Solid phase synthesis and conformation of sequential glycosylated polytripeptide sequences related to antifreeze glycoproteins

Solid phase synthesis and conformation of sequential glycosylated polytripeptide sequences related to antifreeze glycoproteins F. Filira, L. Biondi, B. Scolaro, M. T. Foffani, S. Mammi, E. Peggion and R. Rocchi Biopolymer Research Center, Department of Organic Chemistry, University of Padua, Ha Marzolo I, 35131 Padua, Italy

(Received 8 April 1989; revised 2 September 1989) Sequential glycopeptides [Thr(fl-o-galactose)-Ala-Ala] n, with n ranging from 2 to 7, as models of natural antifreeze glycoproteins were synthesized by the continuous flow, solid phase procedure. The conformational properties of these materials in solution were investigated by c.d. and I H-n.m.r. spectroscopy. In aqueous solution the c.d. pattern is practically independent of chain length and is very similar to that of natural antifreeze #lycoproteins. The results are interpreted in terms of random coil structure. The absence of ordered structures is further confirmed by n.m.r, data. A small amount of ordered conformation can be induced either by increasing the temperature of the aqueous solution or by addition of TFE. The c.d. pattern of all glycopeptides in water at temperatures higher than 50°C are compatible with the presence of a small amount of or-helix or 31o helix. Since the glyco-hexapeptide is too short to form an ~-helix, the hypothesis is made that in the glycopeptides in water at high temperature a small amount of 31o helix is formed. The same is observed for the 21-residue #lycopeptide in presence of 85% (v/v) TFE. In this medium, the c.d. data on the glyco-hexapeptide are more compatible with the presence of a small amount of t-structure. Keywords: Glycopeptides;conformation;random coils; n.m.r, spectroscopy;c.d. spectroscopy

Introduction The primary structure of antifreeze glycoproteins (AFGP), isolated from the antarctic polar fish B. saida, consists of a repeating glycotripeptide sequence Thr-Ala-Ala, with the disaccharide fl-Gal(1-3)-ct-GalNAc O-glycosydically linked to the threonyl side-chains L2. It has been shown that the antifreezing properties are not due to a colligative effect but to a kinetic effect on the growth of the ice crystals 1. The glyco-tripeptide Ala-Thr[fl-Gal(1-3)-~-Gal]-Ala as a model of the simple repeating unit of antifreeze glycoproteins has been recently synthesized in solution 3. Undoubtedly, the synthesis of sequential polyglycotripeptide analogues of A F G P and the study of their conformational properties would provide some insight on the relationship between specific structural features and antifreezing effect~'2. In the synthesis of glycosylated peptides, a number of procedures have been reported 4. Strategies involving direct glycosylation of the desired peptide backbone 3,s a or stepwise elongation of the peptide chain in solution 9-17 or in solid phase 1s'19, utilizing suitably protected glycosylated amino acid derivatives have been described. In general, better results have been obtained utilizing a glycosylated amino acid derivative for the stepwise construction of the peptide Presentedin part at the 20th European PeptideSymposium,Tubingen, FRG, September 1988 (Scolaro, B., Biondi,L., Filira, F., Peggion,E. and Rocchi, R., Peptides 1988, 322-324 (Eds. G. Jung and E. Bayer) W. de Gruyter, Berlin, 1989). 0141-8130/90/0100414)9 © 1990 Butterworth & Co. (Publishers) Ltd

chain, but, in some cases 3'5'l° glycosylation of a preformed peptide sequence was also satisfactorily achieved. For recent advances on the synthesis of glycopeptides see Kunz et al. 2°'21, Paulsen et al. 22, and references cited therein. In the present paper we report the synthesis and conformational analysis, by c.d. and high resolution 1H-n.m.r., of sequential glycopeptides [Thr(fl-Gal)-AlaAla],, with n ranging from 2 to 7. The sequential glycopeptides can be considered A F G P analogues in which the a-linked disaccharides have been replaced by t-linked galactose residues. The fl linkage mimics the glycosidic bond connecting the terminal non-reducing galactose residue in the native structure. The potential of the continuous flow solid phase procedure to obtain peptide sequences containing several glycosylated amino acid residues is explored.

Abbreviations. The amino acid residues are of L-configuration.Standard abbreviations for amino acid derivatives and peptides are used according to the IUPAC-IUB Commissionof BiochemicalNomenclature, publishedin Eur. J. Biochem. 1984,138,9-37. Other abbreviations: Fmoc,9-fluorenylmethyloxycarbonyl;Z, benzyloxycarbonyl;Pfp, pentafluorophenyl;Su, succinimidyl;Bzl,benzyl;Gal, D-galactopyranosyl; GalNAc, 2-amino, 2-deoxy-D-galactopyranosyl;Gal(OAc)4, 2,3,4,6tetra-O-acetyl-D-galactopyranosyl; HO-Bt, 1-hydroxybenzotriazole; SDS, sodium dodecyisulphate;DMF, dimethylformamide;TFE, trifluoroethanol;TOCSY, total correlationspectroscopy;COSY, homonuclear correlation spectroscopy;ROESY, rotating frame Overhauser effectspectroscopy.

Int. J. Biol. Macromol., 1990, Vol 12, February

41

Glycosylated polytripeptide sequences: F. Filira et al.

Experimental Materials and methods Sephadex G 15 was purchased from Pharmacia. 4Hydroxymethylphenoxyacetyl-norleucylderivatized polydimethylacrylamide-kieselguhr supported resin (Pepsyn KA) and Fmoc-Ala-OPfp were Cambridge Research Biochemicals Ltd products. Boron trifluoride ethyl etherate, 4-dimethylaminopyridine,TFE spectroquality grade, Fmoc-OSu, and 1,2,3,4,6-penta-O-acetyl-fl-D-galactopyranose were obtained from Fluka. D20 (99.96% deuterated) was a Stohler Isotopes Inc. product. All chemicals were reagent grade or the best commercially available grade. Fmoc-Thr[fl-Gal(OAc)4]-OH was prepared from Z-Thr[fl-Gal(OAc)4]-OBzl and converted in the usual way23 to the corresponding symmetrical anhydride. The continuous flow variant of the 'Fmocpolyamide' method 23, with a manually controlled Pepsynthesizer apparatus (Cambridge Research Biochemicals Ltd) was used for the solid phase synthesis. The continuous spectrophotometric monitoring at 312nm of both the amino acid addition and deprotection steps was carried out on a Varian 635 spectrophotometer by inserting a u.v. flow cell (pathlength 0.1 cm) into the recirculation stream. Esterification of the first amino acid (5-fold molar excess) to the hydroxy resin was carried out in the presence of 4-dimethylaminopyridine catalyst. Residual active sites were terminated with acetic anhydride. Fmoc-amino acid derivatives used as the acylating agents were then added sequentially in 4-fold molar excess. Intermediate Fmoc-peptide resins were deprotected with 20% piperidine-DMF solution. Samples were removed at each acylation step for qualitative ninhydrin 24 and trinitrobenzenesulphonic acid tests 25. Resin samples for amino acid analyses were taken after removal of the Fmoc-protecting group. Amino acid analyses were carried out with a Carlo Erba model 3A 28 amino acid analyser equipped with a Perkin-Elmer Sigma 10 Chromatography Data Station following hydrolysis of peptide samples for 22 h at 110°C in sealed evacuated vials in constant boiling hydrochloric acid. In the continuous flow solid phase synthesis, DMF was treated with phosphorous pentoxide and freshly distilled under reduced pressure in the presence of ninhydrin. Piperidine was distilled from potassium hydroxide pellets and dichloromethane was distilled from phosphorous pentoxide less than 48 h before use. All other solvents were freshly distilled. Evaporations were carried out under reduced pressure at 40~5°C using a rotary evaporator. Yields are based on the weight of lyophilized product. C.d. measurements were performed using a Jasco model J-600 automatic recording spectropolarimeter equipped with a personal computer. The signal-to-noise ratio was improved accumulating from 4 to 32 scans. The spectropolarimeter was equipped with a thermostatically controlled cell assembly and with a sample alternator, which permitted subtraction of the baseline immediately after each scan 26. The c.d. spectra reported in this paper are in ellipticity units [0], per mole of peptide linkage. Aqueous solutions of the glycosylated peptides were prepared directly by dissolving a weighed amount of material in 10ram sodium phosphate buffer, pH6.96. Solutions in T F E - H 2 0 mixtures were prepared by addition of TFE to the aqueous solution, All glycopeptides were insoluble at TFE content higher than 85%. The peptide concentration was always determined by weight

42

Int. J. Biol. Macromol., 1990, Vol 12, February

and peptide content and by quantitative amino acid analysis. The agreement between concentration values obtained by the two methods was within 3%. N.m.r. spectra were obtained on a Bruker AM-400 instrument equipped with an Aspect 3000 computer and operating at 400MHz for protons. The solutions of glyco-hexapeptide were 5.3 mra in 90% H20, pH 6.9 (10 mra sodium phosphate), and 5.0 mM in DzO, pD 6.8 (uncorrected, 10mM sodium phosphate). The COSY experiment in H20 was recorded in the magnitude mode 2v, collecting 256 t~ increments of 72 scans each with pre-irradiation of the water resonance. In D20, the phase sensitive version was used zS, and 512 t 1 increments of 32 scans each were collected. The TOCSY experiment in H20 was performed using the sequence described by Bax and Davis 29 with preirradiation of the water resonance. The MLEV-17 spin locking sequence was cycled 20 times and the 'trim' pulses were 3 ms each for a total mixing time of 74.6 ms. The spin locking power was 7B2/2/~= 4.8 kHz; 320 t~ increments of 128 scans each were collected. The ROESY experiments 3° were conducted using a continuous spin locking pulse, 500 ms long. In H/O, 216 t~ increments of 80 scans each were collected and the strength of the spin locking field was yBJ2n = 10.6 kHz. In D20, 256 t 1, increments of 80 scans each were collected. A spin lock power of 7Bz/2n=4.8kHz was used. In all cases, a final matrix of 1K by 1K real points was obtained by zero-filling in the t~, dimension only. Temperature dependence studies were carried out between 25 and 49°C, using the 133T pulse sequence to eliminate the water resonance3~.. In this temperature range the NH chemical shifts are linearly dependent upon temperature. At temperatures higher than 50°C the NH resonances became very broad due to fast exchange with the solvent.

Fmoc-Thr[fl-Gal(OAc)4]-OH. 1,2,3,4,6-Penta-Oacetyl-fl-o-galactopyranose (38.64g, 99mmol) and ZThr-OBzl 32 (17g, 49.5mmol) were dissolved in anhydrous dichloromethane (50 ml). The solution was cooled to 0°C and ice-cold boron trifluoride ethyl etherate (6.09 ml, 49.5 mmol) was added through a refrigerated separatory funnel33. Moisture had to be avoided during the reaction. After 4h stirring at room temperature the reaction mixture was poured into ice water (500 ml), the aqueous phase was separated, re-extracted with dichloromethane (2 x 100 ml) and the combined extracts were washed with ice-cold saturated sodium hydrogen carbonate aqueous solution (250 ml) and water, dried over sodium sulphate, and evaporated to dryness. The syrupy residue was dissolved in glacial acetic acid (140 ml) and hydrogenated in the presence of palladium catalyst for 20 h. The catalyst was then removed by filtration and the mixture diluted with water and extracted with ethyl acetate (4 x 150ml). The aqueous phase was evaporated to dryness and the residue dried in vacuo at 30°C in the presence of phosphorous pentoxide and potassium hydroxide pellets. The crude H-Thr[fl-Gal(OAc)4]-OH [17.8 g, 39.6 mmol, 80%, some minor contaminants by t.l.c, in butan1-ol/acetic acid/water (3:1:1 by vol) and light petroleum/ethyl acetate (l:lv/v)] was dissolved in water cooled to 0°C (250ml) and 0.1N sodium hydrogen carbonate was added to adjust the pH to 7.5-8.0. An

Glycosylated polytripeptide sequences: F. Filira et al. 0 II Fmoc-Ata-OPfp + HO-CHa-~O-CHa-C-NIe-NH-CH2-CHz-NH-CO~)

i calatyst 4-d~methytaminopyridine Fmoc-A la-O-CHa-~

0 0 - CHa- d_ Nle- NH- CHa-CHa-NH - C0-(~)

20 % piperidine/OHF 0 H- Ala- O-CH2-~ O-CHz-~- Nta-NH- CHa-CH2-NH-CO-(~ i

~-Gat(0A0, I

cycles of Fmoc-amino acid t Fmoc-Ata-OPfp/or rmoc- Thr ['fiat (OAc)4]-symmetricat anhydride addition and deprotection (20 % piperidine/OMr) o

~

H- ~T hr-AIa- Ala)~ O - C H 1 ~

H

O- CHf C -NIe-NH- CH I-CH I- NH - C 0--(~)

95 ",1, TFA aq. H-(Thr-Ata-Ata)~OH

~ aeacety(ation /~- Gal I

H-(T hr- Ata-Ata);OH

Figure 1 Assembly of sequential glycosyl~tedpolytripeptide sequences by continuous flow solid phase procedure. P, kieselguhr-supported polydimethylacrylamideresin

ice-cold solution of Fmoc-OSu (13.4g, 39.6mmol) in acetone (400 ml) was added, the reaction mixture stirred overnight at 4°C, the organic solvent removed and the aqueous solution first extracted with ethyl acetate and then acidified to pH 2 with 0.1 N KHSO 4. The resulting oily precipitate was extracted with ethyl acetate (2 x 150ml) and the extracts pooled, washed with 0.1 N KHSO 4 and water, dried over sodium sulphate and evaporated to dryness (19.7 g, 72%); m.p. 80082°C dec.; [~]D-4.5 ° (c 1.12, methanol). Anal. Calcd. for C33H37N1014.H20 (689.65): C, 57.47; H, 5.70; N, 2.03. Found: C, 57.63; H, 5.63; N, 1.88.

Synthesis of O-protected sequential glycosylated polytripeptides Assembly of the different sequential glycosylated polytripeptide sequences by the continuous flow solid phase procedure was achieved as depicted in Figure 1. The Pepsyn KA resin (2 g, 0.2 mEq) was swollen in DMF for 2h, packed in the reaction column (1 x 10cm) of the Pepsynthesizer and esterified with Fmoc-Ala-OPfp (0.496g, 1.0mmol) dissolved in DMF (2ml) in the presence of 4-dimethylaminopyridine catalyst (24.5 mg, 0.2mmol). After 30min recirculation the column was washed with DMF for 10min and residual hydroxy groups on the resin were blocked by circulating for 30 min acetic anhydride (0.038ml, 0.4mmol) in DMF (2ml) containing 4-dimethylaminopyridine (24.5 mg, 0.2 mmol) followed by DMF for 20rain. Amino acid analysis of a sample of resin indicated that, in the various syntheses, the incorporation of alanine to the resin was in the range from 63 to 77% with respect to the norleucine as the internal standard. The Fmoc group was then cleaved from the resin with 20% piperidine-DMF solution (10min) and the resin washed with DMF for 20 min. The synthesis was then continued by using Fmoc-Ala-OPfp (0.4g, 0.8 mmol) and freshly prepared Fmoc-Thr[fl-Gal(OAc)4]

symmetrical anhydride (1.06 g, 0.8 mmol) as the acylating agents. The coupling rate of both reagents decreased with the increase of the peptide length but negative ninhydrine 2~ and trinitrobenzenesulphonic acid 2s colour tests for residual amine were obtained within the first 30°60 min of the acylation reaction period in the case of Fmoc-Ala-OPfp and 60090min in the case of FmocThr[fl-Gal(OAc)4] symmetrical anhydride. Recirculation was continued for 10min and the resin was then washed with DMF for 10min. Removal of the Fmoc protecting group from the intermediate peptide-resin was achieved within 10-20 min by recirculating 20% piperidine-DMF solution. The resin was then washed with DMF (10min) and samples for amino acid analysis were removed. Ratios between alanine and threonine residues in the different sequential glycosylated polytripeptides calculated from the amino acid analyses are shown in Table I. After completion of the synthesis of the desired sequences, the Fmoc protecting group was removed and the peptide resin was successively washed on a sintered glass funnel with DMF, 2-methyl-butan-2-ol, acetic acid, 2-methyl-butan-2-ol, and ethyl ether and dried in vacuo. For cleaving from the resin the sequential glycosylated polytripeptides {Thr[fl-Gal(OAc)4]-Ala-Ala}, containing, respectively, 6, 9, 12, and 21 amino acid residues (n = 2, 3, 4, and 7) a sample of the final peptide-resin (1 g) was treated with aqueous 95% trifluoroacetic acid (100ml). After 90min stirring, the resin was filtered, washed with trifluoroacetic acid (3 x 5 ml) and dichloromethane (3 x 5 ml), and dried. Amino acid analysis of a sample of resin after cleavage indicated about 2% residual peptide. The filtrate and washings were combined and evaporated to dryness. The residue was redissolved in water (20 ml), extracted with ethyl acetate (2 x 20 ml) and the aqueous phase was lyophilized to constant weight; yields: n = 2 (glycohexapeptide), 48mg; n = 3 (glycononapeptide), 70mg; n = 4 (glycododecapeptide), 78 mg. In the case of n=7, the 21-residue glycopeptide was insoluble in water and was thus obtained from the aqueous phase by filtration, washed with ethyl acetate, and dried (110 mg). Samples of peptide-resin (25-30 mg) were withdrawn during the synthesis after incorporation of each threonine residue and removal of the Fmoc protecting group from the growing peptide chain. The peptides were then cleaved from the resin in the conditions described above, and analysed by analytical h.p.l.c. Fractions containing the main peak were combined and evaporated to dryness. Results of amino acid analyses are shown in Table 2. Table 1 Alanine/threonine ratios in the crude H-{Thr[fl-

Gal(OAc)4]-Ala-Ala},-Pepsyn KA n r

1 1.96

2 1.94

3 2.00

4 1.92

5 2.00

6 2.00

7 1.96

Table 2 Amino acid analysis for synthetic sequential glycosylated polytripeptides after cleavage from the resin and h.p.l.c, purification n Ala Thr

1 1.98 1.02

2 3.91 2.09

3 6.09 2.93

4 8.03 3.97

5 9.91 5.09

6 7 11.92 13.75 6.08 7.25

Int. J. Biol. Macromol., 1990, Vol 12, February

43

Glycosylated polytripeptide sequences: F. Filira et al. /

!

n=2 20

//

/i n=3

n=4

n=7

80

70 E

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60

50

12

40 .~

I11 Q: 0

m 0,8

30

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04

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i

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Time

lS

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15

i

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Cmin)

Figure 2 Analytical h.p.l.c, elution profile of crude sequential glycosylated polytripeptides after deacetylation (procedure b). Column: Aquapore octyl RP-300 (4.6 x 220 mm, Brownlee Labs Inc., Santa Clara, CA, USA). Eluent A, aqueous 0.1% trifluoroacetic acid; B, 90% acetonitrile in A. Flow rate 1.5 mi/min; n = 2 (load 120 pg, elution isocratic 0.3% B over 5 min, linear gradient 0.3-5% B over 10 min, 5-90% B over 20 min); n ---3 (load 180/tg, elution isocratic 0,1% B over 5 min, linear gradient 0.1-15% B over 15 rain, 15-90% B over 15 min; for n = 4 (load 270 #g) and n = 7 (load 450/zg) elution was carried out as indicated for n = 3

Removal of the O-acetyl protecting groups Procedure a 34. The glyco-nonapeptide {Thr[flGal(OAc)4]-Ala-Ala}3 (40rag, 0.023mmol) was suspended in methanol (2.5ml) and magnesium oxide (100 mg) was added. After 4 h stirring the suspension was centrifuged, the residue taken up with methanol, and the centrifugation was repeated. The supernatants were combined and evaporated to dryness. The residue was dissolved in water, filtered to remove some insoluble material, and lyophilized (23.6mg, 83%). The same procedure was used for the deacetylation of the glycohexapeptide (n = 2, yield 81%) and the glycododecapeptide (n = 4, yield 79%). H.p.l.c. analysis of crude products indicated that deacetylation was partially satisfactory only in the case of the glyco-nonapeptide, but some major by-products were present in the crude deacetylated glyco-hexapeptide and glyco-dodeeapeptide. Procedure b 35. A sample (30mg, 0.026mmol) of the glyco-hexapeptide {Thr[fl-Gal(OAc)4]-Ala-Ala}2 was suspended in methanol (0.5 ml), hydrazine hydrate (50 pl) was added and the reaction mixture was kept for 3 h at room temperature. A 5-fold ratio of hydrazine to acetyl group was used. Acetylacetone (125#1, 1.2mmol) was then added and after 20 min the solution was diluted with water (10ml), and extracted with ether (6 x 10ml) and chloroform (6x 10ml). The aqueous phase was lyophilized and the residue was dissolved in 5% aqueous acetic acid (1 ml) and applied to a Sephadex G-15 column (1.9x95cm; eluent 5% aqueous acetic acid, flow rate 16.2 ml/h, fraction volume 2 ml). The product-containing fractions were combined and lyophilized yielding 17 mg (79%) of deacetylated glycopeptide. The same procedure was used for deacetylation of glyco-nonapeptide (n = 3, 45mg, yield 25mg, 78%), glyco-dodecapeptide (n=4,

44

Int. J. Biol. Macromol., 1990, Vol 12, February

25 mg, yield 11 mg, 61%), and 21-residue glycopeptide (n=7, 30mg, yield 19mg, 90%). The h.p.l.c, elution profiles of the isolated products are shown in Figure 2. N.m.r. experiments showed the residual presence of some acetyl groups in the debloeked peptides: 1.5% (n=2); 0.1% (n=3); 2.5% (n=4); 1.5% (n=7).

Results and discussion

Synthesis Glycosylation of the threonine residue was carried out by reacting the per-O-acetylated sugar with Z-Thr-OBz131 and boron-trifluoride ethyl etherate in dichloromethane 33. In this modified form of the neighbouring-group-assisted procedure 36 (by which fl-glycosidic linkages in the D-gluco and D-galacto series are synthesized via formation of a stabilized cyclic acyloxonium intermediate) the 1-O-acetate is the leaving group 3v. Catalytic hydrogenation of the resulting O-glycosylated amino acid derivative removed simultaneously both amino and carboxyl protecting groups. Reaction of the crude 3-0-(2,3,4,6tetra-O-acetyl-fl-D-galactopyranosyl)-threonine with 9-fluorenylmethyl succinimidyl carbonate yielded the desired Fmoc-Thr[fl-Gal(OAc)4]-OH which was isolated and characterized. The presence of a number of chemically sensitive O-glycosidic linkages which must be retained during the synthesis and the removal of the final glycopeptides from the resin suggested the choice of the continuous flow variant of the Fmoc-polyamide method 23 for solid phase synthesis. Fmoc chemistry achieves amine deprotection and final peptide-resin cleavage under mild conditions and thus seemed suitable for glycopeptide synthesis. The O-glycosylated threonine residue was incorporated into the polypeptide chain using

Glycosylated polytripeptide sequences: F. Filira et al.

-8 -12,n

-16 -

O at

20-

- 24 -

28-

-3,7

-36

-

4 190

0 200

_ 210

220

~ 230

240

250

~. (,,,,,)

Figure 3 Far u.v.c.d, spectra of [Thr(fl-Gal)Ala-Ala]2 in aqueous buffer at pH 6.96 at various temperatures: 1, -2.5°C; 2, 23°C; 3, 52°C; 4, 62°C. The glycopeptide concentration was 9.64 x 10-SM and the optical pathlength was 0.1 cm Fmoc-Thr[fl-D-Gal(OAc)4]-symmetrical anhydride as the acylating agent. Fmoc-Ala-OPfp was used to introduce alanine residues. In order to avoid possible problems in the attachment to the resin of a sterically hindered O-glycosylated threonine residue, alanine was selected as the first and the second amino acid. The resulting polytripeptide sequences {Thr[fl-D-Gal(OAc)4]-Ala-Ala},, with n ranging from 2 to 7 were then amino deprotected, removed from the resin, deacetylated and characterized by amino acid analysis and h.p.l.c. C.d. results in aqueous solution The c.d. spectra in the far-u.v, absorption region of the glyco-hexapeptide and of the 21-residue glycopeptide in aqueous buffer at neutral pH and at various temperatures are reported in Figures 3 and 4. Identical results were contained with the glyco-nonapeptide and glycododecapeptide (data not shown). At temperatures near 0°C, the c.d. patterns of all fragments are characterized by a strong, negative band at 193nm and by a weaker positive one at 217 nm. The intensity of the negative band is almost independent of chain length, ranging from - 3 5 500 ellipticity units (per peptide linkage) in the hexamer to - 38 000 units in the 21-residue peptide, while the intensity of the positive band remains constant. These c.d. characteristics are very similar to those of poly(Llysine) in the random coil conformation and suggest that we are dealing with random structures. When the temperature is increased, a spectral change is observed with the same trend for all glycopeptides of the series. The high energy negative band decreases in intensity and slightly shifts to the red; the positive band at 217nm disappears and a weak negative shoulder is formed around 220 nm. These spectral variations are consistent with the formation of a small amount of ordered structure at high temperatures. For all fragments, the spectra recorded at different temperatures match the same, well defined isodichroic point at about 204 nm, indicating that

only two conformations are present in equilibrium. The high temperature c.d. spectrum is almost identical for all fragments thus implying that they tend to adopt the same ordered conformation. For all glycopeptides the temperature-induced conformational change is completely reversible. In no case have we observed a concentration-dependence of the chiroptical properties in the concentration range from 10 -4 to 10 -6 molar residue. The low temperature c.d. pattern is quite similar to that reported by Bush et a l . 3 8 ' 3 9 for antifreeze glycoproteins in water in spite of the different glycosidic side-chains. Their results also show that the contribution of the sugar moiety to the optical activity is a minor one. On the basis of these chiroptical properties they suggested the presence of a left-handed 3-fold helix of the collagen type as the most likely conformation of the peptide backbone. This hypothesis is based on the assumption that the positive band at 218 nm is diagnostic for the presence of a 3-fold left-handed polyproline II type helical structure. This matter has been the subject of long and controversial discussions and the whole problem of the chiroptical properties of random coiled polypeptides has been exhaustively discussed several years ago by Woody 4° in an authoritative review. From the available literature results, the proposition that the two band spectrum is diagnostic of conformations containing significant amounts of 3-fold helical regions has never been proved. Moreover, our experimental results show that the hexamer and the 21-residue glycopeptide exhibit almost identical c.d. patterns, implying that they have the same conformation, and the possibility of a 3-fold helix of polyproline II type at the level of the hexamer seems to be rather unlikely. The most recent n.m.r, results of Narasinga-Rao and Bush 41 do not provide convincing evidence that a left-handed, 3-fold helical structure exists for antifreeze glycopeptides in aqueous solution. They concluded that the strong NOE between the a-protons

0

-8-

-12-

-16' o

,~

-20-24-28-

-36-

-40 190

200

210

220

230

240

250

,I. (,,,,,)

Figure 4 Far u.v.c.d, spectra of [Thr(fl-Gal)Ala-Ala]7 in aqueous buffer at pH 6.96 at various temperatures: 1, -2.2°C; 2, 23°C; 3, 52°C; 4, 62°C. The peptide concentration was 1.97 x 10 -5 molar and the optical pathlength was 0.1 cm

Int. J. Biol. Macromol., 1990, Vol 12, February

45

Glycosylated polytripeptide sequences: F. Filira et al. !

1 3 C n.m.r, relaxation measurements on antifreeze glycoproteins, which indicate the presence of an 'extended random conformation '42 or of 'a flexible random coil '43.

1 ..

.~.f2

0 4 8 12 'o

C.d. results in TFE-water mixtures

43 6,5

6

16 -

x ac

24 28

1

32 36 40

i

190

i

i

i

~

i

200

210

220

230

240

250

4(-,-)

Figure 5 Far u.v.c.d, spectra of [Thr(fl-Gal)Ala-Ala]2 in HEO-TFE mixtures at 25°C. The water content (v/v) in the solvent was the following: 1,100%; 2, 86%; 3, 74%; 4, 47.7%; 5, 24.4%; 6, 15%. The peptide concentration was from 5 × 10- 5 to 6 x 10-5 ~t and the optical pathlength was 0.1 cm

4

,

T

1

0 4

4

3

8 12 o x

The conformational properties of the glycopeptide series were investigated in water-TFE solvent mixtures of various composition. The results of solvent titration experiments are reported in Figures 5-8. The effect of T F E on the c.d. properties of the glycopeptides depends on chain length. In the glycohexapeptide the intensity of the high energy negative band decreases in presence of T F E but its position does not appreciably shift to the red. The isodichroic point is located at 208-209 nm, and the intensity of the negative shoulder is - 3 0 0 0 molar ellipticity units in 85% TFE. When the chain length increases from the hexamer to the 21-residue peptide, the red-shift of the negative band upon increasing the T F E concentration becomes more and more evident, and the isodichroic point progressively shifts to the blue, being at 204 nm in the longer fragment. Comparison with the results reported in Figures 3 and 4 indicates that temperature or addition of T F E have the same effect on the c.d. properties of the 21-residue glycopeptide in water. The isodichroic point is located at the same wavelength, 204nm, in both cases. Actually, it is possible to find a solvent composition (27% TFE) at which the c.d. spectrum is the same as that observed in water at 52°C. These observations lead to the conclusion that there is a change of conformational preference with chain elongation and that the longer glycopeptides tend to assume the same ordered conformation either in water at high temperature or in 85% T~.E. In 85% TFE, no temperature effect was observed, and the c.d. properties are independent ' o f the peptide concentration in the range from 10 -4 to 10 -6 molar residue. 4

16

2

0

20" 4543

24 6

8

28 12-

32

6

o

36

-16-

204O

i

t

L

i

i

i

190

200

210

220

230

240

k(-,-)

250

Figure 6 Far u.v.c.d, spectra of [-Thr(fl-Gal)Ala-Ala]3 in H20-TFE mixtures at 25°C. The water content (v/v) in the solvent was the following: 1,100%; 2, 86%; 3, 74%; 4, 47.7 %; 5, 24.4%; 6, 15%. The peptide concentration was from 4.8 × 10- 5 to 6 x 1 0 - S M and the optical pathlength was 0.1 cm and the amide protons of the following residue, and the high-temperature coefficients of the N H resonances are all indicative of an extended conformation, not containing a-helix, turns, or bends. From all n.m.r, results we are unable to deduce that convincing experimental evidence exists for the postulated structure. Our interpretation of the c.d. data is consistent with the results of quasi-elastic light scattering experiments and of

46

Int. J. Biol. Macromol., 1990, Vol 12, February

24-

28t

32

36 40

I

190

t

I

I

I

I

200

210

220

230

240

250

4(-,")

Figure 7 Far u.v.c.d, spectra of I-Thr(fl-Gal)Ala-Ala]4 in H20-TFE mixtures at 25°C. The water content (v/v) in the solvent was the following: 1,100%; 2, 86%; 3, 74%; 4, 47.7 %; 5, 24.4%; 6, 15%. The peptide concentration was from 3.7 x 10- 5 to 4.7 × 10-5M and the optical pathlength was 0.1 cm

Glycosylated polytripeptide sequences: F. Filira et al. I

1216-

x

24~

28t

32

36 40 190

200

210

220

230

240

250

;.(.m)

Figure 8 Far u.v.c.d, spectra of [Thr(fl-Gal)Ala-Ala]7 in H~O-TFE mixtures at 25°C, The water content (v/v) in the solvent was the following: 1,100%; 2, 89%; 3, 73.5%; 4, 49%; 5, 24.4% ; 6, 15% . The peptide concentration was from 1.9 x 10-~M and the optical pathlength was 0.1 cm

¢

"rz ZZ

Z Z

Z

'~ ZZ

Z Z

¢W

•

•

(I-

1[

Ala z is likely to be broadened by fast exchange with the water protons, as found in other peptides ~ . The spin systems of the alanines were identified by the TOCSY (Fi#ure 9) and COSY (not shown) experiments. Sequential assignment was accomplished with the ROESY spectrum in H 2 0 (Fi#ure 9). Although this experiment was performed with a continuous spin locking pulse and therefore both scalar and dipolar couplings give rise to cross peaks, the comparison with the TOCSY spectrum (in which the MLEV-17 spin locking train allows only scalar cross peaks) gives the desired information. Sequential C~H,)-NH(~+~ cross peaks were found from Ala 2 through Ala 6. In addition, a cross peak was found between Thr 4 Call,) and Ala 5 N H , + t r These results allow the unambiguous assignment of all the peptide backbone resonances. Phase-sensitive COSY in D 2 0 (not shown) was used to assign the resonances of the sugar protons. Not all the peaks could be assigned, because of extensive overlapping of all the resonances. The two anomeric protons are readily recognized around 4.45 ppm. Both give a cross peak in the COSY spectrum to the respective H2 protons, which in turn show coupling to the H3 protons, and these to the H4 protons. No cross peak is present between H4 and H5 protons, because of the very low coupling constants, due to the roughly orthogonal arrangement of these protons 45. Cross peaks are present between H5 and H6 protons. With a ROESY in D 2 0 (500 ms mixing) the sequential assignment of the H1 protons was possible (Figure 10). The almost perfect overlap of the two H2 protons did not permit further assignment.

.I., j

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.

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.

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Figure 9 Aliphatic (F1 axis)-NH (F2 axis) region of the two-dimensional TOCSY (left) and ROESY (right) spectra of the glyco-hexapeptide in 90% H20, with assignment of the resonances. Only positive and only negative peaks are plotted for the TOCSY, and for the ROESY, respectively

N.m.r. results The results of n.m,r, experiments on the glycohexapeptide in aqueous solution at 25°C are reported in Figures 9 and 10. The two threonines form unique spin systems: Thr 1 is an AMXa, while Thr 4 is an AMQX a. Their cross peak patterns in the TOCSY spectrum are therefore readily distinguished from one another and from those of the alanines. In the amide region, only four resonances are present (Figure 9). The amide proton of

I~

1

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.

I

|

i

i,

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Figure 10 Two-dimensional ROESY spectrum of the glycohexapeptide in DzO, with assignment of the resonances. The negative peaks are filled

Int. J. Biol. Macromol., 1990, Vol 12, February

47

G l y c o s y l a t e d p o l y t r i p e p t i d e s e q u e n c e s : F . F i l i r a et al,

Table 3 Temperature coefficients (Appb/K) of amide protons of the glycohexapeptide in water Assignment

Appb/K

AIa3-NH Thr4-NH AlaS-NH Ala6-NH

-9.4 + 1.1 - 7.4 _+0.7 - 7.1 + 0.9 - 8.2 +_0.7

The chemical shifts and the coupling constants of the two anomeric sugar protons are in agreement with a fl-configuration at these sites. The synthesis did not cause a rearrangement of the anomeric configuration. Temperature dependence of amide protons are reported in T a b l e 3. Both the value of the temperature coefficients and the complete lack of ROESY effects other than the sequential ones indicate a flexible conformation for this glycopeptide in water in the examined temperature range. No further n.m.r, experiments have been performed on the longer glycopeptides, since the c.d. properties (and therefore conformation) in water are not sensitive to chain length. Measurements in 85% T F E were prevented by solubility problems.

Conclusions The results presented in this work allow us to conclude that the continuous flow solid phase procedure, based on the Fmoc-polyamide chemistry, appears to be suitable for the synthesis of glycopeptide sequences containing a number of glycosylated amino acid residues. The mild basic conditions required for removal of the Fmoc protecting group during the synthesis do not affect the chemically sensitive O-glycosidic linkage and the anomeric configuration is maintained even during the final cleavage of the sequential glycopeptides from the resin with 95% trifluoroacetic acid. Methanolic hydrazine appears to be effective in removal of acetylprotecting groups from the sugar moieties. C.d. measurements indicate that the conformation of our glycopeptides in aqueous solution at temperatures lower than 25°C are essentially random. N.m.r. measurements performed on the hexamer show very high temperature coefficients of the amide protons and do not provide any evidence for the presence of a folded conformation. Since the c.d. properties of all fragments are very similar, the conclusions from the n.m.r, data can be extended to all fragments. A small amount of ordered structure can be induced either by increasing the temperature of the aqueous solution or by addition of TFE. In the aqueous medium all oligomers tend to form the same ordered structure at high temperature. The shape of the c.d. patterns and the fact that they cross an isodichroic point at 203-204 nm suggest the presence of a small amount of either a-helix or 31 o-helix in equilibrium with a random structure 46. In fact it has been shown that these two ordered structures cannot be really distinguished on the basis of their chiroptical properties 47. Since in water such a conformational preference is observed even at the level of the hexamer, we suggest that the ordered structure in the conformer population is a type III fl-bend, which is a turn of a 31o helix. In fact, the hexamer seems too short to form

48

Int. J. Biol. Macromol., 1990, Vol 12, February

an a-helix. The same conformation can be found also in the presence of TFE, but for the longer glycopeptides only. Thus, there is the possibility of formation of a 31o-helix in sufficiently long glycopeptides in a lipophilic environment. For the hexamer in water-TFE mixtures the shape of the c.d. spectra and the presence on an isodichroic point at 208-209 nm are better compatible with the presence of a small amount of fl-structure in equilibrium with a random coil conformation 46. Feeney, Bush et al. 1'38 proposed a very attractive model for the physiological action of antifreeze glycopeptides. They suggested that in water the glycoproteins form a flexible, left-handed 3-fold helix of polyproline II type. In this structure all sugar moieties are on one side of the helix and allow hydrophilic interactions with water molecules, particularly through the action of hydroxyl groups, thus blocking the growth of ice crystal. As noted previously, we do not agree with the existence of such a structure in aqueous solution, at least in the case of the glycosylated peptides described here. However, the c.d. data presented in this work suggest that an ordered conformation and, more specifically, a right-handed 3 lo -helix might be formed in sufficiently long fragments of glycosylated peptides in an organic solvent. In such a case the model proposed by Feeney and Bush for the antifreezing action of glycoproteins remains valid since also in the 31o-helix all sugar side chains are located on one side of the structure.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Feeney,R. E. and Yeh, Y. in 'Adv. Prot. Chem.' (eds C. B. Anfinsen,J. T. Edsall, and F. M. Ridaards) AcademicPress, New York, 1978, 32, p 191 De Vries, A. L. Phil. Trans. R. Soc, London "Set. B 1984,3045575 Anisuzzaman, A. K. M. Anderson, L. and Navia, J. L. Carbohydr. Res. 1988, 174, 265 Garg,H. G. and Jeanloz, R. W. Adv. Carbohydr. Chem. Biochem. 1985, 43, 135 Garg,H. G., Hasenkamp, T. and Paulsen, H. Carbohydr. Res. 1986, 151,225 Garg,H. G. and Jeanloz, R. W. Carbohydr. Res. 1979, 76, 85 Bucholz,M. and Kunz, H. Liebigs Ann. Chem. 1983, 1859 Maeji,N. J., Inoue, Y. and Chujo, R. Carbohydr. Res. 1986, 146, 174 Lacombe,J. M. and Pavia, A. A. J. Org. Chem. 1983,48, 2557 Paulsen,H., Schultz,M., Klamann,J. D., Waller, B. and Pall, M. Liebigs Ann. Chem. 1985, 2028 Hoogerhout,P., Guis, C. P., Erkelens, C., Bloemhoff, W., Kerling, K. E. T. and Van Boom, J. H. Reel. Trat,. Chim. Pays-Bas 1985, 104, 54 Gobbo,M., Biondi, L., Filira, F., Rocchi, R. and Lucchini, V. Tetrahedron 1988, 44, 887 Ferrari,B. and Pavia, A. A. Int. J. Pept. Protein Res. 1983, 22, 549 Bencomo,V. V. and Sinay, P. Glyconjugate J. 1984, 1, 5 Kunz,H. and Kauth, H. Liebigs Ann. Chem. 1983, 337 Kauth,H. and Kunz, H. Liebigs Ann. Chem. 1983, 360 Rocchi,R., Biondi, L., Filira, F., Gobbo, M., Dagan, S. and Fridkin, M. Int. J.Pept. Protein Res. 1987, 29, 250 Lavielle,S., Ling, N. C., Saltman, R. and Guillemin, R. C. Carbohydr. Res. 1981, 89, 229 Lavielle,S., Ling, N. C. and Guillemin, R. C. Carbohydr. Res. 1981, 89, 221 Kunz,H. Angew. Chem. Int. Ed. Engl. 1987, 26, 294 Kunz,H. and Dombo, B. Angew. Chem. Int. Ed. Engl. 1988, 27, 711 Paulsen,H., Merz, G. and Weichert, U. Angew. Chem. Int. Ed. Engl. 1988, 27, 1365 Dryland,A. and Sheppard, R. C. J. Chem. Soc. Trans. 1 1986, 125 and references cited therein Kaiser,E., Colescott, R. C., Bossinger, C. D. and Cook, P. J. Anal. Biochem. 1970, 34, 595

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25 26 27 28 29 30 31 32 33 34 35 36 37 38

Hancock, W. S. and Battersby, J. E. Anal. Biochem. 1976, 21,260 Peggion, E. and Foffani, M. T. Suppl. J. Bioscience 1986, 8, 179 Bax, A. in 'Two Dimensional NMR in Liquids', Riedel, Delft, The Netherlands, 1981, p 69 Marion, D. and Wiithrich, K. Biochem. Biophys. Res. Commun. 1981, 113, 967 Bax, A. and Davis, D. J. Magn. Reson. 1985, 65, 355 Bax, A. and Davis, D. J. Magn. Reson. 1985, 63, 207 Hore, P. J. Magn. Reson. 1983, 55, 283 Tilak, M. A. Tetrahedron Lett. 1968, 611,6323 Magnusson, G., Noori, G., Dahmen, J., Frejd, T. and Lave, T. Acta Chem. Scand. 1981, 35, 213 Herzig, J. and Nudelman, A. Carbohydr. Res. 1986, 153, 162 Schultheiss-Reimann, P. and Kunz, H. Angew. Chem. Int. Ed. Engl. 1983, 22, 62 Paulsen, H. Chem. Soc. Rev. 1984, 13, 15 Paulsen, H., Paal, M. and Schultz, M. Tetrahedron Lett. 1983, 24, 54 Bush, C. A., Feeney, R. E., Osuga, D. T., Ralapaty, S. and Yeh,

Y. Int. J. Pept. Protein Res. 1981, 20, 125

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Bush, C. A., Ralapaty, S., Matson, G. M., Yamasaki, R. B., Osuga, D. T., Yeh, Y. and Feeney, R. E. Arch. Biochem. Biophys. 1984, 232, 624 Woody, R. W. J. Polym. Sci., Macrom. Rev. 1978, 12, 181 Nagarasinga Rao, B. N. and Bush, C. A. Biopolymers 1987, 26, 1277 Ahmed, A. I., Feeney, R. E., Osuga, D. T. and Yeh, Y. J. Biol. Chem. 1975, 2511, 3344 Berman, E., Allerhand, A. and Vries, A. L. J. Biol. Chem. 1981, 255, 4407 Mammi, S., Mammi, N. J. and Peggion, E. Biochemistry 1988, 27, 1374 Inagaki, F., Kodama, C., Suzuki, M. and Suzuki, A. FEBS Lett. 1987, 219, 45 Greenfield, N. and Fasman, G. D. Biochemistry 1969, 8, 4108 Sudha, T. S., Vijayakumar, E. K. S. and Balaram, P. Int. J. Pept. Protein Res. 1983, 22, 464

I n t . J. Biol. M a c r o m o l . , 1990, Vol 12, F e b r u a r y

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