Bioconjugate Chem. 1995, 6, 316-31 8
316
Strategies for the Synthesis and Screening of Glycoconjugates. 1. A Library of Glycosylamines Dirk Vetter a n d Mark A. Gallop* A f f p a x Research Institute, 4001 Miranda Avenue, Palo Alto, California 94304. Received September 28, 1994@
A simple one-step procedure is found to be highly effective for the “functionalization”of glycodiversity. This study encompasses 50 unprotected mono- and oligosaccharides, which are subjected to Kochetkov aminations in saturated aqueous ammonium carbonate. The reaction allows for the stereo- and regioselective introduction of an amino group into all oligosaccharides tested, as well as into a great variety of monosaccharides including charged species. The resulting unprotected glycosylamines are stable compounds, and the inherent amino group provides a convenient site for chemoselective conjugation and modification as described in the following paper in this issue.
INTRODUCTION
Carbohydrates have recently attracted significant attention from the pharmaceutical industry (1). Natural sources provide a great wealth of structurally diverse and biologically relevant carbohydrates, many of which are available as commercial products. Rare native structures may be routinely obtained by polysaccharide and glycosaminoglycan hydrofluorolysis or automated hydrazinolysis of glycoproteins. Nevertheless, a systematic screening of the carbohydrate pool for drug discovery purposes is complicated by synthetic difficulties associated with the chemical modification of sugars. More widespread utilization of glycodiversity in ligand discovery would be facilitated by general strategies for straightforward functional group manipulation. Reductive amination is one such versatile reaction, though this transformation results in disruption of the pyranose or furanose ring structures of monosaccharides or the reducing termini of oligosaccharides. This paper describes the one-step conversion of a variety of unprotected mono- and oligosaccharides to their corresponding glycosylamines. Subsequent acylation with spacer molecules carrying a biotin moiety or succinimidyl ester for immobilization to streptavidincoated microtiter wells or amino-functionalized resin, respectively, will be described in future papers. EXPERIMENTAL SECTION
General. Saccharides were from Sigma, Boehringer Mannheim, or Oxford Glycosystems, lyxosylamine was from Toronto Research Chemicals. Ammonium carbonate was purchased from Fluka. Glycosylamine Preparations. A solution of the saccharide (1%, wiv, 5-50 mg, up to 1g for less expensive saccharides) in saturated aqueous ammonium carbonate was stirred a t room temperature for 5 days. Solid (NH4)ZCOa (ca. 40 mgimg saccharide) was added in fractions during the course of the reaction to ensure saturation. Kinetics were followed by TLC (K 60, 1-propanoVethy1 acetateiwater, 6:1:3, detection with orcinol and ninhydrin reagents, respectively). After the conversion, samples were frozen and lyophilized for 3 days. Gravimetrically determined yields were typically in the order of 100-
* To whom correspondence should be addressed. Phone: (415) 812-8706. Fax: (415) 424-9860. Abstract published in Advance ACS Abstracts, April 15, 1995. @
1043-1802/95/2906-0316$09.00/0
110%. Samples showing yields > 120% were repeatedly lyophilized. Excess solid (NH4)&03 was most effectively removed by dissolving the crude glycosylamine in warm methanol (ca. 70 “C). After termination of COz evolution, the methanol was slowly evaporated and the residual material dried in uacuo. Batches where gravimetry indicated large amounts of residual ammonium carbonate (yields >> 150%) were discarded. This was the case for reactions with 3-methylglucose and 6-methylgalactose. The reason for the persistance of ammonium carbonate with these sugars is unclear. IH-NMRs of glycosylamines were run in DzO, and conversions were found in the range 50-90%. However, by comparing DzO spectra with &-DMSO spectra for identical samples it became apparent that the hydrolytic stability of different glycosylamines varied significantly. In DzO the half-life of some glycosylamines fell within the time frame of a lH-NMR experiment (5 min), making the lower conversions artifactual. A striking example is the difference between the 1-amines of lactose and 2’methyl-lactose. Both reactions afforded over 95%conversions when monitored in &-DMSO. In DzO the lactosylamine gave the same result, but its 2’-methyl derivative showed 50% hydrolysis. Diagnostic analytical data as well as yields for 54 glycosylamines are summarized in Table 1. RESULTS AND DISCUSSION
Recently, a simple derivatization procedure (Kochetkov reaction) for unprotected mono- and oligosaccharides was disclosed and subsequently employed in a number of representative reactions (2). The anomeric hydroxyl group of a reducing sugar is converted to an amino group upon treatment with aqueous ammonium carbonate. The literature describes the successful amination of 25 different carbohydrates (3-7). Realizing that the Kochetkov amination should be applicable to a much broader range of saccharide structures, we decided to subject a large number of commercially available carbohydrates to these reaction conditions. We confirm the findings of other groups and extend the range of substrates for this reaction to oligosaccharides with the reducing termini Gal, Man, Ara, GalA, and GalNAc (8). We add AllNAc, Al13NAc, GlcGNAc, GlcSNAc, and Xyl to the list of neutral monosaccharides. Also, we describe for the first time the glycosylamine derivatives 0 1995 American Chemical Society
Bioconjugate Chem., Vol. 6, No. 3, 1995 317
Technical Notes
Table 1. Analytical Data for 1-Amino-1-deoxySugars Prepared in This Study (Glycosylamines Are Characterized by FAB-MS and 300 MHz 'H-NMR in DzO. If Glycosylamines Were Obtained from a Commercial Source or Attempted Conversions Failed, This Is Indicated) glycosylamine: m / z (calcdlfound for low or high resolution MS), 'H-NMR (6 and 4 for the anomeric proton of the hemiaminal, yield" (%) saccharide neutral monosaccharides 1.Lyx 2. Ara 3. Glc 4. Gal 5. Fuc 6. Man 7. Rib 8. Xyl 9. GlcNAc 10. GalNAc 11. ManNAc 12. Glc6NAc 13. Glc3NAc 14. Gal6Me 15. Glc3Me 16. AllNAc 17. Al13NAc charged monosaccharides 18. GlcA 19. GalA 20. GlcNAc3S0321. GlcNAc6SO322. GlcNAcGP03'23. GlcN2,3(S03-)2 24. GlcN2,6(S03-)~ 25. Man6P03'26. Ga16P03'27. Gal6S0328. Rib5P03'neutral disaccharides 29. Glc(a1-4)Glc 30. GalZMe(pl-4)Glc 31. Glc(pl-4)Glc 32. Gal(/31-4)Glc 33. GlcNAc(P1-4 )GlcNAc 34. Gal(/3-4)GlcNAc 35. GlcNAc(pl-6)GlcNAc 36. Gal(p1-3)GlcNAc 37. Gal(p1-6)GlcNAc 38. GlcNAc(pl-6)Gal 39. Gal(a1-4)Gal 40. Gal(/31-6)Gal 41. Gal(/31-3)GalNAc 42. Gal(p1-4)Man 43. Gal(p1-3)Ara 44. Man(a1-3)Man 45. Fuc(a1- 2 )Gal(P1-4)Glc 46. Gal/31-4(Fucal-3)Glc 47. Glc(al-4)Glc(al-4)G1c 48.Fucal-4Gal~l-4(Fucal-3)Glc 49. Gal~l-3(Fucal-4)-GlcNAc~l-3Gal~l-4Glc charged oligosaccharides 50. GalA(a1-4)GalA 5 1. Neu5Ac(a2-3)Gal(~l-4)Glc 52. Neu5Ac(a2 - 6)Gal(p1-4)Glc 53. Neu5Ac(a2-3)Gal(L?l-4)GlcNAc 54. Neu5Ac(a2-6)Gal~jB1-4)GlcNAc(~l-3)Gal(~1-4)Glc
glycosylamine obtained from Toronto Research, nd, 4.019 ppm (d, 1.2 Hz) failed glycosylamine obtained from Sigma, nd, 4.093 ppm (8.7 Hz) glycosylamine obtained from Sigma, nd, 4.035 ppm (8.7 Hz) glycosylamine obtained from Sigma, nd, 4.023 ppm (8.7 Hz) nd, 4.343 (s), 80 failed 150.0766/150.0761 (M H+), 4.169 ppm (d, 9.1 Hz), 50 glycosylamine obtained from Sigma, nd, 4.156 ppm (d, 9.0 Hz) 221.1138/221.1142 (M H+), 4.081 ppm (d, 9.4 Hz), 90 221.1137/221.1135 (M H+), 4.153 ppm (d, 4.4 Hz), 90 221.1138/221.1133 (M H+), 4.074 ppm (d, 8.8 Hz), 50 221.1138/221.1135 (M H+), 4.177 ppm (d, 8.7 Hz), 70 failed failed 221.1137/221.1139 (M + H+), 4.385 ppm (d, 9.6 Hz), 70 220/221.0 (M + H+), 4.294 ppm (d, 9.4 Hz), 50
+ + + + +
2151238.0 (Msodmmsalt + Na+),4.111 ppm (d, 8.8 Hz), 60 nd, 4.032 ppm (d, 8.8 Hz), 50 nd, 4.281 ppm (d, 9.4 Hz), 50 322/345.1 (Msodmmsalt + Na+), 4.192 ppm (d, 8.8 Hz), 90 344/345.1 (Mdlsodlumsalt + H+), 4.169 ppm (d, 8.8 Hz), 90 3821405.1 (Mdlsodlum salt Na+), 4.226 ppm (d, 8.81, 50 382/405.1 (Mdlsodium salt + Na'), 4.221 ppm (d, 8.4 Hz), 70 nd, 4.361 (s), 70 nd, 4.066 ppm (d, 8.8 Hz), 90 282.0260/282.0255 (Msodlumsalt H+), 4.068 ppm (d, 8.6 Hz), 70 failed
+
+
342.1400/342.1391 (M + H+), 4.117 ppm (d, 8.7 Hz), 90 356.1557/356.1563 (M + H+),4.136 ppm (d, 8.3 Hz), 50 342.1400/342.1407 (M + H+),4.125 ppm (d, 8.7 Hz), 40 34U342.1 (M + H+),4.129 ppm (d, 8.9 Hz), 90 424.1931/424.1930 (M + H+), 4.157 ppm (d, 8.7 Hz), 90 383.1666/383.1661 (M + H+), 4.189 ppm (d, 8.7 Hz), 90 424.1931/424.1930 (M + H+), 4.148 ppm (d, 9.4 Hz), 90 383.1666/383.1669 (M + H+), 4.042 ppm (d, 8.7 Hz), 70 383.1666/383.1665 (M H+), 4.183 ppm (d, 9.4 Hz), 90 383.1666/383.1665 (M + H+), 4.024 ppm (d, 8.7 Hz), 90 nd, 4.104 ppm (d, 8.6 Hz), 50 342.1400/342.1404 (M+ H+), 4.065 ppm (d, 8.9 Hz), 50 3821383 (M H+), 4.137 ppm (d, 9.0 Hz), 90 342.1400/342.1404 (M + H+), 4.374 (SI, 50 312.1295/312.1300 (M + H+), 4.165 (SI, 50 342.1400/342.1407 (M + H+), 4.361 ppm (s), 50 488.1979/488.1975 (M + H+), 4.103 ppm (d, 8.8 Hz), 90 488.1979/488.1975 (M + H+),4.134 ppm (d, 8.8 Hz), 90 503604.2 (M H+),4.121 ppm (d, 8.8 Hz), 90 634.25581634.2551 (M + H+),4.092 ppm (d, 8.9 Hz), 90 853.330U853.3283 (M + H+), 4.123 ppm (d, 8.7 Hz), 90
+
+
+
369/370 (Mdiacid+ H'), 4.189 ppm (d, 8.8 Hz), 90 633.23541633.2350 (Msadlumsalt+ H+),4.064 ppm (d, 9.1 Hz), 90 salt + H+), 4.023 ppm (d, 9.0 Hz), 90 655.2174/655.2170 (Msodmm 696.2439/696.2391 (Msodium salt + H+),nd nd, 4.120 ppm (d, 8.7 Hz), 90
a Yields were determined from 'H-NMR spectra in DzO and are based on the integral ratio for the anomeric proton of the glycosylamine and underivatized saccharide, respectively. It should be borne in mind that the yields determined in DzO may be artificially low due to hydrolysis of the glycosylamines, and more likely are a measure of hydrolytic susceptibility (see Experimental Section). n.d.: not determined.
of a variety of charged monosaccharides, comprising GalA, GlcA, Gal-6P032-, GlcNA~6P03~-, Man6P032-, GlcNAc6S03-, G l ~ N 2 , 3 ( S 0 ~ - )G~l, ~ N 2 , 6 ( S 0 ~ - )and ~, Ga16S03-. In all cases, products are white, fluffy solids and are stable for months under anhydrous conditions. Compounds encompassed in this study are listed in Table 1. We failed to obtain glycosylamines from some furanosidic or pentopyranosidic monosaccharides (Rib, Rib5P032-,
h a ) . Substantial browning and side-product formation in these reactions is attributed to Amadori rearrangements and Maillard reactions, due to enhanced reactivity of the immonium ion intermediate (9). On the other hand, glycosylamine derivatives of Gal(b1-4)Ara and Xyl could be prepared without any problems. This suggests that only a few furanosidic or pentopyranosidic l-amino1-deoxy sugars are either not accessible via this route or might require optimization of the amination protocol.
Vetter and Gallop
318 Bioconjugate Chem., Vol. 6,No. 3, 1995
NMR investigation of the obtained glycosylamines reveals that the Kochetkov amination yields stereochemically well-defined products, with '95% P-anomer obtained in all cases (10). The extent of conversions of the saccharides to the glycosylamines are typically 80-95%. One typical side reaction during the amination procedure is condensation of the product glycosylamine with the initial saccharide. The resulting diglycosylamines can make up to 10% of the mass fraction of product. They cannot be separated by size exclusion chromatography in aqueous media as many of the glycosylamines are highly susceptible to hydrolysis. However, since neither the diglycosylamines nor the parent saccharides are substrates for subsequent chemoselective acylation reactions, conjugates of the desired monoglycosylamines are readily isolated and purified. Another constituent of the products from the amination procedure is residual ammonium carbonate which can usually not be completely removed by lyophilization without lowering the yield of glycosylamine. Ammonia can be detrimental to subsequent acylation reactions by scavenging the activated acid of the conjugation reagent. If the active ester is present in stoichiometric or substoichiometric ratios, measures must be taken to remove all traces of ammonium salts. This is achieved by warming the glycosylamine in methanol until termination of gas evolution and subsequent evaporation in uucuo. LITERATURE CITED (1) Alper, J. (1993) Carbohydrates surge through clinical trials. BIOPTECHNOLOGY 11, 1093. (2) Likhosherstov, L. M., Novikova, 0. S., Derevitskaja, V. A., and Kochetkov, N. K. (1986) A new simple synthesis of amino sugar /?-D-glyCOSylamineS.Carbohydr. Res. 146, Cl-C5.
(3) Kallin, E., Lonn, H., Norberg, T., and Elofsson, M. (1989) Derivatization procedures for reducing oligosaccharides, Part 3: Preparation of oligosaccharide glycosylamines, and their conversion into oligosaccharide-acrylamide copolymers. J . Carbohydr. Chem. 8 , 597-611. (4) Urge, L., Kollat, E., Hollosi, M., Laczko, I., Wroblewski, K., Thurin, J., and Otvos, L., Jr. (1991) Solid-phase synthesis of glycopeptides: synthesis of Na-fluorenylmethoxycarbonylLasparagine Nb-glycosides. Tetrahedron Lett. 32, 3445-3448. (5) 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. (6) Manger, I. D., Rademacher, T. W., and Dwek, R. A. (1992) 1-N-Glycyl P-oligosaccharide derivatives as stable intermediates for the formation of glycoconjugate probes
