Synthesis and biological evaluation of new mannose derived immunomodulating adamantyltripeptides

Article abstract of DOI:10.5562/cca1827

A novel class of mannosylated adamantyltripeptides, general formula of which is α-Man-OCH₂CH(CH₃)CO-AdGly-L-Ala-D-isoGln, were prepared, characterized, and their possible immunomodulatory properties investigated in some preliminary tests in vivo. All newly synthesized glycopeptides comprise in their structure the adamantylglycyl moiety linked to the dipeptide L-Ala-D-isoGln, as well as mannose. Adjuvant activity of mannosylated adamant-2-yl tripeptides was tested in the mouse model using ovalbumin as an antigen and in comparison to the peptidoglycan monomer (PGM, β-D-GlcNAc-(1→4)-D-MurNAc-L-Ala-D-isoGln-mesoDAP(εNH ₂)-D-Ala-D-Ala).

Full text of DOI:10.5562/cca1827

CROATICA CHEMICA ACTA
CCACAA, ISSN 0011-1643, e-ISSN 1334-417X
Croat. Chem. Acta 84 (2) (2011) 233–244.
CCA-3470
Original Scientific Article
Synthesis and Biological Evaluation of New Mannose Derived Immuno-
modulating Adamantyltripeptides†
Rosana Ribić,a Lidija Habjanec,b Branka Vranešić,b Ruža Frkanec,b and Srđanka Tomića,
*
aDepartment of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia
bInstitute of Immunology Inc., Rockefellerova 10, HR-10000 Zagreb, Croatia
RECEIVED FEBRUARY 25, 2011; REVISED MAY 15, 2011; ACCEPTED MAY 31, 2011
Abstract. A novel class of mannosylated adamantyltripeptides, general formula of which is α-Man-
OCH2CH(CH3)CO-AdGly-
dulatory properties investigated in some preliminary tests in vivo. All newly synthesized glycopeptides
comprise in their structure the adamantylglycyl moiety linked to the dipeptide -Ala- -isoGln, as well as
mannose. Adjuvant activity of mannosylated adamant-2-yl tripeptides was tested in the mouse model us-
ing ovalbumin as an antigen and in comparison to the peptidoglycan monomer (PGM, β- -GlcNAc-
(1→4)- -MurNAc-L-Ala- -isoGln-mesoDAP(εNH2)- Ala- -Ala). (doi: 10.5562/cca1827)
L-Ala-D-isoGln, were prepared, characterized, and their possible immunomo-
L
D
D
D
D
D-
D
Keywords: O-mannopyranosides, adamantyltripeptides, peptidoglycan monomer, immunomodulation
INTRODUCTION
towards desmuramyl peptides in which the N-acetyl-
muramic portion of MDP was replaced with the lypo-
Peptidoglycans, the essential and unique components of
bacterial cell walls, possess diverse biological activities
that depend upon the size and/or structure of peptidogly-
can fragments. They are generally built of large polysac-
charide chains and short peptide units. Natural and syn-
thetic peptidoglycans as well as their fragments of dif-
ferent molecular mass exhibit remarkable biological ac-
tivites. It was established that they in particular affect the
immune system of mammalian hosts.1,2 Smaller size pep-
tidoglycans as well as some peptidoglycan fragments of
well defined structures, the best known of which are
muramyl peptides, had been extensively studied as poss-
ible adjuvants for human and animal vaccines.3,4,5 Mu-
ramyl peptides are considered to be structural fragments
of the monomeric peptidoglycans (e.g. PGM, 1,
philic adamantylglycine. Thus, previously synthesized
diastereoisomers of admant-1-yl tripeptides 2a, 2b6
(Figure 1) and adamant-2-yl tripeptides 3a, 3b7 were
tested and shown to differ in biological activity in sev-
eral model systems.6−10
Compounds containing adamantyl residues are
known to exhibit various biological activities. They are
mostly used in the prevention of viral diseases, and in
the treatment of Parkinson disease and depression.11−13
Some glycoconjugates of adamantane are also used as
drugs. The most often used one is gludantan, the ada-
mantamine conjugate of glucuronic acid, which is used
as an antiviral agent as well as a drug in the treatment
of Parkinson disease and depression.11 It is also known
that introducing an adamantyl moiety into substances
with known biological activity improves their pharma-
cological properties mostly due to its pronounced lipo-
philic character.12,13 Furthermore, the role of adaman-
tane conjugates with mannose in the process of pre-
venting microbial adhesion to host cells was recently
discussed as well.14 Therefore, it seems to be of inter-
est to further explore the influence of the adamantyl
moiety on biological properties of the dipeptide build-
ing block of MDP, with emphasis on its adjuvant ac-
tivity.
GlcNAc-MurNac-
Ala- -Ala, Figure 1) of bacterial cell walls, and MDP
(muramyl dipeptide; N-acetyl-muramyl- -alanyl-
L-Ala-D-isoGln-mesoDAP(εNH2)-D-
D
L
D-
isoglutamine) is known as the smallest synthetic adju-
vant molecule capable of replacing whole Mycobacteria
in Freund’s adjuvant.5
Several hundred chemically defined MDP analogs
and derivatives were synthesized, in order to modulate
the properties of the parent molecule. Up to now our
research in the field of potential adjuvants was directed
†
This article belongs to the Special Issue Chemistry of Living Systems devoted to the intersection of chemistry with life.
* Author to whom correspondence should be addressed. (E-mail: stomic@chem.pmf.hr)

234
R. Ribić et al., Mannose Derived Adamantyltripeptides
Figure 1. Structures of PGM (1), adamant-1-yl tripeptides (2a, 2b) and adamant-2-yl tripeptides (3a, 3b).
In continuation of our interest and research of
monosaccharide conjugates,15 their enzymatic trans-
formations16−19 and synthesis,20,21 especially of those
with biological activity8,22 we now report the synthesis
of mannose conjugates with admant-1-yl tripeptides 2
and adamant-2-yl tripeptides 3 containing the L-alanyl-
D-isoglutamine dipeptide sequence as in PGM and
MDP. Furthermore, the study was designed to investi-
gate how mannosylation of adamantyl tripeptides
through a chiral spacer of defined configuration (R or
S) might affect the immunostimulating activity in
mice. Mannose receptors (MR) present on the cell
surface of different macrophages and dendritic cells
are considered to be pattern-recognition receptors
binding compounds comprising mannose (Man), N-a-
cetylglucosamine (GlcNAc) and fucose. Therefore,
they are responsible for the binding, among others, of
mannosylated antigens or relevant biologically active
molecules containing mannose, thus affecting the im-
mune reactions. Previous reports on conjugates of
mannosylated proteins with MDP showed enhanced
immunostimulating and antitumor properties in com-
parison to conjugates without mannose.23,24 A previous
report was also given on the effects of mannosylated
PGM on immune reactions in mice and the susceptibil-
ity of these derivatives to the enzyme N-acetylmu-
ramyl-L-alanine amidase.22
EXPERIMENTAL
General Remarks
Bovine serum albumin (BSA), Tween 20, monoclonal
anti-chicken egg albumin (clone OVA-14, mouse IgG1
isotype), avidin-peroxidase and o-phenylenediamine
dihydrochloride (OPD) were from Sigma. Ovalbumin
(OVA) was purchased from Serva. Horseradish peroxi-
dase conjugated goat anti-mouse IgG (HRP-anti-mouse
IgG) was from Bio-Rad Laboratories. Biotin-conjugated
rat anti-mouse IgG1 and anti-mouse IgG2a monoclonal
antibodies and streptavidin-peroxidase were purchased
from PharMingen, Becton Dickinson. Chemicals for
buffers and solutions were from Kemika, unless stated
otherwise. Peptidoglycan monomer was prepared in
PLIVA, Chemical and Pharmaceutical Works (Zagreb,
Croatia), according to the previously described proce-
dure.25 Racemic (adamant-2-yl)glycine26 was prepared at
the Institute of Immunology (Zagreb, Croatia), as de-
scribed earlier. Racemic (adamant-1-yl)glycine hydroch-
loride was prepared as previously described.27 Most of
the chemical reagents used in syntheses were obtained
from Fluka and Aldrich Corp. All solvents were purified
using standard procedures. Column cromatography (sol-
vents and proportions are given in the text) was per-
formed on Merck silica gel 60 (size 70−230 mesh
ASTM) and TLC monitoring on Fluka silica gel (60 F
254) plates (0.25 mm). Visualization was effected mostly
by use of UV light and/or by charring with H2SO4. Opti-
cal rotations were measured at room temperature using
the Schmidt + Haensch Polartronic NH8. NMR spectra
were recorded using Bruker Avance spectrometer at 300
or 600 MHz. C, H and N analyses were provided by the
Analytical Services Laboratory of Ruđer Bošković Insti-
tute, Zagreb. Mass spectra were recorded with Waters
MS − Quattro micro instrument.
Our current study in regard to biological activity
of novel mannosylated adamantyltripeptides should
provide information on their adjuvant effect on humoral
IgG immune response in mice. Ovalbumin was used as
a customary antigen for studying the adjuvant effect.
The comparison of induced anti-OVA IgG levels was
carried out quantitatively and the subclasses of IgG,
IgG1 and IgG2a, as indicator of Th1 and Th2 type of
immune response were also determined.
Croat. Chem. Acta 84 (2011) 233.

R. Ribić et al., Mannose Derived Adamantyltripeptides
235
Chromatographic separations were carried out us-
ing the Waters HPLC System equipped with 2996 PDA
detector and Empower software (Milford, MA, USA). A
LiChrosorb RP-18 column, 244 mm × 4 mm, 5 m, and
a LiChrospher guard column 100 RP-18 (5 m) were
used (Merck, Darmstadt, Germany). Analyses were run
at a flow rate of 1.0 mL/min at room temperature and
the eluate was monitored at 200 nm. The gradient sol-
vent system used was made of acetonitrile containing
0.035 % TFA and water containing 0.05 % TFA.28 The
percentage of acetonitrile at 0, 15 and 20 min was 10,
30 and 10, respectively and a running time was 25 min.
Acetonitrile and trifluoroacetic acid (TFA) were of
HPLC – grade from Merck (Darmstadt, Germany). A
daily supply of water was obtained from Millipore Sim-
plicity – Personal ultra pure water system (Bedford,
MA, USA). The percentage of each peak in the respec-
tive chromatograms was calculated by the integration of
the UV response (peak area).
OH), 4.39 (m, 2H, CH2; Fmoc), 4.12 (d, 1H, J = 7.0 Hz,
CH-α), 4.06 (t, 1H, J = 5.9 Hz, CH; Fmoc), 2.04−1.51
(m, 15H, 5CH, 5CH2; Ad); 13C NMR (CDCl3) δ / ppm:
143.90, 143.70, 143.65, 143.54 (Carom; Fmoc), 127.57,
126.99, 125.05, 119.86 (CHarom; Fmoc), 65.18 (CH2;
Fmoc), 50.31 (CH-α), 47.12 (CH; Fmoc), 38.55, 37.90,
31.69, 31.59 (CH2; Ad), 29.51, 28.13, 27.75, 27.50 (CH;
Ad). ESI-MS: calcd. for C27H29NO4: 431.52; found
[M+H]+ at m/z 432.30.
General Procedure for Synthesis of Adamantyltripep-
tides
To a solution of Fmoc-AdGly 5 or 6 (562 mg, 1.30
mmol) in dry DCM (10 mL) at 0 °C EDCxHCl (300 mg,
1.56 mmol, 1.2 equiv) and HOBt (176 mg, 1.30 mmol, 1
equiv) were added. The mixture was stirred for 0.5 h at
the same temperature and then added to a solution of L-
alanyl-D-isoglutamine tert-butyl ester 4 (355 mg, 1.30
mmol, 1 equiv) in dioxane (5 mL). Subsequently
N-ethylmorpholine (329 μL, 2.60 mmol, 2 equiv) was
added to the reaction mixture. The mixture was stirred
for 1 h at 0 °C and then for 48 h at room temperature.
The solution was diluted with AcOEt and washed with
water. The organic layer was dried over MgSO4 and
after filtration, evaporated. The residue was purified by
column chromatography on silica gel (AcOEt). White
solid foam was afforded as a mixture of diastereomers
(only one spot by TLC).
Characterization was performed on the mixture of
diasteromers. Whenever distinguishable, values given
are for one isomer with those of the second one listed in
square brackets.
Synthesis
General Procedure for 9-Fluorenylmethyloxycarbonyl
Protection of Adamantylglycine
L,D-(Adamant-1-yl)glycine or L,D-(adamant-2-yl)glyci-
ne (350 mg, 1.67 mmol) was suspended in a 2:1 mixture
of dioxane/H2O (12 mL). 9-Fluorenylmethyloxycarbo-
nyl chloride (518 mg, 2.0 mmol, 1.2 equiv) and Na2CO3
(531 mg, 3 equiv) were added. The reaction mixture
was stirred 1 h at 0 ºC and then overnight at room tem-
perature. The mixture was acidified (pH 3) with satu-
rated KHSO4 (aq), and extracted twice with AcOEt. The
organic layers were washed with water and dried over
Na2SO4.
9-Fluorenylmethyloxycarbonyl-L,D-(adamant-1-yl)gly-
cyl-L-alanyl-D-isoglutamine tert-butyl ester (7): 723 mg
(81 %); Rf = 0.43 (AcOEt); H NMR (CDCl3) δ / ppm:
1
7.78−7.26 (m, CHarom; Fmoc), 6.93 (br s, NH), 6.80 (br
s, NH), 6.08−5.57 (m, NH), 4.52−3.90 (m; 3CH-α;
isoGln, Ala, AdGly and CH, CH2; Fmoc), 2.52−2.25
(m, CH2-β, CH2-γ; isoGln), 1.96 (br s, 3CH; Ad),
1.65−1.44 (m, 6CH2; Ad), 1.41 (s, 3CH3; t-Bu), [1.39 (s,
3CH3; t-Bu)], 1.40 (d, J = 7.1 Hz, CH3; Ala), [1.39 (d, J
= 7.1 Hz, CH3; Ala)]; 13C NMR (CDCl3) δ / ppm:
173.81, 173.75, 172.67, 172.58, 172.55, 172.37, 170.30,
170.26 (C=O), 156.75, 156.64 (CO; Fmoc), 144.35,
143.88, 143.61, 141.47, 141.24 (Carom; Fmoc), 127.65,
127.52, 127.02, 125.10, 125.05, 124.69, 120.00, 119.93,
119.90 (CHarom; Fmoc), 80.92, 80.87 (C; t-Bu), 66.98,
65.07 (CH2; Fmoc), 63.44, 63.36 (CH; AdGly), 52.35,
52.10 (CH; isoGln), 50.33, 47.14 CH; Fmoc), 49.23,
48.91 (CH; Ala), 38.67, 38.54, 36.64, 36.54, 36.20,
31.67 (CH2; γ-isoGln, CH2; Ad), 28.16, 27.99 (CH; Ad,
CH3; t-Bu), 27.69, 27.37 (CH2; β-isoGln), 18.25, 18.13
(CH3; Ala). ESI-MS: calcd. for C39H50N4O7: 686.84;
found [M+H]+ at m/z 687.40.
9-Fluorenylmethyloxycarbonyl-L,D-(adamant-1-yl)gly-
cine (5): White solid foam (684 mg, 95 %); Rf = 0.62
(CH3CN/H2O = 5:1). 1H NMR (MeOH-d4) δ / ppm: 7.77
(d, 2H, J = 7.4 Hz, CHarom; Fmoc), 7.64 (d, 2H, J = 7.4
Hz, CHarom; Fmoc), 7.53 (t, 2H, J = 7.2 Hz, CHarom
;
Fmoc), 7.28 (t, 2H, J = 7.3 Hz, CHarom; Fmoc), 4.01 (t,
1H, J = 6.8 Hz, CH; Fmoc), 3.87 (d, 2H, J = 11.2 Hz,
CH2; Fmoc), 3.82 (s, 1H, CH-α), 1.95 (br s, 3H, Ad),
1.73−1.64 (m, 12H, Ad); 13C NMR (MeOH-d4) δ / ppm:
158.52 (CO; Fmoc), 146.30, 145.45, 145.17, 142.63
(Carom; Fmoc), 128.78, 128.43, 128.16, 128.15, 127.94,
126.14, 120.94, 120.78 (CHarom; Fmoc), 65.88 (CH2;
Fmoc), 51.66 (CH-α), 48.59 (CH; Fmoc), 39.99, 37.99
(CH2; Ad), 29.95 (CH; Ad).
9-Fluorenylmethyloxycarbonyl-L,D-(adamant-2-yl)gly-
cine (6): White solid foam (684 mg, 95 %); Rf = 0.62
(CH3CN/H2O = 5:1), 1H NMR (CDCl3) δ / ppm:
7.81−7.25 (m, 9H, CHarom; Fmoc, NH), 4.59 (br s, 1H,
9-Fluorenylmethyloxycarbonyl-L,D-(adamant-2-yl)gly-
cyl-L-alanyl-D-isoglutamine tert-butyl ester (8): (661
1
mg, 74 %); Rf = 0.45 (AcOEt); H NMR (CDCl3) δ /
ppm: 7.86-7.22 (m, CHarom; Fmoc), 6.80 (br s, NH),
6.73 (br s, NH), 6.26 (br s, NH), 6.07 (br s, NH), 5.89
(d, J = 8.1 Hz, NH), 5.83 (d, J = 9.0 Hz, NH), 4.65−4.11
Croat. Chem. Acta 84 (2011) 233.

236
R. Ribić et al., Mannose Derived Adamantyltripeptides
(m; 3CH-α; isoGln, Ala, AdGly and CH, CH2; Fmoc),
2.44−1.53 (m, CH2-β, CH2-γ; isoGln and 5CH, 5CH2;
Ad), 1.41 (s, 3CH3; t-Bu), [1.39 (s, 3CH3; t-Bu)], 1.35
(d, J = 7.1 Hz, CH3; Ala), [1.33 (d, J = 6.7 Hz, CH3;
Ala)]; 13C NMR (CDCl3) δ / ppm: 173.82, 173.80,
172.55, 172.48, 172.38, 172.29, 172.27 (C=O), 156.65,
156.53 (CO; Fmoc), 143.92, 143.80, 143.62, 143.58,
141.22, 141.20 (Carom; Fmoc), 127.66, 127.62, 127.03,
125.13, 125.06, 119.92, 119.90 (CHarom; Fmoc), 80.88,
80.75 (C; t-Bu), 67.05, 66.94 (CH2; Fmoc), 56.06, 52.04
(CH; AdGly), 49.25, 48.87 (CH; isoGln), 52.37, 47.06
CH; Fmoc), 46.57, 46.43 (CH; Ala), 38.65, 38.52,
37.85, 31.77 (CH2; γ-isoGln, CH2; Ad), 29.40, 29.23,
27.87, 27.77, 27.50 (CH; Ad), 28.01, 28.00 (CH3; t-Bu),
27.65, 27.17 (CH2; β-isoGln), 18.07, 17.97 (CH3; Ala).
ESI-MS: calcd. for C39H50N4O7: 686.84; found [M+H]+
at m/z 687.40.
t-Bu), [1.44 (s, 3CH3; t-Bu)], 1.39 (d, J = 7.1 Hz, CH3;
Ala), [1.37 (d, J = 7.1 Hz, CH3; Ala) ]; 13C NMR
(MeOH-d4) δ / ppm: 176.39, 176.26, 175.38, 175.30,
173.82, 173.80 (C=O), 81.91, 81.88 (C; t-Bu), 53.71,
53.53 (CH; AdGly), 51.05, 50.75 (CH; isoGln), 49.50,
48.88 (CH; Ala), 39.86, 39.75, 39.10, 39.02, 32.97,
32.89, 32.75, 32.70, 32.57, 32.53 (CH2; γ-isoGln, CH2;
Ad), 31.11, 30.71, 29.37, 29.33, 29.18, 29.14, 28.95
(CH; Ad), 28.39 (CH3; t-Bu), 28.32, 28.11 (CH2; β-
isoGln), 17.72, 17.60 (CH3; Ala). ESI-MS: calcd. for
C24H40N4O5: 464.60; found [M+H]+ at m/z 465.30.
General Procedure for Condensation of Mannose and
Adamantlytripeptides
To a solution of mannosylated carboxylic acid 11 or 12
(322 mg, 0.52 mmol) in dry DCM (10 mL) at 0 °C
EDCxHCl (119 mg, 0.63 mmol, 1.2 equiv) and HOBt
(70 mg, 0.52 mmol, 1 equiv) were added. The mixture
was stirred for 0.5 h at the same temperature and then
added to a solution of tripeptide 9 or 10 (200 mg, 0.43
mmol) in dioxane (5 mL). Et3N (144 μL, 1.04 mmol, 2
equiv) was added next to the reaction mixture. The
mixture was stirred for 1 h at 0 °C and then for 48 h at
room temperature. The solution was diluted with AcOEt
and washed with water. The organic layer was dried
over Na2SO4 and after filtration, evaporated. The resi-
due was purified by column chromatography on silica
gel (CHCl3/MeOH = 10:1).
N-[(R)-3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl-
oxy)-2-methylpropanoyl]-L,D-(adamant-1-yl)glycyl-L-
alanyl-D-isoglutamine tert-butyl ester (13): White solid
foam (342 mg, 74 %); Rf = 0.58 (CHCl3/MeOH = 10:1);
1H NMR (MeOH-d4) δ / ppm: 8.47 (d, J = 7.4 Hz, NH),
8.42 (d, J = 6.5 Hz, NH), 7.95 (pt, J = 10.4 Hz, J = 7.2
Hz, NH), 7.89 (d, J = 7.8 Hz, NH), 7.86 (d, J = 8.0 Hz,
NH), 7.37−7.13 (m, Harom), 4.90 (s, H-1), 4.81−4.41 (m,
CH; Bn, CH-α; AdGly), [4.76 (d, Jgem = 10.8 Hz, CH;
Bn)], 4.30−4.11 (m, 2CH-α; Ala, isoGln), 3.91−3.27 (H-
2, H-3, H-4, H-5, H-6a, H-6b, OCH2), 2.72−2.61 (m,
CH), 2.18−1.42 (m, CH2-β, CH2-γ; isoGln, 3CH, 6CH2;
Ad), 1.37 (s, 3CH3; t-Bu), [1.36 (s, 3CH3; t-Bu)], 1.22
(d, J = 5.4 Hz, CH3; Ala), [1.18 (d, J = 6.8 Hz, CH3;
Ala)], 0.98 (d, J = 6.8 Hz, CH3), [0.90 (d, J = 6.9 Hz,
CH3)]; 13C NMR (MeOH-d4) δ / ppm: 183.51, 183.13,
182.62, 182.56, 181.52, 181.10, 180.61 (C=O), 148.19,
148.12, 148.06, 148.00, 147.92, 147.89 (Carom),
137.81−135.93 (CHarom), 106.85, 106.61 (C-1), 89.26,
88.98, 84.10, 84.00, 83.94, 83.89, 80.87, 80.79 (C-2, C-
3, C-4, C-5), 89.12, 83.68, 81.89, 81.33, 81.07, 80.94,
80.54, 80.17, 79.28, 78.55 (C-6, CH2; Bn), 75.89, 72.42
(OCH2), 64.13, 62.97 (CH; AdGly), 61.37, 60.93 (CH;
isoGln), 58.46, 57.45 (CH; Ala), 54.92, 54.70 (CH);
47.94, 47.77, 47.66, 47.20, 47.09, 40.82, 40.76 (CH2; γ-
isoGln, CH2; Ad, C; Ad), 36.70, 36.47 (CH2; β-isoGln),
37.25 (CH3; t-Bu), 38.27, 37.85, 36.88, 36.56 (CH; Ad),
27.41, 27.09 (CH3; Ala), 24.07, 23.81 (CH3); ESI-MS:
General Procedure for N-Deprotection of 9-Fluore-
nylmethyloxycarbonyl Adamantyltripeptides
To a solution of 7 or 8 (500 mg, 0.73 mmol) in dry THF
(5 mL) under N2 1-octanthiol (1.26 mL, 7.3 mmol, 10
equiv) and DBU (35.8 μL, 0.24 mmol, 0.33 equiv) were
added. The reaction mixture was stirred at room tem-
perature for 16 h and monitored by TLC (CH3CN/H2O
5:1). The mixture was then triturated with diethyl ether
and a white solid residue was purified by column chro-
matography on silica gel (CHCl3/MeOH = 10:1→1:1).
L,D-(Adamant-1-yl)glycyl-L-alanyl-D-isoglutamine tert-
butyl ester (9): 292 mg (86 %); Rf = 0.59 (CH3CN/H2O
1
5:1). H NMR (MeOH-d4) δ / ppm: 7.90 (s, OH), 7.78
(d, J = 7.5 Hz, NH), 7.65 (dd, J = 7.9 Hz, J = 0.8 Hz,
NH), 7.36 (t, J = 7.5 Hz, NH), 7.29 (dt, J = 6.9 Hz, J =
1.1 Hz, NH), 4.35−4.29 (m; 2CH-α; isoGln, Ala), 3.10
(s, CH-α; AdGly), [3.09 (s, CH-α; AdGly)], 2.35−2.32
(m, CH2-γ; isoGln ), 2.24−2.18 (m, CH-β; isoGln), 2.00
(br s, 3CH; Ad), 1.94−1.82 (m, CH-β’; isoGln),
1.78−1.55 (m, 6CH2; Ad), 1.45 (s, 3CH3; t-Bu), [1.44 (s,
3CH3; t-Bu)], 1.40 (d, J = 7.1 Hz, CH3; Ala), [1.39 (d, J
= 7.1 Hz, CH3; Ala)]. 13C NMR (MeOH-d4) δ / ppm:
176.43, 176.37, 175.38, 175.06, 173.86, 173.81, 173.23,
173.12 (C=O), 81.90, 81.88 (C; t-Bu), 64.71, 64.63
(CH; AdGly), 53.75, 53.61 (CH; isoGln), 51.22, 50.84
(CH; Ala), 39.58, 39.43, 37.83, 36.81, 36.57, 32.76
(CH2; γ-isoGln, CH2; Ad), 29.81, 28.39 (CH; Ad, CH3;
t-Bu), 28.21, 28.07 (CH2; β-isoGln), 17.80, 17.58 (CH3;
Ala). ESI-MS: calcd. for C24H40N4O5: 464.60; found
[M+H]+ at m/z 465.30.
L,D-(Adamant-2-yl)glycyl-L-alanyl-D-isoglutamine tert-
butyl ester (10): 275 mg (81 %); Rf = 0.57 (CH3CN/H2O
1
5:1); H NMR (MeOH-d4) δ / ppm: 7.90 (s, OH),
7.80−7.24 (m, NH), 4.37−4.27 (m; 2CH-α; isoGln, Ala),
3.74 (d, J = 9.1 Hz, CH-α; AdGly), [3.70 (d, J = 9.0 Hz,
CH-α; AdGly)], [3.61 (q, J = 7.1 Hz, CH-α; Ala)],
2.36−2.28 (m, CH2-γ; isoGln), 2.25−2.09 (m, CH2-β;
isoGln), 2.04−1.51 (m, 5CH, 5CH2; Ad), 1.45 (s, 3CH3;
Croat. Chem. Acta 84 (2011) 233.

R. Ribić et al., Mannose Derived Adamantyltripeptides
237
calcd. for C62H81N4O12: 1073.32; found [M+H]+ at m/z
1073.50.
C-5), 89.18, 83.66, 81.87, 81.31, 81.06, 80.93, 80.54,
80.16, 78.58, 78.64 (C-6, CH2; Bn), 75.87, 72.41
(OCH2), 64.11, 62.95 (CH; AdGly), 61.35, 60.91 (CH;
isoGln), 58.44, 57.45 (CH; Ala), 54.89, 54.69 (CH),
47.92, 47.74, 47.66, 47.06, 40.81, 40.75 (CH2; Ad, CH2;
γ-isoGln), 36.67, 36.46 (CH2; β-isoGln), 37.22 (CH3; t-
Bu), 38.27, 37.05, 36.87, 36.54 (CH; Ad), 27.41, 27.09
(CH3; Ala), 24.07, 23.80 (CH3); ESI-MS: calcd. for
C62H81N4O12: 1073.32; found [M+H]+ at m/z 1073.50.
N-[(S)-3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl-
oxy)-2-methylpropanoyl]-L,D-(adamant-2-yl)glycyl-L-
alanyl-D-isoglutamine tert-butyl ester (16): White solid
foam (355 mg, 77 %); Rf = 0.62 (CHCl3/MeOH = 10:1).
1H NMR (MeOH-d4) δ / ppm: 7.93 (d, J = 6.7 Hz, NH),
7.85 (pt, J = 7.2 Hz, J = 8.3 Hz, NH), 7.47 (d, J = 7.0
Hz, NH), 7.38−7.15 (m, Harom), 4.89 (s, H-1), [4.86 (s,
H-1)], 4.81−4.47 (m, CH; Bn, CH-α; AdGly ), [4.52 (d,
N-[(S)-3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl-
oxy)-2-methylpropanoyl]-L,D-(adamant-1-yl)glycyl-L-
alanyl-D-isoglutamine tert-butyl ester (14): White solid
foam (333 mg, 72 %); Rf = 0.50 (CHCl3/MeOH = 10:1);
1H NMR (MeOH-d4) δ / ppm: 8.47 (d, J = 7.0 Hz, NH),
8.32 (s, NH), 7.96 (d, J = 7.7 Hz, NH), 7.92 (d, J = 8.8
Hz, NH), 7.79 (d, J = 8.2 Hz, NH), 7.57 (d, J = 8.0 Hz,
NH), 7.37−7.09 (m, Harom), 4.94 (d, J = 1.1 Hz, H-1),
[4.92 (s, H-1)], 4.77−4.47 (m, CH; Bn, CH-α; AdGly),
4.24 (q, J = 7.2 Hz, CH-α; Ala), 4.20 (q, J = 7.0 Hz,
CH-α; Ala), 4.16 (dd, J = 6.1 Hz, J = 5.0 Hz, CH-α;
isoGln), 4.14 (dd, J = 5.9 Hz, J = 5.0 Hz, CH-α;
isoGln), 3.82−3.55 (m, H-2, H-3, H-4, H-6a, H-6b,
OCH2), 3.46−3.44 (m, H-5), [3.25−3.21 (m, H-5)],
2.76−2.65 (m, CH), 2.18−1.53 (m, CH2-β, CH2-γ;
isoGln, 3CH, 6CH2; Ad), 1.38 (s, 3CH3; t-Bu), [1.37 (s,
3CH3; t-Bu)], 1.09 (d, J = 7.2 Hz, CH3; Ala), [1.06 (d, J
= 7.0 Hz, CH3; Ala)], 0.96 (d, J = 6.9 Hz, CH3), [0.90
(d, J = 6.9 Hz, CH3)]; 13C NMR (MeOH-d4) δ / ppm:
183.49, 182.80, 182.49, 181.50, 181.41, 181.05, 180.94,
180.80 (C=O), 148.08, 148.04, 147.99, 147.94, 147.92
(Carom), 137.76−136.71 (CHarom), 106.39, 105.88 (C-1),
89.52, 89.27, 84.17, 84.13, 83.97, 83.9180.76, 80.56 (C-
2, C-3, C-4, C-5), 89.17, 83.77, 83.56, 81.83, 81.76,
81.29, 81.25, 80.56, 80.37, 78.46, 78.37, 75.87 (C-6,
CH2; Bn, OCH2), 63.67, 62.53 (CH; AdGly), 61.15,
60.76 (CH; isoGln), 58.41, 57.65 (CH; Ala), 55.11,
54.88 (CH), 47.92, 47.80, 47.56, 47.17, 46.95 (CH2; Ad,
CH2; γ-isoGln), 40.78, 40.61 (CH2; β-isoGln), 37.24
(CH3; t-Bu), 38.29, 37.96, 37.00, 36.87, 36.47 (CH;
Ad), 27.38, 27.12 (CH3; Ala), 24.36, 23.79 (CH3); ESI-
MS: calcd. for C62H81N4O12: 1073.32; found [M+H]+ at
m/z 1074.51.
N-[(R)-3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl-
oxy)-2-methylpropanoyl]-L,D-(adamant-2-yl)glycyl-L-
alanyl-D-isoglutamine tert-butyl ester (15): White solid
foam (351 mg, 76 %); Rf = 0.56 (CHCl3/MeOH = 10:1);
1H NMR (MeOH-d4) δ / ppm: 8.47 (d, J = 7.7 Hz, NH),
8.42 (d, J = 6.2 Hz, NH), 8.32 (s, NH), 7.98−7.82 (m,
NH), 7.78 (d, J = 8.2 Hz, NH), 7.37−7.19 (m, Harom),
4.89 (s, H-1), [4.76 (d, Jgem = 11.0 Hz, CH; Bn)],
4.67−4.40 (m, CH; Bn, CH-α; AdGly), 4.29−4.25 (m,
2CH-α; Ala, isoGln), 3.90−3.27 (m, H-2, H-3, H-4, H-5,
H-6a, H-6b, OCH2), 2.69−2.62 (m, CH), 2.20−1.42 (m,
CH2-β, CH2-γ; isoGln, 5CH, 5CH2; Ad), 1.37 (s, 3CH3;
t-Bu), [1.35 (s, 3CH3; t-Bu)], 1.22 (d, J = 6.5 Hz, CH3;
Ala), [1.17 (d, J = 7.0 Hz, CH3; Ala)], 0.97 (d, J = 6.8
Hz, CH3), [0.99 (d, J = 6.8 Hz, CH3)]; 13C NMR
(MeOH-d4) δ / ppm: 183.49, 183.10, 182.59, 182.54,
181.51, 181.47, 181.08, 180.60 (C=O), 148.18, 148.12,
148.05, 147.99, 147.97, 147.92, 147.90, 147.88 (Carom),
137.80−135.92 (CHarom), 106.84, 106.60 (C-1); 88.98,
84.09, 84.00, 83.94, 83.88, 80.85, 80.77 (C-2, C-3, C-4,
Jgem = 11.4 Hz, CH; Bn)], [4.51 (d, Jgem = 10.8 Hz, CH;
Bn)], [4.37 (q, J = 7.1 Hz, CH-α; Ala)], 4.34−4.30 (m,
CH-α; isoGln), [4.25 (q, J = 7.1 Hz, CH-α; Ala)],
3.97−3.28 (m, H-2, H-3, H-4, H-5, H-6a, H-6b, OCH2),
2.75−2.65 (m, CH), 2.30 (pt, J = 7.0 Hz, J = 8.2 Hz,
CH2-γ; isoGln), 2.23 (pt, J = 8.1 Hz, J = 7.4 Hz, CH2-γ;
isoGln), 2.18−1.51 (m, CH2-β; isoGln, 5CH, 5CH2; Ad),
1.424 (s, 3CH3; t-Bu), [1.420 (s, 3CH3; t-Bu)], 1.18 (d, J
= 7.1 Hz, CH3; Ala), [1.11 (d, J = 7.1 Hz, CH3; Ala)],
1.08 (d, J = 7.0 Hz, CH3), [1.03 (d, J = 7.0 Hz, CH3)].
13C NMR (MeOH-d4) δ / ppm: 177.68, 177.60, 177.04,
176.40, 175.94, 174.74, 174.59, 174.33, 174.16, 173.71
(C=O), 140.04, 140.00, 139.94, 139.92, 139.81, 139.77,
139.71, 139.67 (Carom), 129.43−128.57 (CHarom), 99.29,
98.97 (C-1), 82.08, 81.66, 76.50, 76.44, 76.05, 75.92,
73.30, 73.24 (C-2, C-3, C-4, C-5), 76.16, 75.90, 74.44,
74.34, 73.78, 73.71, 73.17, 72.98, 70.91, 70.79, 70.20,
68.16 (C-6, CH2; Bn, OCH2), 56.72, 55.28 (CH;
AdGly), 54.08, 53.41 (CH; isoGln), 50.95, 50.16 (CH;
Ala), 47.26, 46.60 (CH), 39.84, 39.81, 39.79, 39.59,
39.13, 38.91, 32.87, 32.65 (CH2; Ad, CH2; γ-isoGln),
28.60, 28.30 (CH2; β-isoGln), 28.45, 28.42 (CH3; t-Bu),
30.78, 30.46, 29.66, 29.55, 29.42, 29.33, 29.14, 28.91
(CH; Ad), 17.70, 17.63 (CH3; Ala), 14.96, 14.83 (CH3).
ESI-MS: calcd. for C62H81N4O12: 1073.32; found
[M+H]+ at m/z 1073.50.
General Procedure for Hydrolysis of tert-Butyl Esters
Compounds 13−16 (300 mg; 0.28 mmol) were dissolved
in CF3COOH/H2O (3.3 mL; 95:5, v/v). Reaction mix-
tures were stirred at room temperature for 2 h and then
concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (CHCl3/MeOH = 5:1) and
compounds 17−20 were obtained as white solid foams.
N-[(R)-3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl-
oxy)-2-methylpropanoyl]-L,D-(adamant-1-yl)glycyl-L-
alanyl-D-isoglutamine (17): (185 mg, 65%), Rf = 0.47
1
(CHCl3/MeOH = 3:1); H NMR (CDCl3) δ / ppm:
7.52−7.15 (m, Harom), 5.04 (s, H-1), [4.95 (s, H-1)],
Croat. Chem. Acta 84 (2011) 233.

238
R. Ribić et al., Mannose Derived Adamantyltripeptides
4.89−4.28 (m, CH-α; AdGly, Ala, isoGln, CH; Bn),
[4.84 (d, Jgem = 10.7 Hz, CH; Bn)], [4.49 (d, Jgem = 11.5
Hz, CH; Bn)], 4.13−3.44 (m, H-2, H-3, H-4, H-5, H-6a,
H-6b, OCH2), 2.81−2.76 (m, CH), 2.38−2.01 (m, CH2-
β, CH2-γ; isoGln), 1.93 (s, CH; Ad), 1.68−1.56 (m, CH2;
Ad), 1.26 (d, J = 4.9 Hz, CH3; Ala), [1.21 (d, J = 4.7 Hz,
CH3; Ala)], 1.16 (d, J = 6.6 Hz, CH3), [1.07 (d, J = 6.6
Hz, CH3)]; 13C NMR (CDCl3) δ / ppm: 174.87, 173.88,
172.66, 172.33, 172.22, 169.85, 168.60 (C=O), 138.49,
138.41, 138.31, 138.21 (Carom), 128.24−127.37 (CHarom),
98.13, 97.89 (C-1), 80.39, 80.17, 79.89, 74.68, 74.58,
72.05 (C-2, C-3, C-4, C-5), 80.57, 75.10, 75.02, 73.23,
72.58, 71.91, 69.20, 69.09, 68.94, 68.91 (C-6, CH2; Bn,
OCH2), 61.10, 57.29 (CH; AdGly), 51.81, (CH; isoGln),
48.91, 48.72 (CH; Ala), 38.85, 38.64, 36.70, 36.43,
31.54, 29.62, 25.50, 25.38 (CH2; β-isoGln, CH2; γ-
isoGln, CH2; Ad, C; Ad), 28.07, 27.97 (CH, CH; Ad),
14.41, 13.49 (CH3, CH3; Ala). ESI-MS: calcd. for
C58H72N4O12: 1017.21; found [M+H]+ at m/z 1017.50.
N-[(S)-3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl-
oxy)-2-methylpropanoyl]-L,D-(adamant-1-yl)glycyl-L-
alanyl-D-isoglutamine (18): 134 mg (47 %), Rf = 0.48
CH-α; AdGly), 4.41−4.32 (m, 2CH-α; Ala, isoGln,),
[4.25 (q, J = 6.3 Hz, CH-α; Ala)], 4.08−3.54 (m, H-2,
H-3, H-4, H-5, H-6a, H-6b, OCH2), 2.74−2.67 (m, CH),
2.23−2.16 (m, CH-γ; isoGln), 2.07−1.57 (m, CH2-β and
CH-γ; isoGln, CH, CH2; Ad), 1.36 (d, J = 7.1 Hz, CH3;
Ala), [1.32 (d, J = 7.2 Hz, CH3; Ala)], 1.07 (d, J = 6.9
Hz, CH3), [1.00 (d, J = 7.0 Hz, CH3)]; 13C NMR
(MeOH-d4) δ / ppm: 177.63, 177.52, 177.38, 176.51,
176.34, 176.24, 174.89, 174.80, 174.03, 173.93 (C=O),
140.04, 139.90, 139.87, 139.80, 139.68, 139.62, 139.58
(Carom), 129.43−128.62 (CHarom), 100.11, 99.79 (C-1),
81.69, 81.48, 76.48, 76.18, 76.10, 73.39, 73.36 (C-2, C-
3, C-4, C-5), 76.05, 75.99, 74.56, 74.52, 73.84, 73.52,
73.30, 73.06, 71.89, 71.23, 70.60, 65.28 (C-6, CH2; Bn,
OCH2), 54.25, 53.73 (CH; AdGly), 50.13, 50.02 (CH;
isoGln), 47.16, 46.71 (CH; Ala), 42.11, 41.95 (CH),
39.86, 39.78, 39.68, 39.02, 32.83, 32.71, 32.42, 31.34
(CH2; γ-isoGln, CH2; Ad), 28.26, 28.20 (CH2; β-
isoGln), 30.75, 30.39, 29.71, 29.36, 29.07 (CH; Ad),
17.73, 17.50 (CH3; Ala), 14.76, 14.53 (CH3); ESI-MS:
calcd. for C58H72N4O12: 1017.21; found [M+H]+ at m/z
1017.50.
1
N-[(S)-3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl-
oxy)-2-methylpropanoyl]-L,D-(adamant-2-yl)glycyl-L-
alanyl-D-isoglutamine (20): 154 mg (54 %), Rf = 0.50
(CHCl3/MeOH = 3:1); H NMR (MeOH-d4) δ / ppm:
8.30 (d, J = 6.3 Hz, NH), 8.21 (d, J = 6.9 Hz, NH), 7.89
(d, J = 9.0 Hz, NH), 7.84 (d, J = 7.9 Hz, NH), 7.71 (d, J
= 9.2 Hz, NH), 7.39−7.03 (m, Harom), 4.97 (s, H-1),
[4.93 (s, H-1)], 4.75−4.41 (m, CH2; Bn, CH-α; AdGly),
4.30−4.11 (m, 2CH-α; Ala, isoGln), 4.03−3.35 (m, H-2,
H-3, H-4, H-5, H-6a, H-6b, OCH2), 2.93−2.84 (m, CH),
2.06 (t, J = 7.0 Hz, CH2-γ; isoGln), 1.89−1.46 (CH2-β;
isoGln, CH, CH2; Ad), 1.19 (d, J = 7.0 Hz, CH3; Ala),
[1.08 (d, J = 7.0 Hz, CH3; Ala)], 1.00 (d, J = 6.8 Hz,
CH3), [0.91 (d, J = 6.8 Hz, CH3)]; 13C NMR (MeOH-d4)
δ / ppm: 183.46, 183.34, 182.80, 182.56, 182.42, 182.21,
181.54, 181.42, 180.98, 180.82 (C=O), 148.44, 148.33,
148.09 (Carom), 138.24−136.74 (CHarom), 106.42, 105.88
(C-1), 89.54, 89.27, 84.14, 83.97, 80.57, 80.19, 76.19
(C-2, C-3, C-4, C-5), 81.83, 81.77, 81.29, 80.45, 80.39,
78.38, 78.26, 75.89, 72.42 (C-6, CH2; Bn, OCH2),
63.59, 62.55 (CH; AdGly), 61.24, 60.86 (CH; isoGln),
58.44, 57.68 (CH; Ala), 55.16, 55.01 (CH), 47.94,
47.86, 47.52, 47.20, 46.96, 40.87, 40.69, 39.65, 39.49
(CH2; Ad, CH2; β-isoGln, CH2; γ-isoGln), 38.32, 37.96,
36.94, 36.60, 36.50 (CH; Ad), 27.41, 27.13 (CH3; Ala),
24.40, 23.79 (CH3); ESI-MS: calcd. for C58H72N4O12:
1017.21; found [M+H]+ at m/z 1017.40.
1
(CHCl3/MeOH = 3:1); H NMR (MeOH-d4) δ / ppm:
8.02 (d, J = 8.3 Hz, NH), 7.96 (d, J = 8.1 Hz, NH), 7.83
(d, J = 7.7 Hz, NH), 7.75 (pt, J = 6.9 Hz, J = 9.3 Hz,
NH), 7.57 (d, J = 7.4 Hz, NH), 7.52−7.04 (m, Harom),
5.21 (d, J = 1.7 Hz, H-1), [5.17 (s, H-1)], 5.03−4.34 (m,
CH2; Bn, 2CH-α; AdGly, Ala), [4.69(d, Jgem = 11.1 Hz,
CH; Bn)], [4.65 (d, Jgem= 12.0 Hz, CH; Bn)], 4.27 (q, J
= 6.4 Hz, CH-α; Ala), 4.12−4.00 (m, CH-α; isoGln,),
3.95−3.31 (m, H-2, H-3, H-4, H-5, H-6a, H-6b, OCH2),
2.74−2.65 (m, CH), 2.24−2.17 (m, CH-γ; isoGln),
2.07−1.57 (m, CH2-β, CH-γ; isoGln, CH, CH2; Ad),
1.33 (d, J = 6.6 Hz, CH3; Ala), [1.32 (d, J = 7.2 Hz,
CH3; Ala)], 1.02 (d, J = 6.7 Hz, CH3), [1.00 (d, J = 6.9
Hz, CH3)]; 13C NMR (MeOH-d4) δ / ppm: 177.58,
177.38, 176.30, 176.16, 174.82, 174.74, 174.22, 173.90
(C=O), 139.99, 139.84, 139.77, 139.65, 139.61, 139.56,
139.54 (Carom), 129.41−127.98 (CHarom), 100.07, 99.76
(C-1), 81.86, 81.45, 76.46, 76.16, 76.08, 73.36 (C-2, C-
3, C-4, C-5), 76.02, 75.96, 75.91, 74.53, 74.48, 73.81,
73.81, 73.51, 73.27, 73.18, 73.03 (C-6, CH2; Bn,
OCH2), 53.70, 53.54 (CH; 2-AdGly), 52.22, 51.05 (CH;
isoGln), 47.10, 46.68 (CH; Ala), 42.07, 42.03 (CH),
39.84, 39.74, 39.65, 38.99, 32.80, 32.68 (CH2; Ad, CH2;
γ-isoGln), 30.71, 30.37, 29.68, 29.32, 29.03 (CH; Ad),
28.26, 28.17 (CH2; β-isoGln), 17.74, 17.52 (CH3; Ala),
14.77, 14.54 (CH3); ESI-MS: calcd. for C58H72N4O12:
1017.21; found [M+H]+ at m/z 1017.48.
N-[(R)-3-(2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl-
oxy)-2-methylpropanoyl]-L,D-(adamant-2-yl)glycyl-L-
alanyl-D-isoglutamine (19): 151 mg (53 %), Rf = 0.47
1
(CHCl3/MeOH = 3:1); H NMR (MeOH-d4) δ / ppm:
8.52 (d, J = 7.5 Hz, NH), 8.44 (d, J = 5.5 Hz, NH), 8.06
(d, J = 7.8 Hz, NH), 8.02 (d, J = 7.6 Hz, NH), 7.95 (d, J
= 8.1 Hz, NH), 7.82 (d, J = 7.2 Hz, NH), 7.75 (d, J = 6.2
Hz, NH), 7.41−7.17 (m, Harom), 4.91 (d, J = 1.6 Hz, H-
1), [4.90 (d, J = 1.3 Hz, H-1)], 4.75−4.46 (m, CH2; Bn,
General Procedure for O-debenzylation
To a solution of compounds 17−20 (100 mg, 0.132
mmol) in 3:1 mixture of CHCl3/MeOH (5 mL) 50 mg of
Croat. Chem. Acta 84 (2011) 233.

R. Ribić et al., Mannose Derived Adamantyltripeptides
239
10 % Pd/C and 20 mL of 50% EtOH in water were
added. The mixtures were subjected to hydrogen atmos-
phere under 4 bars at room temperature and stirred for
24 h. Then catalysts were filtrated and filtrates concen-
trated under reduced pressure. The residues were puri-
fied by column chromatography on silica-gel
(CHCl3/MeOH = 1:1) and compounds 21−24 were ob-
tained.
calcd. for C30H48N4O12: 656.72; found [M+H]+ at m/z
657.50.
N-[(R)-3-(α-D-mannopyranosyloxy)-2-methylpropano-
yl]-L,D-(adamant-2-yl)glycyl-L-alanyl-D-isoglutamine
(23): 42 mg (48 %); Rf = 0.29 (CHCl3/MeOH = 1:1). 1H
NMR (D2O/dioxan) δ / ppm: 8.44 (s, OH), 8.06 (d, J =
7.8 Hz, NH), 7.87 (d, J = 7.5 Hz, NH), 7.71 (d, J = 7.5
Hz, NH), 7.64 (pt, J = 7.7 Hz, J = 7.4 Hz, NH),
7.44−7.32 (m, NH) 5.16 (d, J = 1.0 Hz, H-1), [4.88 (s,
H-1)], 4.76 (d, J = 6.7 Hz, CH-α; AdGly), [4.64−4.63
(m, CH-α; AdGly)], 4.40 (q, J = 7.1 Hz, CH-α; Ala),
[4.34 (q, J = 7.1 Hz, CH-α; Ala)], 4.25 (dd, J = 5.0 Hz, J
= 6.6 Hz, CH-α; isoGln ), [4.16−4.14 (m, CH-α;
isoGln)], 3.93−3.45 (m, H-2, H-6a, OCH2, H-3, H-4, H-
6b, H-5), 2.78−2.71 (m, CH), 2.65−2.60 (m, CH),
2.26−1.60 (m, CH2-γ and CH2-β; isoGln, Ad),
1.41−1.37 (m, CH3; Ala), 1.09−1.02 (m, CH3); 13C
NMR (D2O/dioxan) δ / ppm: 177.60, 177.58, 176.12,
174.83, 174.56, 173.90, 173.79, 173.55 (C=O), 100.08,
100.06 (C-1), 72.63, 72.55, 70.60, 70. 57, 70.48, 70.46,
66.76, 66.71 (C-2, C-3, C-4, C-5), 69.32, 69.30 (C-6);
60.87 (OCH2), 55.09, 53.04 (CH; AdGly), 49.55, 49.36
(CH; isoGln), 45.37, 45.34 (CH; Ala), 40.74, 40.71
(CH), 38.05, 37.83, 37.20, 31.25, 31.18, 31.13, 31.02
(CH2; γ-isoGln, CH2; Ad), 29.01, 28.97, 27.91, 27.39,
27.12 (CH; Ad), 26.76 (CH2; β-isoGln), 17.07, 16.76
(CH3; Ala), 13.36, 12.78 (CH3). ESI-MS: calcd. for
C30H48N4O12: 656.72; found [M+H]+ at m/z 657.30.
N-[(S)-3-(α-D-mannopyranosyloxy)-2-methylpropano-
yl]-L,D-(adamant-2-yl)glycyl-alanyl-D-isoglutamine
(24): 59 mg (68 %); Rf = 0.34 (CHCl3/MeOH = 1:1);
1H NMR (D2O/dioxan) δ / ppm: 8.44 (s, OH), 7.87 (d,
J = 7.7 Hz, NH), 7.71 (pt, J = 7.4 Hz, J = 7.5 Hz, NH),
7.63 (pt, J = 7.5 Hz, J = 7.3 Hz, NH), 7.43−7.38 (m,
NH), 5.16 (s, H-1), [4.88 (s, H-1)], 4.64 (d, J = 6.5 Hz,
CH-α; AdGly), [4.59 (d, J = 5.6 Hz, CH-α; AdGly)],
4.36 (q, J = 7.5 Hz, CH-α; Ala), [4.32 (q, J = 7.1 Hz,
CH-α; Ala)], 4.24 (dd, J = 4.7 Hz, J = 7.0 Hz, CH-α;
isoGln ), [4.20−4.11 (m, CH-α; isoGln)], 3.97−3.33
(m, H-2, H-6a, OCH2, H-3, H-4, H-6b, H-5), 2.78−2.73
(m, CH), 2.63−2.59 (m, CH), 2.25−1.56 (m, CH2-γ and
CH2-β; isoGln, Ad), 1.39−1.36 (m, CH3; Ala),
1.06−1.01 (m, CH3). 13C NMR (D2O/dioxan) δ / ppm:
177.97, 177.93, 177.40, 177.23, 176.08, 174.65,
173.87, 173.56 (C=O), 99.34, 99.28 (C-1), 72.62,
72.33, 70.63, 70.55, 70.17, 70.02, 66.80 (C-2, C-3, C-
4, C-5), 68.91, 68.88 (C-6), 60.91, 60.82 (OCH2),
53.19, 53.00 (CH; AdGly), 49.87, 49.82 (CH; isoGln),
45.55, 45.52 (CH; Ala), 40.22, 39.89 (CH), 38.09,
38.05, 37.98, 37.86, 37.23, 31.28, 31.26, 31.15, 31.08
(CH2; γ-isoGln, CH2; Ad), 28.97, 28.82, 28.72, 27.87,
27.74, 27.41, 27.16, 27.11, 27.15 (CH; Ad), 27.26,
27.06 (CH2; β-isoGln), 16.24, 16.21 (CH3; Ala), 13.28,
13.05 (CH3). ESI-MS: calcd. for C30H48N4O12: 656.72;
found [M+H]+ at m/z 657.30.
N-[(R)-3-(α-D-mannopyranosyloxy)-2-methylpropano-
yl]-L,D-(adamant-1-yl)glycyl-L-alanyl-D-isoglutamine
(21): 66 mg (76 %); Rf = 0.31 (CHCl3/MeOH = 1:1); 1H
NMR (D2O/dioxan) δ / ppm: 8.34 (s, OH), 7.86 (d, J =
7.2 Hz, NH), 7.64 (d, J = 7.2 Hz, NH), 7.58 (d, J = 7.9
Hz, NH), 7.41−7.31 (m, NH), 5.10 (d, J = 1.4 Hz, H-1),
[4.82 (d, J = 1.4 Hz, H-1)], 4.64 (br s, CH-α; AdGly),
4.30−4.09 (m, CH-α; Ala, isoGln), [4.27 (q, J = 7.1 Hz,
CH-α; Ala)], [4.19 (dd, J = 4.4 Hz, J = 7.7 Hz, CH-α;
isoGln)], 4.04−3.42 (m, H-2, H-3, H-4, H-5, H-6a, H-6b,
OCH2), 2.85−2.75 (m, 1H, CH), 2.22−2.00 (m, CH2-β,
CH2-γ; isoGln), [2.18 (q, 1H, J = 6.9 Hz, CH2-β;
isoGln)], 1.91 (br s, CH; Ad), 1.66−1.52 (m, CH2; Ad),
1.33 (d, J = 6.9 Hz, CH3; Ala), [1.32 (d, J = 7.0 Hz,
CH3; Ala)], 1.06 (d, J = 7.2 Hz, CH3), [0.98 (d, J = 6.9
Hz, CH3)]; 13C NMR (D2O/dioxan) δ / ppm: 177.97,
177.79, 176.79, 176.33, 175.69, 174.81, 171.54 (C=O),
100.16, 100.05 (C-1), 76.09, 72.97, 72.60, 72.32, 71.15,
70.59, 70.15, 69.90 (C-2, C-3, C-4, C-5), 69.44 (C-6),
66.83, 66.77 (CH; AdGly), 60.89, 60.84 (OCH2), 53.43,
53.35 (CH; isoGln), 49.87, 48.80 (CH; Ala), 40.61,
40.52 (CH), 38.62, 38.26, 36.02, 35.72, 33.77, 33.57
(CH2; γ-isoGln, CH2; Ad, C; Ad), 28.04, 28.00 (CH;
Ad), 27.70, 27.61 (CH2; β-isoGln), 16.73, 16.19 (CH3;
Ala), 12.90, 12.83 (CH3); ESI-MS: calcd. for
C30H48N4O12: 656.72; found [M+H]+ at m/z 657.40.
N-[(S)-3-(α-D-mannopyranosyloxy)-2-methylpropano-
yl]-L,D-(adamant-1-yl)glycyl-L-alanyl-D-isoglutamine
(22): 44 mg (50 %); Rf = 0.30 (CHCl3/MeOH = 1:1); 1H
NMR (D2O/dioxan) δ / ppm: 8.08 (d, J = 7.7 Hz, NH),
7.71 (s, NH), 7.55 (d, J = 8.7 Hz, NH), 7.47-7.44 (m,
NH), 7.21 (s, NH), 7.12 (s, NH), 7.03 (s, NH), 5.16 (s,
H-1), [4.89 (s, H-1)], 4.50−3.98 (m, 3CH-α; AdGly,
Ala, isoGln), [4.33 (br s, CH-α; AdGly)], 3.92−3.33 (m,
H-2, H-6a, OCH2, H-3, H-4, H-6b, H-5), 2.88−2.19 (m,
CH, CH2-γ and CH2-β; isoGln), 1.99−154 (m, Ad), 1.40
(d, J = 5.6 Hz, CH3; Ala), [1.38 (d, J = 6.2 Hz, CH3;
Ala)], 1.11 (d, J = 6.6 Hz, CH3), [1.04 (d, J = 6.4 Hz,
CH3)]; 13C NMR (D2O/dioxan) δ / ppm: 179.20, 173.58,
173.50, 173.43, 172.31 (C=O), 93.93, 93.54 (C-1),
76.01, 72.93, 72.30, 71.14, 70.61, 70.15, 70.00, 66.80
(C-2, C-3, C-4, C-5), 62.49, 60.89, 60.77 (C-6, OCH2),
49.64, 49.30 (CH; AdGly), 46.50, 46.45 (CH; isoGln),
44.60, 44.07 (CH; Ala), 40.22, 39.89 (CH), 38.32,
38.23, 38.21, 35.96, 29.89, 29.85 (CH2; β-isoGln, CH2;
γ-isoGln, CH2; Ad), 27.89, 27.87, 27.84 (CH; Ad),
18.43, 18.42 (CH3; Ala), 16.51, 16.49 (CH3); ESI-MS:
Croat. Chem. Acta 84 (2011) 233.

240
R. Ribić et al., Mannose Derived Adamantyltripeptides
Experiments in vivo
between experimental groups was evaluated by Kruskal-
Wallis ANOVA, followed by multiple Mann-Whitney
U-nonparametric tests. A probability values less than
0.05 (p < 0.05) were considered significant.
Experiments in vivo were performed on CBA (H-2k)
inbread mice strains. All mice used were females 2 to
2.5 months old. Commercial food and water were pro-
vided ad libitum. During the experiments animals were
kept at the animal facility at the Institute of Immunolo-
gy and all experiments were performed according to the
Croatian Law on Animal Welfare (The Official Gazette
“NN” 135/06).
Experimental groups of five mice were immunized
and boosted two times subcutaneously (s.c.) into the tail
base at 21-days intervals. Mice were anesthetised prior
to blood collection on 7th day after the second booster.
Sera were collected, decomplemented at 56 C for 30
minutes and stored at −20 C until tested.
RESULTS AND DISCUSSION
Immunomodulating characteristics of natural peptidog-
lycan molecules are well known,2,32 and some of these
compounds were considered for use as adjuvants for
human and animal vaccines. Smaller, synthetic mole-
cules, such as muramyldipeptides and tripeptides were
prepared and their immunomodulating properties inves-
tigated as well. These investigations led to a number of
structure-activity studies which include synthetic mod-
ifications, possibilities of which are numerous since the
molecules are multifunctional, and both saccharide and
peptide parts may be modified. Synthetic modifications
described so far were mostly on smaller molecules since
the protection and deprotection of multifunctional com-
pounds with groups of similar activity represent a conti-
nuous synthetic problem. Thus, several synthetically
modified MDP, syntheses of which were patented, have
already been included in clinical trials.33 Despite nu-
merous synthetic problems encountered in transforma-
tions of larger molecules several successful attempts to
synthetically modify PGM monomer 1 as a parent com-
pound were made. The previous reports describe the
synthesis of PGM modified with Boc-tyrosine,34 ada-
mant-1-yl,35 as well as with mannopyranosyl22 residues.
This was possible because the preferential conformation
of PGM in aqueous and dimethylsulfoxide solutions
showed that the amino group of meso-diaminopimelic
acid is exposed and can therefore be manipulated with-
out the interference of other reactive groups.36
The dose of OVA (antigen) was 10 µg per mouse.
The dose of PGM and adamantyltripeptides was 200 µg
per mouse. OVA and tested substances were dissolved
in saline and the injection volume in all experimental
groups was 0.1 mL per mouse.
Enzyme immunoassays for qualitative and quantita-
tive determination of OVA-specific IgG
(anti-OVA IgG) in mice sera
Enzyme immunoassays (ELISA) were performed on
flat-bottomed high binding microtitre plates (Costar,
USA) according to previously described procedures.22,29
The relative quantities of anti-OVA IgG were deter-
mined by parallel line assay comparing each serum to
monoclonal anti-chicken egg albumin, declared as stan-
dard preparation, to which 20,000 arbitrary units per
milliliter (AU/mL) were voluntarily assigned.30
Enzyme immunoassays for qualitative and quantita-
tive determination of OVA-specific IgG subtypes
anti-OVA IgG1 and anti-OVA IgG2a respectively, in
mice sera
In the present work a novel class of mannosyla-
ted adamantyl tripeptides, general formula of which is
α-Man-OCH2CH(CH3)CO-AdGly-L-Ala-D-isoGln, were
prepared, characterized, and their possible immunomo-
dulatory properties investigated in some preliminary
tests in vivo. All newly synthesized glycopeptides com-
prise in their structure the adamantylglycyl moiety
linked to the dipeptide L-Ala-D-isoGln, characteristic of
the peptide portion of natural peptidoglycans and syn-
thetic muramyldipeptides. They also comprise mannose,
an important sugar whose receptors on immunocompe-
tent cells (such as macrophages and dendritic cells) are
considered to be pattern-recognition receptors biding
compounds comprising mannose, N-acetylglucosamine
and fucose. Previous investigations showed that ada-
mant-1-yl tripeptides 2a, 2b6 (Figure 1) and adamant-2-
yl tripeptides 3a, 3b7 can be prepared as pure diastereoi-
somers and they were tested and shown to differ in
biological activity in several model systems.6−10 Synthe-
sis of mannosylated adamantyltripeptides reported in
ELISA for determination of anti-OVA IgG1 and anti-
OVA IgG2a were performed as described previously.31
The relative quantities of antibody subtypes were de-
termined by parallel line assay using appropriate stan-
dard preparation. The monoclonal anti-OVA IgG1 was a
standard for relative quantification of anti-OVA IgG1 to
which 400,000 AU/mL was assigned, while polyclonal
mouse serum containing high levels of anti-OVA IgG2a
was used as a standard for relative quantification of
IgG2a specific antibodies with voluntarily assigned
20,000 AU/mL. The ratio of anti-OVA IgG1 and anti-
OVA IgG2a (IgG1/IgG2a) was used as indication of the
Th1/Th2-bias of induced immune response.
Statistics
Statistical analyses were performed using Statistica 6.0
for Windows, StatSoft Inc. The significant difference
Croat. Chem. Acta 84 (2011) 233.

R. Ribić et al., Mannose Derived Adamantyltripeptides
241
Scheme 1. i) EDCxHCl, HOBtxH2O, N-ethylmorpholine, Fmoc-(adamant-1-yl)Gly 5 or Fmoc-(adamant-2-yl)Gly 6, dio-
xane:CH2Cl2=1:1; ii) DBU, 1-octanthiol, THF; iii) EDCxHCl, HOBtxH2O, Et3N, dioxane:CH2Cl2 = 1:1; iv) TFA; v) H2, 10 %
Pd/C, 50 % EtOH.
this work led to compounds with fixed anomeric confi-
guration on the sugar ring (α), fixed chirality of the
spacer (R or S) linking the mannose moiety to the rest of
these complex molecules, but in the final step we were
not able to separate diastereoisomers formed due to the
isomeric structures of the tripeptide portion of these
novel molecules. Nevertheless, preliminary investiga-
tion of their adjuvant (immunostimulating) activity in
vivo was conducted in order to obtain results indicating
possible differences in their immunostimulating activi-
ties as a result of the introduced mannose and chiral
spacer parts. Positive results obtained make further
investigations worthwhile especially in view of the
significance of mannose molecules on the cell surfaces,
and further attempts to synthesize pure diasteroisomers
are in progress.
erate overall yields. The hydrolysis of methyl esters of
-anomers was performed by saponification leading to
acids 11 and 12.
Completely protected mannosylated adamantyltri-
peptides 13−16 were obtained in good yields using
EDC/HOBt coupling method. Acid hydrolysis of tert-
butyl protecting group led to compounds 17−20. At-
tempts to separate diastereoisomers were unsuccessful
and compounds 17 ((R)α-Man-OCH2CH(CH3)CO-(Ad-1-
The glycopeptides 21−24 were obtained in a se-
quence of steps (Scheme 1). The amino group of racem-
ic adamant-1-yl or -2-yl glycine was protected with 9-
fluorenylmethyloxycarbonyl (Fmoc) to give protected
compounds 5 and 6. Condensation of 5 and 6 with tert-
butyl ester of L-Ala-D-isoGln 46 followed, to produce
completely protected tripeptides 7 and 8, respectively.
The base labile Fmoc protecting group was them re-
moved using catalytic amounts of dia-za(1,3)bi-
cyclo[5.4.0]undecane (DBU) and 1-octanthiol as a sca-
venger to trap the liberated dibenzofulvene. Tert-butyl
esters of adamantyltripeptides 9 and 10 were obtained
and coupled to perbenzylated -mannosyloxy-2-
methylpropionic acid of R (11) or S (12) configuration.
As previously described the acids 11 and 12 were pre-
pared in a sequence of steps.14,22 Commercially availa-
ble methyl (R) or (S)-3-hydroxy-2-methylpropio-nate
was O-mannosylated using benzyl protected mannosyl
trichloroacetimidate as a good glycosyl donor and cata-
lytic amounts of Lewis acids as reaction promotors. O-
Glycosylations gave anomeric mixtures of the corres-
ponding O-glycosides in different ratios. The obtained
α,β-glycosides were readily separated by column chro-
matography on silica gel giving pure anomers in mod-
Figure 2. HPLC chromatogram of mannosylated adaman-
tyltripeptides diastereoisomeric mixtures: a) 23, R-spacer;
peak l L-L-D of 3b (t = 15.6 min), peak ll D-L-D of 3a (t =
22.0 min); and b) 24, S-spacer; peak l L-L-D of 3b (t = 17.9
min), peak ll D-L-D of 3a (t = 19.9 min); in both cases the
ratio of peak l : peak ll was 6 : 4.
Croat. Chem. Acta 84 (2011) 233.

242
R. Ribić et al., Mannose Derived Adamantyltripeptides
Figure 3. The effect of mannosylated adamantyltripeptides 23 and 24 on the production of anti-OVA IgG (a) and its subtypes
anti-OVA IgG1 (b) and anti-OVA IgG2a (c), respectively, in CBA (H-2k ) mice. Mice were immunized with OVA as an antigen
and sera were analyzed after second booster. Experimental groups: 1-OVA alone; 2-OVA+1; 3-OVA+23; 4-OVA+24.  denotes
group mean value,  denotes each serum separately.
yl)Gly-L-Ala-D-isoGln), 18 ((S)α-Man-OCH2CH(CH3)-
CO-(Ad-1-yl)Gly-L-Ala-D-isoGln),19 ((R) α-Man-
immune response than OVA alone. In comparison to the
control, PGM-injected group, the better immunostimu-
lating effect of tested compounds was not observed
either. However, the measured quantity of anti-OVA
IgG antibodies was markedly higher when compound
23 was applied in comparison to 24.
Similar results were obtained when anti-OVA
IgG1 and anti-OVA IgG2a were measured. Thus, for
manosylated adamantyltripeptide 23 the response was
again a little higher than for 24. Basically, it was the
same pattern as for anti-OVA IgG antibodies.
OCH2CH(CH3)CO-(Ad-2-yl)Gly-L-Ala-D-isoGln) and
20 ((S) α-Man-OCH2CH(CH3)CO-(Ad-2-yl)Gly-L-Ala-
D-isoGln) were obtained as mixtures of diastereoisomers.
Catalytic hydrogenation of 17−20 gave the completely
deprotected compounds 21−24. Diasteroisomeric mix-
tures were not separated but compounds 23 (spacer of R
configuration) and 24 (spacer of S configuration) were
obtained as mixtures of diastereoisomers with identical
ratios (4:6) of tripeptides of D-L-D and L-L-D configura-
tion (Figure 2). Compounds 23 and 24 were then further
tested in vivo for their immunostimulating activity.
The adjuvant activity of mannosylated adamantyl-
tripeptides 23 and 24 in comparison to PGM 1 was
studied. PGM was established as a reference for adju-
vant activity in a previously well-defined mouse model
in vivo, proven suitable for comparison of the observed
effects.37,38 Ovalbumin was used as an antigen for study-
ing the adjuvant effect of mannosylated adamantyltri-
peptides in CBA (H-2k ) mouse strain.
The comparison of induced anti-OVA IgG levels
was carried out quantitatively and the subclasses of IgG,
IgG1 and IgG2a, as indicator of Th1 or Th2 type of
immune response were also determined (Figure 3).
In general, no significant enhancement in total an-
ti-OVA IgG antibody production was observed when
mannosylated adamantyltripeptides 23 and 24 were
applied, in comparison to no adjuvant (OVA alone)
treated group, although glycoconjugate 23 elicited better
From IgG1/IgG2a ratio, calculated for each serum
(obtained after the second booster), it could be observed
that neither of tested glycoconjugates significantly
shifted the IgG1/IgG2a ratio in comparison to no adju-
Table 1. The ratio of anti-OVA IgG1 and anti-OVA IgG2a
(IgG1/IgG2a) in CBA (H-2k ) mice. For each mouse serum,
obtained after the second booster, log10 IgG1/IgG2a was calcu-
lated and the result for each experimental group (n = 5) is
presented as average ± standard deviation (SD)
Average of log10 IgG1/IgG2a ±
Experimental groups
SD
OVA
2.55 ± 0.2
2.23 ± 0.4
2.05 ± 0.6
2.06 ± 0.8
OVA + 1
OVA + 23
OVA + 24
Croat. Chem. Acta 84 (2011) 233.

R. Ribić et al., Mannose Derived Adamantyltripeptides
243
Chem. Biodiv. submitted for publication.
vant treated group of animals (Table 1). Accordingly,
the tested mannosylated adamantyltripeptides 23 and 24
did not stimulate immunomodulatory activity towards
more pronounced Th1 or Th2 type of immune response
in comparison to antigen alone.
7. B. Vranešić, J. Tomašić, S. Smerdel, D. Kantoci, and F.
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CONCLUSION
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Tomašić, Period. Biol. 98 (1996) 305−310.
11. A. A. Spasov, T. V. Khamidova, L. I. Bugaeva, and I. S. Moro-
zov, Pharm. Chem. J. 34 (2000) 1−7.
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hydr. Res. 302 (1997) 13−18.
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10977−10986.
In the present work a novel class of mannosylated ada-
mantyl tripeptides, general formula of which is α-Man-
OCH2CH(CH3)CO-AdGly-L-Ala-D-isoGln, were pre-
pared and characterized. All newly synthesized glyco-
peptides comprise in their structure the adamantylglycyl
moiety linked to the dipeptide L-Ala-D-isoGln, charac-
teristic of the peptide portion of natural peptidoglycans
and synthetic muramyldipeptides. They also comprise
mannose, an important sugar whose receptors on immu-
nocompetent cells are considered to be pattern-
recognition receptors biding compounds comprising
mannose or some other specific saccharides.
The immunostimulating activity of mannosylated
adamant-2-yl tripeptide connected with a spacer of R-
configuration 23 was compared to its analogue 24 con-
nected by a spacer of S-configuration. It was observed
that glycoconjugates 23 exhibited higher activity than
24 in testing of IgG, IgG1 and IG2a levels. The obtained
results indicate that the chirality of a spacer plays a
significant role. Since it is not possible to introduce
mannose directly into the tripeptide molecules due to
sterical reasons the direct influence of mannose on ac-
tivity of the whole molecule, excluding additional chi-
rality in the spacer, can only be determined in further
testing using compounds with achiral spacers. The re-
sults reported so far gave no evidence that novel man-
nosylated tripeptides 23 and 24 bind to mannose recep-
tors. They do not though exclude the possible influence
of mannose since several previous reports suggested
that binding to MR was associated with decreased im-
mune response.39,40 Therefore, further research on this
topic is required.
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M. Monsigny, Res. Virol. 141 (1990) 243−249.
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Ljevaković, B. Vranešić, L. Frkanec, and J. Tomašić, J.
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(2010) 751−759.
Acknowledgements. We wish to thank the Ministry of Science,
Education and Sports of the Republic of Croatia for support of
this work (projects 119-1191344-3121 and 021-0212432-2431).
32. P. H. Seidl and K. H. Schleifer, Biological Properties of Pepti-
doglycans, Walter de Gruyter Inc, Berlin, 1986, pp. 1−436.
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