Macromolecules 1994,27, 875-877
875
Glycopeptide Synthesis by an a-Amino Acid N-Carboxyanhydride (NCA) Method: Ring Opening Polymerization of a Sugar-Substituted NCA
Scheme 1 0
Keigo Aoi, Kaname Tsutsumiuchi, and Masahiko Okada'
OAc 1
Department of Applied Biological Sciences, Faculty of Agricultural Sciences, Nagoya University, Chikusa- ku, Nagoya 464-01, Japan Received June 3, 1993 Revised Manuscript Received December 8, 1993
This paper describes a new general methodology to synthesize glycopeptides by the ring-opening polymerization of sugar-substituted a-amino acid N-carboxyanhydrides (NCAs). This synthetic approach to sugarpolypeptide conjugates differs from conventional methods, Le., polycondensation of amino acids having sugar residues1 and polymer reaction of polypeptide with carbohydrate derivatives.2 Saccharide-carryingNCAs have already been prepared by Rude et al.? whereas a detailed study on their polymerization behavior has never been reported until now. The present study makes it possible to synthesize both stereoregular high molecular weight glycopeptides and monodisperse living glycopeptides by the NCA method. Glycoproteins are present in extra- and intracellular fluids, connective tissues, and cell membranes, in virtually all forms of life: animals, plants, and microorganisms. The diverse biological functions of glycoproteins perform enzymatic catalysis, hormonal control, ion transport, cell adhesion, intercellular interaction and, most important, cell recognition in general.4 Thus, glycotechnology is one of the most attractive fields of life science. Wellcharacterized artificial glycoconjugates are indispensable for the progress of glycotechnology not only as model compounds having biosignals but also as biomedical materials. Biological characteristics of the carbohydrate moiety have been successfully applied to the cell recognition marker of synthetic glycopolymers. For example, N- (2-hydroxypropyl)methacrylamide-sugar-antitumor drug conjugates including biodegradable oligopeptide side chains have been developed as a targetable drug delivery systema5Cell-biologicalinvestigation has been undertaken by using neoglycoprotein, e.g., mannose-bearing poly@lysine).6 Polystyrene having pendant lactose residues was applied as the substratum for a culture of liver cells.7 We have already presented architectural control of sugarcontaining biofunctional polymers by living polymerization.8 However, synthetic examples of sugar-peptide conjugate polymers are just limited.2t57g In the present study, ring-opening polymerization of O-(tetra-O-aCetyl-P-D-glUCOpyan~yl)-L-Serine N-carboxyanhydride (1) was investigated. 0-Glycosylated serine moieties of naturally occurring glycoproteins function in various life phenomena. For example, the N-acetyl-Dglucosamine-linked serine part appears to be highly dynamic and responsive t o cellular stimuli.1° As a representative of saccharide-modified peptides, 0-glycosidically glucose-carrying poly@-serine) was synthesized in this work. A glucose derivative substituted serine NCA 11' was prepared according to a previous paper.3 As shown in Scheme 1, polymerization of 1 was carried out by using primary and tertiary amine initiators. Generally, polymerizations of a-amino acid NCAs proceed via two different mechanisms by the nature of initiators.12 Initiation with primary amines leads to a chain growth via 0024-9297/94/2227-0875$04.50/0
3
4
Table 1. Ringopening Polymerization of 1 with a Tertiary Amine Initiator. run
no. [lld[2alo 1
2 3 4
8.8 16 16 54
product polymer 3 solvent yield, % Mnb X l(r E fi,lMn* dioxane 52 0.48 12 1.5 dichloromethane 50 0.79 19 1.6 acetonitrile 82 1.0 24 1.8 acetonitrile 70 1.9 46 1.5
a Initiator, 2a. At 25 "C, for 3 days, under a nitrogen atmosphere. [llo, 0.10 mol/L. bMeasured by GPC in chloroform at 38 O C (polystyrene standard).
amino end groups. On the other hand, with tertiary amine initiators, propagation proceeds by the so-called "activated monomer mechanism". A first series of polymerization was conducted by using a tertiary amine initiator, Le., triethylamine (2a). The reaction conditions and results are summarized in Table 1. The reaction mixture was homogeneous after the polymerization in dichloromethane and in dioxane. White powdery polymers were obtained by reprecipitation in methanol from chloroform. Both the yield and the molecular weight of the produced polymer 3 were affected by the solvent. Acetonitrile gave the polymer with the highest yield and molecular weight. 3 having a molecular weight of 1.9 X lo4was obtained (run no. 4). Although the number-average molecular weight value was estimated by GPC, the actual molecular weight whould be somewhat higher than this value (vide infra). Broad molecular weight distributions by GPC analysis suggestthat the propagation proceeds via the anionic activated monomer mechanism. The structure of 3 was identified by 'H and 13CNMR and IR analysis.l3 414 was derived in 96% yield by the deacetylation of 3 with hydrazine monohydrate in methanol at 25 "C for 6 h.' Complete deprotection was confirmed by 'H and I3C NMR and IR measurement. Throughout the polymerization deprotection procedures, neither isomerization of the @-glycosidebond nor racemization of the polypeptide was observed. Cleavage of the main chain was negligible by the GPC analysis of 4. 4 showed a randomly coiled conformation in water by circular dichroism measurement. This NCA method with a tertiary amine initiator provides a new route to synthesize stereoregular sugar-polypeptide conjugates having relatively high molecular weight. In primary amine initiator systems, a monodisperse glycopeptide was synthesized. The results are listed in Table 2. In the polymerizations initiated by n-hexylamine (2b), especially in run nos. 5 and 6, the average degrees 0 1994 American Chemical Society
Macromolecules, Vol. 27, No. 3, 1994
876 Communications to the Editor
Table 2. Ring-Opening Polymerization of I with Primary Amine Initiators. product polymer 3 initiator [lld[2lo
run no. 5 6 7 8
2b 2b 2b 2c
21
20 17 23
DP
solvent dioxane dichloromethane acetonitrile dichloromethane
yield, % 81 92 78 87
0.82 0.74
GPCc 14 15 12
1.1
17
f i n bX 10-4
0.80
NMRd 19 20 17 25
VPOb 19 19 17 26
Mw/Mnc 1.i6 1.16 1.16
1.17
At 25 O C , for 6 days, under a nitrogen atmosphere. [llo, 0.10mol/L. Determined by VPO in chloroform at 35 "C. Measured by GPC in chloroform at 38 "C (polystyrene standard). Measured in chloroform-d at 25 O C .
of polymerization determined by 'H NMR and VPO showed good agreement with each other as well as with the feed molar ratios. Molecular weight distributions estimated by GPC were reasonably narrow by considering a Poisson distribution. These findings imply the polymerization of 1with 2b proceeds accordingto the nucleophilic addition mechanism, although NCA 1 has a bulky substituent of a protected glucose unit. In the polymerization with tert-butylamine (2c),production of a small amount of hydantoic acid was suggested by the slightly higher DP determined by 'H NMR and VPO and by the M,/M,, value (run no. 8). In run nos. 5-8, the apparent values estimated by GPC tend to be observed lower than those by NMR and VPO. The significant feature of the 2b initiator system is the living nature of the polymerization of a carbohydratebearing monomer. This is the first application of living polymerization of a carbohydrate-derivatized monomer to synthesize a glycopeptide having a sugar-peptide linkage. As reported in a previous paper, synthetic strategy using living polymerization should be an ideal approach to the precisely controlled structure of artifical glycoconjugates.8 Living polymerization of sugar-carrying NCAs can be utilized to design various types of glycoprotein models and synthetic glycopolymers such as block and graft copolymers. The living character in the primary amine initiator system was further demonstrated by the AB-type block copolymer synthesis with sequential monomer additions. Block copolymerization between 1 and alanine NCA (5) with initiator 2b was performed by using a "one-pot twostage feeding" technique. After completion of the firststage polymerization of 1,the second monomer 5 was added to the reaction mixture. The second-stage polymerization was initiated by the propagating amino end group of the A block of the first monomer to produce the B block (Scheme 2). The results are shown in Table 3. The molecular weight of the first-stage homopolymer 3, which was determined by lH NMR, was in good agreement with the calculated value from the feed ratio. The unit ratio of the block copolymer 6 also coincides with the feed molar ratio of the second monomer to the first monomer. The formation of block copolymer was further confirmed by GPC analysis. Both the block copolymer 6 and the correspondinghomopolymerof 1indicated unimodal peaks in their GPC profiles. Deacetylation of 6 was achieved in the same manner as described above. A block-type glycopeptide7 having a regulated structure can be regarded as a-helical oligo(alanine1 having a random-coiled glycopeptide moiety at the end.15 In this report, widely applicable synthetic utilities of the polymerization of sugar-substituted NCAs were proposed both for high molecular weight glycopeptides and for monodisperse glycopeptides having a well-defined structure. Conventional polycondensation and polymer reaction have some difficulties in the synthesis of these
Scheme 2
2b
-
5
1
*
OAc
.N
H
OR
6,R=Ac 7,R=H
Table 3. "One-Pot Two-Stage" Block Copolymerization of 1 and 5 product polymer 6 lsts e* 2ndstageb X 10-9 unit rat& n:md M,/Z&,e [Ild%lo [51d[2blo yield, % 10.1 0.0 91 4.1 9.7:O.O 1.10 10.1 9.8 91 4.8 9.7:9.4 1.06 b Initiator, 2b. In dichloromethane, under a nitrogen atmosphere. [ l ] ~0.10 , mol/L. b At 25 OC, for 6 days. Estimated by 'H NMR in chloroform-d at '25 OC. d See Scheme 2. e Measured by GPC in chloroform at 38 "C (polystyrene standard).
types of multiply glycosylated peptides. A variety of sugarbearing NCAs can be used in this NCA method. For example, the 0-acetylated 0-(@-glycopyranosy1)-L-serine derivatives Of D-gdaCtOSe,lactose, cellobiose,andN-acetylD-glucosamine have been prepared by Rode et a1.16 Recently, Fuller et a l . I 7 have reported the synthesis of urethane-protected amino acid NCAs and their use in stepwise peptide synthesis. Urethane-protected sugarcarrying amino acid NCAs may be highly effective reagents for glycopeptide synthesis. Further investigations on sugar-polypeptide conjugate synthesis using the NCA method and their application to biomedical carbohydratebased material are now in progress.
References and Notes (1) For example: Kunz, H. Angew. Chem., Znt. Ed. Engl. 1987,
26, 294. (2) For example: Stowell,C. P.; Lee, Y. C. Adu. Carbohydr. Chem. Biochem. 1980,37, 225. ( 3 ) Rade, E.;Westphal, 0.; Hurwitz, E.; Fuchs, S.; Sela, M. Immunochemistry 1966,3, 137. (4) (a) Bahl, 0. P. An Introduction to Glycoproteins. Glycoconjugates; Allen, H. J., Kisailus, E. C., Eds.; Marcel Dekker, Inc.: New York, 1992;Chapter 1. (b) Sharon, N. Complex Carbohydrates. Their Chemistry, Biosynthesis and Functions.; Addison-Wesley Publishing Co. Inc.: Reading, MA, 1975. (5) (a) Duncan, R.; Hume, I. C.; Yardley, H. J.; Flanagan, P. A.; Ulbrich, K.; Subr, V.; Strohalm, J. J.ControtledRelease 1991,
Macromolecules, Vol. 27, No. 3, 1994 16,121. (b) Duncan, R.; KopeEkovB-RejmanovB,P.; Strohalm,
J.; Hume, I.; Cable, H. C.; Pohl, J.; Lloyd, J. B.; KopeEek, J.
Br. J. Cancer 1987,55,165. Tomoda, H.; Kishimoto, Y.; Lee, Y. C. J. Biol. Chem. 1989, 264, 15445. (a) Kobayashi, K.; Sumitomo, H.; Kobayashi, A.; Akaike, T. J. Macromol. Sci. Chem. 1988, A25,655. (b) Kobayashi, A.; Akaike, T.; Kobayaehi, K.; Sumitomo, H. Makromol. Chem., Rapid Commun. 1986, 7,645. Aoi, K.; Suzuki, H.; Okada, M. Macromolecules1992,25,7073. (a) Kobayashi, K.; Zhou, S. H.; Sumitomo, H.; Okada, M.; Akaike,T.KobuMhiRonbunshu 1991,48,253. (b)Kuroyanagi, Y.; Kubota, T.; Miyata, T.; Seno, M. Znt. J. Biol. Macromol. 1986,8,52. (c)Kuroyanagi,Y.; Kobayashi,H.; Seno,M.; hhida, M.; Tominaga, N.; Akaike, T.; Sakamoto, M.; Ebert, G. Znt. J. Biol. Macromol. 1984, 6, 266. (d) Hatten, M. E. J. Cell. Biol. 1981, 89, 54. Haltiwanger,R. S.;Kelly, W. G.;Roquemore, E. P.; Blomberg, M. A,; Dong. L.-Y. D.; Kreppel, L.; Chou, T.-Y.; Hart,G. W. Biochem. SOC. Tram. 1992,20, 264. 0-(Tetra-O-acetyl-@-Dglucopyranosyl)-bserine N-carboxyanNMR (CDsCN): 8 20.7hydride (1). See ref 3. 67.8-MHz 20.9 (methyl carbons of acetyl groups), 59.4 ((2-4 of the NCA ring), 62.6 (CHzOAc),68.3 (methylene carbon of serine NCA), 69.2 (C-4 of the pyranose ring), 71.6 ((2-2of the pyranose ring), 72.6 (C-5 of the pyranose ring), 73.0 ((3-3 of the pyranose ring), 101.9 (C-1 of the pyranose ring), 152.9 (carbonyl carbon C-2
Communications to the Editor 877
of the NCA ring), 169.9, 170.3, 170.5, 170.9, 171.4 (carbonyl carbons of acetyl groups and carbonyl carbon C-5 of the NCA ring). (12) For example: Imanishi,Y. Ring-OpeningPolymerization;Ivin, K., Saeguaa, T., E&.; Elsevier Applied Science Publishers: New York, 1985; Vol. 2, Chapter 8. (13) P o l y [ O - ( ~ t r ~ O - a c e t y l - ~ ~ g l u c o p y r a n o e y l(3). ) - ~67.8 ~~el MHz 1% NMR (CDCh): 8 20.5 (methyl carbons of acetyl groups),53.0 (a-carbonof polyb-serine)), 62.2 (CHzOAc),68.8 ((2-4 of the pyranose ring), 71.5 (C-5 and methylene carbon of poly(L-serine), 72.0 (C-2),73.3 (C-3), 102.3 (C-l), 168.9-170.9 (carbonyl carbons). IR (KBr disk): 2960 (VGH), 1760 (v(ester)), 1660 (u-(amide)), 1510 ( ~ N - H ) crn-'. (14) Poly[O-(@-~lucop~anosyl)-b~~el(4). 67.8-MHz 'BC NMR (DzO): 854.5 (a-carbon of poly(L-serine)),61.8 (CHzOH),69.7 (methylenecarbon of poly(L-serine)),70.6 ((2-4of the pyranose ring), 74.0 (C-51, 76.5 (C-21, 77.0 (C-3), 103.5 (C-11, 172.0 (carbonyl carbons). IR (KBr disk): 3360 (UGH), 2910 (VGH), 1640 (v-), 1610 ( ~ N - H ) cm-I. (15) Otoda, K.; Kmura, S.; Imaniehi, Y. Bull. Chem. SOC. Jpn. 1990, 63, 489. (16) (a) Riide, E.; Meyer-Delius, M. Carbohydr. Res. 1968,8,219. (b) Riide, E.; Meyer-Delius, M.; Gundelach, M.-L. Eur. J. Zmmunol. 1971,1, 113. (17) Fuller, W. D.; Cohen, M. P.; Shabankareh, M.; Blair, R. K. J. Am. Chem. SOC. 1990,112,7414.
