Dimeric analogs of immunosuppressive decapeptide fragment of ubiquitin

Article abstract of DOI:10.1002/psc.2416

Our previous studies revealed that ubiquitin and its decapeptide fragment with the LEDGRTLSDY sequence, located on the exposed molecule loop, strongly suppressed the immune response. This suggested that the loop may serve as a functional epitope of ubiquitin molecule and that a possible mechanism of biological action of the synthesized peptides is associated with interfering in interactions of ubiquitin with other molecules. Ubiquitin is known to exist in oligomeric forms, which can interact with various oligomeric receptors. We designed and synthesized new dimeric analogs of the ubiquitin fragment, to probe whether dimeric peptides may have higher affinity towards the ubiquitin receptors responsible for immunosuppression, which are believed to form oligomeric structures. Three dimerization strategies, N-terminus to N-terminus, C-terminus to C-terminus, and N-terminus to C-terminus (head-to-tail) via PEG derivatives were used to synthesize the dimeric peptides on solid support. In the course of our research, we developed a new and straightforward procedure of dimerization where α-amino groups of the C-terminal lysine residues of two peptide fragments were linked by PEG spacer directly on solid support. The effect of dimeric analogs on the immunological response was tested in the AFC in vitro experiment. The immunological tests showed that the head-to-tail dimerization caused a more profound increase in the biological activity than other tested dimerization methods. Our results suggest that such orientation of peptide components may correspond to orientation of the hypothetic ubiquitin receptors responsible for the immunomodulatory activity.

Full text of DOI:10.1002/psc.2416

Research Article
Received: 21 December 2011
Revised: 30 March 2012
Accepted: 30 March 2012
Published online in Wiley Online Library: 25 May 2012
(wileyonlinelibrary.com) DOI 10.1002/psc.2416
Dimeric analogs of immunosuppressive
decapeptide fragment of ubiquitin{
Alicja Kluczyk,a Marzena Cydzik,a Monika Biernat,a Remigiusz Bąchor,a
Paweł Pasikowski,a Piotr Stefanowicz,a Jolanta Artym,b Michał Zimeckib
and Zbigniew Szewczuka*
Our previous studies revealed that ubiquitin and its decapeptide fragment with the LEDGRTLSDY sequence, located on the
exposed molecule loop, strongly suppressed the immune response. This suggested that the loop may serve as a functional
epitope of ubiquitin molecule and that a possible mechanism of biological action of the synthesized peptides is associated
with interfering in interactions of ubiquitin with other molecules. Ubiquitin is known to exist in oligomeric forms, which
can interact with various oligomeric receptors. We designed and synthesized new dimeric analogs of the ubiquitin fragment,
to probe whether dimeric peptides may have higher affinity towards the ubiquitin receptors responsible for immunosuppres-
sion, which are believed to form oligomeric structures. Three dimerization strategies, N-terminus to N-terminus, C-terminus to
C-terminus, and N-terminus to C-terminus (head-to-tail) via PEG derivatives were used to synthesize the dimeric peptides on
solid support. In the course of our research, we developed a new and straightforward procedure of dimerization where
a-amino groups of the C-terminal lysine residues of two peptide fragments were linked by PEG spacer directly on solid support.
The effect of dimeric analogs on the immunological response was tested in the AFC in vitro experiment. The immunological
tests showed that the head-to-tail dimerization caused a more profound increase in the biological activity than other tested
dimerization methods. Our results suggest that such orientation of peptide components may correspond to orientation of
the hypothetic ubiquitin receptors responsible for the immunomodulatory activity. Copyright © 2012 European Peptide
Society and John Wiley & Sons, Ltd.
Keywords: dimerization strategies; dimerization on solid support; PEG linker; linker length; ubiquitin fragments
significantly enhanced tumor uptake in vivo compared with PEG4-
E[c(RGDfK)]2 without the two PEG4 spacers between the two RGD
Introduction
Dimerization or oligomerization of receptors is an essential step
in various cellular signal transduction processes. Understanding
of the multivalent character of some bioreceptors may be helpful
in designing new compounds with high affinity and enhanced
selectivity for a given receptor. Substances that are able to mod-
ulate the receptor dimerization may control such a process and
therefore affect the biological outcome.
motifs [5]. The influence of dimerization and linker length on the
activity of Smac fragments, binding to XIAP, an important regulator
of apoptosis, was analyzed by biological and physicochemical
methods [6,7].
A combinatorial approach based on dipeptide linkers attached to
a central scaffold (bis-3,5-aminomethyl benzoic acid) was applied to
the design of bivalent glutathione S-transferase inhibitors, resulting
in compounds with increased affinity and selectivity, and better sol-
ubility [8]. Two peptide fragments of discontinuous HLA-DQ epitope
were linked by g-Abu residue to increase their immunosuppressory
Dimerization of an active biomodulator often results in
enhanced binding and improved pharmacological properties.
Compounds consisting of two ligands connected by a proper
spacer have a potential for bridging vicinal receptors. Therefore,
the synthesis and biological evaluation of dimeric analogs of
biologically active compounds is of great interest in biomedical
applications [1]. Many biological ligands are also composed of
clustered binding epitopes [2]. Properly designed dimeric analogs
of bioligands should enhance interactions between neighboring
receptors, possibly by reducing unfavorable entropic effects. The
dimerization strategy was successfully applied to antagonists of
chemokine receptor CXCR4, responsible for regulation of inflamma-
tion and immune response, with linkers composed of oligoglycine
and dicarboxylic acids [3]. Multimeric forms of cyclic RGD analogs
attached to rigid scaffolds presented increased binding avidity
towards avb3 integrin, although addition of oligo(ethylene glycol)
spacers reduced the effect, presumably by enthropic effect [4].
On the other hand, the 18F-labeled PEG4-E[PEG4-c(RGDfK)]2
construct showed increased in vitro receptor binding affinity and
*
Correspondence to: Zbigniew Szewczuk, Wydział Chemii, Uniwersytet
Wrocławski, ul. F. Joliot-Curie 14, 50-383 Wrocław, Poland. E-mail: zbigniew.
szewczuk@chem.uni.wroc.pl
Dedicated to Prof. Ignacy Z. Siemion on the occasion of his 80th birthday.
{
A preliminary report of this work ‘Dimerization strategies of the immunosup-
pressory decapeptide ubiquitin fragment’ was presented at the European
Peptide Symposium in 2010.
a Faculty of Chemistry, University of Wrocław, Wrocław, Poland
b Institute of Immunology and Experimental Therapy, Polish Academy of
Sciences, Wrocław, Poland
Abbreviations used: HLA, human leukocyte antigen; AFC, antibody-forming
cells; SRBC, sheep red blood cells
J. Pept. Sci. 2012; 18: 456–465
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

DIMERIZATION OF THE UBIQUITIN FRAGMENTS
activity [9]. Several short linkers, ranging from single Asp residue to
PEG diacid, were used in the synthesis of N-to-N and N-to-C dimers
in search for more efficient antagonists of the human IgG–human
FcRn interaction [10].
potency [19,20] and prompted us to use such approach to
ubiquitin fragments.
Ubiquitin is known to exist in an oligomeric form that can
interact with various oligomeric receptors [21,22]. It was
established that the ubiquitin system is involved in the regulation
of immune response by the activation of the NF-kB pathway [23],
with linear polyubiquitination and linear ubiquitin chain assem-
bly complex playing a role in B cell activation and differentiation,
regulation of type I interferon production, genotoxic stress
response, and osteogenesis [24]. However, the biological signifi-
cance of some interactions of the oligomeric ubiquitin still
remains unknown. It is feasible that the dimeric analogs of
ubiquitin fragments (Figure 1) are able to interact with the
dimeric receptors, which may cause the modulation of the inter-
action of oligoubiquitin with the oligomeric receptors. We
decided to synthesize dimeric analogs of the immunosuppressive
decapeptide fragment of ubiquitin, utilizing three dimerization
strategies N-terminus to N-terminus, C-terminus to C-terminus,
and N-terminus to C-terminus (head-to-tail) (Figure 1) to investi-
gate a possibility of multivalency of the receptors.
It may be expected that properly designed dimeric analogs of
biologically active bioligands are more selective in their affinity
towards dimeric receptors. Therefore, the dimeric ligands may serve
as a tool to examine the phenomenon of receptor dimerization.
Ubiquitin is a conservative 76-amino acid polypeptide present
in all eukaryotic cells. Apart from its key function in proteasomal
degradation [11], this polypeptide is involved in many intracellu-
lar signal pathways [12] and also plays an important role as the
extracellular immunomodulator and antimicrobial agent [13]. It
has been postulated that cryptides originating from ubiquitin
could interfere in the ubiquitination process, by disruption of
some ubiquitin activities. Moreover, ubiquitin-derived peptides
were shown to express various important biological activities [14].
We pointed out some topological similarities between 50–59 loop
of ubiquitin and b164–172 loop of HLA-DQ molecule with
TPQRGDVYT sequence, known for suppression of the humoral
and cellular immune response and inhibition of integrins activity
[15,16]. We found that the 50–59 ubiquitin fragment
LEDGRTLSDY, containing the retro-RGD sequence, exhibited
strong immunosuppressive effects on the cellular and humoral
immune responses, comparable with that of cyclosporine [17].
This finding suggested that ubiquitin fragment with
LEDGRTLSDY sequence could interact with similar receptor type
as the immunosuppressive fragments of HLA. The dimerization
of the nonapeptide VPRSGEVYT, designed to mimic the discon-
tinuous immunosuppressive epitopes of the HLA-DR superdi-
mers [18], resulted in the enhancement of immunosuppressive
Materials and Methods
Reagents
The derivatives of amino acids for peptide synthesis, the coupling
reagent 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tet-
rafluoroborate N-oxide (TBTU) and N-hydroxybenzotriazole (HOBt)
were purchased from IrisBiotech (Marktredwitz, Germany) and
NovaBiochem (as Merck, Darmstadt, Germany). The side chain pro-
tecting groups for Fmoc-amino acids were t-butyl for Glu, Asp, Thr,
Figure 1. The synthetic analogs of the immunosuppressive ubiquitin fragment.
J. Pept. Sci. 2012; 18: 456–465 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

KLUCZYK ET AL.
Ser, and Tyr; Pmc for Arg; and Mtt for Lys. The MBHA Rink
amide resin (0.69 mmol/g) was purchased from NovaBiochem.
The HOOC-CH2CH2O(CH2CH2O)12CH2CH2-COOH and Fmoc-NH-
CH2CH2O(CH2CH2O)5CH2CH2-COOH were obtained from Aldrich
(as Sigma-Aldrich, St. Louis, MO, USA) and NovaBiochem, respec-
tively. TFA was from IrisBiotech. Thioanisole, pentafluorophenol,
and triisopropylsilane (TIS) were purchased from Fluka (as Sigma-
Aldrich, St. Louis, MO, USA); phenol from Aldrich; piperidine from
Merck (Darmstadt, Germany); and dicyclohexylcarbodiimide (DCC)
from Riedel-de Haën (as Sigma-Aldrich, St. Louis, MO, USA). The
solvents for peptide synthesis were obtained from Aldrich (DMF
and DCM) and IrisBiotech (DIPEA). Other chemicals were purchased
from POCH (Gliwice, Poland).
Synthesis
General peptide synthesis
Peptides were prepared by manual solid-phase technique by using
the standard Fmoc (9-fluorenylmethoxycarbonyl) synthesis proce-
dure. The MBHA Rink amide resin was functionalized with Fmoc-
Tyr(But)-OH (for compounds 1, 2, 5, and 6) and Fmoc-Lys(Mtt)-OH
(for compounds 3 and 4). Fmoc-protecting groups were removed
in the presence of 25% piperidine in DMF. The e-amino groups of
lysine were deprotected using 1% TFA in DCM (TFA/TIS/DCM 1/4/
95 v/v). Single coupling by TBTU was performed in DMF. The reac-
tion was monitored by Kaiser test [26]. Lithium chloride was used
during synthesis of dimeric compounds as chaotropic agent. All
synthesized compounds were cleaved from the resin and depro-
tected using modified reagent K (TFA/water/phenol/thioanisole/
TIS 82.5/5/5/5/2.5 v/v) at room temperature for 2 h, the resin was
rinsed with TFA, and the products were precipitated with cold
diethyl ether (Et2O). The yield of synthesis is presented according
to the participation of the peak representing the synthesized
compound in HPLC profile of crude product at 220 nm.
General Methods
Mass spectrometry
MS and MS/MS experiments were carried out on micrOTOF-Q and
Apex-Qe Ultra 7T FT-ICR instruments (Bruker Daltonic, Bremen,
Germany) equipped with an electrospray source. Spectra were
recorded using 50 : 50 acetonitrile–water mixture containing
0.1% HCOOH at the peptide concentration 0.5 mM. The potential
between the spray needle and the orifice was set to 4.5 kV. In the
MS/MS mode, the quadrupole was used to select the precursor
ions, which were fragmented in the hexapole collision cell generat-
ing product ions that were subsequently mass analyzed by the
mass analyzer. Argon was used as the collision gas. In the mass
spectrometric (MS/MS) experiments, the collision energy was
optimized for the best fragmentation.
PfpOOC-CH2CH2O(CH2CH2O)12CH2CH2-COOPfp (reagent A). HOOC-
CH2CH2O(CH2CH2O)12CH2CH2-COOH (3g, 4.34mmol) and penta-
fluorophenol (1.6 g, 8.69 mmol) were dissolved in 20 ml of ethyl acetate
at 0 ꢀC, and DCC (1.8 g, 8.69 mmol) was added. The reaction mixture
was stirred for 1.5 h at 0 ꢀC. Dicyclohexylurea was filtered off, and the
filtrate was evaporated in vacuo. The obtained product was used for
synthesis of dimeric compounds 2 and 4 without further purification.
ESI-MS: m/z 1023.345, calculated for [M + H]+ 1023.343 (mono-
isotopic mass); m/z 1045.327, calculated for [M + Na]+ 1045.325
(monoisotopic mass); m/z 1061.301, calculated for [M + K]+
1061.298 (monoisotopic mass).
Purification
PfpOOC-CH2CH2O(CH2CH2O)12CH2CH2-CONHC4H9 (reagent B).
Reagent A (3g, 3 mmol) was dissolved in DMF (10 ml). Then, n-buty-
lamine (148 ml, 1.5 mmol) was added to the solution in three portions
at 15-min intervals. The reaction was completed after 1.5 h as judged
by the ninhydrin test. The obtained product was used for synthesis of
monomeric compounds 1 and 3 without further purification.
ESI-MS: m/z 912.430, calculated for [M + H]+ 912.437 (monoiso-
topic mass).
Analytical HPLC was performed on Vydac C18 column (4.6 mm Â
250 mm) (Grace, Deerfield, IL, USA). Solvent system: S1, 0.1% aque-
ous TFA; S2, 80% acetonitrile + 0.1% TFA, linear gradient from 0%
to 100% of S2 for 60 min, flow rate 1.0 ml/min, UV detection at
220 nm. For compounds 1, 2, 4, 5, and 6, additional HPLC analyses
were performed, using YMC-Pack ODS-AQ column (4.6 mm  250
mm) (YMC, Kyoto, Japan) and Varian Microsorb-MV 100-5 CN
(4.6 mm  250 mm) (Varian, Palo Alto, CA, USA) in linear gradient
from 0% to 80% of S2 for 40 min, flow rate 1.0 ml/min (retention
times for analyses are given as Rt1, Rt2, and Rt3, respectively).
Preparative reversed-phase HPLC was performed on Tosoh TSKgel
ODS-120T column (21.5 mm  300 mm) (Tosoh, Tokyo, Japan) using
the same solvent system (gradient 0.5%/min, flow rate 7 ml/min).
Purified compounds (purity determined by HPLC >98%) were trans-
formed into acetate salts by dissolving in 50% acetic acid, dilution,
and lyophilization [25].
Peptide 1. a-Amino group of protected peptide H-Leu-Glu(OBut)-
Asp(OBut)-Gly-Arg(Pmc)-Thr(But)-Leu-Ser(But)-Asp(OBut)-Tyr(But) at-
tached to the resin (200 mg, 0.14 mmol) was acylated by Reagent B
(1.3 g, 1.4 mmol) in the presence of HOBt (190 mg, 1.4 mmol) dissolved
in DMF. The reaction was performed for 24 h. The peptide was cleaved
from the resin and purified by reversed-phase HPLC. The fractions were
collected, lyophilized, and transformed into acetate forms.
Yield 52%, Rt1 20.8 min, Rt2 25.6 min, Rt3 27.7 min.
ESI-MS: m/z 948.002, calculated for [M + 2H]2+ 948.003 (mono-
isotopic mass); m/z 632.339, calculated for [M + 3H]3+ 632.338
(monoisotopic mass).
CD spectroscopy
MS/MS on parent ion 632.34 ([M+3H]3+) (m/z value for the most
abundant peaks): 857.96+2 (calc. for b9 857.96+2); 421.27+2 (calc. for b1
421.27+2); 1054.48+1 (calc. for y9 1054.48+1); 383.1+1 (calc. for y3 383.1+1).
CD spectra were recorded on a Jasco J-600 spectropolarimeter
(Easton, MD, USA). Peptides were dissolved at concentrations of
approximately 70 mg/ml, and the spectra were recorded in water,
10% aqueous sodium chloride, TFE, and aqueous solution of TFE
(20% and 50%) at room temperature, at pH 6. The spectrum of
the solvent was recorded under identical conditions and subtracted
during data analysis. A rectangular quartz cuvette of 1 mm
path length was used. Each spectrum represents the average of
eight scans. Data are presented as molar ellipticity (Y) per amino
acid residue.
Peptide 2. The strategy of the synthesis of the dimeric compound
2 (Figure 2) was similar to the one described by us previously [20].
Two adjacent resin-bound peptide chains (H-Leu-Glu(OBut)-Asp
(OBut)-Gly-Arg(Pmc)-Thr(But)-Leu-Ser(But)-Asp(OBut)-Tyr(But)-) were
linked through their N-termini by reagent A in DMF at room
temperature in the presence of HOBt. A solution of reagent A
(51 mg, 0.05 mmol) and HOBt (33.8mg, 0.25 mmol) in 1 ml DMF
wileyonlinelibrary.com/journal/jpepsci Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2012; 18: 456–465

DIMERIZATION OF THE UBIQUITIN FRAGMENTS
(monoisotopic mass). MS/MS on parent ion 620.68 ([M + 3H]3+
)
(m/z value for the most abundant peaks): 494.24+2 (calc. for b9
494.24+2); 393.21+2 (calc. for b7 393.21+2); 437.29+2 (calc. for y1
437.29+2).
Peptide 4. Fmoc-Lys(Mtt) was attached directly to the MBHA
Rink amide resin (200 mg, 0.14 mmol). The Fmoc-protecting
group was removed, and the dimerization was performed by
linking the a-amine groups of resin-bound lysine residues by
reagent A. The mixture of reagent A (71 mg, 0.07 mmol) and
HOBt (47 mg, 0.35 mmol), dissolved in 1 ml DMF, was added to
the resin suspended in DMF in several portions during 1 h, and
the reaction mixture was stirred for 24 h. Next, the Mtt-protecting
group from the e-amine group of Lys was removed with 1% TFA
in DCM (TFA/TIS/DCM 1/4/95 v/v). Further synthesis of peptide
chain, attached to the e-amine group of Lys, was continued
according to the Fmoc synthesis procedure. The peptide was
cleaved from the resin, and the crude product was characterized
and purified by reversed-phase HPLC. The fractions were collected,
lyophilized, and transformed into acetate forms.
Yield 70%, Rt1 19.1 min, Rt2 19.2 min, Rt3 17.3 min.
ESI-MS: m/z 1459.779, calculated for [M + 2H]2+ 1459.769 (mono-
isotopic mass); m/z 973.512, calculated for [M+ 3H]3+ 973.515
(monoisotopic mass); m/z 730.386, calculated for [M + 4H]4+
730.388 (monoisotopic mass). MS/MS on parent ion 730.39
([M + 4H]4+) (m/z value for the most abundant peaks): 494.24+2
(calc. for b9 494.24+2); 436.73+2 (calc. for b8 436.73+2); 393.21+2
(calc. for b7 393.21+2); 336.67+2 (calc. for b6 336.67+2); 745.35+1
(calc. for y8b9 745.35+1); 630.32+1 (calc. for y8b8 630.32+1).
Figure 2. The synthesis strategy of compound 2. In synthesis of mono-
meric analog 1, reagent B was used instead of di-pentafluorophenyl ester
of PEG diacid (reagent A). Grey balls indicate MBHA Rink amide resin bead.
Peptide 5. The Fmoc-NH-CH2CH2O(CH2CH2O)5CH2CH2-COOH
(79 mg, 0.14 mmol) was added to the synthesized decapeptide
fragment attached to the resin (100 mg, 0.07 mmol) suspended
in DMF. The reaction was performed for 5 h in the presence of
DIPEA (51 ml, 0.28 mmol) and TBTU (44 mg, 0.14 mmol). After
removing the Fmoc-protecting group, the synthesis of the next
decapeptide fragment was continued according to the Fmoc
synthesis procedure, each coupling was repeated twice, and
lithium chloride was used as chaotropic agent. The peptide was
cleaved from the resin, and the crude product was characterized
and purified by reversed-phase HPLC. The fractions were collected,
lyophilized, and transformed into acetate forms.
was added to the peptidyl resin (150 mg, 0.1mmol) suspended in
DMF in a few portions during 24 h. The peptide was cleaved from
the resin and purified by reversed-phase HPLC. The fractions were
collected, lyophilized, and transformed into acetate forms.
Yield 61%, Rt1 17.6 min, Rt2 23.7 min, Rt3 22.6 min.
ESI-MS: m/z 1495.720, calculated for [M+ 2H]2+ 1495.722 (mono-
isotopic mass); m/z 997.481, calculated for [M + 3H]3+ 997.484
(monoisotopic mass); m/z 748.365, calculated for [M+ 4H]4+
748.365 (monoisotopic mass).
Yield 76%, Rt1 19.3 min, Rt2 21.6 min, Rt3 20.7 min.
MS/MS on parent ion 997.48 ([M + 3H]3+) (m/z values for the
most abundant peaks): 1304.15+2 (calc. for b7 1304.15+2);
968.49+2 (calc. for b1 968.49+2); 528.24+2 (calc. for y9 528.24+2);
384.14+1 (calc. for y3 384.14+1); 297.11+1 (calc. for y2 297.11+1).
ESI-MS: m/z 1326.647, calculated for [M + 2H]2+ 1326.648
(monoisotopic mass); m/z 884.765, calculated for [M + 3H]3+
884.768. MS/MS on parent ion 884.77 ([M + 3H]3+) (m/z value for
the most abundant peaks): 824.73+3 (calc. for b20 824.73+3);
799.91+2 (calc. for b12 799.91+2); 751.88+2 (calc. for y11 751.88+2).
Peptide 3. Fmoc-Lys(Mtt) was attached to the MBHA Rink amide
resin. Then Fmoc-protecting group was removed, and the
mixture of reagent B (1.3 g, 1.4 mmol) and HOBt (190 mg,
1.4 mmol) dissolved in DMF was added to the resin with attached
Lys(Mtt) residue (200 mg, 0.14 mmol) suspended in DMF. The
reaction was performed for 24 h. Next, the Mtt-protecting group
from the e-amine group of Lys was removed with 1% TFA in
DCM (TFA/TIS/DCM 1/4/95 v/v), and the synthesis of peptide
chain was continued. The peptide was cleaved from the resin,
and the crude product was characterized and purified by
reversed-phase HPLC. The fractions were collected, lyophilized,
and transformed into acetate forms.
Peptide 6. The compound was synthesized by the method
described for peptide 5. In order to increase the linker length,
coupling of a second Fmoc-NH-CH2CH2O(CH2CH2O)5CH2CH2-
COOH molecule was executed in the presence of DIPEA and TBTU
after the removal of the Fmoc-protecting group of the immobi-
lized peptide already containing one residue of PEG-amino acid.
Yield 82%, Rt1 20.4 min, Rt2 22.0 min, Rt3 20.9 min.
ESI-MS: m/z 1494.251, calculated for [M + 2H]2+ 1494.245
(monoisotopic mass); m/z 996.497, calculated for [M + 3H]3+
996.499 (monoisotopic mass); m/z 747.625, calculated for
[M + 4H]4+ 747.626 (monoisotopic mass). MS/MS on parent ion
747.63 ([M + 4H]4+) (m/z value for the most abundant peaks):
936.48+3 (calc. for b21 936.48+3); 869.12+3 (calc. for b19 869.12+3);
636.02+3 (calc. for a12 636.02+3).
Yield 64%, Rt1 23.5 min.
ESI-MS: m/z 930.515, calculated for [M + 2H]2+ 930.518 (mono-
isotopic mass); m/z 620.681, calculated for [M + 3H]3+ 620.682
J. Pept. Sci. 2012; 18: 456–465 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

KLUCZYK ET AL.
Biological Tests
lysine residue attached to solid support. Such dimers with oligogly-
cyl bridges evoked high immunosuppressive activity; however,
poor solubility in physiological fluids excluded their further applica-
bility. To overcome this problem, we selected PEG derivative to
serve as a flexible linker in the C-terminus to C-terminus strategy.
The formation of dimeric analogs, where two peptide sequences
were covalently linked through their N-termini (Figure 2), was per-
formed according to procedure described by us previously [28].
The dimerization was achieved by cross-linking the N-terminal
amino group of the immobilized peptides with the dicarboxylic
derivative of PEG, activated by esterification with pentafluorophenol.
Development of such derivative of PEG in the synthesis of peptide
dimers was presented by us previously [20]. It is worth noting that
gradual addition of reagent (0.5 equivalent in respect to resin
loading) is required for efficient dimerization.
Animals
Twelve-week-old male CBA mice were used for the experiments.
The mice were kept at the Animal Facility of the Institute of
Immunology and Experimental Therapy, Wroclaw, and fed com-
mercial granulated food and water ad libitum. The local ethics
committee approved the study.
Reagents
SRBC used for priming mice and in vitro culture were provided by
the Wroclaw University of Life and Environmental Sciences and
were stored in Alsever’s solution until use.
Determination of the AFC numbers in the cell culture
The second strategy of formation of dimeric analogs directly
on solid support was based on linking the a-amino groups of
two C-terminal lysine residues by PEG derivative (reagent A)
and continuing the peptide synthesis on the e-amino groups.
Previously, we found that the Tyr residue in the ubiquitin50–59
immunosuppressive LEDGRTLSDY sequence was not crucial for
its immunomodulatory activity [17]. Therefore, for dimerization
purposes, we replaced tyrosine by lysine residue, using orthogo-
nally protected Fmoc-Lys(Mtt)-OH derivative.
After the removal of the Fmoc-protecting groups, the two
lysine residues were linked through their a-amino groups using
the di-pentafluorophenyl ester derivative of PEG (reagent A)
(Figure 3). This dimerization procedure was similar to that
presented for the N-terminus to N-terminus dimer (compound 2)
but was performed on the C-terminal amino acid residues
directly attached to the resin. Next, the Mtt-protecting group from
the e-amine group of the dimerized lysine residues was removed,
and the synthesis of the immunosuppressive peptide fragment
of ubiquitin was continued on resin directly at the e-amine groups
of cross-linked lysine residues.
Relatively high yield of the synthesis of dimeric compounds 2
and 4 indicates that cross-linking of two peptide fragments
attached to solid support can be easily performed not only on
their N-terminal groups, where the amino group is located far
away from the solid support, but also on a-amino groups of
the residues attached directly to the resin, despite the sterical
interference of the support. To the best of our knowledge, this
paper describes the first cross-linking reaction between two
a-amino groups of amino acids directly attached to a resin, to
produce the desired dimeric products. We propose that this
method could be general and may be applied for synthesis of
various peptide conjugates.
Mice were sensitized with 0.2 ml of a 1% SRBC suspension in 0.9%
NaCl, intravenously. Four days later, the spleens were used for
generating the secondary immune response in vitro. The spleens
were isolated aseptically and pressed through a plastic screen
into Hanks’ medium. The cells were then centrifuged and treated
for 5 min at room temperature with 0.83% ammonium chloride to
lyze erythrocytes. Subsequently, the cells were washed twice
with Hanks’ medium and passed through cotton wool columns
to remove cell debris and dead cells. Finally, the splenocytes
were resuspended in a culture medium (RPMI 1640, supple-
mented with 10% fetal calf serum, glutamine, sodium pyruvate,
2-mercaptoethanol, and antibiotics) and distributed to 24-well
culture plates (5 Â 106 per well). The cell cultures were immu-
nized with 50 ml of 0.001% SRBC, and the peptides were added
at the dose range of 1–100 mg/ml. To control cultures, 0.1 ml of
the culture medium was added. After 4 days of incubation in a
cell culture incubator, the AFC numbers were determined
according to Mishell and Dutton [27]. The results are presented
as mean AFC numbers from four wells, calculated per 106 viable
splenocytes Æ standard deviation.
Statistics
The results were presented as mean values Æ standard deviation
(SD). The Levene’s test was used to determine the homogeneity
of variance between groups. Analysis of variance was applied
to estimate the significance of the difference between groups.
Significance was determined at p ≤ 0.05. The statistical analysis
was performed using STATISTICA for Windows statistical package.
Results and Discussion
We synthesized four new dimeric analogs consisting of two
immunosuppressive peptide fragments of ubiquitin connected
by selected derivatives of PEG as linkers (compounds 2, 4, 5,
and 6), as well as two monomeric analogs with PEG attached to
N-terminus or C-terminus (compounds 1 and 3).
The immunosuppressive peptide sequences were connected
in various combinations: N-terminus to N-terminus (compound
2), C-terminus to C-terminus (compound 4), and N-terminus to
C-terminus (compounds 5 and 6). Synthesis of all analogs was
performed directly on solid support.
Previously, we used the a and e di-oligoglycinated lysine residue
(–(Gly4-6)2–Lys-NH2) as an effective spacer in preparation of dimeric
analogs of immunosuppressive HLA fragments, utilizing C-terminus
to C-terminus strategy [19]. The synthesis of the linker was per-
formed simultaneously on the a-amino and e-amino groups of
To evaluate the influence of the dimerization process on the
structural and biological properties of ubiquitin fragment, we syn-
thesized two conjugates, in which the PEG derivative was attached
to the N-terminal amino group of the decapeptide, to compare
with N-terminal dimer 2 (compound 1), or the a-amino group of
C-terminal Lys residue, to compare with dimer 4 (compound 3)
(Figure 1). Reagent B, developed for this procedure, was obtained
by reaction of pentafluorophenyl diester of PEG diacid with
butylamine. By optimizing the reaction conditions during the
synthesis of reagent B (see Materials and Methods), a product
containing monoester–monoamide derivative (the reactive form
of reagent B) and diamide (non-reactive form) was obtained.
For synthesis of monomeric compounds, a significant excess
(ten equivalents) of reagent B could be used, as the non-reactive
component is removed during resin washing.
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DIMERIZATION OF THE UBIQUITIN FRAGMENTS
Figure 3. The synthesis procedure of compound 4. In synthesis of monomeric analog 3, reagent B was used instead of di-pentafluorophenyl ester of
PEG diacid (reagent A). Grey balls indicate MBHA Rink Amide resin beads.
The synthesis of head-to-tail dimers (compounds 5 and 6)
was performed using commercially available Fmoc-protected
PEG-amino acid residue (Fmoc-NH-CH2CH2O(CH2CH2O)5CH2CH2-
COOH), according to the standard Fmoc synthesis procedure.
Two dimeric analogs were obtained by incorporation of one
(compound 5) or two (compound 6) Fmoc-PEG-amino acid resi-
dues between two peptide chains (Figure 4).
All of the obtained dimeric analogs of the immunosuppressive
peptide fragment of ubiquitin were purified by preparative
RP-HPLC. The presence of Asp-Gly motif in the investigated
peptides did not cause significant synthetic problems. In
crude compounds 5 and 6 (21 and 22 synthetic steps, respec-
tively, as compared with other dimers requiring only ten Fmoc
protection removal steps), the presence of piperidide by-
products, which could indicate the level of succinimide forma-
tion and opening during removal of Fmoc protection [29], was
practically negligible according to MS analysis, and by-products
containing two piperidide modifications were not detected
(data not shown). The structure of the decapeptide LEDGRTLSDY
was also confirmed by NMR [30]. The molecular weights and
structures of the synthesized compounds were confirmed by
ESI-MS and ESI-MS/MS analysis, respectively. The ESI-MS/MS
spectra (one example is presented in Figure 5) contain peaks
derived from typical an, bn, and yn series and some internal
fragments. We found that the fragmentation occurs on peptide
bonds only, but not in the PEG linker, which facilitates the
interpretation of the spectrum.
The effects of the peptides on the secondary humoral immune
response were analyzed by determining the number of AFC in
the culture of mouse splenocytes (Figure 6). There are two effects
that may be anticipated as a result of the dimerization. First, the
modification may have a potential for bridging vicinal receptors,
which in turn may affect the binding potency. Second, the
introduction of the PEG linker may alter the chemical profile of
the peptide. To separate the contributions of these two effects,
we synthesized the peptide monomers pegylated on their N- or
J. Pept. Sci. 2012; 18: 456–465 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

KLUCZYK ET AL.
Figure 4. The synthesis strategy of compound 5. Compound 6 was synthesized using two PEG-amino acid linkers. Grey balls indicate MBHA Rink
Amide resin.
Figure 5. MS/MS spectrum of triply charged peptide 2 (parent ion m/z 997.43).
C-terminal amino acid residue (compounds 1 and 3, respectively).
The comparison of the immunomodulatory potencies of the
ubiquitin fragment with LEDGRTLSDY-NH2 sequence with its
analogs 1 and 3 suggests that the PEG-containing chain has
nearly no effect on the immunosuppressive effect of the ubiqui-
tin fragment. However, the dimeric compounds 2, 4, 5, and 6
generally exhibited higher immunosuppressive potencies than
their monomeric counterparts, 1 and 3. A significant increase in
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DIMERIZATION OF THE UBIQUITIN FRAGMENTS
oligomers. The polyubiquitin chains formed in vivo can adopt
different topologies that are defined by the lysine residue
involved in the formation of the isopeptide bond between
ubiquitin subunits. The arrangement of the polyubiquitin chain
influences its interaction with ubiquitin receptors and conse-
quently determines its signaling role [38].
To specify the potential molecular target for the most active
dimeric analog of fragment LEDGRTLSDY (compound 5), we
compared structures of diubiquitin units formed by linking
specific Lys residues (Figure 7). Crystal structures are available
for Lys-6-linked and Lys-48-linked dimers [21,39], whereas Lys-
11-linked diubiquitin is the dimeric fragment of the ubiquitin
oligomer crystal structure [39]. For Lys-63-linkage, the NMR
solution structure of ubiquitin dimer interacting with yUIMs
receptor was described [40].
The PEG linker utilized in the synthesis of compound 5 is 28 Å
long, assuming its fully stretched conformation, which could be
rarely adopted in solution. It is very unlikely that this dimeric
compound could mimic the interaction of ubiquitin loops located
in farther distance. Therefore, the only orientation of two
LEDGRTLSDY fragments that can be mimicked by compound 5
is that of diubiquitin connected via Lys-48. Interestingly, similarity
to the Lys-48 dimer can be achieved in two ways, as the distances
between C- and N-termini of two fragments are 21.0 and 22.3 Å,
respectively (Figure 7). Together, this suggests that dimeric
compound 5, which possesses the highest immunosuppressory
activity in this study (Figure 6), may act through a signaling
pathway connected with recognition of Lys-48 polyubiquitin
chains, which are mainly used as signals for targeting proteins
to the proteasome [41]. Compound 6 (with linker of 56 Å) also
expresses high immunosuppressive effect, which can be
explained by the flexibility of the PEG linker. Still, there is a possi-
bility that Lys-6-linked, Lys-11-linked, or Lys-63-linked ubiquitin
chains can adopt bent conformation, different from the observed
in crystal form, in order to interact with the receptor.
Figure 6. Effect of the synthesized peptides 1–6 on the humoral im-
mune response. AFC numbers in the mouse spleen cell cultures of CBA/
Iiw mice immunized with SRBC and treated in vitro with the peptides.
The results are expressed as a mean Æ SD of six wells. The results were
elaborated by the analysis of variance: control versus all concentrations
of UB50-59, 2, 5, and
6 (p = 0.0001); control versus 1: 1 mg/ml
(p = 0.0008), 10 mg/ml (p = 0.0001), 100 mg/ml (p = 0.0001); control versus
3: 1 mg/ml (NS), 10 mg/ml (NS), 100 mg/ml (p = 0.0021); control versus 4:
1 mg/ml (NS), 10 mg/ml (p = 0.0001), 100 mg/ml (p = 0.0001); NS – not
significant.
immunological activity in the case of the head-to-tail dimers (5
and 6) was observed, particularly for small concentration (1 mg/
ml) of peptide 5. When examined on molar basis, the immuno-
logical activity of dimeric compounds seems actually much
higher than that of monomeric compounds 1 and 3, as nearly
the same effect is evoked by only 60% of molecules (the average
formula weight of dimers is approximately 3 kDa as compared
with 2 kDa of compounds 1 and 3). However, the examined com-
pounds in general show small changes in activity with increasing
dose, and a ten times increase in concentration of the dimer does
not result in a respective change of activity. The suppressive
effect of ubiquitin-derived peptides in vitro at 100 mg/ml may
be not spectacular; however, we recently showed that a ubiqui-
tin-derived peptide prolonged allogeneic skin transplant survival
in mice almost as potently as FK-506 [31].
Our earlier studies revealed that the immunomodulatory
properties of the ubiquitin fragments were similar to that of
intact ubiquitin [30,31]. Although mechanism of the immunosup-
pressive action of the ubiquitin fragments remains unknown, it
may interrupt the interactions of ubiquitin with its hypothetical
receptors. Because ubiquitin is known to exist in the oligomeric
form, a possibility of interaction of oligoubiquitin with some
oligomeric receptors cannot be excluded.
Ubiquitin has been reported to interact with numerous pro-
teins. These interactions regulate various processes, including
DNA repair, endocytosis, and vesicular trafficking [32]. Recogni-
tion is mediated by binding to different ubiquitin binding
domains (reviewed by Dikic et al. [33]). Although the interactions
of ubiquitin with ubiquitin binding domains are rather weak, the
affinity is usually increased by polymerization of ubiquitin and
multimerization of the ubiquitin receptor [34]. There are reports
that tandem repeats of ubiquitin binding domains may also
interact with variously linked ubiquitin chains [35,36].
It is known that a linker length may dramatically affect the
biological potencies of bivalent bioligands [37]. The bivalent
analog should therefore consist of two monomers – ubiquitin
fragments – covalently linked by a spacer with a sufficient length,
able to position them in the same orientation as in the ubiquitin
We performed conformational investigations of the synthe-
sized peptides by CD in water, to examine the possible effect of
the PEG linker and dimerization on overall conformation of the
studied peptides. CD spectra of investigated peptides are pre-
sented in Figure 8 as molar ellipticity per amino acid residue to
compensate for different lengths of peptides.
CD spectra obtained for nonpegylated peptide LEDGRTLSDY-
NH2 and its pegylated analogs 1–6 dissolved in water (at neutral
pH) were characterized by a negative band at 195 nm and a
weak negative shoulder at 220 nm (Figure 8). The ellipticity of
investigated peptides has the same order of magnitude; how-
ever, two conjugates – compounds 2 and 5 – were characterized
by significantly lower dichroic intensity of the negative band.
The spectra were typical for open conformation [42]. The simi-
larity of the tested compound spectra in water implies that
the dimerization has little effect on the stabilization of their
structure in water. The unstructured conformation of the pep-
tides in water may also suggest that the PEG linkers may easily
adopt extended conformation, which enables them to situate
the immunosuppressory peptides in a desired position.
We also found that addition of sodium salt does not signifi-
cantly affect the CD spectra (results not shown), which indicates
that possible interaction of sodium cations with polyoxyethylene
chain has little or no effect on conformation of PEG-linked
peptide segments.
The enhancement in the immunosuppressory activity of our
dimeric analogs could be explained by microaggregation of the
J. Pept. Sci. 2012; 18: 456–465 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

KLUCZYK ET AL.
Figure 7. Orientation of immunosuppressory fragment LEDGRTLSDY in the ubiquitin dimers connected by different lysine residues. X-ray structures
(Lys-6-linked (A) [39], Lys-48 (B) [21], and Lys-11 (C) [39]) and NMR solution structure (Lys-63 [40] (D)) are presented. The distances between amide
nitrogen atom of the N-terminal residue (blue) of the one 50–59 fragment and carbonyl carbon atom of the C-terminal residue (red) of the second
50–59 fragment were shown.
increase the immunomodulatory effect [20]. The dimerization
presented in this paper causes limited increase in immunosup-
pressory effect, which could be interpreted by different molecular
targets for ubiquitin and HLA class II fragments.
Conclusions
We synthesized new analogs of the ubiquitin fragment, exploring
all dimerization strategies: N-terminus to N-terminus, C-terminus
to C-terminus, and N-terminus to C-terminus via PEG derivatives.
In the course of our research, we developed a new and straight-
forward procedure for the direct dimerization of the C-terminal
residues of peptide on a solid support using a PEG spacer. Results
of the performed immunological tests indicate that the N-terminus
to C-terminus dimerization enhances the immunosuppressive
Figure 8. CD spectra of compounds 1–6 and the ubiquitin 50–59 fragment
dissolved in water.
activity of the conjugated decapeptide fragments of ubiquitin.
Taking into account that differentially linked polyubiquitin chains
may be regarded as independent posttranslational modifications
evoking various biological effects [44], dimeric fragments of
ubiquitin could interact with the hypothetic ubiquitin receptors
responsible for the immunomodulatory activity.
receptors at the cell surface induced by the dimeric antagonists,
which would prevent the access of the proper ligands to the
receptors. Nevertheless, it is known that increased affinity of
dimeric analogs of the ligands may be also independent of
receptor density because bivalent ligands are known to produce
thermodynamically more favorable binding interaction with their
receptor than the monovalent ligands [43]. Their potencies may
be attributed to a higher concentration of pharmacophores in
the proximity of their recognition sites by a simple statistical
advantage. However, this effect may not depend on the orienta-
tion of the ligands in their dimer analog, nor the linker length.
The observed dependence of the immunosuppressory potency
on the dimer structure (orientation and length) supports rather
a possibility that dimeric structures may be capable of bridging
independent recognition sites of the receptor.
Acknowledgement
This work was supported in part by grant no. N N401 222734
from the Ministry of Science and Higher Education (Poland).
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