Bloconjugate Chem. 1882, 3, 391-390
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A Novel Method for the Incorporation of Glycoprotein-Derived Oligosaccharides into Neoglycopeptides Stephen J. Wood and Ronald Wetzel' Macromolecular Sciences Department, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, Pennsylvania 19406. Received April 6,1992
We describe a new method for the transfer of carbohydrate moieties to polypeptides in which complex carbohydrate, in the form of glycosyl amino acid, is removed from an available glycoprotein, derivatized, and reacted with a polypeptide via an iodoacetylated a-amino group. A family of oligomannose chains, N-linked to the side chain of Asn, was obtained from ovalbumin by pronase digestion and purified as previously described. A reactive sulfhydryl group was specifically placed on these molecules by reaction of 2-iminothiolane with the Asn a-amino group. Separately, the a-amino group of the peptide GGYR was specifically iodoacetylated by reaction with iodoacetic anhydride at pH 6. Reaction of the thiolcontaining carbohydrate with iodoacetylated peptide at pH 8 gave in high yield the corresponding oligomannosyl-peptides, whose structures were confirmed by mass spectrometry. A peptide inhibitor of HIV protease was also oligomannosylated by this procedure. The principle advantage of this method is the efficiency of the reaction even when performed with stoichiometric amounts of the two molecules at low concentration. It should be feasible to extend this chemistry to larger polypeptides.
INTRODUCTION
There is increasing interest in the biological roles and potential therapeutic exploitation of the carbohydrate of glycoproteins, and many methods have been described for preparing conjugates of proteins and carbohydrate (1, 2). The available methods, however, are of limited value in carrying out a number of described transformations. In particular, there appear to be no methods available for efficiently attaching scarce, chemically complex carbohydrate to equally scarce polypeptide, with or without selectivity. Although biological methods are feasible in principle, our ignorance of the details of the cellular glycosylation process makes recombinant biosynthesis an unreliable approach to novel glycoproteins (3). Of the many chemical methods which have been described, each suffers from one or more limitations. In many conjugation reactions, the amino groups of polypeptides are used as the attachment site, but it is often difficult to control the point of attachment and the extent of modification in a polypeptide containing a number of amino groups (4). Many of the available condensation reactions are relatively inefficient, requiring substantial excesses of carbohydrate (5, 6 ) or polypeptide (7) for significant conversion of a limiting, costly molecule. For example, for modification of scarce or costly polypeptides, reductive amination and similar methods are only feasible for simple, readily available saccharides. Bulk preparation of more complexcarbohydrates by total chemical synthesis is not currently feasible. An alternative is to obtain complex carbohydrate of the desired structure from a plentiful glycoprotein. Classical methods which utilize available carbohydrate, however, suffer from relatively low yields of recovery of the required form of the carbohydrate from source glycoprotein,as well as low yields in the condensation reaction unless a large molar excess of carbohydrate or protein is used to achieve efficient utilization of the other molecule (7). Recently methods have been described for synthesis of neoglycoproteins which exploit the high reactivity and selectivity of the reaction between sulfhydryl groups and iodo- or bro-
* Author to whom correspondence should be addressed.
moacetyl groups-the same reaction used in the method described here-for efficient cross-linking ( 4 9 ) . One of these methods uses as a starting material oligosaccharide derived from soy meal (B), while the other utilizes a monosaccharide derivative (9). It is not clear whether either of these methods will allow efficient use of carbohydrate derived from a natural glycoprotein. We describe here a novel method for the synthesis of neoglycopeptides by the chemical conjugation of polypeptide to a carbohydrate moiety derived from a natural glycoprotein. In the method, a glycosylated amino acid is obtained by total digestion of a source glycoprotein with pronase followed by chromatographic purification, as previously described (10). The glycosyl-aminoacid is then conjugated via its a-amino group in two high-yield steps. The chief advantages of the method are that both N- and 0-linked carbohydrates can be utilized and the conjugation step involves highly efficient chemistry which can be conducted under mild conditions in small or large scale. In addition, the attachment point can be directed to the N-terminus of the polypeptide, especially in the case of peptides. To illustrate the method we describe the conjugation of a family of related oligomannosyl-NAGZ-Asn' molecules to the N-terminal a-amino groups of the peptide glycylglycyltyrosylarginine (GGYR, 1)and the HIV-I protease inhibitor (4S,5S)-[4-hydroxy-5-[(alanylalanyl)aminol-6phenylhexanoyllvalylvaline methyl ester (AAF'qGWOMe, 2, SKF# 107457) (11). EXPERIMENTAL PROCEDURES
Materials and General Methods. The tetrapeptide GGYR (1) was synthesized by the standard Merrifield solid-phase method and supplied by Mike Burke of SmithKline Beecham Pharmaceuticals. The proteaseinhibitor SKF AAFqGVV-OMe (2) (11)was supplied by Geoff Dreyer, SmithKline Beecham Pharmaceuticals. ~
Abbreviations used: NAG = N-acetylglucosamine; 2-IT = 2-iminothiolane; TFA = trifluoroacetic acid; DTNB = 5,5'dithiobis-2-nitrobenzoicacid; MES = 2-(N-morpholino)ethanesulfonic acid; EDTA = ethylenediaminetetraacetic acid; FAB = fast atom bombardment. 0 1992 American Chemical Society
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Chicken egg ovalbumin (Grade V), dithiothreitol, DTNB and Protease type XV (Pronase E) were purchased from Sigma. Iodoacetic anhydride was from Aldrich Chemical Co. and 2-ITwas from Pierce Chemical Co. HIV-I protease was provided by James Stickler of SmithKline Beecham. HIV-I protease inhibition was determined as previously described (12). HPLC was performed on a protein and peptide C18 column (Vydak) using linear gradients of buffer B (0.1% TFA in acetonitrile) in buffer A (0.1% TFA in water). Isolation and Purification of Glycosyl-Asn. Asparagine-linked carbohydrate components of ovalbumin were isolated and purified according to the method of Huang et al. (10). Briefly, ovalbumin at pH 7.8 was heated to 80 "C for 15min, cooled, and then digested with pronase at pH 7.4 for 24-h at room temperature. OligomannosylNAG-NAG-Asn was separated from incompletely digested material by gel filtration on a Sephadex G-25 column (3.5 X 50 cm), and the incompletely digested ovalbumin fractions were pooled and digested further with pronase. This was repeated twice. Carbohydrate-containing fractions were identified with the phenol-sulfuric acid carbohydrate assay (13). Oligomannosyl-NAG-NAG-Asn containing fractions were pooled, desalted on a G-25 (3.5 X 50 cm), and fractionated on aDowexAG-50W-X4 cation exchange column (1.6 X 90 cm). Glycosyl-Asn/2-Iminothiolane Reaction. A pool of oligomannosyl-NAG-NAG-Asn fractions was prepared by mixing peaks A-E of the Dowex column, which behaved as described (10). A stock solution of 2-IT was freshly prepared by dissolving 2-IT in 50 mM potassium acetate buffer, pH 5, to a concentration of 100 mM, and an aliquot of this stock used to prepare the reaction mixture. A 1550pL reaction mixture consisting of 1 mM oligomannosylNAG-NAG-Asn (6) and 33 mM 2-IT in 1mM EDTA, 33 mM potassium phosphate buffer, pH 7.0, was incubated at 37 "C. After 24 h, 500 pL of 100 mM DTT was added and incubation at 37 "C continued. After 4 h the entire reaction mixture was chromatographed at room temperature on a G-10 gel filtration column (1.5 X 17 cm) equilibrated with 0.1 % acetic acid. Fractions were assayed for carbohydrate using the phenol-sulfuric acid assay (13) and for the presence of free sulfhydryl using DTNB (14). Fractions testing positive for both carbohydrate and free sulfhydryl were freeze-dried and stored at -20 "C in 0.1% acetic acid (Figure 1). Iodoacetic Anhydride Reactions. A stock solution of iodoacetic anhydride was prepared by dissolving iodoacetic anhydride in dry THF at a concentration of 250 mM (15). This solution was stored at -20 "C in microcentrifuge tubes which were stored inside 50-mL conical tubes with calcium carbonate dessicant and sealed with parafilm. Derivatization of AAFQGVV-OMe (2) with iodoacetic anhydride was accomplished at 0 "C as follows. To a 550pL reaction mixture containing 5 mM AAFQGVV-OMe (2),3.6mM EDTA, and 100 mM MES, pH 6, were added three additions (30 pL), with vortexing, of iodoacetic anhydride (0.25 M) at 3-min intervals. Followingthe final addition, 700 pL of a 10% (v/v) methanol in chloroform solution was added and the mixture was vortexed for 1 min and then centrifuged for 5 min. The organic phase was removed into a fresh microcentrifuge tube, spun to dryness in a rotary evaporator, and stored at -20 "C. Iodoacetylation of the tetrapeptide GGYR (1) was accomplished in the same manner with the exception that the organic extraction of the reaction product was not
performed. Instead, the iodoacetylated GGYR reaction product (4)was purified by reverse-phase HPLC (15). Condensation Reactions. AAF#G VV-OMe. To 500 pL of a 4 mM solution of Na-iodoacetylated AAFqGVVOMe (5) in DMSO was added 1000pL of an approximately 2 mM solution of oligomannosyl-NAG2-Asn-SH (8) in 20 mM acetic acid, 0.2 mM EDTA. The solution was brought to pH 8 by adding 500 pL of 1 M potassium phosphate, pH 8.0, and the reaction incubated at 37 "C for 6 h. GGYR. To 750 p L of an approximately 50 pM solution of oligomannosyl-NAGZ-Asn-SH (8) in 20 mM acetic acid, 1mM EDTA was added 100 pL of an aqueous solution 750 pM in N"-iodoacetyl-GGYR (4). The solution was brought to pH 8 by addition of 500 pL of 1M potassium phosphate, pH 8.0, and the reaction incubated at 37 "C for 20 h. Mass Spectrometry. Reaction products were analyzed by fast atom bombardment mass spectrometry on the first portion of a double focusing VG ZAB SE-4F tandem magnetic deflection mass spectrometer, which employs an accelerating voltage of 10 kV and a mass range of 12 500, or on a VG ZAB-HF magnetic deflection mass spectrometer, with an accelerating voltage of 8 kVand a mass range of 3000. The VG ZAB SE-4F was equipped with a flow FAB ion source and a Cs ion gun operated at 35 kV and a 2-4 pA emission current. The VG ZAB-HF was also equipped with a standard flow FAB ion source, but employed an ION Tech FAB gun (operating voltage of 8 kV) which utilized a discharge current of 1mA. All data was processed on a VG 11-2505data system. Most samples were analyzed by FAB-MS in a matrix of either thioglycerol or 1:l thioglycerol-m-nitrobenzyl alcohol plus 1% trifluoroacetic acid. RESULTS
The reactions described in this paper are summarized in Scheme 1. To simplify the scheme, the family of carbohydrates derived from ovalbumin, the major components of which are identified in the product as shown in Table I, is represented in a single structure, 6. Digestion of ovalbumin with pronase generated a mixture of glycosylated asparagine derivatives giving a chromatogram on Dowex very similar to the published distribution (10). Fractions A-E (IO),reported to contain glycosyl groups composed of 4-7 mannose units (plus the NAG-NAG bridge to the Asn side chain) (161,were pooled. This mixture was reacted with 2-iminothiolane (7) (13, which reacts with amino groups of proteins, peptides, and amino acids to generate a spacer terminating with a sulfhydryl group (15,18-20). The reaction mixture was incubated with DTT to insure against loss of reactive thiolated carbohydrate by disulfide formation under reaction conditions. The desired product (8) was separated from 2-IT breakdown products and DTT by chromatography on Sephadex G-10. Figure 1 shows that the carbohydrate-containingfractions comigrate with the only macromolecular peak of DTNB-positive fractions. Further, there is good agreement between the estimated concentrations of Man,-NAG-NAG-Asn-SH as assessed by both sulfhydryl and carbohydrate contents. This close agreement suggests that most of the Man,-NAG-NAGAsn a-amino groups have reacted. The DTNB estimate was taken as the correct concentration for setting up the condensation reactions. The results of these reactions, however, suggest that the DTNB-determined concentration may be an underestimate by approximately 50%. Figure 2a shows the HPLC elution position of the peptide (4S,5S)-[4-hydroxy-5-[(alanylalanyl)amino1-6phenylhexanoyl]valylvaline methyl ester (AAF9GVV-
Incorporation of Oligosaccharides into NeoglycopeptMes
Bioconlugate Chem., Vol. 3, No. 5, 1992 993
Scheme I pH 6.0
00 c
+
H,N+-R
-
2
1,2
0 11 ICH,CNH -R
4.5
+ 1
6
Table I. FAB-MS Parent Ions of the Major Components of Conjugates o1i gomannosy 1ated oligomannosylated AAFIGVV-OMe (10) GGYR (9) carbohydrate M+, M+, component m/z (*1.5) mlz (*2) 2070 MmsNAGz 1943.3 MmNAG2 2105.5 2232 2394 ManaNAGd 2349.7 ManbNAGs 2553.2 2556 a Man = mannose, NAG = N-acetyl-8-D-glucosamine.
carbohydrate componenta ManaNAG2 ManeNAG2 Mm7NAG2 ManaNAG2
801
.
I
-
I
-
I
*
I
- I
Eluate (ml)
Figure 1. Estimate of 2-iminothiolane-derivatizedcarbohydrate eluting from a G-10 column based on the phenol-sulfuric acid carbohydrate assay (13) (+) and the DTNB assay for free thiols (14) (a).
OMe) (2). Figure 2b shows the chromatogram of the reaction mixture of AAFQGVV-OMe with iodoacetic anhydride at pH 6, conditions previously described to be highly selective for iodoacetylation of the Nu-aminogroup of peptides (15,21,22).The major product peak elutes later than starting material, as expected for an iodoacetylated derivative (15).The yield of iodoacetylated peptide (5) is about 80 % . Figure 2c shows the reaction mixture between the iodoacetylated peptide and the pool of Man,-
8
NAG-NAG-Asn-SH (8)(which does not absorb at 215 nm) immediately after mixing at pH 2. Carbohydrate analysis (13) (not shown) indicates that M&,-NAG-NAG-AsnSH elutes in the injection peak. Figure 2d shows the chromatogram of the same mixture after the pH was adjusted to 8 and the mixture allowed to stand for 6 h at room temperature. The figure shows that the iodoacetylated peptide has disappeared and been replaced by an earlier-eluting peak of similar size. Earlier elution in an aqueous TFA, reversed-phase HPLC system is expected for the addition of carbohydrate to a peptide ( 4 ) . Closer examination of the peak shows it to be asymmetric, consistent with its being a mixture of related structures. Figure 2d suggests that conversion of iodoacetylated peptide to the mannosylated peptide is essentially quantitative at relatively low reactant concentrations at a ratio of about 1:l. Table I shows the MS analysis of this peak, which confirms that it is a mixture of glycosylated conjugates of AAFQGVV-OMe in the anticipated Man,-NAG-NAG series. Three of the four carbohydrate structures found in the product are as expected from previous analysis of the heterogeneous oligosaccharide of ovalbumin (16). Analysis of inhibition of HIV-I protease by the conjugate mixture showed it to be only modestly reduced in inhibitory activity compared to the unmodified inhibitor (KI= 50 and 10 nM, respectively, for oligomannosylated and unmodified inhibitor). Figure 3a shows the chromatogram of the reaction mixture containing Na-iodoacetyl-GGYR (4) and Man,NAG-NAG-Asn-SH(8)before adjusting the pH to8. Figure 3b shows the same reaction mixture 20 h after adjusting the pH to 8 as described in the methods. The figure shows some residual, unreacted iodoacetylated peptide, which is expected given that the reaction mixture was adjusted to give a stoichiometry of about 1:2 carbohydrate to peptide. As mentioned above, the fact that the reaction appears to have progressed well beyond 50% suggesb that the concentration of Man,-NAG-NAG-Asn-SH was underestimated. The figure also clearly shows the heterogeneity in the product, which is expected on the basis of the known
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a)
0
5
IO 15 Elution Time (min)
20
25
Figure 3. Reverse-phase HPLC chromatograms of (a) reaction mixture of Na-iodoacetyl-GGYR(4) with oligomannosylNAGZ-Asn-SH (8) at pH 2 and (b) reaction mixture of the Nuiodoacetyl-GGYR (4) with oligomannosyl-NAG*-Asn-SH (8) at pH 8 after 20 h at 37 "C.
DISCUSSION
*
:
0
5
:
:
10
15
:
20
:
25
:
30
:
35
:
40
I
45
Elution Time (min)
Figure 2. Reverse-phase HPLC chromatograms (A215) of (a) pure HIV protease inhibitor, (b) reaction mixture from iodoacetylation of AAFQGVV-OMe(2) with iodoaceticanhydride, (c) reaction mixture of iodoacetylated AAFQGVV-OMe(5) and oligomannosyl-NAGZ-Am-SH (8) at pH 2, and (d) reaction
mixture of iodoacetylated AAFQGVV-OMe (5) and oligomannosyl-NAGz-Asn-SH (8) at pH 8 after 6 h at 37 O C . heterogeneity of the carbohydrate. This broad peak was collected and submitted for MS analysis. Table I shows that the four major conjugate peaks are all expected from the previously characterized structures of ovalbumin's oligosaccharide component (16). The MS parent ions listed in Table I are consistent with the molecular weights calculated for the suggested structures in Scheme I. The parent ions of 2-IT reaction products 8 suggest that the imino amide moiety expected in a 2-ITadduct hydrolyzes to the amide during the course of the reaction and workup. The scheme reflects this hydrolysis in the portrayed structures of 8-10. Hydrolysis of the imino amide to an amide is not surprising, based on the described chemistry of imino amides in general (23) and products of 2-IT reactions in particular (24).
The macrophage mannose receptor (25) requires multivalent mannosyl derivatives for endocytosis of the receptor-ligand complex (26). Multivalency can be achieved by modification of multiple lysines of a protein with simple mannose derivatives (5). However, such multivalent conjugates are not feasible for molecules containing only one potential attachment site. Furthermore, proteins hyperglycosylated at many lysine residues display carbohydrate in a significantly different way than do the multivalent, complex carbohydrates which are the presumed normal ligands of receptors like the macrophage mannose receptor. Other carbohydrate receptors might prove to have even higher requirements for native glycosyl chain structure than the mannose receptor. For these reasons, we set out to develop a general, efficient method which would allow one to borrow the glycosyl chains from a glycoprotein in hand and chemically transfer the intact carbohydrate to another protein or peptide with some control over the point of attachment. Because of the difficulty in obtaining the carbohydrate moieties of glycoproteins in large amounts, we put a premium on conversion efficiency in choosing a condensation chemsitry. We chose the reaction between the a-iodocarbonyl group and the thiol group, chemistry which can be controlled by pH and which can proceed with excellent yields under mild conditions without using large excesses or high concentrations of either reactant (14,15). The results described here support the merit of this choice of chemistry. The reaction of oligomannosyl-Asn with 2-IT appears to proceed in high yield, given the good agreement between measured SH and carbohydrate levels in the macromolecular fraction of the reaction mixture (Figure 1). Similarly, the condensation of the product Man,,-NAG-NAG- Asn-SH with iodoacetylated peptides also proceeds in high yields (Figures 2 and 3), even at low concentrations and 1:1 stoichiometries of reactants. These features should allow efficient use of available carbohy-
Incorporation of Oligosaccharides into Neoglycopeptides
drate as well as polypeptide. Since the reaction with 2-IT simply requires any active amino group, the conjugation strategy should work equally well for 0- and N-linked carbohydrate. Similarly, carbohydrate attached to Asn, Thr, or Sei which are incorporated into peptides (for example, as in the products of incomplete proteolytic digestion of a glycoprotein) should react equally well to give neoglycopolypeptides, albeit with longer, less welldefined spacer arms. The chemistry also allows for varying degrees of homogeneity of the carbohydrate moiety. It should be possible to isolate and conjugate particular molecular species. In developing this chemistry we chose to use a mixture of glycosyl-Asn molecules obtained by pooling the chromatographic peaks from the separation of the products of pronase digestion of ovalbumin. Carbohydrate hydroxyl groups can also react with 2-IT, especially at high pH values, at a rate estimated to be about 1% that of the reaction of 2-IT with amino groups (24). If a significant proportion of the product of 2-IT with oligomannosyl-NAG-NAG-Asn is modified at hydroxyl groups, it would thus be expected to produce a disubstituted molecule (with the amino group also modified), which would in turn produce a conjugate with two peptide groups per molecule of carbohydrate. We see no evidence for the formation of such conjugates in the MS data. However, the formation of a small amount of hydroxyl-modified conjugate cannot be discounted. In contrast to one-step conjugation reactions such as reductive amination, the procedure described here also affords a degree of control over the attachment point, especially in the case of peptides. Reaction of peptides with iodoacetic anhydride at pH 6 has been shown previously to greatly favor the acylation of the N-terminal a-amino group, even in the presence of a number of unprotected lysine €-amino groups (15). Furthermore, further control of the attachment site is possible since iodoacetylated peptides can be HPLC-purified, stored, and characterized before use in an alkylation reaction. (However, some care should be taken with iodoacetylated peptides containing Met, His, or Lys in the sequence, since, under appropriate conditions, such peptides can cyclize (22).) Absolute specificity of attachment is more difficult to achieve with proteins containing many lysine groups, unless one of them is uniquely reactive. In cases where specificity is not possible, the iodoacetylation mediated attachment of carbohydrate described here still provides the advantage of finer control over the number of carbohydrate molecules bound per protein molecule. Where greater control over attachment point is desired, protein engineering methods might be utilized to construct mutants with uniquely reactive side chains which can be modified for specific reaction with a derivitized carbohydrate. Although logic dictates that additional steps in a synthetic route should generally decrease final yield, the strategy presented here, based on synthesis of reactive intermediates of both polymers, should lead to increased efficiency of use of these polymers, because of the high yields of all of the steps involved, including the final coupling step. The cell-type specificity in the distribution of carbohydrate receptors makes drug targeting via added carbohydrate an attractive possibility (27,28). Our results provide a new means to attachment of complex carbohydrates to potential drug molecules. In the example, attachment of a family of branched mannose oligosac-
B/oconiugate Chem., Vol. 3, No. 5, 1992 395
charides to the N-terminus of an HIV-I protease inhibitor allowed good retention of biological activity. ACKNOWLEDGMENT
We thank Geoff Dryer for the gift of SKF# 107457, Jim Strickler, and Tom Meek for help with the HIV-I protease inhibition assay, Steve Carr and Gerald Roberts for mass spectrometry, and Kalyan Anamula, Rich Kirsh, and Harma Ellens for helpful discussions. LITERATURE CITED (1) Stowell, C. P., and Lee, Y. C. (1980) Neoglycoproteins: The
preparation and application of synthetic glycoproteins. Adu. Carbohydr. Chem. Biochem. 37, 225-281. (2) Aplin, J. D., and Wriston, J. C., Jr. (1981) Preparation, properties and applications of carbohydrate conjugates of proteins and lipids. CRC Crit. Rev. Biochem. 10, 259-306. (3) Smith, P. L., Kaetzel, D., Nilson, J., and Baenziger, J. U. (1990) The sialylated oligosaccharidesof recombinant bovine lutropin modulate hormone bioactivity. J. Biol. Chem. 265, 874-881. (4) Morehead, H., McKay, P., and Wetzel, R. (1982) Highperformance liquid chromatography analysis in the synthesis, characterization, and reactions of neoglycopeptides. Anal. Biochem. 126, 29-36. (5) Lee, Y. C., Stowell, C. P., and Krantz, M. J. (1976) 2-imino2-methoxyethyl l-thioglycosides: New reagents for attaching sugars to proteins. Biochemistry 15, 3956-3963. (6) Marsh, J. W., Denis, J., and Wriston, J. C., Jr. (1977) Glycosylation of Escherichia coli L-asparaginase. J. Biol. Chem. 252,7678-7684. (7) Mencke, A. J., and Wold, F. (1982) Neoglycoproteins: Preparation and in uiuo clearance of serum albumin derivatives containing ovalbumin oligosaccharides. J. Biol. Chem. 257, 14799-14805. (8) Colon, M., Staveski, M. M., and Davis, J. T. (1991) Mild conditionsfor the preparation of high-mannoseoligosaccharide oxazolines: Entry point for &glycoside and neoglycoprotein synthesis. Tetrahedron Lett. 32, 4447-4450. (9) Davis, N. J., and Flitach, S. L. (1991)A novel method for the specificglycosylationof proteins. Tetrahedron Lett. 32,67936796. (10) Huang, C.-C., Mayer, H. E., Jr., and Montgomery, R. (1970) Microheterogeneity and Paucidispersity of Glycoproteins. Carbohydr. Res. 13, 127-137. (11) Dreyer, G. B., Lambert, D. M., Meek, T. D., Carr, T. J., Tomaszek,T. A., Jr., Fernandez, A. V., Bartus, H., Cacciavillani, E., Hassell, A. M., Minnich, M., Petteway, S. R., Jr., Metcalf, B. W., and Lewis, M. (1992) Hydroxyethylene isostere inhibitors of human immunodeficiencyvirus-1protease: Structureactivity analysis using enzyme kinetics, X-ray crystallography, and infected T-cell assays. Biochemistry 31, 664643659, (12) Meek, T. D., Dayton, B. D., Metcalf, B. W., Dreyer, G. B., Strickler, J. E., Gorniak, J. G., Rosenberg, M., Moore, M. L., Magaard, V. W., and DeBouck, C. (1989) Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease. Proc. Natl. Acad. Sci. U.S.A. 86, 1841-1845. (13) Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Colorimetric Method for Determination of Sugars and Related Substances. Anal. Chem. 28,350-356. (14) Means, G. E., and Feeney, R. E. (1971) Chemical Modification of Proteins, p 220, Holden-Day, San Francisco. (15) Wetzel, R., Halualani, R., Stulta, H. T., and Quan, C. (1990) A General Method for Highly Selective Cross-Linking of Unprotected Polypeptides via pH-Controlled Modification of N-Terminal a-Amino Groups. Bioconjugate Chem. 1, 114122.
(16) Kobata, A. (1984) The carbohydrates of glycoproteins. Biology of Carbohydrates (V. Ginsburg, and P. W. Robbins, Eds.), pp 87-161, John Wiley & Sons, New York.
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(17) Lambert, J, M., McIntyre, G., Gauthier, M. N., Zullo, D., Rao, V., Steeves, R. M., Goldmacher, V. S., and Blaettler, W. A. (1991) The galactose binding sites of the cytotoxic lectin ricin can be chemically blocked in high yield with reactive ligands prepared by chemical modification of glycopeptides containing triantennary N-linked oligosaccharides. Biochemistry 30, 3234-3247. (18) Traut, R. R., Bollen, A., Sun, T.-T., Hershey, J. W. B., Sundberg, J., and Pierce, L. R. (1973) Methyl 4-mercaptobutyrimidate as a cleavable cross-linking reagent and its application to the Escherichia coli 30s ribosome. Biochemistry 12, 3266-3273. (19) Jue, R., Lambert, J. M., Pierce, L. R., and Traut, R. R. (1978) Addition of sulfhydryl groups to Escherichia coli ribosomes by protein modification with 2-iminothiolane (methyl 4-mercaptobutyrimidate). Biochemsitry 17, 53995406. (20) Goff, D. A., and Carroll, S. F. (1990) Substituted 2-iminothiolanes: reagents for the preparation of disulfide cross-linked conjugates with increased stability. Bioconjugate Chem. I , 381-386. (21) Stults, J. T., Lai, J., McCune, S., and Wetzel, R. (1992) Simplification of high energy collision spectra of peptides by amino-terminal derivatization. Submitted for publication.
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(22) Wood, S. J., and Wetzel, R. (1992) A novel cyclization chemistry especially suited for biologicallyderived, unprotected peptides. Int. J . Pept. Protein Res. 39, 533-539. (23) De Wolfe, R. H. (1975)Kinetics and mechanisms of reactions of amidines. The Chemistry of Amidines and Imidates (S. Patai, Ed.) pp 349-387, Wiley, New York. (24) Alagon, A. C., and King, T. P. (1980) Activation of polysaccharides with 2-iminothiolane and its uses. Biochemistry 19, 4341-4345. (25) Taylor, M. E., Conary, J. T., Lennartz, M. R., Stahl, P. D., and Drickamer, K. (1990) Primary structure of the mannose receptor contains multiple motifs resembling carbohydraterecognition domains. J. Biol. Chem. 265, 12156-12162. (26) Hoppe, C. A., and Lee, Y. C. (1983) The binding and processing of mannose-bovine serum albumin derivatives by rabbit alveolar macrophages. J . Biol. Chem.258,14193-14199. (27) Poznansky, M. J., and Juliano, R. L. (1984) Biological approaches to the controlled delivery of drugs: A critical review. Pharm. Rev. 36, 277-336. (28) Roche,A. C., Midoux, P., Pimpaneau, V., Negre, E., Mayer, R., and Monsigny, M. (1990) Endocytosis mediated by monocyte and macrophage membrane lectins-Application to antiviral drug targeting. Res. Virol. 141, 243-249.
