Nα-Dmc protected amino acid aglycones: Versatile hydrogenolysis stable acceptors for O-glycosylation

Article abstract of DOI:10.1016/j.tetlet.2005.01.055

N^α-(4,4-Dimethyl-2,6-dioxocyclohexylidenemethylene) (Dmc) protected L-serine, L-threonine and L-homoserine have been prepared as tert-butyl esters in excellent yields. These hydrogenolysis stable acceptors underwent efficient α-O-glycosylation

Full text of DOI:10.1016/j.tetlet.2005.01.055

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 1703–1706
Na-Dmc protected amino acid aglycones:
versatile hydrogenolysis stable acceptors for O-glycosylation
Barrie Kellam,* Paul A. Ward, Aisyah S. Abdul Rahim and Siri Ram Chhabra
School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK
Received 1 December 2004;accepted 12 January 2005
Available online 28 January 2005
Abstract—Na-(4,4-Dimethyl-2,6-dioxocyclohexylidenemethylene) (Dmc) protected L-serine, L-threonine and L-homoserine have
been prepared as tert-butyl esters in excellent yields. These hydrogenolysis stable acceptors underwent efficient a-O-glycosylation
with an L-fucopyranosyl bromide donor and also allowed convenient protecting group manipulations to ultimately deliver novel
glycoamino acid building blocks suitable for Fmoc based solid-phase glycopeptide synthesis.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The majority of proteins observed in Nature bear di-
verse O- and N-linked oligosaccharide structures,1 and
it has become increasingly clear that glycosylation
modulates both the physicochemical properties and bio-
logical functions of these glycoproteins. For example,
glycosylation influences uptake, distribution, excretion
and proteolytic stability, yet is also implicated in com-
munication between cells and in attachment of bacteria
and viruses to host cells.2 One such carbohydrate entity,
sialyl Lewis X (sLeX) is located on the cell-surface of
many important celltypes,3 and plays a key role in both
inflammatory responses and metastasis of cancer cells.4
In addition, other key O-linked fucosylated sequences
have been identified in human factor IX, insect neuro-
peptides and epidermal growth factor (EGF) domains
of assorted coagulation and fibrinolytic proteins.5
Our interest in the synthesis of novel fucopeptide based
inhibitors of sLeX binding, as potential anti-inflamma-
tory and anti-metastatic agents, revealed a dearth of
established routes for the synthesis of appropriately pro-
tected a-linked fuco-amino acid building blocks suitable
for Fmoc/tBu solid-phase peptide synthesis (SPPS).
Consequently, to investigate their biological function
in detail, efficient access to appropriate glycoamino acid
building blocks is required for the construction of key
fucose-bearing glycopeptides.
Only a handful of synthetic routes have been described
to date. For example, boron trifluoride etherate pro-
moted glycosylation of Fmoc-Thr-OH and Fmoc-Ser-
OH with peracetylated L-fucose afforded the thermody-
namic a-fucosides in 35% and 44% yields, respectively.6
Clearly, this provided a rapid access to the required
building blocks, however, the resulting mixtures re-
quired HPLC purification, thereby opposing scale-up
of the one-step protocol. Traditionally, a-fucosyl amino
acids are obtained by the in situ anomerisation reaction
using nonparticipatory O-benzyl ether protected fucosyl
donors. Since the O-glycosidic linkage of O-benzyl pro-
tected fucose is highly sensitive to acid (e.g., TFA), a
protecting group exchange of O-benzyl for O-acetyl is
paramount if the building blocks are to be utilised in
an Fmoc-based SPPS strategy. Unfortunately, the Fmoc
group itself is not hydrogenolysis stable7 and to circum-
vent this problem, Kunz and co-workers a-fucosylated
Fmoc-Thr-OtBu with a 2,3,4-tri-O-p-methoxybenzyl thio-
fucoside.8 These sugar protecting groups could then be
removed with ceric ammonium nitrate (CAN) and acet-
ylated prior to the acidolytic deprotection of the tert-but-
yl ester (38% over three steps). Using a similar strategy,
glycosylation of Z-Ser-OBn using 2,3,4-tri-O-benz-
ylfucosyl bromide afforded the desired glycoamino
acid.9 From this intermediate, global deprotection using
catalytic hydrogenolysis was followed by Fmoc incorpo-
ration, allyl esterification, sugar hydroxyl acetylation
and allyl ester deprotection. It was observed that direct
incorporation of the sugar acetates was not possible
with the free acid, and thus dictated the excessive pro-
tecting group manipulations outlined. Therefore, in an
Keywords: Protecting groups;Glycosylation;Amino acids and
derivatives.
*
Corresponding author. Tel.: +44 115 9513026;fax: +44 115
9513412;e-mail: barrie.kellam@nottingham.ac.uk
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.01.055

1704
B. Kellam et al. / Tetrahedron Letters 46 (2005) 1703–1706
attempt to overcome some of the problems associated
with the construction of these building blocks, this
letter describes the synthesis and application of piper-
idine labile Na-(4,4-dimethyl-2,6-dioxocyclohexylide-
nemethylene) (Dmc)10 protected amino acids 1–3
as robust hydrogenolysis stable glycosyl acceptors for
a-fucosylation.
bility to hydrogenolysis and lability towards piperidine
would be an asset when used as a protecting group suit-
able for the preparation of a-fucosylated amino acids
for SPPS. To this end, serine 4 and threonine 5 were pro-
tected as their corresponding Na-Dmc derivatives 6 and
7 using 5,5-dimethyl-2-(dimethylaminomethylene)cyclo-
hexane-1,3-dione (DMMD) and subsequently converted
to the corresponding tert-butyl esters 1 and 212 using the
methodology described by Thierry et al.13 (Scheme 1).
Originally, the use of Dmc as an orthogonal amino pro-
tecting group for SPPS became redundant as it was
superseded by its methyl derivative Dde11 due to the lat-
terꢀs improved stability towards the Fmoc deprotection
conditions. We reasoned however, that the formerꢀs sta-
In addition, an efficient synthesis of Na-Dmc-homoser-
ine tert-butyl ester 314 was accomplished in three steps
from the commercially available Z-Asp-OtBu 8 (Scheme
Scheme 1. Reagents and conditions: (i) EtOH, (6;83%, 7;86%);(ii) tert-butyl trichloroacetimidate, EtOAc, (1;42%, 2;47%).
Scheme 2. Reagents and conditions: (i) i-BuOCOCl/Et3N, THF;then NaBH 4/H2O, 81%;(ii) H 2/Pd–C, EtOH;(iii) DMMD, EtOH, rt, 87%.
˚
Scheme 3. Reagents and conditions (i) Et4NBr, DCM, 4 A MS (11;63%, 12;73%, 13;40%);(ii) H 2, Pd/C, EtOH (14;83%, 15;85%, 16;71%);
(iii) Ac2O, Py (17;76%, 18;78%, 19;61%);(iv) TFA:DCM (1:1) 1.5 h ( 20;92%, 21;91%, 22;92%).

B. Kellam et al. / Tetrahedron Letters 46 (2005) 1703–1706
1705
2).15 The b-carboxylic acid in 8 was first activated with
isobutyl chloroformate and then reduced with NaBH4
to yield Z-protected homoserine 9. Replacement of Z
with Dmc was achieved in one pot via hydrogenolysis
in EtOH followed by derivatisation with DMMD to
obtain 3 in high yield.
7. Atherton, E.;Bury, C.;Sheppard, R. C.;Williams, B. J.
Tetrahedron Lett. 1979, 20, 3041–3042.
8. Hietter, H.;Schultz, M.;Kunz, H. Synlett 1995, 1219–
1220.
9. Peters, S.;Lowary, T. L.;Hindsgaul, O.;Meldal, M.;
Bock, K. J. Chem. Soc., Perkin Trans. 1 1995, 3017–3022.
10. Bycroft, B. W.;Chan, W. C.;Chhabra, S. R.;Teesdale-
Spittle, P. H.;Hardy, P. M. J. Chem. Soc., Chem.
Commun. 1993, 777–778.
With the Dmc protected amino acid aglycones in hand,
glycosylation employing 2,3,4-tri-O-benzylfucopyranos-
yl bromide 1016 as the donor, alongside tetrabutylam-
monium bromide promoted in situ anomerisation,
afforded the desired a-glycosides 11–13 in extremely
rewarding yields (Scheme 3). Compared to the equiva-
lent procedures for Fmoc or Z-protected amino acids,
this high yielding glycosylation may be rationalised in
a manner akin to that described for glycosylation of
OꢀDonnell Schiff bases of serine and threonine.17 It
could be reasonably argued that the nucleophilicity of
the aglycones 1–3 may be enhanced due to the absence
of unfavourable intramolecular hydrogen bonding be-
tween the a-NH and side chain-OH groups commonly
observed with acyl or carbamoyl protected equivalents.
In both cases, confirmation of the desired a-linkage
was provided by the observed small coupling constants
11. Bycroft, B. W.;Chan, W. C.;Chhabra, S. R.;Hone, N. D.
J. Chem. Soc., Chem. Commun. 1993, 778–779.
12. (a) Na-Dmc-L-serine tert-butyl ester 1: mp 135–136 ꢁC; m/z
(+ES) 312 (M+1, 100%); mmax (KBr)/cmÀ1: 3426 (O–H, N–
H), 1735 (ester C@O), 1667 (amide C@O), 1606 (C@C);
25
½aꢀD +41.4 (c 0.1, MeOH); 1H NMR d (CDCl3): 0.85 (6H,
s, Dmc C(CH3)2), 1.34 (9H, s, C(CH3)3), 2.11, 2.13 (4H,
2 · s, Dmc 2 · CH2), 3.87 (2H, m, b-CH2), 4.06 (1H, m, a-
CH), 4.96 (1H, t, J 5.9, OH), 7.98 (1H, d, J 14.3, Dmc
C@CH), 11.19 (1H, dd, J 14.1, 8.2, NH); 13C NMR d
(CDCl3): 28.0 (C(C3)3), 28.2, 28.8 (Dmc C(CH3)2), 31.1 (C
(CH3)2), 51.0, 51.3 (Dmc 2 · CH2), 63.4 (b-CH2), 64.8 (a-
CH), 83.9 (C (CH3)3), 108.9 (Dmc C@CH), 158.0 (Dmc
C@CH), 167.1 (CO tert-butyl ester), 196.6, 199.3 (CO
dione);Found: C, 61.12;H, 8.09;N, 4.30. Calcd for
C16H25NO5Æ0.2H2O: C, 61.03;H, 8.14;N, 4.45;(b)
Na-
Dmc-L-threonine tert-butyl ester 2: mp 137–138 ꢁC; m/z
(+ES) 326 (M+1, 100%); mmax (KBr)/cmÀ1: 3423 (O–H, N–
H), 1736 (ester C@O), 1667 (amide C@O), 1605 (C@C);
(d, JH-1 ,H-2 = 3.3 Hz) in the 1H NMR spectra. Catalytic
hydrogenolysis of the glycosides gave the hydroxy sug-
ars 14–16 which were subsequently acetylated to afford
the fully protected adducts 17–19. Final acidolytic cleav-
age of the tert-butyl ester relinquished the desired build-
ing blocks 20–2218 suitable for SPPS.
0
0
25
½aꢀD +21.8 (c 1.0, MeOH); 1H NMR d (CDCl3): 1.04 (6H,
s, Dmc C(CH3)2), 1.28 (3H, d, J 6.4, CH3 Thr), 1.50 (9H,
s, C(CH3)3), 2.32, 2.35 (4H, 2 · s, Dmc 2 · CH2), 3.85 (1H,
dd, J 4.5, 9.3, a-CH), 4.28 (1H, m, b-CH), 8.01 (1H, d, J
14.1, Dmc C@CH), 11.29 (1H, dd, J 9.8, 13.8, NH); 13C
NMR d (CDCl3): 19.9 (CH3 Thr), 27.9 (C(CH3)3), 28.3,
28.7 (Dmc C(CH3)2), 31.1 (C (CH3)2), 51.0, 51.3 (Dmc
2 · CH2), 67.9 (b-CH), 69.2 (a-CH), 83.6 (C (CH3)3),
108.0 (Dmc C@CH), 158.6 (Dmc C@CH), 167.7 (CO tert
butyl ester), 196.6, 199.2 (CO dione);Found: C, 62.35;H,
8.36;N, 4.26. Calcd for C 17H27NO5: C, 62.75;H, 8.36;N,
4.30.
In conclusion we have described the synthesis of
Na-Dmc protected serine, threonine and homoserine
1-O-a-L-fucoside linked building blocks suitable for
Fmoc/tBu SPPS. The stability of Dmc to both hydro-
genolysis and acidolysis has been established and the
employment of these building blocks in the construction
of fucopeptide libraries is now ongoing and will be
reported in due course.
13. Thierry, J.;Yue, C.;Potier, P. Tetrahedron Lett. 1998, 39,
1557–1560.
14. Na-Dmc-L-homoserine tert-butyl ester 3: mp 150–152 ꢁC ;
Rf (acetone/pet.ether bp 40–60 ꢁC, 1:1) 0.41; m/z (+ES)
326.2 (M+H); mmax (KBr)/cmÀ1 3331 (OH), 1718 (ester
25
1
C@O); ½aꢀD + 3.02 (c 0.03, MeOH); H NMR d (CDCl3):
1.05 (6H, s, Dmc C(CH3)2), 1.49 (9H, s, C(CH3)3), 1.97
(1H, ddd, J 14.0, 9.3, 4.5, b-CHa), 2.22 (1H, ddd, J 14.4,
9.7, 4.9, b-CH), 2.34, 2.38 (each 2H, s, Dmc 2 · CH2), 3.19
(1H, m, CH2OH), 3.68 (1H, m, c-CHa), 3.79 (1H, m, c-
CHb), 4.29 (1H, m, J 8.9, 4.4, a-CH), 8.15 (1H, d, J 14.0,
Dmc C@CH), 11.34 (1H, dd, J 12.4, NH). dC (62.9 MHz)
27.94 (hSer-C(CH3)3), 28.50, 28.61 (Dmc (CH3)2), 31.12
(Dmc C(CH3)2), 35.33 (hSer b-CH2), 51.06, 51.42 (Dmc
2 · CH2), 57.52 (hSer c-CH2), 60.11 (hSer a-CH), 83.36
(hSer C(CH3)3), 107.94 (Dmc C@CH), 157.99 (Dmc
C@CH), 167.30 (CO tert butyl ester), 196.80, 199.18
(CO dione);Found: C, 62.46;H, 8.40;N, 4.34;
C17H27NO5 requires C, 62.75;H, 8.36;N, 4.30.
Acknowledgements
Alchemia Pty Ltd, Brisbane, Australia (P.W.) and the
Government of Malaysia (A.S.A.R.) are gratefully
acknowledged for financial assistance.
References and notes
1. (a) Arsequell, G.;Valencia, G. Tetrahedron: Asymmetry
1997, 8, 2839–2876;(b) Taylor, C. M. Tetrahedron 1998,
54, 11317–11362;(c) Arsequell, G.;Valencia, G. Tetra-
hedron: Asymmetry 1999, 10, 3045–3094.
2. (a) Dwek, R. A. Glycobiology 1993, 3, 97–130;(b) Dwek,
R. A. Chem. Rev. 1996, 96, 683–720.
15. Weitz, I. S.;Pellegrini, M.;Mierke, D. F.;Chorev, M.
J. Org. Chem. 1997, 62, 2527–2534.
3. Simanek, E. E.;McGarvey, G. J.;Jablonowski, J. A.;
Wong, C.-H. Chem. Rev. 1998, 98, 833–862.
4. Sawada, R.;Tsuboi, S.;Fukuda, M. J. Biol. Chem. 1994,
269, 1425–1431.
5. Harris, R. J.;Spellman, M. W. Glycobiology 1993, 3, 219–
224.
16. Dejter-Juszynski, M.;Flowers, H. M. Carbohydr. Res.
1971, 18, 219–226.
17. OꢀDonnell, M. J.;Polt, R. L. J. Org. Chem. 1982, 47,
2663–2666.
18. (a)
Na-Dmc-O-(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-
L-serine 20: mp 121–122 ꢁC; mmax (KBr)/cmÀ1: 1745 (ester
C@O), 1668 (amide C@O), 1601 (C@C); dH (250 MHz,
CDCl3): 1.05 (6H, s, Dmc (CH3)2), 1.11 (3H, d, J 6.3, Fuc
6. Elofsson, M.;Roy, S.;Salvador, L. A.;Kihlberg, J.
Tetrahedron Lett. 1996, 37, 7645–7648.

1706
B. Kellam et al. / Tetrahedron Letters 46 (2005) 1703–1706
CH3), 1.97, 2.11, 2.15 (9H, 3 · s, 3 · COCH3), 2.37 (4H, s,
(Fuc C2), 68.4 (Fuc C4), 69.8 (Thr b-C), 70.8 (Fuc C3),
93.2 (Fuc C1), 108.3 (Dmc C@CH), 158.9 (Dmc C@C H),
169.7, 170.1, 170.7 (3 · acetyl C@O), 170.9 (COOH),
196.3, 199.6 (Dmc dione);Found: C, 54.45;H, 6.59;N,
2.39;Calcd for C 25H35NO12: C, 54.45;H, 6.51;N, 2.59;(c)
Na-Dmc-O-(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-L-homo-
serine 22: Oil, mmax (KBr)/cmÀ1: 1744 (ester C@O), 1662
(amide C@O), 1601 (C@C); dH (400 MHz, CDCl3): 1.07
(6H, s, Dmc (CH3)2), 1.12 (3H, d, J 6.5, Fuc CH3), 2.00,
2.08, 2.18 (9H, 3 · s, 3 · COCH3), 2.26 (1H, m, hSer
b-CHa), 2.42 (4H, s, Dmc 2 · CH2), 2.46 (1H, m, hSer
b-CHb), 3.55 (1H, m, hSer c-CHa), 3.86 (1H, m, hSer c-
CHb), 4.10 (1H, q, J 6.8, Fuc H5), 4.39 (1H, m, hSer a-
CH), 5.05 (1H, d, J 3.5, Fuc H1), 5.13 (1H, dd, J 10.8, 3.6,
Fuc H3), 5.27 (1H, d, J 2.8, Fuc H4), 5.35 (1H, dd, J 10.8,
3.8, Fuc H2), 8.26 (1H, d, J 14.2, Dmc C@CH), 11.46
(1H, dd, J 13.8, 8.5, Dmc NH); dC (100.6 MHz, CDCl3):
15.82 (Fuc CH3), 20.68, 20.72, 20.81 (3 · acetyl CH3),
28.39, 28.52 (Dmc C(CH3)2), 31.33 (Dmc C (CH3)2),
32.18 (hSer b-CH), 51.25, 50.90 (Dmc 2 · CH2), 59.21
(hSer a-CH), 62.58 (hSer c-CH2), 65.14 (Fuc C4), 68.01
(Fuc C3), 68.10 (Fuc C2), 71.17 (Fuc C5), 96.34 (Fuc C1),
107.81 (Dmc C@CH), 158.80 (Dmc C@CH), 170.72,
170.91, 170.97 (3 · acetyl C@O), 171.64 (COOH),
198.43, 200.01 (Dmc dione); m/z HRMS (TOFES-)
540.2056 (MÀH). Calcd for C25H34NO12 (MÀH)
540.2080.
Dmc 2 · CH2), 3.77 (1H, m, Ser b-CHa), 4.08 (1H, q, J
6.3, Fuc H5), 4.25 (1H, dd, J 10.0, 4.0, Ser b-CHb), 4.42
(1H, m, Ser a-CH), 5.09–5.25 (4H, m, Fuc H1, H2, H3 and
H4), 8.23 (1H, d, J 14.0, Dmc C@CH), 11.45 (1H, dd, J
14.0, 8.0, Dmc NH); dC (62.9 MHz, CDCl3): 16.3 (Fuc
CH3), 20.20, 21.00, 21.20 (3 · acetyl CH3), 28.3, 28.6
(Dmc C(CH3)2), 31.07 (Dmc-C (CH3)2), 50.8, 51.2 (Dmc
2 · CH2), 62.44 (Fuc C5), 64.8 (Ser a-CH), 67.0 (Ser b-
CH2), 68.0 (Fuc C2), 68.2 (Fuc C4), 71.1 (Fuc C3), 96.2
(Fuc C1), 108.00 (Dmc C@CH), 158.9 (Dmc C@CH),
170.0,170.5, 171.4 ( 3 · acetyl C@O), 171.9 (COOH),
195.6, 198.8 (Dmc dione);Found: C, 54.60;H, 6.52;N,
2.43;Calcd for C 24H33NO12: C, 54.64;H, 6.31;N, 2.66;(b)
Na-Dmc-O-(2,3,4-tri-O-acetyl-a- L-fucopyranosyl)- L-threo-
nine 21: mp 129–130 ꢁC; mmax (KBr)/cmÀ1: 1745 (ester
C@O), 1668 (amide C@O), 1601 (C@C); dH (250 MHz,
CDCl3): 1.06 (6H, s, Dmc (CH3)2), 1.08 (3H, d, J 6.3, Fuc
CH3), 1.19 (3H, d, J 6.1, Thr CH3), 1.98, 2.12, 2.14 (9H,
3 · s, 3 · COCH3), 2.39 (4H, s, Dmc 2 · CH2), 4.14 (2H,
m, Thr a-CH and Fuc H5), 4.49 (1H, m, Thr b-CH), 5.05
(1H, dd, J 10.5, 3.5, Fuc H3), 5.24 (3H, m, Fuc H1, H2,
and H4), 8.19 (1H, d, J 14.3, Dmc C@CH), 11.33 (1H, dd,
J 12.4, 8.8, Dmc NH); dC (62.9 MHz, CDCl3): 15.3 (Fuc
CH3), 15.8 (Thr CH3), 20.30, 20.60, 20.8 (3 · acetyl CH3),
28.2, 28.4 (Dmc C(CH3)2), 31.2 (Dmc C(CH3)2), 51.3, 51.7
(Dmc 2 · CH2), 65.2 (Fuc C5), 65.9 (Thr a-CH), 68.0