J . Am. Chem. SOC.1990, 112, 9308-9309
9308
High-Efficiency Synthesis of Sialyloligosaccharides and Sialoglycopept ides Carlo Unverzagt,s Horst Kunz,+ and James C. Paulson*s* Contribution from the Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90024- 1737. Rereired April 23, I990 Abstract: Sialyllactosaminc structures were synthesized in high yield from carbohydrates and glycopeptides containing terminal G l c N A c with a two-step enzymatic glycosylation in a “one-pot” reaction. A key improvement is the use of alkaline phosphatase to destroy nucleotide phosphate inhibitors generated in the glycosyltransferase reaction.
The biological importance of sialosides has been well documented,’ and t h e need for model compounds has posed synthetic challenges to carbohydrate chemists. Despite recent progress in t h e chemical synthesis of sialosides,* t h e enzymatic approach3 is potentially more efficient in t h e rapid formation of naturally o c c u r r i n g sialyloligosa~charidcs.~ Increasing availability of glycosyltransferases through cloning techniquesSwill have a strong influcncc o n oligosaccharidc synthcsis in t h e foreseeable future. A niultigram syntlicsis for C M P - N - a c c t y l n c u r a m i n i c acid6 has been published. E n z y m a t i c glycosylation is still limited because enzymes and s u g a r nucleotides are expensive and should be used efficiently. This has been recognized by Wong et al.’ w h o used a sophisticated rccyclirig system t h a t g c n c r a t c d U D P - g a l a c t o s e (2) from t h e product UDP. which is also a potent inhibitor of t h e reaction. ticrc wc rcport t h c high-yicld synthcsis of sialyllactosamine s t r u c t u r c s 6a-d a s diagrammcd in Figure 1. Experimental Section
acetylglucosamine (3b)’“ was reacted according to the procedure for the synthesis of 6a: yield 1 I .4 mg (16.3 pmol; 72% calculated from starting material 3b); ‘ H N M R 6 4.45 (d, 1 H, J1,,2,= 7.6 Hz. H-I’)9 2.67 (dd, 1 H, J3qpf.4f, = 4.7 Hz, J3cgfc.3arf = 12.2 Hz, H-3eq”), 2.09, 2.03 ( 2 s, 6 H, NAc, NAc”), 1.72 (1, I H, J3ait,,4,, = J3qr,,3ax,, = 12.2 Hz, H-3ax”). N4-[(J-Acetamido-3,5-dideoxy-a-~-g/ycefo -~-ga/aclo -2-nonulopyran1,4)-2-acetamido-Z-deoxy-Bulosonic acid)-(2,6)-8-~-galactopyranosyl-( D - g ~ u c o p y r a n o s y l ] - N2 - [ (allyloxy)carbonyl- phenyla alanyl]-^asparaginyl-i.-~ryl-L-threonyl-L-isoCucine(6c). A 19.7 mg portion (22.7 pmol) of N4-(2-acetamido-2-deoxy-~-~-glucopyranosyl)-N~-[ [ (allyloxy)carbonyl]-~-phenylalanyl]-~-asparaginyl-~-seryl-~~ threonyl-~-isoleucine (3c)’“ and 4 mg of bovine serum albumin were reacted according to the procedure for the synthesis of 6a: yield 24.7 mg ( I 8.7 pmol, 82.4% calculated from starting material 3c); IH N M R 6 7.33 (m, 5 H, aromat), 5.9 (m, 1 H, =CH- allyl), 2.68 (m, 2 H, P-CH2b Asn, H-3eq”), 2.05, 2.03 (2 S , 6 H, NAc, NAc”). 1.73 (t. 1 H, J3sxr,,4n = J3cg,,,3ax,, = 12.1 Hz, H-3ax”), 1.24 (d, 3 H,J = 6.1 Hz, C H I Thr), 0.91 (m, 6 H, C H 3 He). N4-[(5-Acetamido-3,5-dideoxy-a-~-g/ycero -o-galacto-2-nonulopyranulosonic acid)-( 2,6)-8-~-galactopyranosyl-(1,4)-2-acetamido-Z-deoxy-& o-glucopyranosyl~N2-(glycylglycyl)-L-asparaginylglycylglycine (6d). A 12.8 mg portion (22.7 pmol) of N4-(2-acetamido-2-deoxy-P-~-glucopyranosyl)-N2-(glycyIglycyl)-L-asparaginylgiycylglycine(a) was reacted according to the procedure for the synthesis of 6a: yield 19.8 mg (19.5 pmol; 86% calculated from starting material 3d); IH N M R 8 5.1 (d, 1 H, J1,2= 9.4 Hz, H-I), 4.44 (d, 1 H, JlJ.2r = 8.3 Hz, H-l’), 2.85 (m, 2 = 12.2 Hz, H, @-CHI Asn), 2.66 (dd, 1 H, J,q,,,4f,= 4.6 Hz, J3cqrr,3ax,, H - 3 ~ 9 2.03, , 2.01 (2 S, 6 H, NAc, NAc”). 1.7 (t. 1 H, J3ax,,C,, = Jw,3ax,, = 12.2 Hz, H-3eq”).
Materials. UDP-glucose, bovine serum albumin, UDPglucose 4’epimerase (EC 5.1.3.2), and bovine galactosyltransferase ( E C 2.4.1.22) were purchased from Sigma. Calf intestinal alkaline phosphatase ( E C 3.1.3.I ) was obtained from Boehringer Mannheim. The GalPl.4GlcNAc tu-2.6-si;ilyltransfcriisc ( E C 2.4.99.1 ) was purified as described prcviously.’ CMP-N-acctylncuraminic acid was preparcd according to rcfcrcncc.6 IH N M R spectra were recorded in deuterium oxide on a Bruker Results A M 360/Wb. Thin-layer chromatography was performed on silica gcl It has been s h o w n previouslyg t h a t N-acetylglucosamine (3a) plates (60-FX4; E. Mcrck, Darmstadt) and visualized by spraying with I N H2S04 in cthiinol containing 0.1% orcinol. c a n be converted t o sialyllactosamine (6a) in a single s t e p by subsequent e n z y m a t i c transfer of galactose and sialic acid. The (5-Acetamido-3,5-d~deoxy-cr-~-~/ycefo-~-ga/aclo -2-nonulopyranulosonic acid)-( 2,6)-B-D-g;alactopyranosyl-( 1,4)-2-acetamido-Z-deoxyrcaction efficiency could be increased by two improvements: First, glucopyranose (6a).A 5-mg portion (22.7 pmol) of N-acetylglucosamine t h e use of alkaline phosphatase to destroy nucleotide p h o s p h a t e (3a) was dissolved in 565 p L (50 m M ) of sodium cacodylate (pH 7.4) inhibitors that are released during the glycosyltransferase reaction containing 0.5 mg of bovine serum albumin, 1. I pmol of MnCI2. 3.4 pmol a n d , second, the use of conditions t h a t minimize t h e hydrolysis of IVaN,, 2 8 . 3 pmol of UDP-glucosc. 200 milliunits of GlcNAc 0-1,4of nucleotide sugars. ga1actoayltransfcr;isc ( E C 2.4.1.22). I unit of UDPglucose 4’-epimerase ( E C 5.1.3.2), and 4 u n i t a of calf intestinal alkaline phosphatiisc (EC ( 1 ) (a) Kawasaki, T.; Ashwell, G.J . Biol. Chem. 1977,252,6536-6543. 3.1.3.1). Thc rcaction mixturc was incubated at 37 O C . and the pH was (b) Schauer, R. Ado. Curbohydr. Chem. Biochem. 1980, 40, 131-234. (c) maintained at 7.4 by periodic addition of 0.25 N NaOH. After 48 h, I .7 Paulson, J. C. In The Receptors; Conn, M., Ed.; Academic Press: New York, mL of H 2 0 . 30 pmol of CMP-NcuAc. I .4 mg of bovine serum albumin, 1985; Vol. 2, p 131-219. I O pmol of NaN,, 100 milliunits of &galactoside a-2.6-sialyltransferase (2) (a) Murase, T.;Ishida, H.; Kiso, M.; Hasegawa, A. Curbohydr. Res. (EC 2.4.99.1). and 6 units of calf intestinal alkaline phosphatase wcrc 1989, 188, 71-80. (b) Ito, Y.; Numata, M.; Sugimoto, M.; Ogawa, T. J . Am. addcd. Incubation was continucd for 2 days at 37 OC with the pH at 7.4. Chem. Sor. 1989, 111, 8508-8510. (3) Toone, E. J.; Simon, E. S . ; Bednarski, M. D.; Whitesides, G. M. Sialosidc 6a w x isolated by gcl chromatography on a Scphadcx G-25 Tetrahedron 1989, 45, 5365-5422. supcrfinc column ( 2 . 3 X 32 cm) by eluting with 0.1 M N H 4 H C 0 3 . The (4) (a) Sabesan, S.; Paulson, J. C . J . A m . Chem. SOC. 1986, 108, fractions ( 3 ml.) containing 6a (TLC in I M NH40Ac/isopropyl alcohol, 2068-2080. (b) Thiem, J.; Treder. W. Angew. Chem., fnr. E d . Engl. 1986, 1/2.4) were pooled and lyophilized. ‘H N M R data were in accord with 25, 1096-1097. (c) De Heij, H. T.;Kloosterman, M.; Koppen, P. L.; van thosc rcportcd? yicld 11.4 mg (16.9 pmol; 74.4’% calculatcd from Boom, J. H.; van den Eijnden, D. H. J . Curbohydr. Chem. 1988, 7,209-222. starting material 3a). (d) AugB, C.; Gautheron, C.; Pora, H. Curbohydr. Res. 1989,193,288-293. Azido(S-acetamido-3,5-d~deoxy-cr-D-~/ycero-~-~a/aclo -2-nonulo(e) Nilsson, K. G . Curbohydr. Res. 1989, 188, 9-17. (fj Palcic, M. M.; Venot, pyranulosonic acid)-(2,6)-~-~-ga~actopyranosyl-(1,4)-2-deoxy-~-~- A. P.: Ratcliffe, R. M.; Hindsgaul, 0. Curbohydr. Res. 1989, 190, 1-11. ( 5 ) Review: Paulson, J. C.; Colley, K. C. J . Biol. Chem. 1989, 264, glucopyranose (6h). A Z.6-mg portion (22.7 pmol) of @-azido N 17615-1 761 8. (6) Simon, E. S.; Bednarski, M. D.; Whitesides, G . M. J . Am. Chem. SOC. 1988, 110. 7159-7163. ’ Prcscnt ilddrcss: Institut fur Orpanischc Chcmic der Univcrsitiit. J.(7) Wong. C. H.; Haynie. S. L.;Whitesides. G . M. J . Org. Chem. 1982. J.-Bcchcr-Wcg 18-20, 6500 Mainz. Gcrmuny. 47, 5416-5418. Prcscnt uddrcss: Cytcl Corp. and Dcpartmcnt of Chcmistry, Scripps (8) Weinstein, J.; de Souza-e-Silva, U.; Paulson. J. C. J . Biol. Chem. 1982. Rescarch Institutc. I1099 North Torrcy Pincs Rd., La Jolla, CA 92037. 257, 13835-13844. Prcscnt addrcss: lnstitut fur Organische Chemie dcr Tcchnischen (9) Paulson. J. C.; Rearick, J. 1.; Hill, R . J. J . Biol. Chem. 1977, 252, Univcrsitlit. I.ichtcnbcrgstrassc 4. 8046 Garching, Germany. 2363-2371. ~~
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0002-7863/90/
I5 I2-9308$02.50/0
0 1990 A m e r i c a n C h c m i c a l Society
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J . Am. Chem. Soc., Vol. 112, No. 25, 1990 9309
Sialyloligosaccharides and Sialoglycopeptides
in
ID
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0-UDP
0-UDP
)
tD
2
1
-
Galactosyltransferase. R
CIAP, pH 7.4
I
3a-d
+ Undine + 2 P,
'Io
4a.d
I
0-CMP
a2.6-Sialyitransferase, CIAP, pH 7.4 ID
+ Cytidine
+ P,
'lo
6a-d
Figure 1 . Calf Alkaline Phosphalase (CIAP) Improves the Yleld of the Calactosyltranslerase Reaction
"/--C-.i 80
4 5
-
s
E 20 0
4 0
.
3 20
.
I
40
-
s
60
wilh CIAP
noClAP
.
r
80
.
4
100
Reaction Time I Hours
Figure 2.
The UDP-galactose ( 2 ) required for the galactosyltransferase reaction is gcncrated in situ by UDPgalactose 4'-epimerase (UDPGE), an enzyme that converts inexpensive UDP-glucose (1) into UDP-galactose (2). while an equilibrium of Glc/Gal = 3.5/1 is maintaincd. Since UDP-galactose ( 2 ) is subject to degradation by Mn2+ in alkaline media.I0 the concentration of Mn2+was kept low ( 2 mM) and ncutral pH (7.4) was maintained. A 25% excess of nucleotide sugar (50 mM) over the acceptor (40 mM) assured thc prcscncc of sufficient amounts of donor substrate even toward thc cnd of thc reaction. To cvuluntc the inhibitory effect of UDP on the final yields of disacchardide 4a, the galactosyltransferase reaction was examined with and without the presence of calf intestinal alkaline phosphatase (CIAP, molecular biology grade). As seen in Figure 2, the use of nlknlinc phosphatase gives a dramatic improvement, leading to a highcr rcaction vclocity ovcr thc entire time course and permits near quantitative conversion of the acceptor employed. Sincc nuclcotidc phosphatcs arc gcnerally product inhibitors of glycosyltransfcrascs. alkaline phosphatase may be of general use to acccleratc enzymatic oligosaccharide synthesis. Following galactosylation, sialic acid addition could be achieved without intcrmediatc purification. For optimal yields, the reaction mixture was diluted 4-fold and adjusted to pH 7.4 with 0.25 N N a O H . This was necessary since the cu-2,6-sialyltransferase is inhibited by salt concentrations higher than 100 mM" and there is significant hydrolysis of CMP-NeuAc at pH
