Novel glycosylated endomorphin-2 analog produces potent centrally-mediated antinociception in mice after peripheral administration

Article abstract of DOI:10.1016/j.bmcl.2013.10.041

We report the synthesis and pharmacological characterization of a novel glycosylated analog of a potent and selective endogenous μ-opioid receptor (MOP) agonist, endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂, EM-2), obtained by the introduction in positio

Full text of DOI:10.1016/j.bmcl.2013.10.041

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6673–6676
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel glycosylated endomorphin-2 analog produces potent
centrally-mediated antinociception in mice after peripheral
administration
c
d
e
a
Jakub Fichna a,b, , Marzena Mazur , Daria Grzywacz , Wojciech Kamysz , Renata Perlikowska ,
Justyna Piekielna a, Marta Sobczak a, Maciej Sałaga a, Geza Toth f, Anna Janecka a, Chunqiu Chen b,
Jacek Olczak c
⇑
a Department of Biomolecular Chemistry, Faculty of Medicine, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland
b Department of Gastroenterological Surgery, Tenth People’s Hospital of Shanghai, School of Medicine, Tongji University, Shanghai, China
c TriMen Chemicals Ltd, Lodz, Poland
d Research and Development Laboratory, Lipopharm.pl, Zblewo, Poland
e Department of Inorganic Chemistry, Faculty of Pharmacy, Medical University of Gdansk, Poland
f Institute of Biochemistry, Biological Research Centre of Hungarian Academy of Sciences, Szeged, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and pharmacological characterization of a novel glycosylated analog of a potent
and selective endogenous l-opioid receptor (MOP) agonist, endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM-
2), obtained by the introduction in position 3 of the tyrosine residue possessing the glucose moiety
attached to the phenolic function via a b-glycosidic bond. The improved blood–brain barrier permeability
and enhanced antinociceptive effect of the novel glycosylated analog suggest that it may be a promising
template for design of potent analgesics. Furthermore, the described methodology may be useful for
increasing the bioavailability and delivery of opioid peptides to the CNS.
Received 6 August 2013
Revised 19 October 2013
Available online 30 October 2013
Keywords:
Endomorphin-2
l-opioid receptor (MOP)
Ó 2013 Elsevier Ltd. All rights reserved.
Antinociception
Peripheral administration
Opiates, such as morphine and other opium-derived alkaloids,
have been used for centuries to alleviate moderate to severe pain.
However, the side effects associated with their administration,
such as respiratory depression, inhibition of gastrointestinal motil-
ity and development of physical dependence, in particular when
extended in time to treat chronic conditions, severely limit the
opiate application as analgesics. Recently, much effort has been
put into the design of the new molecules targeting the opioid
system, with potent antinociceptive action in the central nervous
system (CNS) and limited effect in the periphery after systemic
administration.
Most of the rational drug design studies have focused on the
endogenous opioid peptides, which are important neurotransmit-
ters and play a major role in the maintenance of homeostasis in
the CNS and in the periphery. The aim is to maintain the pharma-
cological profile of the opioid peptides, while improve biodistribu-
tion by increasing their permeability through the blood–brain
barrier (BBB) and ameliorate their stability against enzymatic deg-
radation. The BBB, situated at the level of the endothelial cells of
the brain microvascular capillaries coupled with tight junctions,1,2
is characterized by a reduced vesicular transport, high electrical
resistance and proteolytic activity, and low paracellular diffusion,3
excluding most of the peptides from reaching the brain.
Of several approaches, which have been proposed to improve
peptide delivery through the BBB, for example, increasing lipophil-
icity4 or serum stability,1 glycosylation seems to be the most prom-
ising. Glycosylation increases metabolic stability,5 attenuates
in vivo clearance,6 and enhances pharmacological effect compared
to non-glycosylated compounds, as shown for deltorphin,7,8
cyclized Met-enkephalin analogs,9,10 and linear Leu-enkephalin
analogs.1 However, the attachment of the carbohydrate was also
shown as detrimental for opioid receptor binding affinity (Ref. 1
and citations therein).
Abbreviations: AgOTf, silver triflate; BBB, blood–brain barrier; CDMT, 2-chloro-
4,6-bis[3-(perfluorohexyl)propyloxy]-1,3,5-triazine; CNS, central nervous system;
DMF, dimethylformamide; EM-2, endomorphin-2; Fmoc, N-(9-fluorenylmethyloxy-
carbonyl); GPI assay, guinea pig ileum assay; HATU, 1-[bis(dimethylamino)meth-
ylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; HR-ESI-
MS, high-resolution electrospray ionization mass spectrometry; iv, intravenous;
MOP,
l-opioid receptor; RP HPLC, reversed-phase high-performance liquid chro-
matography; TBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluo-
roborate; TFA, trifluoroacetic acid; NALME, naloxone methiodide.
⇑
Corresponding author. Tel.: +48 42 272 57 07; fax: +48 42 272 56 94.
E-mail address: jakub.fichna@umed.lodz.pl (J. Fichna).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.041

6674
J. Fichna et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6673–6676
Here we report the synthesis of a novel glycosylated analog of a
potent and selective endogenous -opioid receptor (MOP) agonist,
endomorphin-2 (Tyr-Pro-Phe-Phe-NH2, EM-2, 1), obtained by the
introduction in position 3 of the tyrosine residue possessing the
glucose moiety attached to the phenolic function via a b-glycosidic
RO
l
RO
RO
O
O
RO
bond (Tyr(b-D-glucopyranose)). In this study we have also investi-
gated the pharmacological properties of the new analog by using
in vitro and in vivo techniques.
H2N
O
O H
O
O
H
N
Fmoc-Tyr(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)-OH was
N
obtained in three consecutive steps: allyl ester protection,11 glyco-
sylation12 and allyl ester deprotection13 (Scheme 1). Glycosylated
EM-2 analogs were assembled on the Rink resin using N-(9-fluore-
nylmethyloxycarbonyl) (Fmoc)-protected amino acids and O-(ben-
N
H
NH2
O
zotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
tetrafluoroborate
(TBTU)/N-methylmorpholine as coupling reagents.14 The final
peptide resin was N
-deprotected,15 thoroughly washed with
2a R = Ac
2b R = H
a
Figure 1. Structures of glycosylated endomorphin-2 analogs, 2a and 2b.
dichloromethane, dried and divided into two portions. One portion
was directly cleaved from the resin to give glycosylated EM-2 with
acetylated hydroxyl groups on the glucopyranosyl moiety (2a)
(Fig. 1), as described earlier.14 The second portion of the peptide-
resin was subjected to the action NaOCH3 in DMF/MeOH, resulting
in de-acetylation of the hydroxylic groups of the glucose moiety.16
The fully de-protected peptide was then cleaved from the resin to
give 2b (Fig. 1). Glycopeptides were further purified by semi-pre-
parative reversed-phase high-performance liquid chromatography
(RP HPLC).17 Calculated values for protonated molecular ions were
in agreement with those determined by high-resolution electro-
spray ionization mass spectrometry (HR-ESI-MS) (see Supplemen-
tary data for analysis results).
The pharmacological profiles of EM-2 (1) and newly synthe-
sized glycosylated peptides 2a and 2b were characterized in vitro
and in vivo. Receptor binding study was performed as described
earlier,18 using [3H]DAMGO as a selective MOP ligand. The func-
tional potency at MOP was characterized in the guinea pig ileum
(GPI) assay, as previously reported.19 Antinociception was
measured by the hot plate test in mice after intravenous (iv)
administration of the peptides as a bolus injection at the dose of
3 mg/kg. Additionally, the peripherally restricted opioid antagonist
naloxone methiodide (NALME, 1 mg/kg, ip) was used to elucidate
the action of 2b in the CNS. Serum content of 2b in mice was
analyzed using mass spectrometry.20
weakly reactive towards acylation of the amino acid residue,
Phe(4), bound directly to the solid support. Having this in mind
we decided to prepare the Fmoc-protected tyrosine derivative
with free carboxylic group that would allow for the use of the
more reactive coupling reagents like uronium salts (TBTU,
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridi-
nium 3-oxid hexafluorophosphate, HATU) or 2-chloro-4,6-bis[3-
(perfluorohexyl)propyloxy]-1,3,5-triazine (CDMT).
Therefore, the tyrosine derivative suitable for the solid-phase
peptide synthesis was prepared in three steps from the commer-
cially available Fmoc-Tyr(tBu)-OH (Scheme 1). The carboxylic
group of the Fmoc-Tyr(tBu)-OH was protected with the allyl ester
via the reaction of the carboxylate with the allyl bromide in high
yield (92%). The glycosylation of the Fmoc-Tyr(tBu)-OAll was then
accomplished by reacting it with the commercially available
2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide in presence of
silver triflate (AgOTf) and 3Å molecular sieves in dichloromethane.
Although the actual configuration of the glycosidic center was of
no importance to us at this moment, we wanted the glycosylation
reaction to deliver as much homogenous product as possible. It is
known that the solvent strongly affects the stereochemical out-
come of the glycosylation of the tyrosine: the glycosidic bond of
b-configuration prevails in dichloromethane whereas acetonitrile
The data are expressed as mean SEM. Statistical analysis was
performed using Prism 5.0 (GraphPad Software Inc., La Jolla, CA,
USA). Student’s t-test or ANOVA followed by Bonferroni post-hoc
testing was used. P Values <0.05 were considered statistically
significant.
promotes formation of the a
-glycosidic bond.22,23 Furthermore, it
is also known that the tert-butyl protection of the phenol hydrox-
ylic group actually enhances its nucleophilicity towards the glyco-
syl donor, what results in the increased yield of the glycosylation
reaction. It is believed that the bulkiness of the tert-butyl group
forces out the phenolic oxygen lone-pairs electrons out of their
conjugation with the aromatic ring. Also, it was observed that
the presence of the tert-butyl protection further increases the b/
Fmoc-tyrosine pentafluorophenyl esters carrying the sugar
moieties (glucose and maltose, either of an
a and b-glycosydic
bond configuration) were earlier prepared by Jansson and collabo-
rators.21 However, we envisaged that the Pfp-ester may prove too
a
-anomer ratio in comparison to the glycosylations run on the
phenol-unprotected tyrosine residue.24 The actual yield of the gly-
cosylation of Fmoc-Tyr(tBu)-OAll with the glucosyl donor in our
hands was 76% and we were not able to detect the
product.
a-glycosylation
AcO
AcO
O
The final deprotection of the carboxylic functionality was
AcO
tBuO
accomplished by treating the Fmoc-Tyr(2,3,4,6-tetra-O-acetyl-b-
AcO
O
D-glucopyranosyl)-OAll with Pd(0) catalyst and morpholine as an
i., ii., iii.
allyl group scavenger. Workable, though somewhat disappointing
38% yield was encountered.
Fmoc-HN
CO2H
Receptor studies of the fully assembled peptides revealed a
dramatic difference in the binding affinity of the new analogs at
the MOP. The acetylated analog 2a did not bind to the receptor
(IC50 >1000 nM), while 2b displayed a potent MOP affinity in the
nanomolar range, although approximately 70-fold lower than that
of the parent compound (IC50 73.23 3.85 vs 0.99 0.08 for 2b and
Fmoc-HN
CO2H
Scheme 1. Synthesis of Fmoc-Tyr(2,3,4,6-tetra-O-acetyl-b-
Reagents and conditions: (i) allyl bromide, DIPEA; DCM, 35 °C, 4 h; 92%; (ii) 2,3,4,6-
tetra-O-Ac-
-glucopyranosyl bromide, AgOTf, 3A MS; DCM, ꢀ10 °C to rt, 1 h; 76%;
(iii) Pd(PPh3)4, morpholine; DCM, rt, 1 h; 38%.
D-glucopyranosyl)-OH.
a-D
1, respectively; data for
1
from18). However, 2b showed

J. Fichna et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6673–6676
6675
significantly higher affinity at MOP compared to Tyr(3)-substituted
EM-2 (1100 113 nM, data from Ref. 25). In the GPI assay, 2b was
Recently, Varamini and collaborators28 showed that the modifica-
tion of EM-1 with a lactose residue in position 1 resulted in a
significant pain relief after iv and oral administration in rats. Inter-
estingly, similarly to our compound 2b, modification of EM-1 with
lactose led to a decrease in MOP binding affinity and agonist activ-
ity, but was still in the nanomolar range.28 Conjugation of EM-1
with glucose produced similar results.29 Moreover, in our study
the acetylation of hydroxyl groups of the sugar moiety, which
increases lipophilicity, decreased the CNS entry of 2a, what is in
line with previous observations for glycosylated dermorphin deriv-
atives.30 This observation strongly argues for the presence of
unsubstituted carbohydrate moiety necessary for good pharmaco-
logical activity of glycosylated analogs.
It was postulated previously that the enhancement of the BBB
permeability for glycosylated compounds results from an in-
creased uptake by the transmembrane glucose transporter GLUT1
(Ref. 9 and citations therein). However, this hypothesis has been
proven incorrect30 and the exact mechanism by which glycosyla-
tion improves BBB transport has yet to be elucidated. Among
others, interaction with specific transporters, promotion of the
negative membrane curvature on the surface of endothelial cells,
which may increase endocytosis and result in enhanced transport
through membranes via transcytosis, as well as shifting from he-
patic to renal clearance have been suggested.9,28,30
only about threefold less potent than
1 (IC50 16.1 1.3 vs
4.7 0.5 nM, respectively), while 2a did not activate MOP (IC50
>1000 nM).
As shown in Figure 2A, only 2b (3 mg/kg) produced a potent
antinociception after iv administration in the hot plate test in mice.
Peripherally restricted opioid receptor antagonist naloxone
methiodide (1 mg/kg, ip) did not block the effect of the glycosyl-
ated EM-2, suggesting central site of action of 2b (Fig. 2B). How-
ever, an ‘off-target’ activity, although unlikely, might also be
suggested.
The analysis of mouse blood samples for 2b contents produced
interesting results. We have detected the peptide (M + 39 = 787.2),
but we also obtained two signals that may signify the presence of
its metabolites (M = 588.4 for deglycosylated and M + 23 = 759.4
for glucuronic derivative of 2b, respectively) (see Supplementary
data for analysis results). We have not observed any signals from
2b-derived di- or tripeptides, which were expected based on previ-
ously reported degradation pathways for EM-2.
Our data show that the glycosylation of the Phe(3) aromatic
moiety in EM-2 (1) may be an interesting approach to enhance
its antinociceptive activity in the CNS, despite lower binding affin-
ity at MOP observed in the in vitro studies. We may suggest that
the augmentation of the central effect of 2b in comparison with
the parent peptide results from an increased stability against pro-
teolytic degradation, as well as improved BBB permeability.
EMs are endogenous opioid ligands with high affinity and good
selectivity at MOP compared with other opioid receptors.26 There-
fore they are regarded as good candidates for the design of novel
opioid-based analgesics with potent biological activity, yet without
undesired side effects in the periphery, in particular in the gastro-
intestinal tract (for review, see Ref. 27). Although several glycosyl-
ated analogs of endogenous opioids have been reported,1,7–10 only
some attempts have been made to increase the passage of EMs
through the BBB by attachment of the carbohydrate moiety.
In conclusion, the improved BBB permeability and enhanced
antinociceptive effect of 2b suggest that this glycosylated EM-2
analog is a promising template for design of potent analgesics.
Furthermore, the described methodology may be useful for
increasing the bioavailability and delivery of opioid peptides to
the CNS.
Disclosures
The authors have nothing to disclose.
Author contributions
Study concept and design: J.F., J.O.
(A)
240
Peptide design and synthesis: J.F., M.M., J.P., M. S., J.O.
Acquisition and analysis of pharmacological data, statistical
analysis: J.F., D.G., R.P., M.S.
200
160
120
80
**
Control
1
2a
2b
Drafting of the manuscript: J.F., J.O.
Obtained funding: J.F., W.K., G.T., A.J., C.C., J.O.
Study supervision: J.F., J.O.
40
Acknowledgments
0
Supported by the Iuventus Plus program of the Polish Ministry
of Science and Higher Education (0119/IP1/2011/71 and IP2012
010772 to J.F.) and National Natural Science Foundation of China
(NSFC) Research Fund for International Young Scientists
(81250110087 to J.F.).
(B)
240
200
160
120
80
nsp
**
Control
2b
+ NALME
The authors wish to thank Jozef Cieslak for his excellent techni-
cal assistance.
Supplementary data
40
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
10.041.
0
Figure 2. Pharmacological characterization of 2b in vivo. (A) Glycosylated EM-2
analog 2b (3 mg/kg, iv) produced a potent antinociceptive action in the hot plate
test in mice as shown by the prolonged time to jump compared with vehicle-
(controls) and EM-2 (3 mg/kg, iv)-treated animals. (B) The antinociceptive effect of
2b was not blocked by the peripherally-restricted opioid antagonist naloxone
methiodide (NALME, 1 mg/kg, ip), suggesting a central site of action. Results are
shown as mean SEM of n = 5–6 mice for each experimental group. ⁄⁄p <0.01, as
compared with control (vehicle treated mice).
References and notes
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Jones, H.; Yamamura, H. I.; Janders, J.; Davis, T. P.; Porreca, F.; Hruby, V. J.; Polt,
R. J. Med. Chem. 2000, 43, 2586.

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J. Fichna et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6673–6676
2. Reese, T. S.; Karnovsky, M. J. J. Cell Biol. 1967, 34, 207.
J = 13.7, 9.6 Hz), 1.98 (s, 3H), 1.97 (s, 3H), 1.96 (s, 3H), 1.94 (s,3H).
13C NMR (DMSO, 600 MHz) d: 170.05, 169.68, 169.40, 169.20, 155.88, 155.03,
143.83, 140.72, 130.44, 127.69, 127.13, 125.38, 125.30, 120.16, 116.19, 97.44,
72.01, 70.79, 68.06, 65.57, 61.60, 56.31, 46.64, 36.07, 20.35. LC/MS
M + 1 = 734.8.
3. Jones, H. C.; Keep, R. F.; Butt, A. M. Prog. Brain Res. 1992, 91, 123.
4. Bodor, N.; Prokai, L.; Wu, W. M.; Farag, H.; Jonalagadda, S.; Kawamura, M.;
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Broeck, J. V.; Costentin, J.; Janecka, A. J. Med. Chem. 2007, 50, 512.
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16. 8 mM NaOCH3 in DMF/MeOH (17:3), 2 ꢁ 3 h.
17. Glycopeptides were further purified by semi-preparative reversed-phase high-
performance liquid chromatography (RP HPLC) on a Vydac C18 column (10 lm,
8. Negri, L.; Lattanzi, R.; Tabacco, F.; Orru, L.; Severini, C.; Scolaro, B.; Rocchi, R. J.
Med. Chem. 1999, 42, 400.
22 ꢁ 250 mm), equipped with a Vydac guard cartridge. A solvent system of
0.1% trifluoroacetic acid (TFA) in water (A)/80% acetonitrile in water containing
0.1% TFA (B) and a linear gradient of 0–100% B over 15 min was used. Peptide
9. Egleton, R. D.; Mitchell, S. A.; Huber, J. D.; Janders, J.; Stropova, D.; Polt, R.;
Yamamura, H. I.; Hruby, V. J.; Davis, T. P. Brain Res. 2000, 881, 37.
10. Polt, R.; Porreca, F.; Szabo, L. Z.; Bilsky, E. J.; Davis, P.; Abbruscato, T. J.; Davis, T.
P.; Harvath, R.; Yamamura, H. I.; Hruby, V. J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
7114.
purity was verified by analytical HPLC employing a Vydac C18 column (5
lm,
4.6 mm ꢁ 250 mm) and
a
solvent system of 0.1% TFA in water (A)/80%
acetonitrile in water containing 0.1% TFA (B). A linear gradient of 0–100%
solvent B over 25 min at a flow rate of 1 mL/min was used for the analysis.
Final purity of both peptides was >98%.
11. To a solution of Fmoc-Tyr (tBu)-OH (10 g; 21,76 mmol) in DCM, DIPEA (37 ml;
217,6 mmol) and allyl bromide (9,4 ml; 108,8 mmol) were added. The reaction
was slightly heated to 35 °C for 4 h, TLC (AcOEt/hexane 1/2) indicated
disappearance of the starting material. The reaction mixture was diluted
with DCM, washed with 2 M HCl and brine, dried over MgSO4 and
concentrated. Residue was purified by column chromatography AcOEt/
hexane gradient 1/10 to 1/5. Product was obtained as an yellow oil. Yield
10.0 g (92%).
18. Fichna, J.; Perlikowska, R.; Wyrebska, A.; Gach, K.; Piekielna, J.; do-Rego, J. C.;
Toth, G.; Kluczyk, A.; Janecki, T.; Janecka, A. Bioorg. Med. Chem. 2011, 19, 6977.
19. Fichna, J.; Rego, J. C.; Costentin, J.; Chung, N. N.; Schiller, P. W.; Kosson, P.;
Janecka, A. Biochem. Biophys. Res. Commun. 2004, 320, 531.
20. Blood samples were withdrawn from mice which received 2B (3 mg/kg, iv)
10 min after administration of the peptide. After 4 h of precipitation at 4 °C
supernatants were collected and centrifuged (3000 rpm, 1 min).
1H NMR (CDCl3, 200 MHz) d: 7.79–7.76 (m, 2H), 7.60–7.57 (m, 2H), 7.45–7.27
(m, 4H), 7.04–6.89 (m, 4H), 5.94–5.77 (m,1H), 5.35–5.22 (m, 2H), 4.73–4.60 (m,
3H), 4.49–4.31 (m, 2H), 4.25–4.18 (m, 1H), 3.10 (d, 2H, J = 5.5 Hz), 1.33 (s, 9H).
13C NMR (CDCl3, 600MHz) d: 171.20, 155.47, 154.39, 143.77, 144.65, 141.22,
131.33, 130.36, 129.74, 127.64, 126.98, 125.02, 124.11, 119.90, 118.98, 78.34,
66.86, 65.97,47.07, 37.57, 28.74. LC/MS M+1 = 500.7.
Proteins from obtained supernatants were precipitated by addition of two
volumes of acetonitrile. Briefly, to 100 ll of plasma, 200 ll of acetonitrile was
added with gentle vortexing. The samples were then left at room temperature
for 30 min. The supernatants were retained after ultracentrifugation at 13,000g
for 15 min and lyophilized.
The samples were then diluted with MeOH 1:1 (V/V) and acidified with 20 ll
12. To a mixture of Fmoc-Tyr(tBu)-OAll (3.0 g; 6 mmol), AgOTf (2 g; 9 mmol) and
3Å molecular sieves in dry DCM under argon at ꢀ10 °C was added a solution of
HCOOH. After preparation the samples were analyzed by electrospray
ionization (ESI) and the time of flight (TOF) in micrOTOF-Q II (Bruker) mass
spectrometer equipped with a quadrupole mass analyzer. Analyses were
2,3,4,6-tetra-O-acetyl-
a-
D
-glucopyranosyl bromide (4.9 g, 12 mmol) in DCM.
White suspension was formed. After 1 h at rt, TLC (AcOEt/hexane 1/2) showed
no staring material present. The reaction mixture was neutralized with 2 equiv
of DIPEA and filtered through a pad of Celite. The filtrate was concentrated and
the residue was crystallized from Et2O. Product was obtained as a white solid.
Yield 3.5 g (76%).
performed with the following spectrometer parameters: mass range:
50 ꢂ 1200 m=z; polarity: positive; end plate offset: ꢀ500 V; capillary:
4500 V; nebulizer: 1 bar; dry gas: 6 l/min; dry temperature: 180 °C. The
strong signal and low background allowed the easy identification of peptide
metabolite. The experiments were repeated at least twice.
1H NMR (CDCl3, 600 MHz) d: 7.77 (d, 2H, J = 7.5 Hz), 7.56 (d, 1H, J = 7.5 Hz),7.53
(d, 1H, J = 7.5 Hz), 7.42–7.39 (m, 2H), 7.32–7.30 (m, 2H), 7.04 (d, 2H, J = 8.2 Hz),
6.89 (d, 2H, J = 8.2 Hz), 5.92–5.84 (m, 1H), 5.34–5.22 (m, 5H), 5.16–5.12 (m,
1H), 4.96 (d, 1H, J = 6.8 Hz), 4.69–4.62 (m, 2H), 4.46–4.42 (m, 1H), 4.33–4.30
(m,1H), 4.27–4.23 (m,1H), 4.20–4.17(m,1H), 4.12–4.10 (m,1H), 3.78–3.72
(m,1H), 3.16–3.04 (m, 2H), 2.05 (s, 9H), 2.03 (s, 3H). LC/MS M + 1 = 774.5.
21. Jansson, A. M.; Jensen, K. J.; Meldal, M.; Lomako, J.; Lomako, W. M.; Olsen, C. E.;
Bock, K. J. Chem. Soc., Perkin Trans. 1 1996, 1001.
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23. Jensen, K.; Meldal, M.; Bock, K. J. Chem. Soc., Perkin Trans. 1 1993, 2119.
24. Vargas-Berenguel, A.; Meldal, M.; Paulsen, H.; Jensen, K. J.; Bock, K. J. Chem. Soc.,
Perkin Trans. 1 1994, 3287.
13. Solution of Fmoc-Tyr(2,3,4,6-tetra-O-acetyl-b-
D
-glucopyranosyl)-OAll (3,1 g;
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4 mmol) in DCM was treated with Pd(PPh3)4 (120 mg) followed by addition of
morpholine (1,3 ml; 15 mmol). After 1 h deprotection was completed. The
reaction was diluted with AcOEt, washed with 10% citric acid, brine, dried over
MgSO4 and concentrated yielding the yellow oil. Column chromatography
(DCM/MeOH, gradient elution 16/1 to 4/1) and crystallization from Et2O gave
pure product as a white solid. Yield 1.1 g (38%).
1H NMR (DMSO, 600 MHz) d: 7.86 (d, 2H, J = 7.5 Hz), 7.63 (d, 2H, J = 2.5 Hz),
7.61 (d, 2H, J = 2.5 Hz), 7.40–7.37 (m, 2H), 7.31–7.25 (m, 2H), 7.18 (d, 2H,
J = 8.3 Hz), 6.85 (d, 2H, J = 8.5 Hz), 5.43–5.34 (m, 2H), 5.02–4.94 (m, 2H), 4.22–
4.12 (m, 5H), 4.05–3.97 (m, 2H), 3.03 (dd, 1H, J = 13.7, 4.0 Hz), 2.8 (dd, 1H,
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