Synthesis of a Tumor-Associated2,3-Sialyl-T Glycododecapeptide Antigen from the Tandem Repeat Regionof the Mucin MUC1

Abstract

As a cell surface antigen for the development of selective antitumorvaccines, a tumor-associated glycododecapeptide from epithelialMUC1 carrying the 2,3-sialyl-T antigen was synthesized. The 2,3-STbuilding block was assembled from a T_N-threonine conjugateby stepwise saccharide chain extension and utilized for the solid-phaseglycopeptide synthesis on the fluoride-labile PTMSEL linker.

2,3-sialyl-T antigen - glycopeptides - MUC1 - regio­selective sialylation - solid-phasesynthesis

References

1a Feizi T. Nature (London)  1985,  314:  53 

1b Springer GF. Science  1984,  224:  1198 

2a Taylor-Papadimitriou J. Burchell JM. Plunkett T. Graham R. Correa I. Miles D. Smith M. J. Mammary Gland Biol. Neoplasia  2002,  2:  209 

2b Hanisch F.-G. Müller S. Glycobiology  2000,  10:  439 

2c von Mensdorff-Pouilly S. Snijdewint FGM. Verstraeten AA. Verheijen RHM. Kenemans P. Int.J. Biol. Markers  2000,  15:  343 

2d Taylor-Papadimitriou J. Burchell JM. Miles DW. Dalziel M. Biochim.Biophys. Acta  1999,  301 

3a Gum JR. Biochem. Soc. Trans.  1995,  23:  795 

3b Carlstedt I. Davies JR. Biochem. Soc. Trans.  1997,  25:  214 

4 Brockhausen I. Yang J.-M. Burchell J. Whitehouse C. Taylor-Papadimitriou J. Eur.J. Biochem.  1995,  233:  607 

5 Vlad AM. Müller S. Cudic M. Paulsen H. Otvos L. Hanisch F.-G. Finn OJ. J.Exp. Med.  2002,  11:  1435 

6a Nakahara Y. Nakahara Y. Ito Y. Ogawa T. Carbohydr.Res.  1998,  309:  287 

6b Komba S. Meldal M. Werdelin O. Jensen T. Bock K. J.Chem. Soc., Perkin Trans. 1  1999,  415 

6c Schwarz JB. Kuduk SD. Chen X.-T. Sames D. Glunz PW. Danishefsky SJ. J.Am. Chem. Soc.  1999,  121:  2662 

7a Liebe B. Kunz H. Angew.Chem. Int. Ed. Engl.  1997,  36:  618 

7b Liebe B. Kunz H. Helv. Chim. Acta  1997,  80:  1473 

7c Dziadek S. Espínola CG. Kunz H. Aust. J. Chem.  2003,  56:  519 

8 George SK. Holm B. Reis CA. Schwientek T. Clausen H. Kihlberg J. J. Chem. Soc., Perkin Trans.1  2001,  880 

9 Helferich B. Wedemeyer F. Liebigs Ann. Chem.  1949,  563:  139 

10 Lergenmüller M. Ito Y. Ogawa T. Tetrahedron  1988,  54:  1381 

11a Marra A. Sinaӱ P. Carbohydr.Res.  1989,  187:  35 

11b Keil S. Claus C. Dippold W. Kunz H. Angew. Chem. Int. Ed.  2001,  40:  366 

12 Martichonok V. Whitesides GM. J. Org. Chem.  1996,  61:  1702 

13

9: [α]_D ²⁷ = 32.01(c = 1, CHCl₃); R_t = 30.07min [Phenomenex Luna C18 (2), grad.: CH₃CN/H₂O(60:40) → (90:10), 40min]; ¹HNMR (400 MHz, CDCl₃, COSY, HMQC): δ (ppm) = 7.74(d, 2 H, H4-, H5-Fmoc, J _H4,H3 = J _H5,H6 = 7.84 Hz),7.58 (dd, 2 H, H1-, H8-Fmoc, J _H1,H2 = J _H8,H7 = 7.80 Hz),7.49 (d, 2 H, H_ar-Bzn, J _Ha,Hb = 5.84),7.42-7.35 (m, 2 H, H3-, H6-Fmoc), 7.35-7.24 (m,15 H, H2-, H7-Fmoc, H_ar-Bzn (3 H), H_ar-Bn(10 H)), 6.59 (d, 1 H, NH-GalNAc, J _NH,H2 = 9.40Hz), 6.12 (d, 1 H, NH-T, J _{NH,T α} = 9.76 Hz),5.45 (s, 1 H, CH-Bzn), 5.40 (t, 1 H, H8′′, J _{H8 ′′ ,H9 ′′} = 6.64 Hz),5.28-5.13 (m, 2 H, H7′′, NH-NeuNAc),5.16 (s, 2 H, CH₂-Bn), 4.98 (d, 1 H, H1, J _H1,H2 = 3.52 Hz),4.92-4.81 (m, 1 H, H4′′), 4.74-4.63(m, 1 H, H2), 4.55 (s, 2 H, CH₂-Bn), 4.52-4.03(m, 10 H, H9_a′′{4.46},CH₂-Fmoc {4.39, 4.34}, T^β {4.33},H4 {4.26}, T^α {4.25},H9-Fmoc {4.19}, H1′ {4.15},H6_a {4.13}, H5′′ {4.06}),3.99-3.84 (m, 4H, H6′′ {3.93},H9_b′′ {3.92}, H3′ {3.90},H6_b {3.87}), 3.72-3.55 (m,4 H, H3 {3.67}, H2′ {3.61},H6_a′ {3.59}, H5 {3.57}), 3.54-3.39(m, 2 H, H6_b′{3.87}, H5′{3.41}),3.24 (s, 1 H, H4′), 2,78 (s_b, 1 H, OH), 2.71(dd, 1 H, H3_eq′′, J_{H3eq ′′ ,H3ax ′′} = 12.72Hz, J_{H3eq ′′ ,H4 ′′} = 4.28Hz), 2.36 (s, 1 H, OH), 2.03-2.00 (m, 1 H, H3_ax′′),2.09, 2.07, 2.02, 2.00, 1.92, 1.82 (5*s, 18H, 6* CH₃-Ac),1.42 (s, 9 H, CH₃-t-Bu), 1.26(d, 3 H, T^γ, J _{T γ ,T β} = 5.92Hz); ¹³C NMR (100.6 MHz, CDCl₃,BB, HMQC): δ = 171.53, 170.91, 170.82, 170.24,170.09, 169.76, 167.77 (C=O), 156.83 (C=O-urethane),143.71 (C1a-, C8a-Fmoc), 141.29, 141.15 (C4a-, C5a-Fmoc), 138.07,137.51 (C_q-Bn), 134.21 (C_q-Bn), 128.97, 128.84, 128.78,128.43, 127.51 (C_ar-Bn), 128.78, 128.04, 126.44 (C_ar-Bzn),127.79 (C3-, C6-Fmoc), 127.09, 126.97 (C2-, C7-Fmoc), 125.17, 124.94(C1-, C8-Fmoc), 120.03 (C4-, C5-Fmoc), 106.55 (C1′), 100.96(CH-Bzn), 100.45 (C1), 97.47 (C2′′), 83.13 (C_q-t-Bu), 78.15 (C3), 75.94, 75.15 (T^β,C4, C3′), 73.60 (CH₂-Bn), 73.43, 72.92 (C6′′,C5′),69.72 (C8′′, C6′), 69.07 (C6), 68.61(C4′′), 68.22, 68.11 (CH₂-Bn, C2′,C4′,C7′′),67.25 (CH₂-Fmoc), 63.64 (C9′′), 63.52(C5), 59.24 (T^α), 49.08 (C5′′),47.79 (C2), 47.09 (C9-Fmoc), 37.42 (C3′′), 28.06 (CH₃-t-Bu), 23.16, 23.07(CH₃-NHAc), 21.33, 21.21, 21.01, 20.78, 20.64 (CH₃-OAc),19.47 (T^γ), HR-ESI-TOF-MS (positive ion mode):calcd. for C₇₇H₉₁N₃O₂₇Na: 1512.5737,found: 1512.5729 [M + Na]⁺.

14 Neises B. Steglich W. Angew. Chem., Int. Ed. Engl.  1978,  17:  522 

15

12: [α]_D ²⁷ = 24.75(c = 1, CHCl₃), HR-ESI-TOF-MS (positive ionmode): calcd. for C₇₄H₈₆N₃O₃₁Na₂:1558.5040, found: 1558.5024 [M - H + 2 × Na]⁺.

16

Perkin Elmer ABI 433A (Applied Biosystems).

17a Wagner M. Kunz H. Angew.Chem. Int. Ed.  2002,  41:  317 

17b Wagner M. Kunz H. Z. Naturforsch.  2002,  57b:  928 

18 Knorr R. Trzeciak A. Bannwarth W. Gillessen D. Tetrahedron Lett.  1989,  30:  1927 

19a Carpino LA. J. Am. Chem. Soc.  1993,  115:  4397 

19b Carpino LA. El-Faham AS. Minor C. Albericio F. J. Chem.Soc., Chem. Commun.  1994,  210 

20

16: [α]_D ²⁶ = -40.68(c = 0.86, H₂O), R_t = 14.52min [Phenomenex Jupiter C18, grad.: CH₃CN/H₂O + 0.1% TFA (5:95) → (30:70),40min]; ¹H NMR (400 MHz, D₂O,COSY, TOCSY, HMQC): δ (ppm) = 4.92 (d, 1 H, H1, J _H1,H2 = 3.72 Hz),4.69 (t, 1 H, D^α, J_{D α ,D β} = 6.36Hz), 4.65-4.58 (m, 2 H, T_ST ^α {4.63},R^α {4.62}), 4.57-4.49(m, 1 H, A₁ ^α), 4.49-4.41 (m,3 H, H1′ {4.47}, S^α {4.45},A₂ ^α {4.44}), 4.40-4.34(m, 3 H, 3 × P^α), 4.33-4.26(m, 3 H, T_ST ^β {4.31},V^α {4.29}, T₁ ^α {4.28}),4.22-4.14 (m, 3 H, H4 {4.21}, H2 {4.19},T₁ ^β {4.18}), 4.08-3.95(m, 3 H, H3′ {4.04}, H7′′ {4.03},H3 {4.00}), 3.94-3.87 (m, 3 H, G^α {3.91},H4′{3.90}), 3.86-3.52 (m, 20H, H5′, H8′′ {3.83},H6_a {3.80}, S^β {3.76},3 × P_a ^δ {3.73},H5′′ {3.71}, H9_a′′,H9_b′′, H4′′ {3.67},3 × P_a ^α {3.63},H6_b {3.62}, H6′′,H5, H6_a′, H6_b′), 3.48 (dd,1 H, H2′, J _{H1 ′ ,H2 ′} = 7.92Hz), 3.21-3.14 (m, 2 H, R^δ), 2.99-2.83(m, 2 H, D^β), 2.71 (dd, 1 H, H3_eq′′, J _{H3eq ′′ ,H3ax ′′} = 12.20Hz, J _{H3eq ′′ ,H4 ′′} = 4.40Hz), 2.35-2.19 (m, 3 H, 3 × P_a ^β),2.10-1.94 (m, 8 H, V^β {2.03},3 × P^γ {2.01}, R_a ^β),2.02, 1.99, 1.97 (3 × s, 9 H, 3 × CH₃-NHAc),1.93-1.77 (m, 4 H, 3 × P_b ^β {1.88}, H3_ax′′ {1.80}),1.77-1.59 (m, 3 H, R_b ^β{1.70},R^γ {1.63}), 1.38-1.31(m, 6 H, 2 × A^β), 1.24 (d, 3 H, T_ST ^γ, J _{T γ ,T β} = 6.12 Hz),1.15 (d, 3 H, T₁ ^γ, J _{T γ ,T β} = 6.36Hz), 0.94 (d, 6 H, V^γ); MALDI-TOF-MS (dhb, positiveion mode): calcd. for C₇₆H₁₂₄N₁₇O₃₇:1867.9, found: 1868.8 [M + H]⁺,1890.9 ([M + Na]⁺,calcd.: 1889.9), 1907.1 ([M + K]⁺,calcd.: 1905.9), 1912.8 ([M - H + 2 × Na]⁺,calcd.: 1911.9); ESI-MS (positive ion mode): calcd. for [C₇₆H₁₂₁N₁₇O₃₇Na₄]²⁺: 977.88,found: 977.87 [M - 2 H + 4 Na]²⁺.