Orthogonally protected cyclo-β-tetrapeptides as solid-supported scaffolds for the synthesis of glycoclusters

Article abstract of DOI:10.1021/jo052348o

Two novel peptide scaffolds, viz. cyclo[(N^α-Alloc)Dpr- β-Ala-(N^α-Fmoc)Dpr-β-Ala] (1) and cyclo[(N ^α-Alloc)Dpr-α-azido-β-aminopropanoyl-(N ^α-Fmoc)Dpr-/β-Ala] (2), composed of orthogonally protected 2,3-diaminopro

Full text of DOI:10.1021/jo052348o

Orthogonally Protected Cyclo-â-tetrapeptides as Solid-Supported
Scaffolds for the Synthesis of Glycoclusters
Pasi Virta,* Marika Karskela, and Harri Lo¨nnberg
Department of Chemistry, UniVersity of Turku, FIN-20014 Turku, Finland
pasi.Virta@utu.fi
ReceiVed NoVember 14, 2005
Two novel peptide scaffolds, viz. cyclo[(NR-Alloc)Dpr-â-Ala-(NR-Fmoc)Dpr-â-Ala] (1) and cyclo[(NR-
Alloc)Dpr-R-azido-â-aminopropanoyl-(NR-Fmoc)Dpr-â-Ala] (2), composed of orthogonally protected 2,3-
diaminopropanoyl (Dpr) and â-alanyl residues, have been described. Fmoc chemistry on a backbone
amide linker derivatized resin has been used for the chain assembly. Selective removal of the 4-methyltrityl
(Mtt) and 1-methyl-1-phenylethyl protections (PhiPr) exposes the â-amino and carboxyl terminus,
respectively, and on-resin cyclization then gives the desired orthogonally protected cyclo-â-tetrapeptides
(1 and 2). The R-amino groups, bearing the Fmoc and Alloc protections and the azide mask, allow stepwise
orthogonal derivatization of these solid-supported cyclo-â-tetrapeptide cores (1 and 2). This has been
demonstrated by attachments of various sugar units [viz., acetyl- or toluoyl-protected carboxymethyl
R-D-glycopyranosides (13-15) and methyl 6-O-(4-nitrophenoxycarbonyl)-R-D-glycopyranosides (22-
24)] to obtain diverse di- and trivalent glycoclusters (33-42). Acidolytic release (TFA) from the support,
followed by conventional NaOMe-catalyzed transesterification (33-40) or hydrazine-induced acyl
substitution in DMF (41 and 42), gives the fully deprotected clusters (43-52) as final products.
Introduction
where the folding of native proteins has been mimicked by
appropriately aligned amphiphilic units of peptide secondary
structure. The scaffold used in these studies has been a
decapeptide, cyclo(KXKPGKXKPG) (X ) a variable amino
acid residue), incorporating two linked â-turns. Consequently,
the lysine side chains used for attachment of the peptide
branches are orientated to the same face of the peptide ring.
Small cyclic peptides have been used as scaffolds in the
construction of template assembled synthetic proteins (TASP),1-4
(1) Abbreviations used: Alloc, allyloxycarbonyl; DCA, dicloroacetic
acid; DCM, dichloromethane, DIEA, N,N-diisopropylethylamine; Fmoc,
9-fluorenylmethoxycarbonyl; FmocOSu, N-(9-fluorenylmethoxycarbonyl-
oxy)succinimide; HATU, N-[(1H-1,2,3-triazolo[4,5-b]pyridin-1-yloxy)(di-
methylamino)methylene]-N-methylmethanaminium hexafluorophosphate;
HFIP, 1,1,1,3,3,3-hexafluoro-isopropyl alcohol; HOBt, 1-hydroxybenzo-
triazole; Mtt, 4-methyltrityl; NMP, N-methylpyrrolidinone; Dpr, 2,3-
diaminopropanoyl; PhiPr, 1-methyl-phenylethyl; Py, pyridine; PyAOP,
7-azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophos-
phate; PyBOP, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluo-
rophosphate; RP, reverse phase; SPPS, solid-phase peptide synthesis; SPS,
solid-phase synthesis; TASP, template assembled synthetic proteins; TBTU,
1-[bis(dimethylamino)methylene]-1H-benzotriazolium tetrafluoroborate 3-ox-
ide.
The same decapeptide has also been utilized in synthesis of
5-7
glycoclusters,
in which multiple carbohydrate recognition
elements form a well-defined construct and offer a potential
(3) Dumy, P.; Egglestom, I. M.; Cervigni, S.; Sila, U.; Sun, X.; Mutter,
M. Tetrahedron Lett. 1995, 36, 1255.
(4) Xu, Q.; Borremans, F.; Devreese, B. Tetrahedron Lett. 2001, 42, 7261.
(5) Renaudet, O.; Dumy, P. Org. Lett. 2003, 5, 243.
(6) Singh, Y.; Renaudet, O.; Defrancq, E.; Dumy, P. Org. Lett. 2005, 7,
1359.
(2) Mutter, M.; Altman, K.-H.; Tuchscherer, G.; Vuilleumier, S. Tetra-
hedron 1988, 44, 771.
(7) Renaudet, O.; Dumy, P. Bioorg. Med. Chem. Lett. 2005, 15, 3619.
10.1021/jo052348o CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/31/2006
J. Org. Chem. 2006, 71, 1989-1999
1989

Virta et al.
FIGURE 2. 2,3-Diaminopropionic acid derivatives (3-5) used for the
syntheses of the scaffolds (1 and 2).
FIGURE 1. Solid-supported scaffolds 1 and 2.
SCHEME 1. Synthesis of Branching Unit 5a
binding site for lectins. Useful tools for the cell-specific targeting
or artificial receptors for soluble lectins have, hence, been
obtained.
Small cyclic â-peptides (trimers and tetramers) exhibit
interesting conformational properties8-17 that make them at-
tractive scaffolds. The additional methylene group between the
amino and carboxy function in each â-amino acid residue
orientates the amide moieties in a unidirectional manner, with
NH and CO groups lying on opposite faces of the â-peptide
ring. When the cycle is composed of homochiral residues, the
structure adopts a disklike conformation with amino acid side
chains orientating in an equatorial manner on the exterior of
the peptide ring, the axial and interior positions remaining
unobstructed.13 For one cyclo-â-tetrapeptide, a dissenting
conformation in solution has been reported by Seebach and co-
workers.15 There, a transannular H-bond bisects the macrocyclic
16-membered ring to 10- and 12-membered H-bonded rings.
In addition, a homochiral cyclo-â-tetrapeptide may adopt a boat-
shaped structure in solution, which fluctuates with the disklike
conformation.17 The appropriately orientated NH and CO groups
in the disklike conformation is an underlying feature of cyclo-
â-peptide structure that leads to vertical stacking in the solid
state via backbone-backbone hydrogen bonding and, hence, to
formation of nanotubular structures.10,13 While this stacking
undeniably is an interesting phenomenon, it at the same time
limits the use of cyclic â-peptides as scaffolds, since problems
associated with the synthesis of the so-called difficult sequences
may arise. Royo et al. used solid-supported cyclo[(NR-Alloc)-
Dpr-â-Ala-(NR-Alloc)Dpr-â-Ala] as a scaffold for a receptor
recognition motif and showed it to be successfully derivatized
with the RGD sequences.18 We have utilized the same backbone
in the present study by describing two novel orthogonally
protected solid-supported cyclo-â-tetrapeptides, viz. cyclo[(NR-
a Reagents and conditions: (i) [bis(trifluoroacetoxy)iodo]benzene, DIEA,
H2O, DMF; (ii) FmocOSu, DIEA, H2O, acetonitrile.
SCHEME 2. Synthesis of PhiPr-Protected â-Alanine 10a
a Reagents and conditions: (i) 1-methyl-1-phenylethyl trichloroacetimi-
date, DCM; (ii) Et2NH, DCM.
Alloc)Dpr-â-Ala-(NR-Fmoc)Dpr-â-Ala] (1) and cyclo[(NR-Al-
loc)Dpr-R-azido-â-aminopropanoyl-(NR-Fmoc)Dpr-â-Ala] (2)
(Figure 1). The applicability of scaffolds 1 and 2 was demon-
strated by the synthesis of 10 diverse di- and trivalent glyco
clusters (33-42), in which the sugar units were attached to the
cyclo-â-tetrapeptide cores by carbamate or amide couplings.
Whereas the previously reported peptide scaffolds have either
been derivatized in solution3-7 or had only several similar units
attached,18 the present methodology allows construction of
conjugates containing two or three different sugars and the
synthesis starting from monomeric constituents can be carried
out entirely on a single solid support.
(8) White, D. N. J.; Morrow, C. J. Chem. Soc., Perkin Trans. 2 1982,
239.
(9) Seebach, D.; Overhand, M.; Ku¨hnle, F. N. M.; Martinoni, B. HelV.
Chim. Acta 1996, 79, 913.
(10) Seebach, D.; Matthews, J.; Meden, A.; Wessels, T.; Baerlocher, C.;
McCusker, L. B. HelV. Chim. Acta 1997, 80, 173.
(11) Matthews, J. L.; Overhand, M.; Ku¨hnle, F. N. M.; Ciceri, P. E.;
Seebach, D. Liebigs Ann./Recueil 1997, 1371.
(12) Matthews, J. L.; Gademann, Jaun, B.; Seebach, D. J. Chem. Soc.,
Perkin Trans. 1 1998, 3331.
(13) Clark, T. D.; Buehler, L. K.; Ghadiri, R. J. Am. Chem. Soc. 1998,
120, 651.
Results and Discussion
(14) Gademann, K.; Seebach, D. HelV. Chim. Acta 1999, 82, 957.
(15) Gademann, K.; Ernst, M.; Seebach, D. HelV. Chim. Acta 2000, 83,
16.
(16) Gademann, K.; Seebach, D. HelV. Chim. Acta 2001, 84, 2924.
(17) Bu¨ttner, F.; Norgren, A. S.; Zhang, S.; Prabpai, S.; Kongsaeree, P.;
Arvidsson, P. I. Chem. Eur. J. 2005, 11, 6145.
(18) Royo, M.; Farrera-Sinfreu, J.; Sole´, L.; Albericio, F. Tetrahedron
Lett. 2002, 43, 2029.
Amino Acid Building Blocks (3-5 and 10). The diamino-
propanoic acid branching units (3-5), affording the shortest
possible orthogonally protected aminoalkyl sidearms to the
cyclo(tetra-â-peptide) cores, are depicted in Figure 2. N2-(9-
Fluorenylmethoxycarbonyl)-N3-(4-methyltrityl)-2,3-diaminopro-
panoic acid [Fmoc-Dpr(Mtt)-OH, 3] was commercially available
1990 J. Org. Chem., Vol. 71, No. 5, 2006

Cyclo-â-tetrapeptides as Solid-Supported Scaffolds
SCHEME 3. Sugar Units (13-15, 22-24) and Their Syntheses (13, 22-24)a
a Reagents and conditions: (i) (1) NaOMe, MeOH, (2) 4-toluoyl chloride, Py; (ii) NaIO4, RuCl3, DCM, MeCN, H2O; (iii) 4-nitrophenylchloroformate,
Py.
and N2-(allyloxycarbonyl)-N3-(9-fluorenylmethoxycarbonyl)-2,3-
diaminopropanoic acid [Alloc-Dpr(Fmoc)-OH] was synthesized
as described in the literature.19 To provide the scaffold (2) with
a third selectively exposable R-amino group, a novel 2,3-
diaminopropanoic acid precursor (5) was prepared, having an
azide mask at the R-position. This protection has recently been
shown to be an attractive choice when a high dimensional
orthogonal protection scheme is required.20,21 The synthesis of
this building block (5) is outlined in Scheme 1. The (2S)-4-
amino-2-azido-4-oxobutanoic acid (6)20 was first obtained in
71% yield from L-asparagine by a copper(II)-catalyzed diazo
transfer method.22 Hoffmann rearrangement of 6 using [bis-
(trifluoroacetoxy)iodo]benzene as an electrophile23,24 gave the
desired â-amino acid (7), which was subsequently protected with
N-(9-fluorenylmethoxycarbonyloxy)succinimide (FmocOSu). By
this simple route, the desired 2-azido-3-[(9-fluorenylmethyloxy-
carbonyl)amino]propanoic acid (5) was obtained in 31% overall
yield from L-asparagine.
however be exercized with this amino acid (5). In our
preliminary attempt, a remarkable 36% racemization of 5
occurred by using HATU25 activation in the presence of DIEA.
This problem was avoided by using collidine instead of DIEA
as a base.26 The coupling procedure (5 equiv of 5, 5 equiv of
HATU, 10 equiv of collidine in DMF, at 25 °C for 1 h) reduced
racemization to a negligible level and gave an acceptable 95%
coupling yield. Evidently the electron-withdrawing azido group
at the R-position is not the only reason for the facilitated
R-proton abstraction, but the presence of the â-carbamate proton
in 5 must somehow be involved in the racemization. Possibly
the â-carbamate proton may stabilize the enolate oxyanion by
intramolecular H-bonding.27
The 1-methyl-1-phenylethyl (PhiPr) protected â-alanine (10)
was synthesized from Fmoc-â-alanine (8) in two steps (Scheme
2). The carboxylic acid (8) was treated with 1-methyl-1-phenyl-
ethyl trichloroacetimidate28-30 to obtain the acid-labile ester (9),
and the Fmoc group was then removed by diethylamine, giving
the desired building block (10) in 79% overall yield.
Coupling of the R-azide masked amino acids has been
reported to take place without racemication.20 Care must
(25) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
(26) Carpino, L. A.; El-Faham, A.; Albericio, F. Tetrahedron Lett. 1994,
35, 2279.
(27) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches
to the Synthesis of Peptides and Proteins; New Directions in Organic and
Biological Chemistry; CRC: Boca Raton, FL, 1997; pp 115-116.
(28) Wessel, H.-P.; Iversen, T.; Bundle, D. R. J. Chem. Soc., Perkin
Trans. 1 1985, 2247
(19) Zhang, W.; Taylor, J. W. Tetrahedron Lett. 1996, 37, 2173.
(20) Lundquist, J. T., IV; Pelletier, J. C. Org. Lett. 2001, 3, 781.
(21) Virta, P.; Leppa¨nen, M.; Lo¨nnberg, H. J. Org. Chem. 2004, 69,
2008.
(22) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett. 1996,
37, 6029.
(23) Radhakrishna, A. S.; Parham, M. E.; Riggs, R. M.; Loudon, G. M.
J. Org. Chem. 1979, 44, 1746.
(24) Waki, M.; Kitajima, Y.; Izumya, N. Synthesis 1981, 266.
(29) Yue, C.; Thierry, J.; Potier, P. Tetrahedron Lett. 1993, 34, 323.
(30) Thierry, J.; Yue, C.; Potier, P. Tetrahedron Lett. 1998, 39, 1557.
J. Org. Chem, Vol. 71, No. 5, 2006 1991

Virta et al.
SCHEME 4. Synthesis of Solid-Supported Scaffolds 1 and 2a
a Reagents and conditions: (i) 10, NaCNBH3, AcOH, DMF; (ii) 4, PyAOP, collidine, DMF, DCM; (iii) piperidine, DMF; (iv) Fmoc-â-Ala-OH, HATU,
DIEA, DMF to obtain 27; (v) 5, HATU, collidine, DMF to obtain 28; (vi) 3, HATU, DIEA, DMF; (vii) (1) 1% TFA in DCM, 6 min, (2) neutralization with
Py, DCM; (viii) PyAOP, DIEA, DMF, 2 h; (ix) 50% TFA in DCM.
Synthesis of the Sugar Units (13-15 and 22-24, Scheme
3). For the synthesis of glycoconjugates, six different sugar units,
bearing either a glycosidic carboxymethyl moiety (13-15) or
an active carbonate ester at the primary hydroxyl function (22-
24), were prepared. These allow attachment to the scaffolds (1
or 2) via amide or carbamate bonds, respectively. In both cases
a four-atomic spacer between the cyclo-â-tetrapeptide core and
the pyranose ring is obtained. Carboxymethyl 2,3,4,6-tetra-O-
acetyl-R-D-mannopyranoside (14)31 and carboxymethyl 2,3,4,6-
tetra-O-acetyl-â-D-galactopyranoside (15)32 were synthesized
according to literature33 by the Ru(III)-catalyzed oxidation of
the corresponding allyl pyranosides.34 Carboxymethyl 2,3,4,6-
tetra-O-toluoyl-â-D-glucopyranoside (13) was obtained similarly.
The acetyl groups of the allyl glucopyranoside (11) were
replaced by the toluoyl groups (12), and the â-glycosidic allyl
group was then oxidized to a carboxymethyl group (13) by
NaIO4 in the presence of a catalytic amount of RuCl3. It is worth
of noting that a toluoylated sugar unit (13) is essential for
reliable detection of the conjugates (33-40) and their released
intermediates, when small aliquots are analyzed by RP HPLC
during the course of the synthesis. The presence of aromatic
toluoyl chromofors is not the only reason for this, but the
corresponding fully acetylated conjugates tend to give broad
FIGURE 3. Released scaffolds 31 and 32. Notation: (i) 31 (19.14
min), (ii) 32 (18.68 min). For the chromatographic conditions, see
protocol A in Experimental Section.
(31) Grandjean, C.; Santraine, V.; Fardel, N.; Polidori, A.; Pucci, B.;
Gras-Masse, H.; Bonnet, D. Tetrahedron Lett. 2004, 45, 3451.
(32) Sasaki, A.; Naoichi, M. JP Patent 06271597, 1994.
(33) Ghosh, M.; Dulina, R. G.; Kakarla, R.; Sofia, M. J. J. Org. Chem.
2000, 65, 8387.
(34) Schmidt, M.; Chatterjee, S. K.; Dobner, B.; Nuhn, P. Chem. Phys.
Lipids 2002, 114, 139.
RP HPLC signals. Methyl 6-O-(4-nitrophenoxycarbonyl)-2,3,4-
tri-O-toluoyl-R-D-glycopyranosides (22-24) were synthesized
from appropriate methyl R-D-glycopyranosides (16-18). A
three-step procedure, including a temporary tritylation of the
6-position, pertoluoylation, and detritylation, gave first the
1992 J. Org. Chem., Vol. 71, No. 5, 2006

Cyclo-â-tetrapeptides as Solid-Supported Scaffolds
SCHEME 5. Syntheses of Glycoconjugates (33-52)a
a Reagents and conditions: (i) piperidine, DMF; (ii) 13, 14, or 15, HATU, DIEA, DMF; (iii) 22, 23, or 24, HOBt, DIEA, NMP; (iv) (Ph3P)4Pd0, PhSiH3,
DCM; (v) TFA, DCM ) release; (vi) Me3P, toluene, H2O, dioxane.
methyl 2,3,4-tri-O-toluoyl-R-D-glycopyranosides (19-21) in
60-85% overall yields, as previously reported.35 The exposed
6-hydroxyl group of 19-21 was then converted to the desired
active carbonate, giving the desired building blocks (22-24)
in 43-76% yield.
Synthesis of the Solid-Supported Scaffolds (1 and 2,
Scheme 4). Building block 10 was first attached to a resin
derivatized with the 4-(4-formyl-3,5-dimethoxyphenoxy)butyrate
linker36 (25, L ) 0.16 mmol/g). Reductive amination in the
presence of NaCNBH3 (4 equiv of 10, 4 equiv of NaCNBH3,
in AcOH-DMF, 1:99 for 1 h at 25 °C) gave a 75% yield (L )
0.12 mmol/g). A small amount of acetic acid was observed to
accelerate the reaction. Peptide chains (27 and 28) were then
assembled using the Fmoc chemistry. HATU was used as an
activator for couplings of 3, 5 and 8 and PyAOP for 4. Due to
the potential racemization, collidine was used as a base in the
coupling of 5 (cf. above). The other branching units (3, 4, and
8) were coupled in the presence of DIEA; 90% overall yields
were obtained for both chain elongations (L ) 0.10 mmol/g,
27 and 28). The next step, i.e., selective removal of Mtt and
PhiPr groups, was carefully optimized, since the backbone amide
linker (25) seemed to be unexpectedly prone to premature
cleavage.37 Several acid treatments, including mixtures of TFA,
DCA, and 1,1,1,3,3,3-hexafluoro-isopropyl alcohol (HFIP) with
or without scavengers were tested. In addition, similar trials
with the 4-(4-formyl-3-methoxyphenoxy) butyrate linker38 were
carried out. Surprisingly, the latter linker turned out to be even
more labile than the former (11), in contrast to what is expected
in the basis of Hammet σ+ values.39,40 The medium of choice
for removal of Mtt- and PhiPr-protections was a short treatment
(35) Katajisto, J.; Heinonen, P.; Lo¨nnberg, H. J. Org. Chem. 2004, 69,
7609.
(36) Jensen, K. J.; Alsina, J.; Songster, M. F.; Va´gner, J.; Albericio, F.;
Barany, G. J. Am. Chem. Soc. 1998, 120, 5441.
(37) Boas, U.; Brask, J.; Christensen, J. B.; Jensen, K. J. J. Comb. Chem.
2002, 4, 223.
(38) Sarantakis, D.; Bicksler, J. J. Tetrahedron Lett. 1997, 38, 7325.
(39) Okamoto, Y.; Brown, H. C. J. Org. Chem. 1957, 22, 485.
J. Org. Chem, Vol. 71, No. 5, 2006 1993

Virta et al.
with 1% TFA in DCM (at 25 °C for 6 min),41 followed by
immediate neutralization with a mixture of Py/DCM (1:5, v/v).
By this procedure the premature cleavage remained at an
acceptable level (20%). The loading of the deprotected linear
peptides (15 and 16) was 0.080 mmol/g. Interestingly, a mixture
of HFIP and DCM (1:3, v/v) cleaved both protections in
solution, but on a solid support, only Mtt group may be
removed.42 Replacement of TFA with DCA did not result in
the required selectivity.
Cyclization (29 and 30) under different conditions (PyAOP43
and PyBOP44 activation in different solvent systems) was then
evaluated to minimize the expected on-resin polymerization.
PyAOP as an activator in DMF (5 equiv of PyAOP, 5 equiv of
DIEA in DMF, at 25 °C for 2 h) gave the best result. Small
aliquots from the resins (1 and 2) were released and analyzed
by analytical RP HPLC (Figure 3). Acceptable purities of crude
mixtures and satisfactory overall isolated yields (15% for 31
and 12% for 32) were obtained for the released scaffolds.
Synthesis of the Glycoconjugates (33-52, Scheme 5).
Protected conjugates 33 and 34. To evaluate the applicability
of amide coupling to construction of glycoconjugates, dipodal
conjugates 33 and 34 were first synthesized on scaffold 1. The
Fmoc group was removed (20% piperidine in DMF, for 30 min
at 25 °C) from the scaffold and carboxymethyl 2,3,4,6-tetra-
O-toluoyl-â-D-glucopyranoside (13) was attached using HATU
as an activator (5 equiv of 13, 5 equiv of HATU, 10 equiv of
DIEA, for 2 h at 25 °C). After the first sugar unit attachment,
the Alloc group was removed by a Pd0 treatment [0.5 equiv of
(Ph3P)4Pd, 24 equiv of PhSiH3 in DCM, 1 h at 25 °C under
argon, the treatment was repeated], and the second carboxy-
methyl glycopyranoside (14 or 15) was attached similarly. The
conjugates were released with TFA in DCM and purified by
RP HPLC (Figure 4). As seen, purity of the crude products (33
and 34) after this multistep synthesis, including construction of
the scaffold (1), was quite high. The yields of the desired
conjugates compared were 77% (33) and 82% (34) of that of
the scaffold (31), and the overall yields were 12% and 13%,
respectively (cf. Table 1). Different orders of deprotection and
coupling steps (i.e., first the Alloc- and then the Fmoc-removal
and first coupling of 15, then 13) were additionally tested, but
no remarkable differences in the RP HPLC profile were
observed.
Protected Conjugates 35-40. Compared to the dipodal
conjugates (33 and 34), construction of tripodal conjugates was
more complex, and the success of the synthesis depended on
the order of conjugation of sugar units. To obtain an optimal
protocol, six tripodal conjugates (35-40), consisting of all
possible combinations of the three different sugar units (13-
15), were synthesized on scaffold 2. The R-amino groups of 2
were exposed in the following order: Fmoc removal, demasking
of the azide group, and Alloc removal. Fmoc and Alloc groups
were removed as described for 33 and 34 above, and the azide
group was demasked using trimethylphosphine in toluene [24
equiv of 1 mol L-1 Me3P in toluene (resin suspended in H2O-
FIGURE 4. RP HPLC profiles of the crude product (33 and 34)
mixtures. Notation: (i) 33 (14.56 min), (ii) 34 (14.52 min). For the
chromatographic conditions, see protocol B in Experimental Section.
[The glycosidic bond in the galactopyranosyl unit (33) is â, and in the
mannopyranosyl unit (34) it is R.]
TABLE 1. Yields of the Clusters (33-42)
relative yield, %
glycoconjugate
(33-42 vs 31 or 32)
overall yield, %
33
34
35
36
37
38
39
40
41
42
77
82
71
36
37
33
57
62
69
78
12
13
9
4
4
4
7
8
10
9
dioxane, 1:4, v/v), for 2 h at 25 °C]. Each R-amino group was
acylated with an appropriate carboxymethyl glycopyranoside
immediately after its exposure. A higher excess of reagents and
a prolonged reaction time was used for these acylations (10
equiv of 13, 14, or 15, 10 equiv of HATU, 20 equiv of DIEA
in DMF, for 15 h at 25 °C), compared to preparation of the
dipodal conjugates (33 and 34). The conjugates were released
with TFA in DCM and then purified by RP HPLC (Figure 5).
As seen, three of the conjugates (35, 39, and 40, iv-vi in Figure
5) were obtained in relatively high purity and good yields [35,
39, and 40 vs 32 being 71-58% and the overall yields 9-7%,
respectively (cf. Table 1)]. However, with conjugates 36-38,
the presence of one glycopyranoside markedly retarded the
subsequent couplings. The low reactivity may result from
increased aggregation of the scaffold (2). These difficult
acylations could not be driven forward, even by repeated
couplings. Addition of chaotropic salts (LiCl, LiClO4, NaClO4,
KSCN, 0.4 M solutions in DMF)45 to the coupling mixture and
applicability of an elevated temperature (55 °C) were also
attempted. Relative yields of the conjugates (36-38 vs. 18)
remained at 33-37% and overall yields at 4%, respectively (cf.
Table 1).
(40) Okamoto, Y.; Inukai, T.; Brown, H. J. Am. Chem. Soc. 1958, 80,
4972.
(41) McMurray, J. S. Tetrahedron Lett. 1991, 32, 7679.
(42) Bollhagen, R.; Schmiedberger, M.; Barlos, K.; Grell, E. J. Chem
Soc., Chem. Commun. 1994, 2559.
(43) Carpino, L. A.; El-Faham. A.; Minor, C. A.; Albericio, F. J. Chem.
Soc., Chem. Commun. 1994, 201.
(44) Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990, 31,
205.
1994 J. Org. Chem., Vol. 71, No. 5, 2006

Cyclo-â-tetrapeptides as Solid-Supported Scaffolds
FIGURE 5. RP HPLC profiles of the crude product (35-40) mixtures. Notation (profiles in a reversed order of synthetic success): (i) 36 (15.64
min), (ii) 38 (15.42 min), (iii) 37 (15.70 min), (iv) 39 (15.59 min), (v) 40 (15.74 min), (vi) 35 (15.76 min). The main side products in the RP HPLC
profiles are related to incomplete couplings. For the chromatographic conditions, see protocol B in Experimental Section. Structure of the conjugate
35 is only shown.
FIGURE 6. RP HPLC profiles of the crude product (41 and 42) mixtures. Notation: (i) 41 (20.91 min), (ii) 42 (13.91 min). For the chromatographic
conditions, see protocol C and D in Experimental Section.
Deprotected Conjugates 43-50. Global deprotection (i.e.,
removal of acetyl and toluoyl groups) by conventional NaOMe/
MeOH treatment46 was first studied using one dipodal (33) and
tripodal conjugate (35) as model compounds. By this base-
catalyzed transesterification (20 mmol L-1 NaOMe in MeOH,
15 h at 25 °C) the desired globally deprotected conjugates were
obtained in 54% (43) and 52% (45) isolated yields (RP HPLC
profiles of the homogenized products, see Figure 7). Using the
same treatment on conjugates 34 and 36-40, which were
synthesized in a small scale, deprotected conjugates 44 and 46-
50 were obtained. No remarkable differences in product
distributions, caused by degradation or incomplete deprotection,
were found.
and amines47 is expectedly a slower reaction than the HATU-
promoted amide coupling, these two model conjugates (41 and
42) were obtained with rather high coupling efficiency resulting
a formation of the desired conjugates in 10% (41) and 9% (42)
overall yields (Figure 6). With the exception of the coupling
procedure (10 equiv of 30, 31 or 31, 10 equiv of DIEA in 0.1
mmol L-1 HOBt/NMP, for 15 h at 25 °C), these conjugates
were synthesized as described for 33-40. However, their global
deprotection by a conventional NaOMe/MeOH treatment re-
sulted in cleavage of the carbamate bond. Sodium methoxide
was found to catalyze nucleophilic attack of one of the ring
amides to the carbamate carbon, which cleaves the sugar unit
and results in formation of a five-membered ring (cf. the
aspartimide formation in SPPS).48,49 The use of hydrazine, being
a weak Brønsted base (pKb ) 6.05) but a strong nucleophile,
suppressed this side reaction. The conjugates (41 and 42) were
treated with hydrazine hydrate in DMF (1:9, v/v, for 15 h at 25
Conjugates 41, 42, 51, and 52. Applicability of the scaffolds
(1 and 2) was further evaluated by the synthesis of two
glycoconjugates (41 and 42), in which carbamate coupling was
utilized. Although coupling between 4-nitrophenoxycarbonates
(45) Stewart, J. M.; Klis, W. A. In InnoVations and PerspectiVes in Solid-
Phase Synthesis, Peptides, Polypeptides and Oligonucleotides; Epton, R.,
Ed.; SPCC (UK) Ltd: Birmingham, 1990; pp 1-9.
(47) Katajisto, J.; Lo¨nnberg, H. Eur. J. Org. Chem. 2005, 3518.
(48) Nicola´s, E.; Pedroso, E.; Giralt, E. Tetrahedron Lett. 1989, 30, 497.
(49) Yang, Y.; Sweeney, W. V.; Schneider, K.; Tho¨rnqvist, S.; Chait,
B. T.; Tam, J. P. Tetrahedron Lett. 1994, 35, 9689.
(46) Zemplen, G.; Kuntz, A. Brit. 1923, 56B, 1705.
J. Org. Chem, Vol. 71, No. 5, 2006 1995

Virta et al.
the acyl protections in solution gives the fully deprotected
conjugates (43-52).
Experimental Section
2-Azido-3-[(9-fluorenylmethyloxycarbonyl)amino]-
propanoic Acid (5). [Bis(trifluoroacetoxy)iodo]benzene (12 g, 27
mmol) was added to a mixture of (2S)-4-amino-2-azido-4-oxobu-
tanoic acid20 (6, 2.83 g, 18 mmol) in aqueous DMF (140 mL, 1:1,
v/v). The mixture was agitated for 15 min, and then DIEA (6.2
mL, 36 mmol) was added. After 3 h of stirring, volatiles were
removed, the residue was dissolved in water, and organic side
products were removed by repeated washings with diethyl ether (4
× 50 mL). The water phase was evaporated to dryness to yield a
reddish oil (crude 7), which was then redissolved in water (15 mL).
DIEA (3.1 mL, 18 mmol) and FmocOSu (5.7 g, 18 mmol) in
acetonitrile (15 mL) were added, and the reaction was allowed to
stir for 1.5 h. The mixture was acidified (to pH 2.0) by addition of
HCl, and the product was extracted in DCM (4 × 50 mL). The
organic phases were combined, dried with Na2SO4, and evaporated
to dryness. The crude product mixture was purified by silica gel
chromatography (10% MeOH in DCM). Hexane was added to the
combined product fractions, and the precipitate formed was filtered,
washed with hexane, and dried to yield 2.8 g (44%) of product as
a white powder: [R]20D -42.1 (c 1.0, MeOH); 1H NMR (DMSO-
d6, 400 MHz) δ 7.87 (d, 2H, J ) 7.6 Hz), 7.68 (d, 2H, J ) 7.6
Hz), 7.42-7.26 (m, 4H), 4.28 (m, 2H), 4.20 (t, 1H, J ) 6.8 Hz),
FIGURE 7. RP HPLC profiles of the homogenized conjugates 43,
45, 51, and 52. Notation: (i) 43 (10.35 min), (ii) 45 (7.86 min), (iii)
51 (13.18 min), (iv) 52 (13.19 min). For the chromatographic conditions,
see protocol E in Experimental Section.
3.62 (dd, 1H, J ) 9.2 and 3.6 Hz), 3.47 (m, 1H), 3.18 (m, 1H); 13
C
NMR (DMSO-d6, 50 MHz) δ 172.2, 156.6, 144.3, 141.2, 128.1,
127.5, 125.7, 120.6, 66.0, 64.3, 47.2, 43.1; HRMS (ESI) [M +
Na]+ requires 375.1057, found 375.1064.
1-Methyl-1-phenylethyl 3-[(9-Fluorenylmethoxycarbonyl)-
amino]propanoate (9). 1-Methyl-1-phenylethyl trichloroacetimi-
date was first prepared according to literature.29 A mixture of
1-methyl-1-phenylethanol (5.1 g, 38 mmol) in diethyl ether (4.0
mL) was added dropwise to a suspension of NaH (0.10 g, 4.2 mmol)
in diethyl ether (4.0 mL). The mixture was stirred at ambient
temperature for 20 min and then cooled to 0 °C. Trichloroaceto-
nitrile (3.8 mL, 38 mmol) was slowly added (upon 15 min), and
the mixture was allowed to warm to ambient temperature. After 1
h of stirring, volatiles were removed, the resulted oil was dissolved
in pentane (4.0 mL), and the solution was filtered. The filtrate was
evaporated to dryness to yield 11 g (quant) of the crude imidate as
a reddish oil, which may be used without further purification. The
freshly prepared 1-methyl-1-phenylethyl trichloroacetimidate (0.9
g, 3.2 mmol) was added to a mixture of Fmoc-â-alanine (8, 0.5 g,
1.6 mmol) in DCM (4.0 mL). After overnight stirring, the
precipitated trichloroacetamide was removed by filtration, and the
filtrate was evaporated to dryness. The resulted crude product
mixture was purified by silica gel chromatography (0-1% MeOH
in DCM) to yield 0.58 g (84%) of 9 as a colorless oil: 1H NMR
(CDCl3, 500 MHz) δ 7.76 (d, 2H, J ) 7.6 Hz), 7.57 (d, 2H, J )
7.5 Hz), 7.41-7.23 (m, 9H), 5.24 (b, 1H), 4.37 (d, 2H, J ) 7.1
Hz), 4.20 (t, 1H, J ) 7.0 Hz), 3.42 (m, 2H), 2.55 (t, 2H, J ) 5.7
Hz), 1.78 (s, 6H); 13C NMR (CDCl3, 100 MHz) δ 171.3, 156.3,
145.5, 144.0, 141.3, 128.4, 127.7, 127.2, 127.1, 125.1, 124.2, 120.0,
82.3, 66.7, 47.3, 36.6, 35.4, 28.7; HRMS (ESI)) [M + Na]+ requires
452.1832, found 452.1824.
1-Methyl-1-phenylethyl 3-Aminopropanoate (10). Diethyl-
amine (6.0 mL) was added to a mixture of 9 (0.53 g, 1.2 mmol) in
DCM (2.0 mL). After 2 h of stirring, toluene (10 mL) was added,
and the mixture was evaporated to dryness. The crude product
mixture was purified by silica gel chromatography (10% MeOH,
2% Et3N in DCM) to yield 0.24 g (94%) of 10 as colorless oil: 1H
NMR (CDCl3, 500 MHz) δ 7.37-7.31 (m, 4H), 7.26-7.23 (m,
1H), 2.94 (b, 2H), 2.47 (t, 2H, J ) 6.2 Hz), 1.83 (b, 2H), 1.77 (s,
6H); 13C NMR (CDCl3, 100 MHz) δ 171.4, 145.8, 128.3, 127.0,
124.2, 81.8, 38.6, 37.8, 28.7; HRMS (ESI) [M + Na]+ requires
230.1152, found 230.1167.
°C) and purified by RP HPLC (Figure 7) to give the globally
deprotected products in 31% (51) and 59% (52) isolated yields.
Shortcoming of this deprotection procedure may be the presence
of toluoylhydrazide in the crude product mixture, which in
contrast to methyl 4-methylbenzoate cannot be cleanly removed
by simple diethyl ether extraction.
The all conjugates synthesized (33-52) were purified by RP
HPLC. Authenticity of the purified products (33-52) was
verified by ESI(MS). In addition, eight of the conjugates (33,
35, 41-43, 45, 51, and 52), which were synthesized in a larger
scale, were characterized by 1H NMR spectroscopy (for the MS
and NMR spectral data, see Supporting Information).
Conclusion
Two novel solid-supported cyclo-â-tetrapeptide scaffolds
{viz., cyclo[(Na-Alloc)Dpr-â-Ala-(Na-Fmoc)Dpr-â-Ala] (1) and
cyclo[(Na-Alloc)Dpr-R-azido-â-aminopropanoyl-(Na-Fmoc)Dpr-
â-Ala] (2)} have been described. The scaffolds (1 and 2) are
composed of â-alanine and orthogonally protected 2,3-diami-
nopropanoic acid residues. These include one novel 2,3-
diaminopropanoic acid precursor (5), i.e., 2-azido-3-[(9-
fluorenylmethyloxycarbonyl)amino]propanoic acid. Orthogonal
derivatization of these cyclo-â-tetrapeptide cores (1 and 2) has
been demonstrated by insertion of acetyl- or toluoyl-protected
carboxymethyl R-D-glycopyranosides (13-15) and methyl 6-O-
(4-nitrophenoxycarbonyl)-R-D-glycopyranosides (22-24) to ob-
tain 10 diverse di- and trivalent glycoclusters (33-42). Both
scaffolds (1 and 2) have been shown to allow efficient sugar
moiety conjugation by either amide or carbamate coupling.
Formation of trivalent glycoclusters (35-40) is, however,
dependent on the order of the sugar unit attachment (13-15).
By these approaches, diverse fully acylated conjugates (33-
42) can be constructed from monomeric constituents entirely
on a solid support in acceptable yield and purity. Removal of
1996 J. Org. Chem., Vol. 71, No. 5, 2006

Cyclo-â-tetrapeptides as Solid-Supported Scaffolds
Allyl 2,3,4,6-Tetra-O-toluoyl-â-D-glucopyranoside (12). Allyl
2,3,4,6-tetra-O-acetyl-â-D-glucopyranoside (11, 1.4 g, 3.6 mmol),
prepared from the commercially available pentaacetyl-â-D-glucose,34
was dissolved in 0.1 mol L-1 NaOMe/MeOH solution. The mixture
was stirred at ambient temperature for 1 h, neutralized by addition
of strongly acidic cation-exchange resin, and filtered. The filtrate
was evaporated to dryness, the residue was dissolved in pyridine
(30 mL), and then toluoyl chloride (3.8 mL, 29 mmol) was added.
After overnight stirring at 50 °C, ice-water was added to quench
the reaction and the product was extracted with DCM. The organic
phases were combined, washed with NaHCO3, dried with Na2SO4,
and evaporated to dryness. The crude product mixture was purified
by silica gel chromatography (20% EtOAc in petroleum ether) to
yield 2.5 g (98%) of 12 as white foam: 1H NMR (CDCl3, 500
MHz) δ 7.93 (d, 2H, J ) 8.2 Hz), 7.88 (d, 2H, J ) 8.2 Hz), 7.81
(d, 2H, J ) 8.2 Hz), 7.75 (d, 2H, J ) 8.2 Hz), 7.22 (d, 2H, J ) 8.1
Hz), 7.20 (d, 2H, J ) 8.1 Hz), 7.15 (d, 2H, J ) 8.0 Hz), 7.09 (d,
2H, J ) 8.0 Hz), 5.90 (dd, 1H, J ) 9.7 and 9.7 Hz), 5.82 (m, 1H),
5.65 (dd, 1H, J ) 9.7 and 9.7 Hz), 5.56 (dd, 1H, J ) 9.8 and 7.9
Hz), 5.24 (m, 1H), 5.14 (m, 1H), 4.90 (d, 1H, J ) 7.9 Hz), 4.62
(dd, 1H, J ) 12.1 and 3.2 Hz), 4.50 (dd, 2H, J ) 12.1 and 5.6
Hz), 4.38 (ddt, 1H, J ) 13.3, 4.9, and 1.5 Hz), 4.19 (ddt, 1H, J )
13.3, 6.4, and 1.3 Hz), 4.15 (m, 1H), 2.43 (s, 3H), 2.39 (s, 3H),
2.37 (s, 3H), 2.31 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 166.2,
165.8, 165.2, 165.1, 144.1, 143.9, 143.8, 133.4, 129.9, 129.9, 129.8,
129.8, 129.1, 129.0, 127.0, 126.7, 126.2, 117.9, 99.9, 72.8, 72.3,
71.7, 70.1, 69.7, 63.2, 21.7, 21.6, 21.6; HRMS (ESI) [M + Na]+
requires 715.2514, found 715.2511.
126.2, 125.9, 125.3, 121.9, 97.2, 71.8, 69.9, 68.8, 67.5, 66.8, 55.9,
21.7, 21.7, 21.6; HRMS (ESI) [M + H]+ requires 714.2181, found
714.2202.
Methyl 6-O-(4-Nitrophenoxycarbonyl)-2,3,4-tri-O-toluoyl-r-
D-mannopyranoside (23). The active carbonate (23) was synthe-
sized from the corresponding methyl 2,3,4-tri-O-toluoyl-R-D-
mannopyranoside35 (8.3 g, 15 mmol) as described for 22. The
product (23) was obtained as a white foam in a 43% yield (4.7 g):
1H NMR (CDCl3, 500 MHz) δ 8.28 (d, 2H, J ) 7.6 Hz), 8.03 (d,
2H, J ) 7.5 Hz), 7.87 (d, 2H, J ) 6.8 Hz), 7.73 (d, 2H, J ) 6.8
Hz), 7.37 (d, 2H, J ) 7.6 Hz), 7.26 (d, 2H, J ) 6.7 Hz), 7.19 (d,
2H, J ) 6.7 Hz), 7.07 (d, 2H, J ) 6.8 Hz), 5.97 [dd, 1H, J ≈ 8.3
Hz (average)], 5.92 (dd, 1H, J ) 8.4 and 2.6 Hz), 5.66 (m, 1H),
5.03 (d, 1H, J ) 0.8 Hz), 4.57 (dd, 1H, J ) 9.9 and 3.9 Hz), 4.53
(dd, 1H, J ) 9.9 and 2.0 Hz), 4.36 (m, 1H), 3.57 (s, 3H), 2.42 (s,
3H), 2.37 (s, 3H), 2.31 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ
165.7, 165.5, 165.5, 155.5, 152.5, 145.4, 144.5, 144.4, 144.0, 130.0,
129.9, 129.8, 129.3, 129.2, 129.0, 126.6, 126.3, 126.0, 125.3, 121.9,
98.7, 70.2, 69.4, 68.6, 67.3, 66.6, 55.7, 21.8, 21.7, 21.6; HRMS
(ESI) [M + H]+ requires 714.2181, found 714.2155.
Methyl 6-O-(4-Nitrophenoxycarbonyl)-2,3,4-tri-O-toluoyl-r-
D-galactopyranoside (24). The active carbonate (24) was synthe-
sized from the corresponding methyl 2,3,4-tri-O-toluoyl-R-D-
galactopyranoside35 (4.2 g, 7.6 mmol) as described for 22. The
product (24) was obtained as a white foam in a 76% yield (4.2 g):
1H NMR (CDCl3, 500 MHz) δ 8.26 (d, 2H, J ) 7.7 Hz), 7.98 (d,
2H, J ) 6.8 Hz), 7.87 (d, 2H, J ) 6.8 Hz), 7.67 (d, 2H, J ) 6.8
Hz), 7.35 (d, 2H, J ) 7.7 Hz), 7.28 (d, 2H, J ) 6.7 Hz), 7.17 (d,
2H, J ) 6.8 Hz), 7.04 (d, 2H, J ) 6.8 Hz), 5.95-5.93 (m, 2H),
5.65 (dd, 1H, J ) 8.6 and 3.0 Hz), 5.33 (d, 1H, J ) 2.9 Hz), 4.55
[dd, 1H, J ≈ 5.2 Hz (average)], 4.45 (d, 2H, J ) 4.9 Hz), 3.50 (s,
3H), 2.44 (s, 3H), 2.35 (s, 3H), 2.29 (s, 3H); 13C NMR (CDCl3,
100 MHz) δ 166.2, 165.7, 165.6, 155.4, 152.2, 145.5, 144.5, 144.2,
144.0, 130.0, 129.9, 129.7, 129.4, 129.2, 129.0, 126.4, 126.4, 126.3,
125.3, 121.8, 97.8, 69.1, 68.9, 68.0, 66.8, 66.6, 55.9, 21.8, 21.7,
21.6; HRMS (ESI) [M + H]+ requires 714.2181, found 714.2205.
Synthesis of the Solid-Supported Scaffolds (1 and 2). The
aldehyde derivatized resin (25) was prepared by the following
procedure. A mixture of Boc- and Fmoc-â-alanine (2.0 equiv of
both, 4.0 equiv of TBTU, 8 equiv of DIEA in DMF, 1 h at 25 °C)
was first coupled to an aminomethyl polystyrene resin (1.0 g, a
loading of 0.5 mmol/g). The Boc groups were removed with 50%
TFA in DCM, and the exposed amino groups were then capped
with an acetanhydride treatment. In this manner loading was reduced
to 0.16 mmol/g (determined by the release of benzofulvene). The
Fmoc groups were then removed with 20% piperidine in DMF and
4-(4-formyl-3,5-dimethoxyphenoxy)butyric acid was attached by
HATU-promoted coupling to obtain resin 25. Resin 25 (1.0 g, a
loading of 0.16 mmol/g) was treated for 1 h at room temperature
with a mixture of 1-methyl-1-phenylethyl 3-aminopropanoate (10,
160 mg, 4 equiv) and NaCNBH3 (48 mg, 4 equiv) in DMF,
containing 1% (v/v) AcOH (16 mL). The resin was washed with
DMF, DCM, and MeOH and dried on a filter. A small aliquot of
26 was acylated with excess of (Fmoc-â-Ala)2O in DCM, to
determine the loading of the acylated secondary amine by the release
of benzofulvene. A loading of 0.12 mmol/g (a 75% yield) was
obtained. The following protocols for the peptide chain elongations
(27 and 28) were then applied. The secondary amine of 26 was
acylated with Alloc-Dpr(Fmoc)-OH (5.0 equiv of 4, 5 equiv of
PyAOP, 10 equiv of DIEA in DMF-DCM, 1:9, v/v, 2 h at 25
°C), the Fmoc group was removed (piperidine-DMF, 1:4, v/v, for
20 min at 25 °C), and then the resin was divided into two batches
(2 × 0.5 g). To obtain resin 27, chain elongation was continued by
couplings of Fmoc-â-Ala-OH and Fmoc-Dpr(Mtt)-OH [5 equiv of
amino acid (8 or 3), 5 equiv of HATU, and 10 equiv of DIEA in
DMF, 1 h at 25 °C]. With the other batch (to obtain resin 28),
chain elongation was continued by couplings of 2-azido-3-[(9-
fluorenylmethyloxycarbonyl)amino]propanoic acid and Fmoc-Dpr-
(Mtt)-OH (5 equiv of 5, 5 equiv of HATU, 10 equiv of collidine
Carboxymethyl 2,3,4,6-Tetra-O-toluoyl-â-D-glucopyranoside
(13). NaIO4 (2.0 g, 9.4 mmol) and a catalytic amount of RuCl3
were added to a stirred biphasic mixture of allyl 2,3,4,6-tetra-O-
toluoyl-â-D-glucopyranoside (12, 1.3 g, 18 mmol), DCM (6 mL),
acetonitrile (6 mL), and water (9 mL). After 5 h of stirring at 40
°C, the mixture was diluted with DCM, washed with water and
brine, dried with Na2SO4, and evaporated to dryness. The crude
product mixture was purified by silica gel chromatography (2%
Py, 10% MeOH in DCM). The product fractions were combined
and evaporated to dryness to give an oil, which was then evaporated
with toluene to yield 0.69 g (53%) of 13 as a yellowish foam: 1H
NMR (CD3OD + CDCl3, 500 MHz) δ 7.85-7.76 (m, 6H), 7.68
(d 2H, J ) 7.9 Hz), 7.15-7.09 (m, 6H), 7.03 (d, 2H, J ) 8.0 Hz),
5.98 (dd, 1H, J ) 9.7 Hz and 9.7 Hz), 5.69 (dd, 1H, J ) 9.8 and
9.8 Hz), 5.59 [dd, 1H, J ) 8.8 Hz (average)], 5.12 (b, 1H), 4.59
(dd, 1H, J ) 12.0 and 3.2 Hz), 4.46 (dd, 1H, J ) 12.1 and 4.6
Hz), 4.39 (d, 1H, J ) 15.0 Hz), 4.30 (m, 1H), 4.16 (d, 1H, J )
15.0 Hz), 2.33, 2.32, 2.30 and 2.22 (each s, each 3H); 13C NMR
(CD3OD + CDCl3, 100 MHz) δ 176.2, 166.7, 166.3, 165.9, 165.4,
144.4, 144.3, 144.0, 129.9, 129.6, 129.5, 129.4, 129.0, 128.9, 128.8,
125.9, 125.9, 125.8, 100.1, 72.6, 72.3, 72.1, 69.5, 67.7, 62.9, 20.8,
20.8, 20.7; HRMS (ESI) [M + Na]+ requires 733.2255, found
733.2247.
Methyl 6-O-(4-Nitrophenoxycarbonyl)-2,3,4-tri-O-toluoyl-r-
D-glucopyranoside (22). 4-Nitrophenylchloroformate (1.1 g, 5.1
mmol) was added to a cooled (0 °C) mixture of methyl 2,3,4-tri-
O-toluoyl-R-D-glucopyranoside35 (2.8 g, 5.1 mmol) in Py (30 mL).
The reaction was allowed to warm to ambient temperature and then
stirred overnight. The mixture was evaporated to dryness and
purified by silica gel chromatography (20% EtOAc in petroleum
ether) to yield 2.2 g (61%) of 22 as a white foam: 1H NMR (CDCl3,
500 MHz) δ 8.30 (d, 2H, J ) 7.6 Hz), 7.88 (d, 2H, J ) 6.8 Hz),
7.85 (d, 2H, J ) 6.8 Hz), 7.77 (d, 2H, J ) 6.8 Hz), 7.43 (d, 2H,
J ) 7.6 Hz), 7.19 (m, 4H), 7.10 (d, 2H, J ) 6.8 Hz), 6.18 (m, 1H),
5.63 (dd, 1H, J ) 8.2 and 8.2 Hz), 5.29-5.27 (m, 2H), 4.55 (dd,
1H, J ) 10.0 and 3.9 Hz), 4.47 (dd, 1H, J ) 10.0 and 1.8 Hz),
4.34 (m, 1H), 3.52 (s, 3H), 2.37 (s, 6H), 2.31 (s, 3H); 13C NMR
(CDCl3, 100 MHz) δ 165.9, 165.8, 165.5, 155.5, 152.4, 145.5,
144.5, 144.3, 143.9, 130.0, 129.9, 129.7, 129.2, 129.2, 129.0, 126.4,
J. Org. Chem, Vol. 71, No. 5, 2006 1997

Virta et al.
in DMF, 1 h at 25 °C, 3 was coupled as above); 90% yields
(loadings of 0.10 mmol/g, 27 and 28) for both chain elongations
were obtained. To remove Mtt and PhiPr protections, the resins 27
and 28 were treated with a solution of TFA in DCM (1:99, v/v, for
6 min at 25 °C), followed by immediate neutralization by washings
with a mixture of Py in DCM (1:5, v/v). After the Mtt/PhiPr
deprotection, loadings of 0.080 mmol/g were obtained for the linear
peptides (29 and 30). Cyclizations were then performed using
PyAOP as an activator (5 equiv of PyAOP, 5 equiv of DIEA in
DMF for 2 h at 25 °C). After each coupling (including the
cyclization step), potentially remaining free amino groups were
capped by an acetic anhydride treatment. Since cyclizations of the
solid-supported scaffolds (1 and 2) could not be monitored by
loading, small aliquots of the resins (10.0 mg samples of 1 and 2)
were treated with TFA in DCM (1:1, v/v, 30 min at 25 °C), and
the released product (31 and 32) mixtures were analyzed by
analytical RP HPLC (Figure 3). Acceptable purities for the crude
released scaffolds (31 and 32) were obtained. Isolated yields of
the released scaffolds (31 and 32) were determined as follows.
Product (31 and 32) fractions were collected, evaporated to dryness,
and then treated with a known (1.0 mL) volume of piperidine in
DMF (1:4, v/v); 22% (31) and 17% (32) yields were obtained,
according to the extent of benzofulvene released (calculated from
29 and 30). The overall yields were 15% (31) and 12% (32),
respectively. Authenticity of the released scaffolds was verified by
HRMS (ESI). 31: [M + H]+ requires 621.2667, found 621.2659.
32: [M + H]+ requires 662.2681, found 662.2644.
Protected Conjugate 33. The Fmoc group of the supported
scaffold (1) (200 mg batch, a loading of 0.080 mmol/g) was
removed with piperidine in DMF (1:4, v/v, 30 min), and the first
sugar unit was coupled to the exposed amino group (5 equiv of
13, 10 equiv of DIEA in DMF, 2 h at 25 °C). The potentially
remaining free amino groups were capped with an acetic anhydride
treatment, and then the Alloc group was removed. The resin was
suspended in DCM, 0.5 equiv of (Ph3P)4Pd and 24 equiv of PhSiH3
were added, and the suspension was stirred for 1 h at 25 °C under
argon. The treatment was repeated, and then the second sugar unit
(15) was coupled in a similar manner. After each step above, the
resin was washed with DMF, DCM, and MeOH and dried on filter.
The protected dipodal glycogluster 33 was released with TFA in
DCM (1:1, v/v, for 30 min at 25 °C), and the crude product mixture
was evaporated to dryness, dissolved in aqueous EtOH (1:1, v/v),
and subjected to RP HPLC [(i) in Figure 4]. The combined product
fractions were lyophilized to give the desired product 33 in a 12%
(3.8 mg) overall isolated yield. The relative yield (33 vs 31) was
77%: 1H NMR (CD3CN, 500 MHz) δ 7.92 (m, 3H), 7.85 (d, 2H,
J ) 8.2 Hz), 7.76 (m, 3H), 7.66 (d, 2H, J ) 8.2 Hz), 7.30 (d, 2H,
J ) 8.0 Hz), 7.26 (d, 2H, J ) 8.1 Hz), 7.24 (d, 2H, J ) 8.1 Hz),
7.19 (d, 2H, J ) 8.1 Hz), 7.14 (b, 1H), 7.08 (b, 1H), 6.94 (b, 1H),
6.89 (b, 1H), 5.90 (dd, 1H, J ) 9.6 and 9.6 Hz), 5.74 (dd, 1H, J )
9.5 and 7.9 Hz), 5.72 (dd, 1H, J ) 9.7 and 9.7 Hz), 5.33 (d, 1H,
J ) 3.5 Hz), 5.26 (dd, 1H, J ) 10.5 and 8.0 Hz), 5.12 (d, 1H, J )
7.9 Hz), 5.06 (dd, 1H, J ) 10.5 and 3.5 Hz), 4.64 (d, 1H, J ) 8.0
Hz), 4.53 (b, 2H), 4.39 (m, 1H), 4.35-4.29 (m, 2H), 4.27 (d, 1H,
J ) 15.6 Hz), 4.20 (d, 1H, J ) 15.6 Hz), 4.19 (dd, 1H, J ) 9.7
and 5.5 Hz), 4.12 (d, 1H, J ) 15.1 Hz), 4.04-3.99 (m, 3H), 3.60-
3.31 (m, 8H), 2.43 (s, 3H), 2.38 (s, 3H), 2.37 (s, 3H), 2.39-2.30
(m, 4H), 2.33 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 2.00 (s, 3H), 1.94
(s, 3H); MS (ESI) [M + H]+ requires 1395.5, found 1395.5.
Protected Conjugate 35. The Fmoc group of the scaffold (2)
(200 mg batch, a loading of 0.080 mmol/g) was removed (as
described for 33), and the first carboxymethyl glycopyranoside (10
equiv of 13, 10 equiv of HATU, 20 equiv of DIEA in DMF, 15 h
at 25 °C) was coupled. The potentially remaining free amino groups
were capped, and the resin was suspended in a mixture of H2O in
dioxane (1:4, v/v). A solution of 1mol L-1 Me3P (24 equiv) was
added, and the suspension was allowed to stir for 2 h at 25 °C
under nitrogen. After filtration and washings, the second sugar unit
(14) was attached as described for the first one (13). The unreacted
free amino groups were capped, the Alloc group was removed (as
described for 33), and the last sugar unit (15) was attached as
described above (13). After each step, the resin was washed with
DMF, DCM, and MeOH and dried on a filter. The protected tripodal
glycocluster 35 was released with TFA in DCM (1:1, v/v, for 30
min at 25 °C), purified by RP HPLC [(vi) in Figure 5], and
lyophilized to give the desired product (35) in a 9% (3.7 mg) overall
isolated yield. Relative yield (35 vs 32) was 71%, respectively:
1H NMR (CD3CN, 500 MHz) δ 7.92 (d, 2H, J ) 8.2 Hz), 7.83 (d,
2H, J ) 8.2 Hz), 7.77 (d, 2H, J ) 8.2 Hz), 7.72 (b, 1H), 7.67 (d,
2H, J ) 8.2 Hz), 7.61 (b, 1H), 7.31-7.19 (m, 12H), 7.11 (b, 1H),
5.90 (dd, 1H, J ) 9.6 and 9.6 Hz), 5.72 (dd, 1H, J ) 9.7 and 9.7
Hz), 5.61 (dd, 1H, J ) 9.6 and 8.0 Hz), 5.42 (m, 1H), 5.35 (b,
1H), 5.30 (dd, 1H, J ) 10.0 and 3.4 Hz), 5.26 (dd, 1H, J ) 10.0
and 10.0 Hz), 5.14 (dd, 1H, J ) 10.5 and 7.8 Hz), 5.14 (d, 1H, J
) 8.0 Hz), 5.08 (dd, 1H, J ) 10.5 and 3.5 Hz), 4.97 (d, 1H, J )
1.2 Hz), 4.67 (d, 1H, J ) 7.8 Hz), 4.54 (b, 2H), 4.49-4.41 (m,
2H), 4.35-4.04 (m, 14H), 3.70-3.28 (m, 8H), 2.43 (s, 3H), 2.38
(s, 3H), 2.37 (s, 3H), 2.32 (s, 3H), 2.40-2.32 (m, 2H), 2.14 (s,
3H), 2.11 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.02 (s, 3H), 2.00 (s,
3H), 1.98-1.96 (s, 3H), 1.95 (s, 3H); MS (ESI) [M + H]+ requires
1798.6, found 1799.7.
Protected Conjugates 34 and 36-40. The other conjugates (34,
36-40), constructed by amide linkages, were synthesized as
described for 33 and 35 using a smaller scale (10 mg resin batches).
Yields of these compounds (listed in Table 1) were determined by
RP HPLC (Figures 4 and 5) comparing the product peak (34, 36-
40) areas to the isolated standards of 33 and 35. Authenticity of
the purified products was verified by MS (ESI). 34: [M + H]+
requires 1395.50, found 1395.49. 36, 38-40: [M + H]+ requires
1798.62, found 1799.70 (36), 1799.70 (38), 1799.69 (39) and
1799.69 (40). 37: [(M + K + H)/2]+ requires 918.79, found 918.80.
Protected Conjugate 41. With the exception of coupling (10
equiv of 23 or 24, 10 equiv of DIEA in 0.1 mol L-1 HOBt/NMP,
15 h at 25 °C), the synthetic details to obtain conjugate 41 were
the same as described for 33. The purified [(i) in Figure 6] conjugate
41 was obtained in a 10% yield. Relative yield (41 vs 31) was a
1
69%, respectively. For H NMR (CD3CN, 500 MHz) spectrum,
see Supporting Information; HRMS (ESI) [M + H]+ requires
1463.5, found 1463.5.
Protected Conjugate 42. Except for coupling (10 equiv of 22,
23, or 24, 10 equiv of DIEA in 0.1 mol L-1 HOBt/NMP, 15 h at
25 °C), the synthetic details to obtain conjugate 42 were the same
as described for 35. The purified [(ii) in Figure 6) conjugate 42
was obtained in a 9% yield. Relative yield (42 vs 32) was 78%,
respectively. For 1H NMR (CD3CN + CDCl3, 500 MHz) spectrum,
see Supporting Information; MS(ESI) [M + H]+ requires 2051.7,
found 2053.7.
Deprotected Conjugates 43 and 45. A 3.0 mg portion of the
conjugate (33 or 35) was treated with a solution of 20 mmol L-1
NaOMe in MeOH (0.5 mL) for 15 h at 25 °C, and then strong
cation-exchange resin was added to neutralize the mixture. The
mixture was filtered, the filtrate was evaporated to dryness, and
the resulted residue was dissolved in a biphasic mixture of Et2O
and water (1:1, v/v, 1.0 mL). The organic layer was removed, the
water phase was lyophilized, and the crude mixture was purified/
desalted by RP HPLC to give the homogenized product (43 or 45)
(Figure 7). The product fractions were combined and lyophilized
to give the desired globally deprotected conjugate in 54% (0.86
mg 43) and 52% (0.86 mg 45) isolated yields. 43: 1H NMR (D2O,
500 MHz) δ 4.46 (d, 1H, J ) 7.5 Hz), 4.43-4.39 (m, 3H), 4.37
(d, 1H, J ) 16.0 Hz), 4.36 (d, 1H, J ) 15.5 Hz), 4.25 (2 × d, b,
2H), 3.86-3.82 (m, 2H), 3.74-3.33 (m, 18H), 2.44-2.34 (m, 4H);
HRMS (ESI) [M + H]+ requires 755.2941, found 755.2927. 45:
1H NMR (D2O, 500 MHz) δ 4.85 (s, 1H), 4.45 (d, 1H, J ) 7.9
Hz), 4.46-4.40 (m, 3H), 4.38 (d, 1H, J ) 7.9 Hz), 4.37 (d, 1H, J
) 16.1 Hz), 4.36 (d, 1H, J ) 16.3 Hz), 4.27 (d, b, 1H), 4.23 (2 ×
d, b, 2H), 4.10 (d, 1H, J ) 15.5 Hz), 4.02 (m, 1H), 3.88-3.80 (m,
1998 J. Org. Chem., Vol. 71, No. 5, 2006

Cyclo-â-tetrapeptides as Solid-Supported Scaffolds
4H), 3.74-3.49 (m, 13H), 3.45-3.26 (m, 7H), 2.40 (m, 2H); HRMS
(ESI) [(M + 2Na)/2]+ requires 517.6673, found 517.6697.
Deprotected Conjugates 44, 46-50. Small samples of the
conjugates (34, 36-40) were treated similarly as described for 43
and 45. According to RP HPLC profiles (comparing to profiles of
crude 43 and 45 mixtures), no remarkable differences in product
distributions were found. Authenticity of the purified products was
verified by MS (ESI). 44: HRMS (ESI) [M + Na]+ requires
777.2761, found 777.2790. 46-50: MS (ESI) [M + Na]+ requires
1012.35, found 1012.32 (46), 1012.33 (47), 1012.33 (48), 1012.32
(49), and 1012.32 (50).
Deprotected Conjugates 51 and 52. A 3.0 mg portion of the
conjugates (41 or 42) was treated with a solution of hydrazine
hydrate in DMF (1:9, v/v, 0.5 mL) for 15 h at at 25 °C. The mixture
was evaporated to dryness, and the residue was subjected to RP
HPLC to give the homogenized product (51 or 52) (Figure 7). The
product fractions were combined and lyophilized yielding the
desired globally deprotected product 51 (0.47 mg) and 52 (0.85
mg) in 31% (51) and 59% (52) yields. 51: 1H NMR (D2O, at 45
°C, 500 MHz) δ 5.00 (d, 1H, J ) 3.1 Hz), 4.91 (s, 1H), 4.52-4.41
(m, 6H), 4.24 (m, 1H), 4.16 (b, 1H), 4.11 (m, 1H), 4.01-3.84 (m,
5H), 3.78-3.54 (m, 8H), 3.55 (s, 3H), 3.55 (s, 3H), 2.61 [2 × (t,
4H, J ) 5.3 Hz, average)]; HRMS (ESI) [M + H]+ requires
755.2941, found 755.2950. 52: 1H NMR (D2O, at 45 °C, 500 MHz)
δ 5.00 (d, 1H, J ) 2.8 Hz), 4.96 (d, 1H, J ) 3.7 Hz), 4.90 (s, 1H),
4.58-4.36 (m, 9H), 4.24 (m, 1H), 4.16 (b, 1H), 4.10 (m, 1H), 4.01-
3.55 (m, 17H), 3.57 (s, 3H), 3.56 (s, 3H), 3.55 (s, 3H), 2.76-2.54
(m, 2H); HRMS (ESI) [(M + 2Na)/2]+ requires 517.6673, found
755.6704.
Acknowledgment. We thank Dr. Jari Sinkkonen for per-
1
forming the H NMR analysis
Supporting Information Available: General procedure and
spectral data for compounds 5, 9, 10, 12, 13 22-24, and 33-52.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO052348O
J. Org. Chem, Vol. 71, No. 5, 2006 1999