Deacetylation of N(α)-methylated glycopeptides reveals that aza- enolates provide protection against β-elimination of carbohydrates O-linked to serine

Article abstract of DOI:10.1016/S0040-4039(00)00611-0

Three glycopeptides Ac-Ala-Ser[β-Gal(OAc)₄]-Phe-NH₂, Ac-Ala-N(α)-Me- Ser[β-Gal(OAc)₄]-Phe-NH₂ and Ac-Ala-Ser[β-Gal(OAc)₄]-N(α)-Me-Phe-NH₂ have been prepared and treated with base in order t

Full text of DOI:10.1016/S0040-4039(00)00611-0

Tetrahedron Letters 41 (2000) 4435±4439
Deacetylation of Na-methylated glycopeptides reveals that
aza-enolates provide protection against b-elimination of
carbohydrates O-linked to serine
Petter Sjolin and Jan Kihlberg*
Ê Ê
Department of Chemistry, Organic Chemistry, Umea University, SE-901 87 Umea, Sweden
Received 25 February 2000; accepted 10 April 2000
Abstract
Three glycopeptides Ac-Ala-Ser[b-Gal(OAc)4]-Phe-NH2, Ac-Ala-Na-Me-Ser[b-Gal(OAc)4]-Phe-NH2
and Ac-Ala-Ser[b-Gal(OAc)4]-Na-Me-Phe-NH2 have been prepared and treated with base in order to
remove the O-acetyl protective groups. The glycopeptide which carried the N-methyl group on the glyco-
sylated serine, was substantially more susceptible to b-elimination than the two others. This reveals that
formation of an aza-enolate from the amide bond to a glycosylated serine provides protection against
b-elimination under basic conditions. # 2000 Elsevier Science Ltd. All rights reserved.
Removal of O-acyl protective groups from the carbohydrate moiety is often the ®nal step in
synthesis of a glycopeptide. This is usually achieved using a moderately strong base such as
sodium methoxide, ammonia1 or hydrazine hydrate2 in methanolic solutions. Since the a-protons
of the amino acid residues are acidic, treatment with base is accompanied by risks of epimerization
of a-stereocenters, or b-elimination if the carbohydrate is attached to serine or threonine.1,3 10
Removal of acetyl groups is usually facile, but the increased concentration of base required for
removal of O-benzoyl groups may cause b-elimination and slight epimerization.1,7 10 It has been
suggested that formation of aza-enolates by removal of the acidic amide protons protects the
glycopeptide from these side-reactions.9,11 To test this hypothesis we have synthesized three
glycotripeptides, two of which are Na-methylated either on the galactosylated serine or on the
C-terminal phenylalanine, in order to prevent aza-enolate formation at these positions. The rate
of b-elimination upon de-O-acetylation of the di€erent glycopeptides was then investigated.
Synthesis of the three glycopeptides required preparation of building blocks 3 and 7 in which a
tetra-O-acetylated galactose moiety is b-linked to serine and N-methylated serine, respectively
(Scheme 1). Building block 3 was synthesized in 55% yield from pentaacetate 1 and Fmoc-Ser-OPfp
(2), using boron tri¯uoride etherate as promoter.12 N-Methylated serine 4 was Fmoc-protected
* Corresponding author.
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00611-0

4436
and then converted into the penta¯uorophenyl (Pfp)-ester giving derivative 6. This was galacto-
sylated using the same conditions as for 3 to give the building block 713 in 71% yield. The higher
yield in the glycosylation of N-methylated serine 6, as compared to 2, is in agreement with pre-
vious observations made by Polt and co-workers.14
.
Scheme 1. Reagents and conditions: (i) BF3 Et2O, CH2Cl2, rt; (ii) Fmoc-Suc, Et3N, H2O:MeCN (1:2), rt; (iii) PfpOH,
DIC, EtOAc, 0ꢀC!rt
Building blocks 3 and 7 were then used for assembly of glycopeptides 8±1015 on a TentaGel
TM
S-NH2
resin functionalized with the Rink linker.16,17 The three glycopeptides were cleaved
from the resin with TFA:water (95:5) and then puri®ed by ¯ash chromatography and reversed-
phase HPLC-chromatography. Treatment of the glycopeptides with 10 mM methanolic sodium
methoxide resulted in rapid (<30 min) removal of the O-acetyl groups to give 11±13 (Scheme
2).18 As revealed by reversed-phase HPLC prolonged exposure to base (0.5±5 h) led to b-elim-
ination of 11±13, giving 14±1619 at di€erent rates. In order that to avoid di€erences in the
experimental conditions a€ected the rates of b-elimination, the non-N-methylated 8 was used as
an internal control which, in two separate experiments, was treated with sodium methoxide in the
same ¯ask as 9 and 10. After deacetylation of 8 and 10 the resulting 11 and 13 both underwent
b-elimination slowly and at equal rates. This established that formation of an aza-enolate at the
Scheme 2.

4437
serine-phenylalanine amide bond did not provide any signi®cant protection against b-elimination.
In contrast, glycopeptide 12, in which the glycosylated serine is N-methylated, underwent
b-elimination much more rapidly than 11. As shown in Fig. 1, only a small amount of non-
methylated 11 had been converted to dehydroalanine 14 when ꢁ50% of 12 had been transformed
to 15. In fact, it was not possible to prepare N-methylated 12 from 9 without formation of 15 by
b-elimination. This is in contrast to glycopeptides 8 and 10, which could be deacetylated without
any detectable b-elimination. Crude 12 could, however, be puri®ed by reversed-phase HPLC, and
NMR spectroscopy of the resulting pure 12 did not reveal any signs of epimerization of
the a-stereocenters.
Figure 1. HPLC-chromatogram of the simultaneous de-O-acetylation of glycopeptides 8 and 9 which leads to forma-
tion of 11 and 12, as well as their respective b-eliminated products 14 and 15 (conditions: Kromasil C-8 column. Linear
gradient of 0±80% B in A over 60 min. A=0.1% aqueous TFA, B=0.1% TFA in MeCN. Flowrate 1.5 ml/min)
The possibility that N-methylation of the central serine residue increased the rate of b-elimination
of 12 by in¯uencing the conformation around the serine Ca±Cb bond was ruled out by inspection
of the coupling constants between the H-a and H-b protons. Glycopeptide 12 populates two
conformations for the alanine-serine amide bond. One rotamer displayed coupling constants of
6.8 Hz between the Sera- and the Serb-protons, thus indicating free rotation around the Ca±Cb
bond just as for non-methylated 11. For the other rotamer the values of the 3Ja,b coupling constants
were found to be 4.8 and 9.4 Hz, respectively. This reveals a predominant population of a
conformation in which Ser Ob and Ha adopt a gauche orientation, i.e. one of the two conformations
which do not allow b-elimination to occur.

4438
In conclusion, we have shown that Na-methylation of a glycosylated serine results in a substantial
increase in the rate of b-elimination on base-catalyzed removal of O-acyl protective groups from
glycopeptides. We interpret this result as being due to the fact that a protective aza-enolate
cannot be formed adjacent to the carbohydrate moiety. Since b-elimination is often encountered
on removal of benzoyl protective groups from O-linked glycopeptides,1,7 10 and cannot be
avoided if the carbohydrate is linked to a N-methylated serine, novel protective groups should
®nd use in the synthesis of glycopeptides. Investigations of such protective groups are now in
progress in our laboratory.
Acknowledgements
This work was funded by grants from the Swedish Natural Science Research Council and the
Swedish Research Council for Engineering Sciences.
References
1. Paulsen, H.; Schultz, M.; Klamann, J.-D.; Waller, B.; Paal, M. Liebigs Ann. Chem. 1985, 2028±2048.
2. Schultheiss-Reimann, P.; Kunz, H. Angew. Chem., Suppl. 1983, 39±46.
3. Kunz, H. Angew. Chem., Int. Ed. Engl. 1987, 26, 294±308.
4. Kunz, H.; Brill, W. K.-D. Trends Glycosci. Glycotechnol. 1992, 4, 71±82.
5. Hoogerhout, P.; Guis, C. P.; Erkelens, C.; Bloemho€, W.; Kerling, K. E. T.; van Boom, J. H. Recl. Trav. Chim.
Pays-Bas 1985, 104, 54±59.
6. Andrews, D. M.; Seale, P. W. Int. J. Peptide Protein Res. 1993, 42, 165±170.
7. Erbing, B.; Lindberg, B.; Norberg, T. Acta Chem. Scand. 1978, B32, 308±310.
8. Reimer, K. B.; Meldal, M.; Kusumoto, S.; Fukase, K.; Bock, K. J. Chem. Soc., Perkin Trans. 1 1993, 925±932.
9. Sjolin, P.; Elofsson, M.; Kihlberg, J. J. Org. Chem. 1996, 61, 560±565.
10. Sjolin, P.; George, S. K.; Bergquist, K. E.; Roy, S.; Svensson, A.; Kihlberg, J. J. Chem. Soc., Perkin Trans. 1 1999,
1731±1742.
11. Seebach, D. Aldrichim. Acta 1992, 25, 59±66.
12. Kihlberg, J.; Ahman, J.; Walse, B.; Drakenberg, T.; Nilsson, A.; Soderberg-Ahlm, C.; Bengtsson, B.; Olsson, H.
J. Med. Chem. 1995, 38, 161±169.
13. Compound 7: H NMR (400 MHz, CDCl3), rotamers (ꢁ2:1), ꢀ (ppm): 5.37±5.42 (m, 2H, H-4,40), 5.21 (dd, 1H,
1
J=7.9, 10.5 Hz, H-20), 5.14 (dd, 1H, J=8.0, 10.5 Hz, H-2), 4.58 (d, 1H, J=7.9 Hz, H-1), 4.27 (d, 1H, J=8.0 Hz,
H-10), 3.05 (s, 1H, Me), 2.99 (s, 1H, Me0); FABMS: (M+Na)+ 860 calcd, 860 obsd.
14. Szabo, L.; Li, Y. S.; Polt, R. Tetrahedron Lett. 1991, 32, 585±588.
15. Glycopeptide 8: yield 97%; 1H NMR (400 MHz, Acetone-d6), ꢀ (ppm): Ala 4.21 (m, 1H, H-a), 1.32 (d, 3H, J=7.2
Hz, Me); Ser 4.36 (m, 1H, H-a), 3.84 (dd, 1H, J=7.3, 10.7 Hz, H-b), 3.76 (dd, 1H, J=4.8, 10.7 Hz, H-b0); Phe 4.51
(m, 1H, H-a), 3.26 (dd, 1H, J=4.4, 14.0 Hz, H-b), 2.99 (dd, 1H, J=9.8, 13.9 Hz, H-b0); Gal 4.74 (d, 1H, J=7.6
1
Hz, H-1); FABMS: (M+H)+ 695 calcd, 695 obsd. Glycopeptide 9: yield 86%; H NMR (400 MHz, Acetone-d6),
major rotamer, ꢀ (ppm): Ala 4.82 (m, 1H, H-a), 1.31 (d, 3H, J=6.9 Hz, Me); Ser 5.16 (dd, 1H, J=4.7, 9.4 Hz, H-
a), 3.9±4.0 (m, 2H, H-b,b0); Phe 4.54 (ddd, 1H, J=3.8, 8.7, 11.2 Hz, H-a), 3.28 (dd, 1H, J=3.7, 14.0 Hz, H-b),
2.92 (dd, 1H, J=11.3, 13.9 Hz, H-b0); Gal 4.75 (d, 1H, J=7.9 Hz, H-1); minor rotamer, ꢀ (ppm): Ala 1.19 (d, 3H,
J=6.8 Hz, Me); Phe 4.62 (m, 1H, H-a), 3.20 (dd, 1H, J=5.1, 14.1 Hz, H-b), 2.95 (m, 1H, H-b0); Gal 4.72 (d, 1H,
J=7.6 Hz, H-1); FABMS: (M+H)+ 709 calcd, 709 obsd. Glycopeptide 10: yield 39%; 1H NMR (400 MHz,
Acetone-d6), rotamers (ꢁ1:1), ꢀ (ppm): Ala 4.38 (t, 1H, J=7.2 Hz, H-a), 4.31 (t, 1H, J=7.2 Hz, H-a); Ser 4.5±4.6
(m, 1H, H-a); Phe 5.20 (dd, 1H, J=6.5, 9.0 Hz, H-a); Gal 4.80 (d, 1H, J=7.9 Hz, H-1), 4.42 (d, 1H, J=7.9 Hz, H-
1); FABMS: (M+H)+ 709 calcd, 709 obsd.
16. Rink, H. Tetrahedron Lett. 1987, 28, 3787±3790.
17. Bernatowicz, M. S.; Daniels, S. B.; Koster, H. Tetrahedron Lett. 1989, 30, 4645±4648.

4439
1
18. Glycopeptide 11: H NMR (400 MHz, MeOH-d4), ꢀ (ppm): Ala 4.25±4.35 (m, 1H, H-a), 1.28 (d, 3H, J=7.2 Hz,
Me); Ser 4.54 (t, 1H, J=5.4 Hz, H-a), 4.09 (dd, 1H, J=5.1, 10.4 Hz, H-b), 3.7±3.8 (m, 1H, H-b); Phe 4.57 (m, 1H,
H-a), 3.21 (dd, 1H, J=4.9, 14.1 Hz, H-b), 2.97 (dd, 1H, J=9.1, 14.0 Hz, H-b); Gal 4.27 (d, 1H, J=7.2 Hz, H-1);
FABMS: (M+H)+ 527 calcd, 527 obsd. Glycopeptide 12: 1H NMR (400 MHz, MeOH-d4), rotamers (ꢁ1:1), ꢀ
(ppm): Ala 4.8±4.9 (m, 1H, H-a), 4.70 (q, 1H, J=7.0 Hz, H-a), 1.36 (d, 3H, J=7.2 Hz, Me), 1.23 (d, 3H, J=7.0
Hz, Me); Ser 5.23 (dd, 1H, J=4.8, 9.3 Hz, H-a), 5.14 (t, 1H, J=6.7 Hz, H-a), 3.9±4.1 (m, 4H, H-b); Phe 4.50±4.65
(m, 2H, H-a), 3.2±3.3 (m, 2H, H-b), 2.9±3.0 (m, 2H, H-b); Gal 4.2±4.3 (m, 2H, H-1); FABMS: (M+Na)+ 563 calcd,
1
563 obsd. Glycopeptide 13: H NMR (400 MHz, MeOH-d4), rotamers (ꢁ3:2), ꢀ (ppm): Ala 4.34 (q, 1H, J=7.1
Hz, H-a), 4.26 (q, 1H, J=7.2 Hz, H-a), 1.29 (d, 3H, J=7.2 Hz, Me), 1.22 (d, 3H, J=7.2 Hz, Me); Ser 5.04 (t, 1H,
J=6.3 Hz, H-a), 4.63 (dd, 1H, J=4.8, 9.3 Hz, H-a), 3.98 (dd, 1H, J=5.9, 10.1 Hz, H-b), 3.75±3.85 (m, 1H, H-b),
3.3±3.4 (m, 1H, H-b), 2.30 (dd, 1H, J=4.0, 10.2 Hz, H-b); Phe 5.25 (dd, 1H, J=5.6, 10.3 Hz, H-a), 5.06 (dd, 1H,
J=4.0, 10.0 Hz, H-a), 3.25±3.40 (m, 2H, H-b), 2.95±3.05 (m, 2H, H-b); Gal 4.24 (d, 1H, J=7.5 Hz, H-1), 3.92 (d,
1H, J=7.3 Hz, H-1); FABMS: (M+Na)+ 563 calcd, 563 obsd.
1
19. Peptide 14: H NMR (400 MHz, MeOH-d4), ꢀ (ppm): Ala 4.30 (q, 1H, J=7.2 Hz, H-a), 1.34 (d, 3H, J=7.2 Hz,
Me); ÁApa 5.76 (s, 1H, H-b), 5.40 (s, 1H, H-a); Phe 4.63 (m, 1H, H-b), 3.27 (dd, 1H, J=5.0, 14.0 Hz, H-b), 2.98
(dd, 1H, J=10.0, 14.0 Hz, H-b); FABMS: (M+H)+ 347 calcd, 347 obsd. Peptide 15: 1H NMR (400 MHz, MeOH-d4), ꢀ
(ppm): Ala 4.41 (m, 1H, H-a), 1.25 (d, 3H, Me); ÁApa 6.19 (s, 1H, H-b), 5.72 (s, 1H, H-b); Phe 4.61 (m, 1H, H-a),
3.3±3.4 (m, 1H, H-b), 3.05 (br t, 1H, J=12.4 Hz, H-b).