Synthesis of novel amino acid carbohydrate hybrids via Mitsunobu glycosylation of nitrobenzenesulfonamides

Article abstract of DOI:10.1016/S0040-4039(01)01062-0

Amino acid derived 2-nitrobenzenesulfonamides were successfully condensed under Mitsunobu conditions with the primary alcohol of various saccharide and nucleoside derivatives to furnish the fully protected hybrids in excellent yield. One such hybrid was i

Full text of DOI:10.1016/S0040-4039(01)01062-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 5763–5767
Synthesis of novel amino acid carbohydrate hybrids via
Mitsunobu glycosylation of nitrobenzenesulfonamides
John J. Turner, Dmitri V. Filippov, Mark Overhand, Gijs A. van der Marel and
Jacques H. van Boom*
Leiden Institute of Chemistry, Gorlaeus Laboratories, PO Box 9502, 2300 RA Leiden, The Netherlands
Received 17 May 2001; accepted 14 June 2001
Abstract—Amino acid derived 2-nitrobenzenesulfonamides were successfully condensed under Mitsunobu conditions with the
primary alcohol of various saccharide and nucleoside derivatives to furnish the fully protected hybrids in excellent yield. One such
hybrid was incorporated into a short peptide sequence in good overall yield. © 2001 Elsevier Science Ltd. All rights reserved.
The most abundant post-translational modification of
polypeptides is glycosylation, where carbohydrates are
covalently attached to specific amino acid residues.1
Such modifications impart structural diversity which
can regulate activity and facilitate involvement in bio-
logical recognition events such as cell adhesion and
infection.2 Consequently, many glycosylated oligopep-
tides have been synthesised in the last couple of
decades2,3 with derivatives that deviate from the natu-
rally occurring N- or O-glycosidic linkage becoming
increasingly widespread as a result of efforts directed
towards enhancing enzymatic stability.4 Furthermore,
the properties and/or conformation of biologically
active peptide sequences have been modified by the
synthesis and incorporation of glycosylated amino acids
with unnatural linkages.5,6 For instance, it was recently
reported that the attachment of a carbohydrate core
onto a cyclic RGD containing pentapeptide resulted in
compounds with improved potency, selectivity and bet-
ter pharmacokinetic properties.6 The cyclic RGD glyco-
peptide derivatives synthesised either possessed a carbo-
hydrate core attached to the side-chain of an amino
acid or one affixed to the peptide backbone via an
a-amino functionality. This latter modification is partic-
ularly interesting because (a)N-alkylated peptides have
been shown to be more resistant to enzymatic
degradation7 however to date, only (a)N-glycosylated
glycine residues have been successfully incorporated
into peptide sequences.5b,6 Moreover, (a)N-alkylated
amino acids in general are often problematic with
respect to their full incorporation (i.e. amino acid
residues connected at both termini to backbone), fre-
quently giving poor yields when extending from the
N-terminus.8
Two years ago, we disclosed a novel route towards the
synthesis of Amadori compounds by the Mitsunobu
glycosylation of amino acid derived ortho-
nitrobenzenesulfonamides9 (path A, Scheme 1). It
occurred to us that successful transference of this
methodology from the anomeric to the primary alcohol
of carbohydrate derivatives would provide glycosylated
amino acids with enzymatically stable artificial linkages
Scheme 1.
Keywords: Mitsunobu glycosylation; nitrobenzenesulfonamides; amino acids; carbohydrates; nucleosides.
* Corresponding author. Tel.: +31 71 5274274; fax: +31 71 5274307; e-mail: j.boom@chem.leidenuniv.nl
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01062-0

5764
J. J. Turner et al. / Tetrahedron Letters 42 (2001) 5763–5767
(path B). Due to the fact that the side-chain glycosyla-
tion of an amino acid does not significantly affect its
incorporation into peptide sequences,2 modifications of
this type were attempted first. To this end, 2,3,4-tri-O-
side-chain glycosylated amino acid 3 in excellent yield
(entry 1, Table 1).12 A more complex example, that of
ornithine/uridine conjugate 5 was also obtained in a
similar yield by reacting uridine derivative 4 with 2
(entry 2). In this case, the pivaloyloxymethyl (POM)
protecting group on N3 proved to be essential for the
prevention of known intramolecular cyclisation.13 Hav-
benzyl-a-
with Na-benzyloxycarbonyl-Nd-(2-nitrobenzenesulfonyl)-
-ornithine tert-butyl ester 211 furnishing the desired
D
-methyl-glucopyranose 110 was condensed
L
Table 1. Mitsunobu condensation of carbohydrates and nucleosides with amino acids

J. J. Turner et al. / Tetrahedron Letters 42 (2001) 5763–5767
5765
ing substrates with side-chain modifications in hand,
attention was turned to the synthesis of (a)N-alkylated
amino acids. Thus, 1 was reacted with ortho-nitroben-
zenesulfonamide derived phenylalanine tert-butyl esters
nishing glycine/guanosine conjugate 18 in excellent
yield.
As previously mentioned, the side-chain attachment of
carbohydrate moieties is unlikely to affect the full
incorporation of an amino acid into a peptide sequence
but (a)N-alkylation often does. Therefore, it was con-
sidered necessary to investigate the assimilation of an
example of the latter. To keep the synthetic route as
concise as possible, the feasibility of performing cou-
pling reactions using the ortho-nitrobenzenesulfonyl
group as a replacement for a urethane protecting group
was investigated.
6a and 6b and the corresponding
L-glutamic di-tert-
butyl ester 7 providing 8a, 8b and 9, respectively, in
excellent yields (entry 3). The more sterically hindered
2,3,4-tri-O-benzyl-a- -methyl-galactopyranose 10 was
D
more sluggish in its reaction with 6a. However,
phenyalanine/galactose conjugate 11 was obtained in
near quantitative yield when the mixture was heated
under reflux (entry 4). Uridine derivative 4 again pro-
vided no problems, smoothly reacting with 6a to fur-
nish the desired product 12 (entry 5). To examine
further the scope of this methodology, ethyl 2,3,4-tri-O-
To this end, both
gates 8a and 8b were treated with neat TFA under dry
conditions followed by coupling of -alanine benzyl
D and L phenylalanine/glucose conju-
benzyl-1-thio-b- -glucopyranose 13 was successfully
D
condensed with alanine derivative 14 to give the
thioethyl donor 15 in 92% yield (entry 6). The thioethyl
functionality of 15 should facilitate a variety of trans-
formations at the anomeric centre. Finally, the ability
to form (a)N-guanosylated amino acids is also demon-
strated (entry 7). Synthetic transformations on
guanosine are notoriously difficult, however the use of
the N,N-dimethylformamidine (dmf) protecting group
for N2 of the nucleobase has previously been shown to
prevent intramolecular condensation under Mitsunobu
conditions.14 Investigations revealed that the secondary
hydroxyl protecting groups employed also play a vital
role in the success of the reaction. For instance, the use
of conformationally constrained isopropylidene diol
protection only yielded intramolecular condensation,15
whereas isobutyryl ester masking of the secondary
hydroxyls allowed intermolecular condensation, fur-
L
ester under the agency of EDC/HOBt/DIPEA to give
the coupled products 20a and 20b in good yield as
depicted in Scheme 2. Comparison of both products
using HPLC revealed that the coupling did not lead to
racemisation.16 In order to fully incorporate the
D-
phenylalanine/glucose conjugate into
a
peptide
sequence, dipeptide 20b was subjected to ortho-
nitrobenzenesulfonyl cleavage conditions9 followed by
coupling of Fmoc protected L-alanine acid chloride in
the presence of 2,6-tert-dibutylpyridine17 to give fully
protected tripeptide 22 in a gratifying 92% yield over
the 2 steps. It should be noted that attempted coupling
of Fmoc-Ala-OH employing established peptide cou-
pling reagents was wholly unsuccessful with no product
formation observed.18
Scheme 2.

5766
J. J. Turner et al. / Tetrahedron Letters 42 (2001) 5763–5767
Having established that the conjugate could be coupled
at both the amino and the acid functionalities we
wished to confirm that standard coupling protocols
could be used again. Therefore, fully protected tripep-
tide 22 was subjected to Fmoc cleavage followed by
coupling of Fmoc protected glycine in a one-pot proce-
dure furnishing the fully protected tetrapeptide 24 in
83% yield. The necessity for the one-pot procedure
arises from the observation that the product resulting
from cleavage of the Fmoc group, slowly forms diketo-
piperazine 23 upon standing. Finally, removal of the
protecting groups of 24 gave the requisite free tetra-
peptide 25 in 75% isolated yield.
35, 20–32; (b) Kessler, H. Angew. Chem., Int. Ed. Engl.
1993, 32, 543.
8. (a) Byk, G.; Scherman, D. Int. J. Pept. Protein Res. 1996,
47, 333–339; (b) Reichwein, J. F.; Liskamp, R. M. J.
Tetrahedron Lett. 1998, 39, 1243–1246 and references
cited therein.
9. Turner, J. J.; Wilschut, N.; Overkleeft, H. S.; Klaffke,
W.; van der Marel, G. A.; van Boom, J. H. Tetrahedron
Lett. 1999, 40, 7039–7042.
10. Compound 1 was synthesised according to Sahai, P.;
Chawla, M.; Vishwakarma, R. A. J. Chem. Soc. Perkin
Trans. 1 2000, 1283–1290. Compound 10 was synthesised
in the same manner. Compound 13 was synthesised
according to Ray, A. K.; Roy, N. Carbohydr. Res 1990,
196, 95–100. Compound 4 was synthesised from O2%,O3%-
isopropylidene-uridine10b utilising standard POM func-
tionalisation conditions according to Brown, J. M.;
Cristodoulou, C.; Jones, S. S.; Modak, A. S.; Reese, C.
B.; Sibanda, S.; Ubusawa, A. J. Chem. Soc. Perkin Trans.
1 1989, 1735–1753. Guanosine derivative 16 was prepared
in 3 steps from dmf protected guanosine10c according to
Scheme 3; (b) Yamane, A.; Inoue, H.; Ueda, T. Chem.
Pharm. Bull 1980, 28, 157–162; (c) Grøtli, M.; Douglas,
M.; Beijer, B.; Garc´ıa, R. G.; Eritja, R; Sproat, B. J.
Chem. Soc. Perkin Trans. 1 1997, 2779–2788.
In summary, we have presented an efficient and facile
method for the synthesis of novel amino acid/carbohy-
drate and amino acid/nucleoside conjugates and fully
incorporated an (a)N-glycated amino acid into a short
peptide sequence in good overall yield. In addition, it
has been shown that the ortho-nitrobenzenesulfonyl
group can be considered as a potential replacement for
the urethane group in peptide synthesis. Finally, the
coupling of hindered secondary amines was also shown
to proceed in excellent yield utilising an Fmoc amino
acid chloride in conjunction with a weak non-nucleo-
philic base.
Acknowledgements
This work was financially supported by Unilever. The
authors would like to thank Fons Lefeber and Kees
Erkelens for recording NMR spectra, Hans van den
Elst for performing the mass spectrometric analyses
and Nico Meeuwenoord for conducting HPLC
analyses.
Scheme 3.
11. (a) Amino acid derived ortho-nitrobenzenesulfonamides
6, 7 and 14 were synthesised as reported previously.9
Compound 17 was obtained by reacting glycine methyl
ester hydrochloride with ortho-nitrobenzenesulfonyl chlo-
ride under Schotten–Baumann conditions. Compound 2
was synthesised by tert-butyl protection11b of the corre-
sponding free carboxylic acid.11c (b) Callahan, F. M.;
Anderson, G. W.; Paul, R.; Zimmerman, J. E. J. Am.
Chem. Soc. 1963, 85, 201–207; (c) Buijsman, R.; PhD
Thesis, Leiden 1998.
References
1. Glycoproteins; Gottschalk, A.; Ed.; Elsevier, 1966.
2. Seitz, O.; Heinemann, I.; Mattes, A.; Waldmann, H.
Tetrahedron 2001, 57, 2247–2277 and references cited
therein.
3. (a) Arsequell, G.; Valencia, G. Tetrahedron: Asymmetry
1999, 10, 3045–3094; (b) Arsequell, G.; Valencia, G.
Tetrahedron: Asymmetry 1997, 8, 2839–2876.
4. (a) Dondoni, A. Pure Appl. Chem. 2000, 72, 1577–1588;
(b) Dondoni, A.; Marra, A. Chem. Rev. 2000, 100, 4395–
4421.
12. All new compounds were fully characterised by 1H and
13C NMR spectroscopy as well as mass spectrometry.
Data for selected examples is as follows. 5: 1H NMR (300
MHz, CDCl3): l 8.00 (m, 1H, oNs), 7.67–7.43 (m, 3H,
&
5. (a) Horvat, S.; Horvat, J.; Varga–Defterdarovic, L.;
Pavelic, K.; Chung, N. N.; Schiller, P. W. Int. J. Peptide.
Protein Res. 1993, 41, 399–404; (b) Cudic, M.; Horvat, J.;
Elofsson, M.; Bergquist, K.-E.; Kihlberg, J.; Horvat, S. J.
Chem. Soc., Perkin Trans. 1 1998, 1789–1795 and refer-
ences cited therein.
oNs), 7.40–7.29 (m, 5Harom.), 7.18 (d, 1H, H5 J5,6=8.1
Hz), 5.90 (AB, 2H, CH2 Z, J=9.5 Hz), 5.74 (d, 1H, H6),
5.52 (app. s, 1H, H1%), 5.35 (d, 1H, NH, JNH,a=7.9 Hz),
5.09 (s, 2H, POM), 5.00 (app. d, 1H, H2%), 4.76–4.73 (m,
1H, H3%), 4.33–4.30 (m, 1H, H4%), 4.28–4.08 (m, 1H, Ha,),
3.59 (m, 2H, H5%), 3.44–3.25 (m, 2H, Hd), 1.86–1.55 (m,
4H, 2×Hb and 2×Hg), 1.52 and 1.32 (2s, 2×3H, CH3
isopropyl), 1.43 and 1.18 (2s, 2×9H, CH3 tBu); 13C NMR
(75 MHz, CDCl3): l 177.4 (CꢀO POM), 171.0 (CꢀO ester
Orn), 161.4 (C4), 155.8 (NH-CꢀO, Z), 150.2 (C2), 147.2
(C-NO2), 141.7 (C5), 136.2 (Cq arom., Z), 133.5 (CHarom.
&
6. Lohof, E.; Planker, E.; Mang, C.; Burkhart, F.;
Dechantsreiter, M. A.; Haubner, R.; Wester, H.-J.;
Schwaiger, M.; Ho¨lzemann, G.; Goodman, S. L.; Kessler,
H. Angew. Chem., Int. Ed. 2000, 39, 2761–2764.
7. (a) Miller, S. M.; Simon, R. J.; Ng, S.; Zuckerman, R.
N.; Kerr, J. M.; Moos, W. H. Drug Develop. Res. 1995,

J. J. Turner et al. / Tetrahedron Letters 42 (2001) 5763–5767
5767
oNs), 133.2 (C-SO2), 131.5, 131.1 (2×CHarom. oNs),
128.5–128.1 (CHarom. Z), 124.2 (CHarom. oNs), 114.5 (Cq
isopropyl), 102.2 (C6), 96.2 (C1%), 85.8 (C4%), 84.2 (C2%),
82.4 (C3%), 81.6 (Cq tBu ester Orn), 66.9 (CH2 POM), 64.6
(CH2 Z), 53.7 (Ca), 49.6 (C5%), 47.9 (Cd), 38.8 (Cq tBu
POM), 29.7 (Cb), 27.9, 26.9 (2×CH3 tBu), 26.9, 25.1
(2×CH3 isopropyl), 23.7 (Cg); MS (ESI): m/z 910.3 [M+
Na]+. 11: 1H NMR (300 MHz, CDCl3): l 7.83 (m, 1H,
oNs), 7.61–7.53 (m, 2H, oNs), 7.47–7.16 (m, 21H:
15Harom. Bn, 5Harom. Phe, 1H oNs), 4.87 (AB, 2H, CH2
Bn), 4.81 (AB, 2H, CH2 Bn), 4.74 (AB, 2H, CH2 Bn),
4.66 (m, 1H, Ha Phe), 4.49 (d, 1H, H1, J1,2=3.4 Hz),
(m, 1H, Ha Ala2), 4.43 (d, 1H, H1, J1,2=3.4 Hz), 4.34–
4.27 (m, 2H, CH2 Fmoc), 4.20–4.18 (m, 1H, CH Fmoc),
4.11–4.09 (m, 1H, 1×H6), 3.92 (app. t, 1H, H3), 3.68–3.65
(m, 1H, H5), 3.46–3.44 (m, 2H, H2, 1×Hb Phe), 3.16–3.14
(m, 1H, 1×Hb Phe), 3.08 (dd, 1H, H4, J=9.8 Hz, J=8.9
Hz), 2.95 (s, 3H, OMe), 2.95–2.92 (m, 1H, 1×H6), 1.32 (d,
3H, Hb Ala2, Jb,a=7.2 Hz), 1.14 (d, 3H, Hb Ala1, Jb,a
=
6.7 Hz). 13C NMR (150 MHz, CDCl3): l 175.6, 172.5,
169.6 (3×CꢀO), 155.7 (NH-CꢀO, Fmoc), 143.7, 143.6,
141.2, 141.2 (4×Cq Fmoc), 138.6, 138.0, 137.9, 137.8
(4×Cq Bn), 135.4 (Cq arom., Phe), 128.7–125.0 (CHarom.
Fmoc, Bn, Phe), 97.6 (C1), 81.6 (C3), 79.8 (C2), 79.5 (C4),
75.5, 75.0, 73.2 (3×CH2 Bn), 69.9 (C5), 67.0 (1×CH2 Bn),
66.8 (CH2 Fmoc), 60.7 (Ca Phe), 55.3 (OMe), 48.5 (C6),
48.4 (Ca Ala1), 47.0 (Ca Ala2), 46.9 (CH Fmoc), 34.1 (Cb
Phe), 18.3 (Cb Ala2), 17.7 (Cb Ala1). MS (ESI): m/z
4.02–3.96 (m, 3H, H2, H4, H5), 3.89 (dd, 1H, H3, J2,3
=
10.1 Hz, J3,4=2.1 Hz), 3.66 (ABX, 2H, H6), 3.25 (s, 3H,
t
OMe), 3.15 (ABX, 2H, Hb Phe), 1.23 (s, 9H, Bu); 13C
NMR (75 MHz, CDCl3): l 168.9 (CꢀO), 148.1 (C-NO2),
138.6, 138.6, 138.4 (3×Cq Bn), 136.7 (Cq arom., Phe), 133.5
(C-SO2), 133.3, 131.5, 131.4 (3×CHarom. oNs), 129.1–
126.8 (CHarom. Bn), 123.8 (1×CHarom. oNs), 98.9 (C1),
82.2 (Cq tBu), 79.2 (C3), 75.9, 75.3 (C2/C4), 74.4, 73.5,
73.2 (3×CH2 Bn), 69.7 (C5), 62.9 (Ca Phe), 55.7 (OMe),
47.9 (C6), 36.9 (Cb Phe), 27.6 (CH3 tBu); MS (ESI): m/z
1
1088.8 [M+Na]+. 25: H NMR (600 MHz, D2O): d 7.35–
7.24 (m, 5H, Harom. Phe), 5.08 (q, 1H, Ha Ala1, Ja,b=6.9
Hz), 4.93 (app. t, 1H, Ha Phe), 4.63 (d, 1H, H1, J1,2=3.7
Hz), 4.17 (q, 1H, HaAla2, Ja,b=7.1 Hz), 3.91 (app. d, 1H,
1×H6), 3.78 (s, 2H, Hb Gly), 3.66 (app. t, 1H, H5), 3.56
(app. t, 1H, H3), 3.45 (dd, 1H, H2, J2,3=9.8 Hz), 3.29–
3.16 (m, 7H, OMe, 2Hb Phe, H4 and 1×H6), 1.27 (d, 3H,
Hb Ala2), 1.04 (d, 3H, Hb Ala1); 13C NMR (150 MHz,
D2O): d 177.8, 176.0, 170.5, 165.6 (4×CꢀO), 136.7 (Cq
arom., Phe), 129.1, 128.8, 127.0 (CHarom. Phe), 99.1 (C1),
72.8 (C3), 71.5 (C4), 71.0 (C2), 70.2 (C5), 61.4 (Ca Phe),
55.3 (OMe), 50.0 (Ca Ala2), 47.5 (C6), 46.4 (Ca Ala1), 40.2
(Cb Gly), 34.0 (Cb Phe), 17.4 (Cb Ala2), 17.0 (Cb Ala1);
MS (ESI): m/z 541.3 [M+H]+.
1
875.5 [M+Na]+. 18: H NMR (300 MHz, CDCl3): l 9.76
(bs, 1H, H1), 8.90 (s, 1H, NꢀC-H dmf), 7.92 (m, 1H,
1×oNs), 7.78–7.51 (m, 4H, 3×oNs, H8), 6.16 (dd, 1H, H3%,
J2%,3%=5.9Hz, J3%,4%=9.2Hz), 6.03 (dd, 1H, H2%, J1%,2%=1.3
Hz), 5.73 (d, 1H, H1%), 4.36 (dt, 1H, H4%, J4%,5%=3.3 Hz),
4.15 (AB, 2H, CH2 Gly), 3.87 (d, 2H, H5%), 3.48 (s, 3H,
OMe), 3.22 and 3.12 (2s, 2×3H, 2×CH3 dmf), 2.68–2.57
(m, 2H, 2×CH isobutyryl), 1.28–1.06 (m, 12H, 4×CH3
isobutyryl). 13C NMR (75 MHz, CDCl3): l 176.0, 175.4
(2×CꢀO isobutyryl ester), 168.8 (CꢀO methyl ester), 159.2
(NꢀCH dmf), 158.0 (C6), 157.2 (C2), 149.5 (C4), 147.8
(C-NO2), 138.3 (C8), 133.7 (CHarom. oNs), 132.5 (C-SO2),
131.5, 130.5, 123.9 (3×CHarom. oNs), 120.9 (C5), 88.7
(C1%), 79.6 (C4%), 73.0 (C2%), 69.4 (C3%), 51.9 (OMe), 48.8
(CH2 Gly), 46.8 (C5%), 41.1, 35.3 (2×CH3 dmf), 33.6, 33.4
(2×CH isobutyryl), 18.7, 18.5 (2×CH3 isobutyryl); MS
(ESI): m/z 735.4 [M+H]+. 22: 1H NMR (600 MHz,
CDCl3): l 7.77–7.75 (m, 2H, 2×Harom. Fmoc), 7.58–7.55
(m, 2H, 2×Harom. Fmoc), 7.41–7.37 (m, 2H, 2×Harom.
Fmoc), 7.33–7.10 (m, 28H: 2×Harom. Fmoc, 20×Harom.
Bn, 5×Harom. Phe, NH Ala2), 5.48 (d, 1H, NH Ala1,
13. Mitsunobu, O. Synthesis 1981, 1–28.
14. Bhat, B.; Swayze, E. E.; Wheeler, P.; Dimock, S.; Per-
bost, M.; Sanghvi, Y. S. J. Org. Chem. 1996, 61, 8186–
8199.
15. As confirmed by mass spectrometry.
16. HPLC detection limit >95%.
17. (a) The use of DIPEA led to rapid decomposition of
Fmoc-Ala-Cl. The amino acid chloride was synthesised as
described by Carpino, L. A.; Cohen, B. J.; Stephens, K.
E., Jr.; Sadat-Aalaee, S.-Y.; Tien, J.-H.; Langridge, D. C.
J. Org. Chem. 1986, 51, 3732–3734; (b) partial racemisa-
tion (5%) of the amino acid chloride was prevented by
starting the reaction at −30°C, instead of at room temper-
ature, and allowing the mixture to warm up after a 30
min period.
JNH,a=6.9 Hz), 5.14 (m, 1H, Ha Phe), 5.08 (AB, 2H, CH2
Bn), 4.86 (AB, 2H, CH2 Bn), 4.70 (AB, 2H, CH2 Bn),
4.70 (m, 1H, Ha Ala1), 4.65 (AB, 2H, CH2 Bn), 4.54–4.52
18. For example: BOP/HOBt, EDC/HOBt, HATU.
.