaction was then warmed to room temperature and stirred for 72 h, before the dark brown solution was cooled to 0 °C and quenched by the cautious addition of a solution of NaHCO3. Dilution of the reaction mixture with EtOAc was followed by washing of the organic layer with a solution of NaHCO3, water, and a saturated solution of NaCl. After drying (Na2SO4), the organic layer was evaporated to provide a dark brown oil that was chromatographed (2:1 petroleum ether/ EtOAc), yielding the product 5 (1.25 g, 81%) as light yellow solid: Rf 0.28 (2:1 petroleum ether/EtOAc); [R]D +26.6° (c 1.1, CHCl3); selected 1H NMR (250 MHz, CDCl3) δ 2.51(1 H), 4.17 (1 H), 5.39 (1 H), 4.98 (1 H), 5.40 (1 H), 3.89 (1 H), 4.11 (2 H); 13C NMR δ 77.82, 75.61, 68.79, 68.16, 71.34, 67.19, 74.51, 61.50. Complete and assigned NMR data are presented in the Supporting Information Tables 1 and 2. Anal. Calcd for C16H20O9 (356.33): C, 53.93; H, 5.66. Found: C, 53.92; H, 5.48. 4,5,6,8-Tetra-O-benzyl-1,1,2,2-tetradehydro-1,2-dideoxy1-C-(trimethylsilyl)-D-mannooct-3-ulopyranose (12). CeCl3‚ 7H2O (1.10 g, 2.95 mmol) was heated under vacuum at 120 °C for 1 h and then 140 °C for 1 h. The flask was then cooled to 0 °C and flushed with argon, and then freshly distilled THF (10 mL) was added. The suspension was allowed to stir at room temperature for 2 h and then cooled to -78 °C. In a separate flask a solution of Li-TMS acetylene was prepared by dissolving TMS acetylene (0.5 mL, 3.54 mmol) in THF (4 mL) and then cooling the mixture to -78 °C before adding 1.6 M n-BuLi in hexane (2 mL). After being stirred for 45 min at -78 °C, the CeCl3 suspension was treated with the freshly prepared acetylene solution (5.25 mL). The resulting yellow slurry was allowed to stir at -78 °C for 30 min before 2,3,4,6tetra-O-benzylmannopyranosyl lactone1 (11) (791 mg, 1.47 mmol) in THF (10 mL) was added dropwise. After being stirred for 2 h while warming to room temperature, the reaction was diluted with EtOAc and then washed with 0.1 HCl, water, and a saturated NaCl solution. The organic layer was then dried (Na2SO4) and evaporated to provide an oil that was chromato- 9664 J. Org. Chem., Vol. 63, No. 26, 1998 graphed (6:1 petroleum ether/EtOAc) to give, as an anomeric mixture, the product 12 (870 mg, 93%) as an oil: Rf 0.26 (6:1 petroleum ether/EtOAc); 1H NMR (250 MHz, CDCl3) δ 6.97.5 (m, 20 H, Ph), 0.002, -0.01 (combined 9H, Si (Si(CH3)3). Anal. Calcd for C39H44O6Si (636.86): C, 73.55; H, 6.96. Found: C, 72.10; H, 7.04. 3,7-Anhydro-4,5,6,8-tetra-O-benzyl-1,1,2,2-tetradehydro1,2-dideoxy-1-C-(trimethylsilyl)-D-glycero-D-galactooctitol (13) and 3,7-Anhydro-4,5,6,8-tetra-O-benzyl-1,1,2,2tetradehydro-1,2-dideoxy-D-glycero-D-talooctitol (17). Ketose 12 (870 mg, 1.37 mmol) was dissolved in 7:3 CH3CN/ CH2Cl2 (25 mL) and cooled to -10 °C. Separately, two stock solutions were prepared: solution A, Et3SiH (1.4 mL) in 2:1 CH3CN/CH2Cl2 (6 mL); solution B, BF3OEt (0.56 mL) in CH3CN (4 mL). To the solution of the ketose was added first solution A (4.7 mL, 5.6 mmol Et3SiH) followed by solution B (3.0 mL, 5.3 mmol BF3OEt). The reaction was allowed to stir at -10 °C for 1 h, before being diluted with EtOAc and washed with water and a saturated NaCl solution. After drying (Na2SO4) and evaporation of the solvent the residue was chromatographed (9:1 petroleum ether/EtOAc) to provide two products. The more polar product, Rf 0.27 (6:1 petroleum ether/EtOAc), was the β-TMS acetylene 13 (465 mg, 55%) obtained as an oil that solidified upon standing: [R]D -39.2° (c 0.4, CHCl3); selected 1H NMR (250 MHz, CDCl3) δ 4.01 (1 H), 3.86 (1 H), 3.38 (1 H), 3.76 (1 H), 3.30 (1 H), 3.63 (1 H), 3.65 (1 H); 13C NMR δ 101.69, 90.87, 69.56, 75.79, 83.34, 74.49, 79.87, 69.34. Complete and assigned NMR data are presented in the Supporting Information Tables 1 and 2. Anal. Calcd for C39H44O5Si (620.86): C, 75.45; H, 7.14. Found: C, 74.98; H, 7.12. The less polar product, Rf 0.35 (9:1 petroleum ether/EtOAc), was a mixture of the R-TMS acetylene 15 and the β-TES acetylene 14. This impure product was then dissolved in 5:1 CH3OH/CH2Cl2 (12 mL), and 1 N NaOH (0.6 mL) was added. After being stirred for 1 h the reaction was neutralized by the addition of 1 N HCl (0.6 mL), the solvent evaporated, and the residue partitioned between EtOAc and water. The organic layer was washed with water and a saturated solution of NaCl, before being dried (Na2SO4) and evaporated to give the partially deprotected product 17 (186 mg, 22%, 2 steps) as an oil: Rf 0.23 (6:1 petroleum ether/EtOAc); [R]D +23.5° (c 0.9, CHCl3); selected 1H NMR (250 MHz, CDCl3) δ 2.41 (1 H), 4.73 (1 H), 3.75 (1 H), 3.94 (1 H), 3.89 (2 H), 3.71 (1 H), 3.64 (1 H); 13 C NMR δ 74.71, 80.68, 68.96, 75.82, 83.33, 74.58, 79.93, 69.36. Complete and assigned NMR data are presented in the Supporting Information Tables 1 and 2. Anal. Calcd for C36H36O5 (548.68): C, 78.81; H, 6.61. Found: C, 78.39; H, 6.85. 3,7-Anhydro-4,5,6,8-tetra-O-benzyl-1,1,2,2-tetradehydro1,2-D-glycero-L-galactooctitol (16). TMS derivative 13 (4.08 g, 6.57 mmol) was dissolved in 5:1 CH3OH/CH2Cl2 (150 mL); then 1 N NaOH (10 mL) was added and the reaction stirred for 1 h before being neutralized by the addition of 1 N HCl (10 mL). The solvent was then evaporated and the residue partitioned between EtOAc and water. Washing of the organic layer with water and a saturated solution of NaCl was followed by drying (Na2SO4) and evaporation of the solvent to give the product 16 (3.54 g, 98%) as an oil that solidified upon standing: Rf 0.15 (6:1 petroleum ether/EtOAc); [R]D -29.1° (c 0.5, CHCl3); selected 1H NMR (250 MHz, CDCl3) δ 2.38 (1 H), 4.05 (1 H), 3.88 (1 H), 3.46 (1 H), 3.84 (1 H), 3.68 (1 H), 3.63 (1 H). Complete and assigned 1H NMR data are presented in the Supporting Information Tables 1 and 2. Anal. Calcd for C36H36O5 (548.68): C, 78.81; H, 6.61. Found: C, 78.10; H, 6.81. 3,7-Anhydro-4,5,6,8-tetra-O-acetyl-1,1,2,2-tetradehydro1,2-D-glycero-L-galactooctitol (18). Benzylated derivative 16 (3.54 g, 6.45 mmol) was dissolved in Ac2O (100 mL) and then cooled to 0 °C before TMSOTf (8 mL, 41.4 mmol) was added. The reaction was then warmed to room temperature and stirred for 72 h, before the dark brown solution was cooled to 0 °C and quenched by the cautious addition of a solution of NaHCO3. Dilution of the reaction mixture with EtOAc was followed by washing of the organic layer with a solution of NaHCO3, water, and a saturated solution of NaCl. After drying (Na2SO4), the organic layer was evaporated to provide a dark Lowary et al. brown oil that was chromatographed (2:1 petroleum ether/ EtOAc), yielding the product 18 (1.65 g, 72%) as light yellow solid: Rf 0.25 (2:1 petroleum ether/EtOAc); [R]D -41.5° (c 1.0, CHCl3); selected 1H NMR (250 MHz, CDCl3) δ 2.51 (1 H), 4.45 (1 H), 5.52 (1 H), 5.06 (1 H), 5.25 (1 H), 3.67 (1 H), 4.25 (1 H), 4.14 (1 H); 13C NMR δ 75.42, 76.81, 68.02, 69.44, 71.41, 65.50, 76.49, 62.59. Complete and assigned NMR data are presented in the Supporting Information Tables 1 and 2. Anal. Calcd for C16H20O9 (356.33): C, 53.93; H, 5.66. Found: C, 53.86; H, 5.45. 3,7-Anhydro-4,5,6,8-tetra-O-acetyl-1,1,2,2-tetradehydro1,2-D-glycero-L-talooctitol (19). Benzylated derivative 17 (164 mg, 0.3 mmol) was dissolved in Ac2O (10 mL) and cooled to 0 °C, and TMSOTf (0.4 mL, 2.07 mmol) was added. The reaction was warmed to room temperature, stirred for 30 h, and then worked up as described for 18. Chromatography (2:1 petroleum ether/EtOAc) yielded the product 19 (65 mg, 61%) as light yellow solid: Rf 0.21 (2:1 petroleum ether/EtOAc); [R]D +30.5° (c 0.8, CHCl3); selected 1H NMR (250 MHz, CDCl3) δ 2.76 (1 H), 4.76 (1 H), 5.32 (1 H), 5.46 (1 H), 5.26 (1 H), 4.17 (1 H), 4.29 (1 H), 4.11 (1 H); 13C NMR δ 79.15, 75.98, 66.93, 70.91, 69.28, 65.91, 71.59, 62.30. Complete and assigned NMR data are presented in the Supporting Information Tables 1 and 2. Anal. Calcd for C16H20O9 (356.33): C, 53.93; H, 5.66. Found: C, 53.75; H, 5.62. N-Acetyl-4-iodo-(DL)-phenylalanine Methyl Ester (24). 4-Iodo-(DL)-phenylalanine (23) (100 mg, 0.343 mmol) was suspended in CH3OH (10 mL), and then Ac2O (65 µL, 0.689 mmol) and NaHCO3 (62 mg, 0.74 mmol) were added. After being stirred for 20 min the solution became clear, and after 45 min the reaction was complete. The solution was then evaporated to give a residue that was immediately resuspended in 2,2-dimethoxypropane (10 mL). Concentrated HCl (350 µL) was added, and after being stirred for 4 h the reaction was evaporated. Chromatography (9:1 CH2Cl2/CH3OH) gave the product 24 (116 mg, 97%) as a white solid: Rf 0.74 (19:1 CH2Cl2/CH3OH); 1H NMR (CDCl3, 250 MHz) δ 7.641 (d, 2 H, 3J 8.2 Hz, H-2, H-6), 6.897 (d, 2 H, 3J 8.2 Hz, H-3, H-5), 5.936 (d, 1 H, JCH,NH 7.0 Hz, NH), 4.894 (ddd, 1 H, JCH,NH 7.0, JR,β 5.9, JR,β′ 5.5 Hz, H-R), 3.759 (s, 3 H, OCH3), 3.145 (dd, 1 H, JR,β 5.9, Jβ,β′ 13.9 Hz, H-β), 3.085 (dd, 1 H, JR,β 5.5, Jβ,β′ 13.9 Hz, H-β′), 2.021 (s, 3 H, NHCOCH3); 13C NMR (CDCl3, 62.5 MHz) δ 171.810, 169.555 (CdO), 137.615 (C-2, C-6), 135.511 (C-4), 131.204 (C-3, C-5), 92.603 (C-1), 52.892 (C-R), 52.413 (OCH3), 37.355 (C-β), 23.116 (NHCOCH3). Anal. Calcd for C12H14INO3 (347.15): C, 41.52; H, 4.04; N, 4.03. Found: C, 41.45; H, 3.99; N, 4.02. Methyl 3′-nitro-r-acetamidocinnamate (28). N-Acetylglycine 26 (5.24 g, 44.7 mmol), 3-nitrobenzaldehyde (25) (10 g, 66.2 mmol), and sodium acetate (2.71 g, 33.1 mmol) were suspended in Ac2O (30 mL), and the reaction was heated at reflux for 1 h. During the course of the reaction the solution turned dark brown and, upon cooling to room temperature, solidified. The yellow-brown solid obtained was then suspended in ice water, filtered off, and washed extensively with cold water before being dissolved in CH2Cl2. After drying (Na2SO4), the organic solution was evaporated to yield 27 as a yellowbrown solid. This product was not purified further but was instead immediately dissolved in CH3OH (200 mL), and the solution was heated at reflux for 9 h. Evaporation of the solvent provided a brown oil that was chromatographed (19:1 CH2Cl2/CH3OH) to give a slightly colored product. Dissolution of this product in CH3OH and then decolorization with activated carbon gave the product 28 (4.3 g, 27%) as a yellowwhite solid, pure by 1H NMR and TLC. The product could be recrystallized from EtOAc: Rf 0.22 (1:1 petroleum ether/ EtOAc); 1H NMR (DMSO-d6, 250 MHz) δ 9.839 (s, 1 H, NH), 8.467 (s, 1 H, H-2), 8.220 (dd, 1 H, 4J 1.9, 3J 8.0 Hz, H-6), 8.046 (d, 1 H, 3J 8.0 Hz, H-4), 7.726 (t, 1 H, 3J 8.0, H-5), 7.315 (s, 1 H, H-β), 3.756 (s, 3 H, OCH3), 2.031 (s, 3 H, NHCOCH3); 13 C NMR (DMSO-d6, 62.5 MHz) δ 170.210, 166.046 (CdO), 148.714 (C-1), 136.836 (C-4), 136.073 (C-3), 130.967 (C-5), 129.486 (C-β), 128.917 (C-R), 124.630 (C-2), 124.369 (C-4), 53.241 (OCH3), 23.281 (NHCOCH3). Anal. Cald. for C12H12N2O5 (264.24): C, 54.55; H, 4.58; N, 10.60. Found: C, 54.50; H, 4.58; N, 10.60. Combinatorial Synthesis of C-Linked Glycopeptides N-Acetyl 3-amino-(DL)-phenylalanine Methyl Ester Acetate Salt (29). Cinnamate 28 (4.2 g, 15.9 mmol) was dissolved in acetic acid (250 mL) and then 10% Pd/C (1.5 g) added. The reaction was allowed to stir for 20 h under H2, before the catalyst was filtered away and the solvent evaporated to yield the product 25 (4.5 g, 96%) as a brown oil. 1H NMR showed the product as pure: Rf 0.24 (19:1 CH2Cl2/CH3OH); 1H NMR (CDCl3, 250 MHz) δ 7.058 (t, 1 H, 3J 7.7 Hz, H-5), 6.573 (m, 1 H, H-4), 6.481 (m, 1 H, H-6), 6.458 (m, 1 H, H-2), 6.110 (d, 1 H, JCH,NH 7.7 Hz, NH), 4.823 (ddd, 1 H, JCH,NH 7.7, JR,β 5.8, JR,β′ 6.1 Hz, H-R), 3.713 (s, 3 H, OCH3), 3.034 (dd, 1 H, JR,β 5.8, Jβ,β′ 13.8 Hz, H-β), 2.963 (dd, 1 H, JR,β 6.1, Jβ,β′ 13.8 Hz, H-β′), 1.957 (s, 3 H, NHCOCH3); 13C NMR (CDCl3, 62.5 MHz) δ 172.149, 169.728 (CdO), 146.141 (C-1), 136.982 (C-3), 129.426 (C-5), 119.506 (C-6), 115.969 (C-2), 114.080 (C4), 52.992 (C-R), 52.206 (OCH3), 37.667 (C-β), 23.000 (NHCOCH3). N-Acetyl-3-iodo-(DL)-phenylalanine Methyl Ester (30). N-Acetyl-3-amino-(DL)-phenylalanine methyl ester (29) (15.2 g, 51.7 mmol) was dissolved in 1 N HCl (130 mL and the solution cooled to 0 °C. To the reaction was added, in 2 mL portions, NaNO2 (4.71 g, 68.3 mmol) as a cold solution in water (150 mL). During the course of the addition the temperature of the mixture was kept below 5 °C by the addition of ice to the reation mixture. After the last aliquot of NaNO2 solution was added, the reaction was allowed to stir for 30 min at 0-5 °C before KI (21.5 g, 129.5 mmol) was added, in 3 g portions. The reaction immediately turned purple, and gas evolution occurred. The flask was then removed from the ice bath and was heated at 80 °C for 1 h. After cooling of the flash to room temperature, ethyl acetate was added and then the aqueous layer was washed with ethyl acetate until no product was observed in the organic layer. The combined organic extracts were then dried (Na2SO4), and the solvent was evaporated. Chromatography (19:1 CH2Cl2/CH3OH) gave the product as a yellow solid. Recrystallization from EtOAc/petroleum ether gave the product 30 (8.6 g, 48%) as white crystals: Rf 0.44 (19:1 CH2Cl2/CH3OH); 1H NMR (CDCl3, 250 MHz) δ 7.589 (m, 1 H, H-6), 7.667 (s, 1 H, H-2), 7.087 (m, 1 H, H-4), 7.026 (t, 1 H, 3J 7.6 Hz, H-5), 6.025 (d, 1 H, JCH,NH 6.2 Hz, NH), 4.855 (ddd, 1 H, JCH,NH 6.2, JR,β 5.8, JR,β′ 5.7 Hz, H-R), 3.740 (s, 3 H, OCH3), 3.109 (dd, 1 H, JR,β 5.8, Jβ,β′ 13.9 Hz, H-β), 3.019 (dd, 1 H, JR,β 5.7, Jβ,β′ 13.9 Hz, H-β′), 2.010 (s, 3 H, NHCOCH3); 13C NMR (CDCl , 62.5 MHz) δ 171.747, 169.619 (CdO), 3 138.338 (C-2), 136.147 (C-6), 136.145 (C.3), 130.205 (C-5), 128.408 (C-4), 94.375 (C-1), 53.038 (C-R), 52.415 (OCH3), 37.352 (C-β), 23.079 (NHCOCH3). Anal. Calcd for C12H14INO3 (347.15): C, 41.52; H, 4.04; N, 4.03. Found: C, 42.02; H, 3.84; N, 4.06. General Procedure for Pd-Coupling Reactions. The acetylene (55 mg, 0.15 mmol) and the iodide (45 mg, 0.13 mmol) were dissolved in piperidine (2 mL) and the flask purged with argon while heating to 80 °C. Following the simultaneous addition of (Ph3P)4Pd (7.5 mg, 6.5 µmol) and CuI (2.5 mg, 13 µmol) the reaction was stirred at 80 °C for 1 h before being cooled and evaporated. The residue was then redissolved in 1:1 acetic anhydride/pyridine (5 mL) and stirred overnight, and the reaction was quenched by cooling the solution to 0 °C and adding methanol (3 mL). Evaporation of the solvent gave a brown oil that was chromatographed (EtOAc) to give the coupled product, Rf 0.45 (EtOAc). N-Acetyl 4-C-(3,7-anhydro-4,5,6,8-tetra-O-acetyl-1,1,2,2tetradehydro-1,2-D-glycero-D-gulooctityl)-DL-phenylalanine methyl ester (33): yield 61 mg, 81%; selected 1H NMR (250 MHz, CDCl3) δ 4.41 (1 H), 5.18 (1 H), 5.16 (1 H), 5.06 (1 H), 3.74 (1 H), 4.28 (1 H), 4.15 (1 H), 7.34 (2 H), 7.04 (2 H), 4.86 (1 H), 3.17 (1 H), 3.05 (1 H); 13C NMR δ 86.79, 82.96, 69.35, 73.49, 71.21, 68.15, 75.91, 62.03, 120.33, 132.13 (2 C), 129.23 (2 C), 137.17, 52.93, 37.77. Complete and assigned NMR data are presented in the Supporting Information Tables 3 and 4. Anal. Calcd for C28H33NO12‚1/2H2O (584.58): C, 57.53; H, 5.86; N, 2.40. Found: C, 57.29; H, 5.62; N, 2.44. N-Acetyl 4-C-(3,7-anhydro-4,5,6,8-tetra-O-acetyl-1,1,2,2tetradehydro-1,2-D-glycero-D-mannooctityl)-DL-phenylalanine methyl ester (35): yield 68.5 mg, 91%; 1H NMR (250 J. Org. Chem., Vol. 63, No. 26, 1998 9665 MHz, CDCl3) δ 4.41 (1 H), 5.18 (1 H), 5.16 (1 H), 5.06 (1 H), 3.74 (1 H), 4.28 (1 H), 4.15 (1 H), 7.34 (2 H), 7.04 (2 H), 4.86 (1 H), 3.17 (1 H), 3.05 (1 H); 13C NMR δ 86.48, 82.21, 69.71, 68.53, 71.41, 67.34, 74.45, 61.57, 120.36, 132.11 (2 C), 129.20 (2 C), 137.13, 52.92, 37.75. Complete and assigned NMR data are presented in the Supporting Information Tables 3 and 4. Anal. Calcd for C28H33NO12‚1/2H2O (584.58): C, 57.53; H, 5.86; N, 2.40. Found: C, 57.31; H, 5.52; N, 2.39. N-Acetyl 4-C-(3,7-anhydro-4,5,6,8-tetra-O-acetyl-1,1,2,2tetradehydro-1,2-D-glycero-D-galactooctityl)-DL-phenylalanine methyl ester (37): yield 68.7 mg, 91%; 1H NMR (250 MHz, CDCl3) δ 4.67 (1 H), 5.60 (1 H), 5.12 (1 H), 5.29 (1 H), 3.72 (1 H), 4.30 (1 H), 4.16 (1 H), 7.11 (2 H), 7.03 (2 H), 4.86 (1 H), 3.15 (1 H), 3.05 (1 H); 13C NMR δ 86.53, 82.29, 69.04, 69.82, 71.52, 65.66, 76.40, 62.65, 120.34, 132.03 (2 C), 129.21 (2 C), 137.11, 52.90, 37.74. Complete and assigned NMR data are presented in the Supporting Information Tables 3 and 4. Anal. Calcd for C28H33NO12‚1/2H2O (584.58): C, 57.53; H, 5.86; N, 2.40. Found: C, 57.35; H, 5.88; N, 2.42. N-Acetyl 4-C-(3,7-anhydro-4,5,6,8-tetra-O-acetyl-1,1,2,2tetradehydro-1,2-D-glycero-D-talooctityl)-DL-phenylalanine methyl ester (39): yield 52.1 mg, 69%; 1H NMR (250 MHz, CDCl3) δ 4.97 (1 H), 5.42 (1 H), 5.54 (1 H), 5.32 (1 H), 4.24 (1 H), 4.32 (1H), 4.15 (1 H), 7.41 (2 H), 7.08 (2 H), 4.87 (1 H), 3.19 (1 H), 3.09 (1 H); 13C NMR δ 90.24, 81.17, 67.70, 71.20, 69.56, 65.99, 71.58, 62.39, 119.95, 132.11 (2 C), 129.33 (2 C), 137.49, 52.94, 37.74. Complete and assigned NMR data are presented in the Supporting Information Tables 3 and 4. Anal. Calcd for C28H33NO12‚1/2H2O (584.58): C, 57.53; H, 5.86; N, 2.40. Found: C, 57.11; H, 5.56; N, 2.40. N-Acetyl 3-C-(3,7-anhydro-4,5,6,8-tetra-O-acetyl-1,1,2,2tetradehydro-1,2-D-glycero-D-gulooctityl)-DL-phenylalanine methyl ester (34): yield 65.6 mg, 87%; 1H NMR (250 MHz, CDCl3) δ 4.40 (1 H), 5.21 (1 H), 5.25 (1 H), 5.12 (1 H), 3.74 (1 H), 4.27 (1 H), 4.15 (1 H), 7.15 (1 H), 7.08 (1 H), 7.23, (1 H) 7.30 (1 H), 4.86 (1 H), 3.13 (1 H), 3.04 (1 H); 13C NMR δ 86.84, 82.94, 69.34, 73.47, 71.19, 68.15, 75.94, 62.02, 121.78, 132.67, 136.23, 130.05, 128.56, 130.77, 52.96, 37.54. Anal. Calcd for C28H33NO12‚H2O (593.58): C, 56.66; H, 5.94; N, 2.36. Found: C, 56.95; H, 5.59; N, 2.37. N-Acetyl-3-C-(3,7-anhydro-4,5,6,8-tetra-O-acetyl-1,1,2,2tetradehydro-1,2-D-glycero-D-mannooctityl)-DL-phenylalanine methyl ester (36): yield 61 mg, 81%; 1H NMR (250 MHz, CDCl3) δ 4.38 (1 H), 5.47 (1 H), 5.05 (1 H), 5.44 (1 H), 3.95 (1 H), 4.14 (1 H), 4.14 (1 H), 7.17 (1 H), 7.07 (1 H), 7.22, (1 H) 7.31 (1 H), 4.85 (1 H), 3.11 (1 H), 3.03 (1 H); 13C NMR δ 86.49, 83.15, 69.67, 68.47, 71.36, 67.32, 74.45, 61.55, 121.80, 132.61, 136.19, 129.96, 128.50, 130.73, 52.92, 37.51. Anal. Calcd for C28H33NO12‚1/2H2O (584.58): C, 57.53; H, 5.86; N, 2.40. Found: C, 57.51; H, 5.62; N, 2.38. N-Acetyl-3-C-(3,7-anhydro-4,5,6,8-tetra-O-acetyl-1,1,2,2tetradehydro-1,2-D-glycero-D-galactooctityl)-DL-phenylalanine methyl ester (38): yield 52 mg, 69%; 1H NMR (250 MHz, CDCl3) δ 4.66 (1 H), 5.60 (1 H), 5.11 (1 H), 5.29 (1 H), 3.72 (1 H), 4.29 (1 H), 4.16 (1 H), 7.12 (1 H), 7.07 (1 H), 7.22, (1 H) 7.28 (1 H), 4.46 (1 H), 3.14 (1 H), 3.03 (1 H); 13C NMR δ 86.56, 82.27, 69.03, 69.80, 71.51, 65.65, 76.41, 62.63, 121.78, 132.55, 136.23, 129.95, 128.54, 130.67, 52.93, 37.53. Anal. Calcd for C28H33NO12‚1/2H2O (584.58): C, 57.53; H, 5.86; N, 2.40. Found: C, 57.39; H, 5.64; N, 2.36. N-Acetyl-3-C-(3,7-anhydro-4,5,6,8-tetra-O-acetyl-1,1,2,2tetradehydro-1,2-D-glycero-D-talooctityl)-DL-phenylalanine methyl ester (40): yield 53.5 mg, 71%; 1H NMR (250 MHz, CDCl3) δ 4.97 (1 H), 5.42 (1 H), 5.53 (1 H), 5.32 (1 H), 4.23 (1 H), 4.33 (1 H), 4.15 (1 H), 7.20 (1 H), 7.12 (1 H), 7.27, (1 H) 7.38 (1 H), 4.86 (1 H), 3.15 (1 H), 3.05 (1 H); 13C NMR δ 90.26, 81.15, 67.68, 71.18, 69.53, 65.98, 71.62, 62.40, 121.39, 132.65, 136.45, 130.23, 128.65, 130.74, 53.02, 37.52. Anal. Calcd for C28H33NO12 (575.57): C, 58.43; H, 5.78; N, 2.43. Found: C, 58.08; H, 5.66; N, 2.43. General Procedure for Deacylation Reactions. The coupled product (ca. 1 mmol) was dissolved in CH3OH (20 mL), a small piece of sodium was added, and the reaction stirred for 2 h and evaporated. The residue was redissolved in water (20 mL), stirred for an additional 2 h, and then brought to pH 9666 J. Org. Chem., Vol. 63, No. 26, 1998 2-3 by the addition of IR 120 (H+) resin. After filtration of the resin the solution was evaporated to give the product, pure by NMR. N-Acetyl 4-C-(3,7-anhydro-1,1,2,2-tetradehydro-1,2-Dglycero-D-gulooctityl)-DL-phenylalanine (41). The conjugate 33 (641 mg, 1.11 mmol) yielded the product 41 (425 mg, 97%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.34 (1 H), 3.51 (1 H), 3.57 (1 H), 3.56 (1 H), 3.53 (1 H), 3.94 (1 H), 3.74 (1 H), 7.50 (2 H), 7.28 (2 H), 4,63 (1 H), 3.24 (1 H), 3.01 (1 H), 1.95 (3 H); 13C NMR δ 89.96, 85.21, 71.01, 69.91, 77.03, 73.77, 80.28, 61.23, 120.18, 132.35 (2 C), 129.76 (2 C), 138.65, 54.67, 37.17. N-Acetyl-4-C-(3,7-anhydro-1,1,2,2-tetradehydro-1,2-Dglycero-D-mannooctityl)-DL-phenylalanine (43). The conjugate 35 (730 mg, 1.27 mmol) yielded the product 43 (485 mg, 97%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.27 (1 H), 3.81 (1 H), 3.68 (1 H), 4.02 (1 H), 3.43 (1 H), 3.58 (2 H), 7.52 (2 H), 7.29 (2 H), 4,64 (1 H), 3.25 (1 H), 3.02 (1 H), 1.95 (3 H); 13 C NMR δ 86.60, 85.45, 71.43, 71.08, 73.74, 69.24, 79.36, 61.51, 120.26, 132.36 (2 C), 129.76 (2 C), 138.51, 54.55, 37.10. N-Acetyl 4-C-(3,7-anhydro-1,1,2,2-tetradehydro-1,2-Dglycero-D-galactooctityl)-DL-phenylalanine (45). The conjugate 37 (640 mg, 1.11 mmol) yielded the product 45 (420 mg, 96%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.51 (1 H), 4.10 (1 H), 3.59 (1 H), 3.66 (1 H), 3.30 (1 H), 3.94 (1 H), 3.76 (1 H), 7.50 (2 H), 7.29 (2 H), 4.52 (1 H), 3.24 (1 H), 3.02 (1 H), 1.95 (3 H); 13C NMR δ 86.71, 84.71, 70.50, 71.72, 73.75, 66.96, 80.55, 61.48, 120.36, 132.31 (2 C), 129.74 (2 C), 138.35, 54.35, 37.00. N-Acetyl 4-C-(3,7-anhydro-1,1,2,2-tetradehydro-1,2-Dglycero-D-talooctityl)-DL-phenylalanine (47). The conjugate 39 (272 mg, 0.47 mmol) yielded the product 47 (183 mg, 98%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.97 (1 H), 4.18 (1 H), 4.09 (1 H), 3.71 (1 H), 3.88 (1 H), 3.86 (1 H), 3.81 (1 H), 7.48 (2 H), 7.27 (2 H), 4,60 (1 H), 3.24 (1 H), 2.99 (1 H), 1.94 (3 H); 13C NMR δ 89.75, 83.08, 70.11, 72.32, 71.54, 67.33, 76.02, 61.33, 120.05, 132.18 (2 C), 129.75 (2 C), 138.81, 54.97, 37.31. N-Acetyl-3-C-(3,7-anhydro-1,1,2,2-tetradehydro-1,2-Dglycero-D-gulooctityl)-DL-phenylalanine (42). The conjugate 34 (647 mg, 1.12 mmol) yielded the product 42 (435 mg, 98%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.35 (1 H), 3.49 (1 H), 3.54 (1 H), 3.53 (1 H), 3.50 (1 H), 3.95 (1 H), 3.75 (1 H), 7.42 (1 H), 7.46 (1 H), 7.38, (1 H) 7.32 (1 H), 4.59 (1 H), 3.23 (1 H), 2.98 (1 H), 1.95 (3 H); 13C NMR δ 86.96, 85.02, 71.00, 77.04, 73.76, 69.91, 80.30, 61.23, 121.68, 132.80, 137.88, 130.76, 129.19, 130.55, 55.09, 37.15. N-Acetyl-3-C-(3,7-anhydro-1,1,2,2-tetradehydro-1,2-Dglycero-D-mannooctityl)-DL-phenylalanine (44). The conjugate 36 (651 mg, 1.13 mmol) yielded the product 44 (435 mg, 98%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.28 (1 H), 3.86 (1 H), 3.68 (1 H), 4.02 (1 H), 3.76 (1 H), 3.79 (2 H), 7.45 (1 H), 7.49 (1 H), 7.40, (1 H) 7.36 (1 H), 4.66 (1 H), 3.24 (1 H), 3.01 (1 H), 1.96 (3 H); 13C NMR δ 86.54, 85.50, 71.40, 71.07, 73.75, 69.24, 79.38, 61.55, 121.87, 132.79, 137.46, 130.87, 129.23, 130.47, 54.34, 36.77. N-Acetyl-3-C-(3,7-anhydro-1,1,2,2-tetradehydro-1,2-Dglycero-D-galactooctityl)-DL-phenylalanine (46). The conjugate 38 (675 mg, 1.17 mmol) yielded the product 46 (447 mg, 97%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.66 (1 H), 4.11 (1 H), 3.79 (1 H), 3.66 (1 H), 3.46 (1 H), 3.95 (1 H), 3.76 (1 H), 7.47 (1 H), 7.41 (1 H), 7.43, (1 H) 7.36 (1 H), 4.63 (1 H), 3.23 (1 H), 3.00 (1 H), 1.96 (3 H); 13C NMR δ 86.70, 84.71, 70.49, 71.71, 73.75, 66.96 80.57, 61.49, 121.91, 132.72, 137.63, 130.77, 129.21, 130.44, 54.65, 36.92. ES-MS: found (M + H)+, m/e 394.3; C19H23NO8 requires M, 393.39. N-Acetyl-3-C-(3,7-anhydro-1,1,2,2-tetradehydro-1,2-Dglycero-D-talooctityl)-DL-phenylalanine (48). The conjugate 40 (272 mg, 0.47 mmol) yielded the product 48 (182 mg, 98%) as a foam: 1H NMR (250 MHz, CDCl3) δ 4.97 (1 H), 4.19 (1 H), 4.09 (1 H), 3.71 (1 H), 3.97 (1 H), 3.96 (1 H), 3.81 (1 H), 7.42 (1 H), 7.45 (1 H), 7.38, (1 H) 7.32 (1 H), 4.61 (1 H), 3.22 (1 H), 2.97 (1 H), 1.95 (3 H); 13C NMR δ 89.72, 83.16, 70.09, 72.31, 71.56, 67.32, 76.06, 61.35 121.68, 132.66, 137.68, 130.62, 129.22, 130.53, 54.68, 36.94. 4-C-(3,7-Anhydro-1,1,2,2-tetradehydro-1,2-D-glycero-Dgalactooctityl)-DL-phenylalanine (49) and 4-C-(3,7-An- Lowary et al. hydro-1-oxo-1,2-D-glycero-D-galactooctit-1-yl)-DL-phenylalanine (51). Method 1: Chemical Deacetylation. The N-acetyl derivative 43 (10 mg, 330 µmol) was dissolved in 30 mL of 1 N HCl and the solution stirred at 90 °C. The reaction was followed by analysis of 10 µL samples on analytical RPHPLC using a linear gradient 0-50% B over 40 min. Two fast eluting products were formed. After 6 h the starting material had disappeared and the reaction mixture was cooled to room temperature and neutralized by addition of 5% NaOH solution. Preparative RP-HPLC using a linear gradient 0-30% B over 90 min gave the product 49 (33.7 mg, 29%): 1H NMR (250 MHz, CDCl3) δ 4.24 (1 H), 3.77 (1 H), 3.64 (1 H), 3.98 (1 H), 3.73 (3 H), 7.54 (2 H), 7.31 (2 H), 4.28 (1 H), 3.34 (1 H), 3.02 (1 H); 13C NMR δ 86.33, 85.88, 71.37, 71.08, 73.74, 69.24, 79.41, 61.55, 121.25, 132.88 (2 C) 129.97 (2 C), 135.78, 54.55, 35.99. ES-MS: (M + H)+, m/z 352.2; C17H21NO7 requires M, 351.355. Furthermore, the compound 51 (52.0 mg, 43%) was isolated: 1 H NMR (250 MHz, CDCl3) δ 3.48 (1 H), 3.23 (1 H), 3.80 (1 H), 3.51 (1 H), 3.58 (1 H), 3.90 (1 H), 3.57 (1 H), 3.59 (2 H), 7.92 (2 H), 7.40 (2 H), 4.26 (1 H), 3.35 (1 H), 3.22 (1 H); 13C NMR δ 202.49, 171.99, 41.73, 76.52, 71.02, 74.31, 69.52, 78.96, 61.54, 141.01, 130.22 (2 C) 129.53 (2 C), 136.28, 54.49, 35.10. ES-MS: found for (M + H)+, m/e 370.5; C17H23NO8 requires M, 369.371. 3-C-(3,7-Anhydro-1,1,2,2-tetradehydro-1,2-D-glycero-Dgalactooctityl)- L-phenylalanine (50). Method 2: AcylaseCatalyzed Reaction. The N-acetyl derivative 43 (102 mg, 259 µmol) was dissolved in 10 mL of PBS buffer (pH ) 7.5). Acylase I (326 mg, 163 units) was added. The mixture was stirred for a total of 48 h, during which the pH was kept at 7.5 by addition of a saturated NaHCO3 solution. The reaction was followed by analytical RP-HPLC of 5 µL samples of the reaction mixture using a gradient buffer A f buffer B 100:0 f 50:50 over 40 min. When no evolution was observed anymore, the mixture was acidified by addition of 1 N HCl solution and filtered. Purification by preparative RP-HPLC yielded a fraction of impure starting material (72.4 mg) and 58.7 mg (62%) of pure product 50: [R]D +5.8° (c 2.0, H2O, pH ) 1); ES-MS (M + H)+ m/z 352.1, C17H21NO7 requires M, 351.355; 1H NMR (250 MHz, CDCl3) δ 4.24 (1 H), 3.76 (1 H), 3.64 (1 H), 3.98 (1 H), 3.72 (3 H), 7.54 (2 H), 7.31 (2 H), 4.35 (1 H), 3.36 (1 H), 3.22 (1 H); 13 C NMR δ 86.28, 85.89, 71.35, 71.06, 73.73, 69.23, 79.40, 61.53, 121.32, 132.88 (2 C) 129.97 (2 C), 135.55, 54.18, 35.83. Nr-(Fluroren-9-ylmethoxycarbonyl)-4-C-(3,7-anhydro1,1,2,2-tetradehydro-1,2-D-glycero-D-galactooctityl)phenylalanine (52, DL Mixture). The glycosyl amino acid 49 (30 mg, 85 µmol) was dissolved in 5 mL of water, and the pH was adjusted to 10 by addition of a 1 M solution of K2CO3. FmocOSu (32 mg, 94 µmol) was added as solution in 2 mL of acetone. After stirring of the reaction overnight TLC (15:5:2.5 DCM/MeOH/AcOH) and analytical C-18 HPLC showed complete conversion of the stating material. The reaction mixture was acidified to pH ) 2 by addition of 1 N HCl thereby precipitating the Fmoc derivative. The solution was extracted 4 times with EtOAc, and the organic extracts were combined, dried over MgSO4, and evaporated. Semipreparative C18HPLC purification yielded 25.1 mg of 52 (DL form, 47%). ESMS: found for (M + Na)+, m/e 596.2; C32H31NO9 requires M, 573.598. Nr-(Fluroren-9-ylmethoxycarbonyl)-4-C-(3,7-anhydro1,1,2,2-tetradehydro-1,2-D-glycero-D-galactooctityl)-L-phenylalanine (52). The amino acid derivative 50 (45 mg, 129 µmol) was dissolved in 10 mL of water, and the pH was adjusted to 10 by dropwise addition of a 1 M solution of K2CO3. A solution of Fmoc-OSu (48 mg, 142 µmol) in 2 mL of acetone was added. The reaction mixture was stirred at room temperature, and every few hours the pH was readjusted to 10 by addition of 1 M K2CO3. After 12 h TLC (15:5:2.5 or 15: 5:2.5 DCM/MeOH/AcOH) showed that the reaction was finished. The reaction mixture was diluted to 20 mL and washed with 10 mL of diethyl ether. The water phase was transferred to two centrifuge tubes and under agitation acidified to pH 2 by addition of 1 N HCl. After centrifugation at 7000 r/min for 10 min the supernatant was decanted from the white precipitate. The precipitate was dispersed in water and collected by Combinatorial Synthesis of C-Linked Glycopeptides centrifugation. This was repeated twice. The precipitate was lyophilized to yield 55.8 mg of white product. Product remaining in the supernatant and washings was recovered by running a preparative RP-HPLC of the combined concentrated solutions, and after lyophilization additional (7.71 mg) white power could be obtained (85% total yield of 52). The two fractions were identical as analyzed by NMR spectroscopy and mass spectrometry: [R]D +6.5° (c 0.8, DMF); 1H NMR (250 MHz, CDCl3) δ 3.99 (1 H), 3.58 (1 H), 3.30 (1 H), 3.71 (1 H), 3.40 (1 H), 3.50 (2H), 7.2-7.6 (4 H), 4.20 (1 H), 3.11 (1 H), 2.90 (1 H); 13 C NMR δ 89.29, 84.72, 72.14, 71.42, 75.11, 69.45, 80.11, 61.59, 121-136 (6 C), 56.05, 37.12; ES-MS found for (M + Na)+ 596.2, C32H31NO9 requires M, 573.598. Nr-(Fluroren-9-ylmethoxycarbonyl)-3-C-(3,7-anhydro1-oxo-1,2-D-glycero-D-galactooctityl)-DL-phenylalanine (53). The amino acid derivative 51 (52 mg, 141 µmol) was dissolved in 10 mL of water, and the pH was adjusted to 10 by dropwise addition of a 1 M solution of K2CO3. A solution of Fmoc-OSu (53 mg, 157 µmol) in 2 mL of acetone was added. The reaction mixture was stirred at room temperature, and every few hours the pH was readjusted to 10 by addition of 1 M K2CO3. After 20 h analytical HPLC showed that the reaction was finished. The reaction was worked up as for compound 52. Product remaining in the supernatant and washings was recovered by preparative HPLC. A total of 66.6 mg (77%) of white product 53 was obtained: 1H NMR (250 MHz, CDCl3) δ 3.27 (1 H), 3.10 (1 H), 3.66 (1 H), 3.35 (1 H), 3.29 (1 H), 3.73 (1 H), 3.27 (1 H), 3.45 (1 H), 3.33 (1 H), 7.35-7.95 (4 H), 4.24 (1 H), 3.17 (1 H), 2.95 (1 H); 13C NMR δ 198.81, 173.91, 42.22, 77.38, 71.37, 75.53, 69.43, 79.63, 61.16, 141.55, 129-130 (4 C), 136.29, 55.88, 37.13; ES-MS (M + Na)+ m/z 596.2, C32H31NO9 requires M, 573.598. Preparation of the PEGA Resin. Syntheses of the peptides were performed in DMF using PEGA resin prepared in the laboratory.36,37 The resin (5 g, loading 0.2 mmol/g) was packed into a 50 cm3 disposable syringe fitted with a Teflon filter. The syringe was connected to a suction flask through a Teflon tube with a manual 2-way Teflon valve, and the resin was swelled in DMF, treated with 20% piperidine in DMF for 10 min, and washed 6 times with DMF. The resin was derivatized with the ((hydroxymethyl)phenoxy)acetic acid (HMPA linker). The HMPA linker (455 mg, 2.5 mmol), O-(1Hbenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) (750 mg, 2.37 mmol), and 4-ethylmorpholine (315 µL, 2.5 mmol) were dissolved in DMF, and after 10 min the solution was added to the resin and left to react overnight. The resin was washed carefully with DMF and dichloromethane. NR-Fmoc-Lys(Boc)-COOH (1.75 mg, 3.75 mmol) and N-methylimidazole (231 mg, 223 µL, 2.81 mmol) were dissolved in dichloromethane (20 cm3), and 1-(mesitylenesulfonyl)-3-nitro-1,2,4-triazole (MSNT) (1.11 g, 3.71 mmol) was added. After 5 min the solution was added to the resin and allowed to react for 3 h. The resin was washed thoroughly with dichloromethane, DMF, and diethyl ether and dried in vacuo. General Procedure for the SPPS of Peptides. Derivatized PEGA resin (0.2 mmol/g capacity, 0.66 equiv compared to the amount of glycosylated building block employed) was transferred into a syringe equipped with a Teflon filter. Apart from the glycosyl-amino acids all amino acids used were NRFmoc-amino acid OPfp (pentafluorophenyl) esters. NR-Fmoc deprotection was effected by successive 2 and 20 min treatments of the resin with 20% piperidine in DMF. The washing procedure (6 times with DMF) was repeated after each coupling/Fmoc deprotection step. Each NR-Fmoc-amino acid OPfp ester (3 equiv) and Dhbt-OH (3,4-dihydro-3-hydroxy-4oxo-1,2,3-benzotriazine) (1 equiv) were dissolved in DMF. The solutions were added to the resins and then left for a minimum of 3 h. After each coupling step the resin was washed, the NRFmoc group removed, and the resin washed as described above. In the case of the glycosyl building blocks 52 and 53 only 1.5 equiv was employed. These were preactivated in DMF by adding 0.95 equiv of TBTU and 1.6 equiv of NEM for 20 min. These solutions were then transferred to the resins and allowed to react for 3 h. In the case of the synthesis using glycosyl-amino acid 53, the resin was washed and the free J. Org. Chem., Vol. 63, No. 26, 1998 9667 Table 1. 1H NMR Data (δ with Hz in Parentheses) the O-linked Glycopeptides 54-56 Measured in CD3COOD/ D2O (1:1) at 600 MHz at 300 K 54 55 56 R-H β-H γ-H3 γ-H3 3.95 2.24 1.01 1.00 Val 3.94 2.23 1.00 0.99 NH R-H β-H γ-H2 γ-H3 δ-H3 8.38 (8.0) 4.34 1.84 1.51/1.16 0.90 0.85 Ile 8.38 (8.0) 4.32 1.84 1.51/1.17 0.87 0.84 8.38 (8.0) 4.40 1.85 1.53/1.18 0.88 0.84 NH R-H β-H γ-H3 8.12 (8.0) 4.40 4.22 1.15 Thr 8.11 (7.5) 4.40 4.21 1.13 8.27 (8.5) 4.30 4.30 1.16 NH R-H β-H3 8.00 (6.5) 4.32 1.28 Ala 8.00 (6.0) 4.31 1.22 8.12 (5.5) 4.30 1.24 NH R-H β-H2 Phenyl 7.27 meta 7.21 para 7.84 (7.0) 4.59 3.02/2.94 7.12 ortho 7.26 meta 7.22 para Phe 7.89 (7.0) 4.57 3.00/2.92 7.11 ortho 7.28 meta 7.24 para NH R-H β-Ha β-Hb 7.97 (8.0) 4.61 3.12 3.04 Phe (Conjugate) 8.02 (8.0) 4.66 3.22 3.09 8.06 (7.0) 4.63 3.10 3.07 NH R-H β-H2 γ-H2 8.01 (7.5) 4.34 2.11/1.95 2.41 Glu 8.04 (7.5) 4.34 2.10/1.96 2.39 8.25 (7.5) 4.37 1.93/1.78 2.03 NH R-H2 7.92 (6.0) 3.96 Gly 8.07 (6.0) 3.94 8.23 (6.0) 3.95 NH R-H β-H2 γ-H δ-H3 7.84 (7.5) 4.43 1.61 1.61 0.90/0.85 Leu 7.85 (7.0) 4.41 1.60 1.60 0.90/0.85 7.90 (7.0) 4.42 1.60 1.60 0.91/0.85 NH R-H β-H γ-H δ-H ǫ-H NH2 8.11 (8.0) 4.43 1.92/1.77 1.45 1.69 3.02 7.46 Lys 8.12 (7.5) 4.42 1.90/1.75 1.43 1.68 3.02 7.46 8.09 (8.0) 4.40 1.89/1.75 1.43 1.68 3.01 7.45 2-Ha 2-Hb 3-H 4-H 5-H 6-H 7-H 8-Ha 8-Hb 2-H′, H-6′ 3-H′, H-5′ Gal-Acetylene 3.51 (1.0) 3.28 (17.0, 9.0) 4.24 (9.5) 3.86 3.84 (9.5) 3.63 3.68 (3.5) 3.66 4.04 3.99 3.71 3.66 3.81 (7.5) 3.69 3.76 (12.0, 4.5) 3.64 7.43 7.95 7.21 7.38 3.95 2.23 1.01 1.00 7.89 (7.0) 4.58 3.03/2.87 7.12 ortho 3.49 (2.0) 3.31 (17.0, 9.0) 3.88 3.64 3.68 (3.0) 4.00 3.68 3.70 3.61 7.97 7.36 amino groups in excess were capped by treatment of the resin with a 20% solution of Ac2O in DMF for 20 min. The resin Lowary et al. 9668 J. Org. Chem., Vol. 63, No. 26, 1998 was washed as usual, and the peptide synthesis was continued using OPfp esters. After cleavage of the final NR-Fmoc group the resin was washed with DMF, CH2Cl2, and ether and dried. Cleavage of the glycopeptides was achieved by treatment with 95% TFA for 2 h. The resin was filtered off and washed 4 times with 95% TFA. The solution was concentrated and the glycopeptides precipitated by addition of ether. The crude glycopeptides were dissolved in water-TFA and analyzed by RPHPLC using a linear gradient from 0 to 100% B over 50 min. Purification by semipreparative RP-HPLC using a linear gradient from 3 to 50% B over 70 min gave the pure glycopeptides. Using the building block 52 (L-epimer) the pure glycopeptide 54 (45.6 mg, 57%) (ES-MS: (M + H)+, m/z 1310.9; C63H97N11O20 requires M, 1310.51) was obtained. Synthesis with the DL-mixture of 52 obtained from 49 gave the mixture of the two epimers of 54 in impure form after preparative RPHPLC. The building block 53 gave the two epimeric peptides 55 (29.6 mg, 34%) (ES-MS: (M + H)+, m/z 1329.0; C63H97N11O20 requires M, 1328.52) and 56 (28.7 mg, 33%) (ES-MS: (M + H)+, m/z 1328.9, C63H97N11O20 requires M, 1328.52), which could be easily separated by RP-HPLC. The capping of free amino functions had avoided the formation of the shorted sequence VITAFEGLK. The 1H NMR spectra of 54-56 were all assigned by 2D-NMR spectroscopy, and the data are presented in Table 1. Acknowledgment. This work was supported with a grant (T.L.) from the Mitzutani Foundation. Supporting Information Available: Tables S1-S12 of the fully assigned 1H and 13C NMR data for compounds 4, 5, 8, 13, 16-19, and 33-53 (12 pages). 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