Design of α-S-Neoglycopeptides Derived from MUC1 with a Flexible and Solvent-Exposed Sugar Moiety

Article abstract of DOI:10.1021/acs.joc.6b00833

The use of vaccines based on MUC1 glycopeptides is a promising approach to treat cancer. We present herein several sulfa-Tn antigens incorporated in MUC1 sequences that possess a variable linker between the carbohydrate (GalNAc) and the peptide backbone. The main conformations of these molecules in solution have been evaluated by combining NMR experiments and molecular dynamics simulations. The linker plays a key role in the modulation of the conformation of these compounds at different levels, blocking a direct contact between the sugar moiety and the backbone, promoting a helix-like conformation for the glycosylated residue and favoring the proper presentation of the sugar unit for molecular recognition events. The feasibility of these novel compounds as mimics of MUC1 antigens has been validated by the X-ray diffraction structure of one of these unnatural derivatives complexed to an anti-MUC1 monoclonal antibody. These features, together with potential lack of immune suppression, render these unnatural glycopeptides promising candidates for designing alternative therapeutic vaccines against cancer.

Full text of DOI:10.1021/acs.joc.6b00833

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Article
Design of alpha-S-neoglycopeptides derived from
MUC1 with a flexible and solvent exposed sugar moiety
Víctor Rojas-Ocáriz, Ismael Compañón, Carlos Aydillo, Jorge Castro-López,
Jesús Jimenez-Barbero, Ramon Hurtado-Guerrero, Alberto Avenoza, María
M Zurbano, Jesús M. Peregrina, Jesus H Busto, and Francisco Corzana
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b00833 • Publication Date (Web): 15 Jun 2016
Downloaded from http://pubs.acs.org on June 18, 2016
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Design of αꢀSꢀneoglycopeptides derived from MUC1 with a flexible and solvent
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exposed sugar moiety
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Víctor RojasꢀOcáriz,‡,1 Ismael Compañón,‡,1 Carlos Aydillo,#,1 Jorge CastroꢀLoṕez,2 Jesús Jiménezꢀ
Barbero,3 Ramón HurtadoꢀGuerrero,2 Alberto Avenoza,1 María M. Zurbano,1 Jesús M. Peregrina,*,1
Jesús H. Busto,*,1 and Francisco Corzana*,1
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1Departamento de Química, Universidad de La Rioja, Centro de Investigación en Síntesis Química.
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Madre de Dios 53, 26006 Logroño, Spain. (i) BIFI, University of Zaragoza, BIFIꢀIQFR (CSIC)
Joint Unit, Mariano Esquillor s/n, Campus Rio Ebro, Edificio I+D, Zaragoza, Spain. (ii) Fundación
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ARAID, 50018, Zaragoza, Spain. (i) Structural Biology Unit, CIC bioGUNE, Parque Tecnológico
de Bizkaia Building 801 A, 48160 Derio (Spain). (ii) IKERBASQUE, Basque Foundation for
Science 48011 Bilbao (Spain). (iii) Department of Chemical and Physical Biology Centro de
Investigaciones Biológicas, CSIC Ramiro de Maeztu 9, 28040 Madrid (Spain).
#Current address: CECB, Department of Chemistry, University of Copenhagen, Universitetsparken
5, 2100 Copenhagen, Denmark.
TOC
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Abstract
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The use of vaccines based on MUC1 glycopeptides is a promising approach to treat cancer. We
present herein several sulfaꢀTn antigens incorporated in MUC1 sequences that possess a variable
linker between the carbohydrate (GalNAc) and the peptide backbone. The main conformations of
these molecules in solution have been evaluated by combining NMR experiments and molecular
dynamics simulations. The linker plays a key role in the modulation of the conformation of these
compounds at different levels, blocking a direct contact between the sugar moiety and the
backbone, promoting a helixꢀlike conformation for the glycosylated residue and favoring the proper
presentation of the sugar unit for molecular recognition events. The feasibility of these novel
compounds as mimics of MUC1 antigens has been validated by Xꢀray diffraction structure of one of
these unnatural derivatives complexed to an antiꢀMUC1 monoclonal antibody. These features,
together with potential lack of immune suppression, render these unnatural glycopeptides promising
candidates for designing alternative therapeutic vaccines against cancer.
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Introduction
Chemistryꢀbased approaches are of paramount importance to battle disease. In the quest of chemical
weapons against cancer, MUC1 is a glycoprotein overꢀexpressed in most of tumors.1ꢀ4 While in
healthy cells, the MUC1 backbone presents complex oligosaccharides, in tumor cells the peptide
backbone is decorated with simple and truncated carbohydrates. As a consequence, different tumorꢀ
associated carbohydrate antigens (TACAs), such as the Tn determinant (αꢀOꢀGalNAcꢀSer/Thr),5 are
exposed to the immune system and can be recognized by different antibodies.6 For this reason,
MUC1 derivatives are attracting great interest as a potential tool in developing therapeutic vaccines
for the treatment of cancer.4,7,8 However, to date, none of these vaccines have succeeded in clinical
trials.9 On the one hand, natural TACAs are tolerated by the immune system.10 To overcome this
problem, chemical modifications of these antigens, generating nonꢀnatural determinants have been
proposed.7,11ꢀ20 On the other hand, most glycan antigens are Bꢀcell epitopes and produce only shortꢀ
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lived and lowꢀaffinity antibodies.4 Induction of a robust immune response that produces high
affinity IgG antibodies is essential and may be achieved through initiation of a Tꢀcellꢀdependent
pathway.
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Figure 1. (a) Crystal structure of a MHCꢀI molecule in complex with a glycopeptide containing a
linker between the peptide backbone and the sugar moiety (PDB id: 1KBG, ref. 21e). (b) Design of
novel glycopeptides containing a linker that favors the presentation of the sugar moiety and
facilitates the interaction with Tꢀcell receptors. (d) General structure of the neoglycopeptides
synthesized and studied in this work. The peptide sequence PDTRP constitutes the most
immunogenic domain of mucin MUC1.6
A key step in this process is the presentation of the antigen through the major histocompatibility
(MHC) molecule to Tꢀcell receptors. In that context, recent studies indicate that glycopeptides can
arbitrate classical MHCꢀmediated immune response.21 The crystal structure of an MHCꢀI molecule
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with a glycopeptide containing a linker between the peptide and the sugar21e showed that while the
aglycone part of the antigen binds to MHC molecule, the carbohydrate moiety can facilitate the
recognition of T cell receptors (TCR) and therefore can stimulate immune response (Figures 1a and
1b).
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In an effort to combine these two concepts, we present herein a set of unnatural Tn antigens
characterized by the incorporation of a flexible and variable linker in their structures, placed
between a peptide fragment and the GalNAc moiety (Figure 1c). These new sulfaꢀTn derivatives
have been prepared as Fmocꢀprotected glycosyl amino acids, ready to use in solid phase peptide
synthesis (SPPS). They have been included in the AlaꢀProꢀAspꢀXaa(αꢀSꢀGalNAc)ꢀArgꢀPro peptide
sequence, which constitutes the key immunogenic epitope of MUC16 when Xaa is a threonine. In
particular, in this work, we have synthesized and studied six neoglycopeptides, being Xaa a
cysteine,
homocysteine,
homohomocysteine,
Oꢀ(mercaptopropyl)serine,
Sꢀ
(mercaptopropyl)cysteine, or Oꢀ(mercaptopropyl)threonine residue. The main conformations of
these molecules in solution have been evaluated. The linker plays a key role in the conformational
behavior of these molecules, from the global 3D structure to the presentation of the glycan part of
these compounds. Indeed, the linker blocks the direct contact between the sugar moiety and the
backbone, promotes a helixꢀlike conformation for the glycosylated residue, provides extra
flexibility to the molecule and favors the presentation of the sugar unit for molecular recognition
events. These novel compounds are indeed mimics of MUC1 antigens as demonstrated by the
analysis of the Xꢀray diffraction data obtained for one of these unnatural glycopeptides complexed
to an antiꢀMUC1 monoclonal antibody. The global behavior of this complex has also been analyzed
by MD simulations.
Results and Discussion
From a synthetic perspective, the existence of Sꢀglycopeptides with important biological properties
has stimulated the development of a variety of glycosylation methods. These methods generally use
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protected carbohydrates as electrophiles and Cys derivatives as nucleophiles. Alternatively,
dehydroalanine (Dha)22ꢀ24 containing peptides and nucleophilic thiocarbohydrate derivatives25 have
been used in 1,4ꢀconjugate additions with poor results in diastereoselectivity.26 Recently, we have
reported a new chiral Dha derivative,27 which has been used as acceptor in stereoselective sulfaꢀ
Michael additions of appropriately protected thiocarbohydrates. This procedure has been applied to
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the synthesis of Sꢀ(2ꢀacetamidoꢀ2ꢀdeoxyꢀαꢀDꢀgalactopyranosyl)ꢀLꢀcysteine, which could be
considered as a mimic of the Tn antigen.28 A method based on the alkene hydrothiolation reaction,29
which is called thiol–ene coupling (TEC), has also been developed. The thermally or
photochemically induced TEC versions occur through a radical mechanism in antiꢀMarkovnikov
regioselective fashion.29 These methods have been applied, in general, to the synthesis of βꢀSꢀ
glycosyl amino acids and βꢀSꢀglycopeptides.29 Nevertheless, the αꢀSꢀglycosidic bond formation in
these motifs,29c especially the αꢀSꢀGalNAc moiety of the Tn antigen,30,31 has received little
attention. In that context, the synthesis of αꢀthioꢀlinked glycosyl amino acids and glycopeptides by
an anomeric thiol group Sꢀalkylation with βꢀiodo(or bromo)alanine has been described.30a Later, this
methodology was modified to a “twoꢀstep oneꢀpot” reaction, generating αꢀthioꢀlinked glycosyl
amino acids by condensation of a βꢀbromoalanine derivative with the corresponding thiolate salt.31
We report herein new stereocontrolled entries to adequately protected Sꢀglycosyl amino acids,
mimics of the Tn antigen, as building blocks for solid phase peptide synthesis of Sꢀlinked
glycopeptides, exploiting the reactivity of the sulfhydryl group of triꢀOꢀacetylꢀ2ꢀacetamidoꢀ2ꢀ
deoxyꢀ1ꢀthioꢀαꢀDꢀgalactopyranose (1, abbreviated as GalNAcꢀαꢀSH) in two types of reactions. The
first one comprises the hydrothiolation reaction of double bonds included in the side chains of
amino acid derivatives. This reaction has been extensively used in glycochemistry.32 Moreover, the
mild reaction conditions, atom economy, and regioselectivity of this process satisfy essential
requirements of the click concept.33 The second type of reaction assayed involves the anomeric thiol
group Sꢀalkylation with bromoꢀamino acid derivatives.
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To know the viability of this project, we started with the synthesis of the shortest glycopeptide (Sꢀ
glycosyl amino acid ended in amide groups) that includes the largest distance between carbohydrate
and peptide backbone moieties. We selected GalNAcꢀαꢀSH (1) as a glycosyl donor, whose synthesis
was made following the procedure described in the literature34a but properly modified to carry out in
a gram scale (Supporting Information). GalNAcꢀαꢀSH (1) was subjected to an alkene
hydrothiolation reaction with Oꢀallyl serine derivative 2bꢀSer, which was prepared according to the
procedure described34b from commercially available NꢀBocꢀserine derivative 2aꢀSer. The
hydrothiolation reaction was carried out using azobisisobutyronitrile (AIBN) as a radical initiator.
The best conditions involved the use of 1.7 equivalents of thiol 1, 1.2 equivalents of AIBN and 1.0
equivalent of alkene 2bꢀSer to afford compound 3 in a 56% yield (Scheme 1). This derivative was
transformed into the required diamide 6. Deprotection of Boc group, followed by acetylation of the
amino group gave compound 4. This compound was subjected to hydrogenolysis of benzyl ester
and the corresponding carboxylic acid was transformed into methyl amide. Diamide derivative 5
was deacetylated to give target compound 6 (Scheme 1).
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AcO OAc
O
OAc
O
AcO
AcO
AIBN
CHCl3
reflux, 7 h
AcO
+
AcHN
O
S
AcHN
3
56%
SH
BocHN
CO2R
2a-Ser
O
1
: R = H
BnOH, DCC,
DMAP, rt, 24 h
BocHN
CO2Bn
2b-Ser: R = Bn
3
AcO OAc
O
RO OR
O
1) H2/Pd-C,
AcO
1) TFA,
CH2Cl2, rt
RO
MeOH, rt
2) MeNH2,
TBTU, DIEA,
CH3CN, rt
AcHN
AcHN
S
S
3
3
2) Ac2O, Py, rt
O
O
O
H
N
70%
46%
AcHN
CO2Bn
N
H
4
O
5: R = Ac
6: R = H
MeONa,
MeOH, rt
90%
Scheme 1. Synthesis of Sꢀglycosyl amino acid 6 as a diamide.
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Encouraged by this result, we synthesized the corresponding Fmocꢀprotected Sꢀglycosyl amino
acids 10ꢀSer/Cys/Thr, ready to use in SPPS and in a gram scale. To this aim, it was necessary to
prepare the (O or S)ꢀallyl NꢀFmocꢀserine/cysteine/threonine tertꢀbutyl esters 9ꢀSer/Cys/Thr, which
had not been described to date. Their synthesis started from commercially available NꢀBocꢀamino
acids 2aꢀSer/Cys/Thr, whose Boc groups were removed, giving amino acids 7ꢀSer/Cys/Thr. The
amino groups of these compounds were then protected as Fmoc carbamates, affording compounds
8ꢀSer/Cys/Thr. Esterifications of acids 8ꢀSer/Cys/Thr with tertꢀbutyl alcohol gave the required
allylic derivatives 9ꢀSer/Cys/Thr (Scheme 2).
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tBuOH,
TFA,
CH2Cl2,
rt
CuCl, DCC,
CH2Cl2, rt
FmocOSu,
Na2CO3
R
R
R
R
X
X
X
X
t
FmocHN
CO2H
FmocHN
CO2 Bu
BocHN
CO2H
H2N
CO2H
8-Ser
8-Cys
8-Thr
9-Ser
9-Cys
9-Thr
(58%, three steps)
(54%, three steps)
(59%, three steps)
2a-Ser
2a-Cys
7-Ser
7-Cys
7-Thr
: X=O, R=H
: X=S, R=H
2a-Thr
: X=O, R=Me
Scheme 2. Synthesis of NꢀFmocꢀSer/Cys/Thr(Oꢀ or Sꢀallyl)ꢀOtBu derivatives 9ꢀSer/Cys/Thr.
As a next step, we assayed hydrothiolation reactions between glycosylꢀthiol 1 and alkenes 9ꢀ
Ser/Cys/Thr, following the conditions above described for compound 2bꢀSer, obtaining good
yields of the required building blocks 10ꢀSer and 10ꢀThr (70% and 71%, respectively). However,
compound 10ꢀCys was obtained only in a 20% yield (Scheme 3). To increase these yields, the
photochemical version of the hydrothiolation reaction29a,b was tested because it is considered as a
lightꢀinduced click reaction,35 with applications to diverse fields of study of biomolecular systems.
We used a photochemical reactor equipped with 16 UVA (350 nm) lamps of 8 W. The reaction was
initiated with 2,2ꢀdimethoxyꢀ2ꢀphenylacetophenone (DPAP) and after testing several conditions,
the best yields involved the use of 1.0 equiv. of alkene, 1.2 equiv. of compound 1 and 0.4 equiv. of
DPAP in dichloromethane (1 mL). In this way, building blocks 10ꢀSer/Cys/Thr were obtained in a
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53%, 30% and 62%, respectively (Scheme 3). It is wellꢀknown that this type of reactions involving
Oꢀ or Sꢀallyl derivatives works with moderate to low yields.36 We also tested a recent method with
9ꢀSer/Cys/Thr, involving the use of BEt3 as an initiator and catechol as a coꢀreagent.37 Although,
only a slight improvement of the yield was achieved, this method provided an improved way to
purify the desired compounds (Scheme 3).
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Scheme 3. Synthesis of Sꢀglycosyl amino acid building blocks 10ꢀSer/Cys/Thr and 12 via
hydrothiolation. TW, PW and RW stand for thermal way, photochemical way and repairing way,
respectively.
Compound 12, which contains a threeꢀmethylene bridge between SꢀαꢀGalNac and amino acid
moieties, was synthesized following the same hydrothiolation protocol, tested again by both thermal
and photochemical ways, starting from αꢀallylglycine 11. This protected compound was obtained
according to the protocol previously published38 and it was reacted with glycosyl donor 1, giving a
70% and 50% yield of desired building block 12 in thermal and phochemical ways, respectively
(Scheme 3). We also tested the repairing way using catechol/BEt3 achieving compound 12 in a
better yield than photochemical way (70%).
To access to GalNAcꢀSꢀglycosylated building blocks 14 and 16, Lꢀhomocysteine (hCys) and Lꢀ
cysteine (Cys) derivatives, respectively, we used a different methodology based on the anomeric
thiol group Sꢀalkylation with bromoꢀamino acid derivatives (Scheme 4). This methodology has been
aboveꢀcommented30a,31 as well as their difficulties to carry out the nucleophilic substitution in a
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basic medium in the presence of Fmoc protecting groups. We followed a recently published mild
synthetic strategy39 that simply requires activated molecular sieves to selectively promote Sꢀ
alkylation. Therefore, activated molecular sieves (4Å) were employed as a base to promote Sꢀ
alkylation of GalNAcꢀαꢀSH 1 with appropriate protected γꢀbromohomoalanine40 13 and βꢀ
bromoalanine30,41 15 using dry DMF as a solvent (Scheme 4).
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AcO OAc
O
AcO
AcHN
AcO OAc
O
S
MS 4Å,
DMF
Br
n
n
+
AcO
t
t
FmocHN
CO2 Bu
FmocHN
CO2 Bu
AcHN
SH
13 (n = 2)
15 (n = 1)
14 (n = 2, 50%)
16 (n = 1, 51%)
1
Scheme 4. Synthesis of Sꢀglycosyl amino acid building blocks 14 and 16 via nucleophilic
substitution.
Although the reactions proceeded with moderate yields, the procedure was easy to perform, fast,
and provided similar overall yields when it was compared with other methods published recently.28
These known bromoꢀderivatives 13 and 15 were obtained from
Lꢀhomoserine and Lꢀserine,
respectively, following the procedure described in the literature, that involved NꢀFmoc protection,
tertꢀbutyl ester formation and bromination under standard conditions.30
These six mimics of the Tn antigen (10ꢀSer/Cys/Thr, 12, 14 and 16), all of Sꢀglycosyl amino acids
whose amino, carboxylic acid and hydroxyl groups are protected as NꢀFmoc, tertꢀbutyl esters and
acetates, respectively, are adequate building blocks for SPPS of glycopeptides. Consequently, they
were used to synthesize different αꢀSꢀneoglycopeptides shown in Scheme 5. The synthesis of these
compounds was performed using the automated SPPS protocol with a Rink amide MBHA resin and
Fmoc and side chain protected amino acids. αꢀSꢀGlycosylamino acid building blocks 10ꢀ
Ser/Cys/Thr, 12, 14 and 16 were manually coupled to the peptide sequence to improve the yield of
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this step. After removal of the acetyl groups of the carbohydrate with a mixture of NH2NH2/MeOH,
αꢀSꢀneoglycopeptides were released from the resin using trifluoroacetic acid (TFA). Under these
conditions, all the acidꢀlabile sideꢀchain protecting groups were also removed. Purification by
preparative HPLC and subsequent lyophilization gave the target αꢀSꢀneoglycopeptides 17ꢀ22 in
good overall yields.
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Scheme 5. αꢀSꢀneoglycopeptides synthesized in this work.
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The conformational behavior of the synthesized neoglycopeptides in solution was then deduced by
using NMR. In particular, NOE data, supported by molecular dynamics (MD) simulations with
timeꢀaveraged restraints (MDꢀtar), were employed.42,43 In a first step, full assignment of the protons
in all derivatives was carried out using standard COSY and HSQC experiments. 2DꢀNOESY
experiments in H2O/D2O (9/1) (25 °C, pH = 5.5) were then performed (Figure 2 and Supporting
Information). Distances involving NH protons were semiꢀquantitatively deduced by integrating the
volume of the corresponding crossꢀpeaks. This experimental data was used as restraints in MDꢀtar
simulations (Figure 2 and Supporting Information).
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Figure 2. Section of the 500 ms 2D NOESY spectrum (400 MHz) in H2O/D2O (9:1) at 25 °C of
derivative 18, showing the amide region. Diagonal peaks and exchange crossꢀpeaks connecting NH
protons and water are negative (green color). The NOE contacts are represented as positive crossꢀ
peaks (blue color). Comparison of the experimental and MDꢀtar derived distances for this
glycopeptide is also shown.
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The structure ensembles obtained through the MDꢀtar simulations indicate that the
neoglycopeptides are rather flexible in solution (Figure 3 and Supporting Information).
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Figure 3. Ensembles obtained through 20 ns MDꢀtar simulations for neoglycopeptides 17ꢀ22. The
backbone is shown in green and the GalNAc in purple.
In general, the studied neoglycopeptides exhibit a randomꢀcoil conformation for the peptide
backbone. This result was corroborated by circular dichroism (CD) spectroscopic studies
(Supporting Information). The CD spectra of these derivatives differed from the one observed for
the natural APDT(αꢀOꢀGalNAc)RP glycopeptide, which adopts mainly a PPII conformation for the
peptide backbone in water44 (Supporting Information). As a point of interest, the contacts between
the sugar and the peptide fragment are almost insignificant. As a consequence, the glycosylated
residue exhibits a helixꢀlike conformation in all studied compounds (Figure 4a for compound 22
and Supporting Information for the other compounds). This feature is corroborated by the mediumꢀ
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size NOE intensities observed for the NHXaa4−HαXaa4, and NHArg5−HαXaa4 crossꢀpeaks, where Xaa is
the glycosylated residue.45 Conversely, it has been shown that GalNAcꢀglycosylation of the natural
Ser or Thr residues forces these amino acids to display extended conformations46ꢀ52 due to the
presence of stabilizing contacts between the sugar and the peptide moieties.46a,50
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Figure 4. (a) Definition of the glycosidic linkage in this work, together with φ/ψ distribution
obtained through 20 ns MDꢀtar simulations for amino acid Thr4 in compound 22. (b) Type I βꢀturn
conformer found in water for glycopeptides 19ꢀ22. (c) φ/ψ distribution obtained through 20 ns MDꢀ
tar simulations for the glycosidic linkage in compound 22. The conformation found in the crystal
structure for the glycosidic linkage of APDC(GalNAc)RP in complex to SM3 antibody (PDB id:
5A2L, ref. 44) is represented as a red circle.
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Of note, compounds 19ꢀ22, which are bearing a longer linker, adopt a low population (<25%) of a
type I βꢀturnꢀlike, stabilized by a hydrogen bond between Pro2 (CO) and Arg5 (NH), which has
been reported for natural MUC1ꢀlike derivatives52 (Figure 4b).
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Concerning the Sꢀglycosidic linkage, as stated by us for other derivatives,34 it is rather flexible in all
compounds, with two main conformations, characterized by ψ ca. 180o or 60o, corresponding to the
local minima calculated for methyl 4ꢀthioꢀαꢀmaltoside.53 The studied glycopeptides display a
conformation centered at φ/ψ ≈ 60o/−60o degrees, also close to that observed in the crystal structure
of a mucinꢀlike glycopeptide incorporating GalNAcꢀCys44 (Figure 4c and Supporting Information).
Regarding the lateral chain, derivatives carrying a serine, homoserine or homohomoserine
(compounds 18, 19 and 20, respectively) present a flexible dihedral angle χ1, with two main values
(Supporting Information). This result is similar to that found for other glycopeptides bearing a
serine residue.49 For the other derivatives, χ1 is fixed around 60 degrees. Glycopeptides 20, 21 and
22 present a similar behavior for the side chain, being derivative 22, with a threonine amino acid,
the one with the more rigid linker in solution (Supporting Information). The result is in agreement
with our previous studies on Thrꢀderived glycopeptides.50 In general, and according to our
simulations, the linker of all glycopeptides is located almost perpendicularly to the peptide
backbone (Figure 3 and Supporting Information).
The viability of these novel compounds as mimics of MUC1 antigen was validated by the analysis
of the crystal structure of one of these unnatural glycopeptides, compound 22, complexed to the
antiꢀMUC1 monoclonal antibody SM3 produced in our lab.44 The analysis of the Xꢀray structure
(PDB id: 5fxc, Figure 5a and Supporting Information) revealed that the hydrogen bonds, as well as
the hydrophobic contacts present in the natural glycopeptide APDT(GalNAc)RP bound to SM3,44
are also conserved in the scFvꢀSM3:22 complex. In detail, Pro2 stacks with Trp91L, Trp96L and
Tyr32L. The sideꢀchains of Asp3 and Arg5 are engaged in hydrophobic contacts with Trp33H and
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Tyr32H, respectively. NH group of Ala1 and carbonyl group of Thr4 are involved in hydrogen
bonds with Tyr32L and Gln97H, respectively. A hydrogen bond between side chain of Arg5 and
carbonyl group of Asn31H was also observed.
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Figure 5. (a) Crystal structure of glycopeptide 22 in complex with scFvꢀSM3, showing the
stabilizing interactions antigenꢀantibody. Carbon atoms of glycopeptide 22 are in green. Carbon
atoms of key residues of SM3 are coloured in yellow. Interactions between the peptidic fragment
and SM3 surface are in pink. (b) Superposition of the peptide backbone of glycopeptide
APDT(GalNAc)RP and glycopeptide 22 in complex with scFvꢀSM3, showing the rootꢀmeanꢀsquare
deviation (rmsd) value for the peptide backbone. (c) Ensembles obtained from the unrestrained 100
ns MD simulations performed on complex scFvꢀSM3:22. The antibody is shown as a grey surface
and the peptide backbone of glycopeptide 22 is shown in green. The carbon atoms of GalNAc unit
are in pink. The conformation of glycopeptide APDT(GalNAc)RP found in the Xꢀray structure is
also shown in purpleꢀblue (PDB id: 5A2K, ref. 44).
The conformation of the peptide fragment of 22 bound to the mAb is almost identical to those
observed for natural glycopeptides in complex with this antibody, indicating the minor impact that
the linker has on the peptide conformation in the bound state (Figure 5b). Unfortunately, the
structure of the linker and the GalNAc, could not be solved due to the lack of electron density. This
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is presumably due to the high degree of flexibility of the linker. Therefore, molecular dynamics
(MD) simulations (100 ns) were performed. All interactions between glycopeptide 22 and the mAb
observed in the Xꢀray structure are also observed though MD simulations. As deduced from these
calculations, the linker is rather flexible and the GalNAc residue is exposed to the solvent (Figure
5c).
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Conclusions
Several sulfaꢀTn antigen derivatives with variable lengths between the glycan and the peptide
backbone have been synthesized using two types of reactions with GalNAc(OAc)3ꢀαꢀSH. The first
one comprised a thiolꢀene coupling of alkenylꢀαꢀamino acids and the second one involved an
anomeric thiol group Sꢀalkylation of bromoꢀαꢀamino acids. The presence of a linker between the
peptide backbone and the sugar precludes the intramolecular interactions between these two
moieties, favoring the presentation of the sugar for molecular recognition events. This property,
together with the potential lack of immunosuppression suffered by the natural derivatives, makes
the studied compounds promising candidates for the design of novel therapeutic vaccines to treat
tumors.
Experimental Section
Reagents and general procedures. Commercial reagents were used without further purification. Analytical thin layer
chromatography (TLC) was performed on MachereyꢀNagel precoated aluminium sheets with a 0.20 mm thickness of
silica gel 60 with fluorescent indicator UV254. TLC plates were visualized with UV light and by staining with
phosphomolybdic acid (PMA) solution (5 g of PMA in 100 mL of absolute ethanol) or sulfuric acidꢀethanol solution.
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Column chromatography was performed on silicagel (230−400 mesh). H and 13C NMR spectra were measured with a
400 MHz spectrometer with TMS as the internal standard. Multiplicities are quoted as singlet (s), broad singlet (br s),
doublet (d), doublet of doublets (dd), triplet (t), or multiplet (m). Spectra were assigned using COSY and HSQC
experiments. All NMR chemical shifts (δ) were recorded in ppm and coupling constants (J) were reported in Hz. The
results of these experiments were processed with MestreNova software. High resolution electrospray mass (ESI) spectra
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were recorded on a microTOF spectrometer; accurate mass measurements were achieved by using sodium formate as an
external reference.
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General procedure for the thiolꢀene coupling by thermal way. To a solution of alkeneꢀamino acid derivative (1.0
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equiv) and triꢀOꢀacetylꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀ1ꢀthioꢀαꢀDꢀgalactopyranose 1 (1.7 equiv) in CHCl3 (5 mL), α,α′ꢀ
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azoisobutyronitrile (AIBN or VAZO 64, Molekula 78ꢀ67ꢀ1) (0.6 equiv) was added and reaction mixture was then heated
at reflux. After stirring for 3 h, another portion of AIBN (0.6 equiv) was added and the mixture was stirred for 4 h more.
The reaction was concentrated and the residue was purified by a silica gel column chromatography to give the
corresponding Sꢀgalactosaminyl amino acid derivative.
General procedure for the thiolꢀene coupling by photochemical way. The reaction was carried out in a glass vial
(diameter: 2 cm), sealed with a natural rubber septum, located 2.5 cm away from the UVA lamp apparatus (LUZCHEM ꢀ
LZCꢀ4Vꢀ, 16 UVAꢀlamps of 350 nm and 8 W). To a solution of alkeneꢀamino acid derivative (1.0 equiv) and triꢀOꢀacetylꢀ
2ꢀacetamidoꢀ2ꢀdeoxyꢀ1ꢀthioꢀαꢀ
Dꢀgalactopyranose
1
(1.2 equiv) in CH2Cl2 (2.5 mL), 2,2ꢀdimethoxyꢀ2ꢀ
phenylacetophenone (DPAP) (0.2 equiv) was added. After stirring for 3 h, another portion of DPAP (0.2 equiv) was added
and the mixture was stirred for 4 h more. The reaction was concentrated and the residue was purified by a silica gel
column chromatography to give the corresponding Sꢀgalactosaminyl amino acid derivative.
General procedure for the thiolꢀene coupling by repairing way. To a solution of triꢀOꢀacetylꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀ1ꢀ
thioꢀαꢀDꢀgalactopyranose 1 (2.0 equiv), catechol (1.2 equiv) and alkeneꢀamino acid derivative (0.75 M in CH2Cl2, 1.0
equiv), BEt3 (1.0 M in hexane, 1.2 equiv) was added. The reaction was stirred at room temperature and BEt3 was regularly
added until complete consumption of the alkenylꢀamino acid derivative (0.1 equiv every 45 min). The reaction was then
concentrated and the residue was purified by a silica gel column chromatography to give the corresponding Sꢀ
galactosaminyl amino acid derivative.
Solidꢀphase (glyco)peptide synthesis (SPPS). All glycopeptides were synthesized by a stepwise solidꢀphase peptide
synthesis using the Fmoc strategy on Rink Amide MBHA resin (128 mg, 0.1 mmol, loading of the resin: 0.78 mmol/g).
The glycosylated amino acid building block (2.0 equiv) was manually coupled using HBTU, while the other Fmoc amino
acids (10.0 equiv) were automatically coupled on an Applied Biosystems 433A peptide synthesizer using HBTU. The Oꢀ
acetyl groups of (AcO)3GalNAc moiety were removed in a mixture of NH2NH2/MeOH (7:3). The glycopeptides were
then released from the resin and all acid sensitive sideꢀchain protecting groups were simultaneously removed using TFA
95%, TIS 2.5%, H2O 2.5%, followed by precipitation with diethyl ether. Finally, all glycopeptides were purified by HPLC
on a Waters Delta Prep 4000 reverse phase HPLC and Waters 2987 Dual Absorbance Detector, using a Phenomenex Luna
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C18(2) column (10 ꢁ, 250 mm × 21.2 mm), 2% (v/v) CH3CN in H2O (containing 0.1% v/v TFA) gradient to 13% CH3CN
(t = 27 min) and then to 60% CH3CN (t = 33 min), 10 mL/min and registered at λ = 212 nm.
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3,4,6ꢀTriꢀOꢀacetylꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀ1ꢀthioꢀαꢀDꢀgalactopyranose (1)
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A stirred suspension of a mixture of α,βꢀanomers of 1,3,4,6ꢀtetraꢀOꢀacetylꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀDꢀgalactopyranose (20.5
g, 52.65 mmol) and Lawesson’s reagent (18.1 g, 44.7 mmol) in (1:1) toluene/1,2ꢀdichloroethane solution (200 mL) was
heated at 80 °C for 12 h. The reaction was then evaporated and the crude used directly in the next reaction. This crude was
dissolved in 200 mL of methanol and stirred at 0 °C. TFA (10 mL) and water (10 mL) were then added, and the reaction
was allowed to warm to room temperature and stirred for 4 h. The reaction was concentrated and the residue was purified
by column chromatography using EtOAc/CH2Cl2 (8:2) as eluent, to give compound 1 (9.55 g, 50%) as a white foam.
Spectroscopic data are consistent with the literature (ref. 26b and 34a).
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NꢀBocꢀOꢀ(Sꢀ((2ꢀacetamidoꢀ2ꢀdeoxy)ꢀ(3,4,6ꢀtriꢀOꢀacetyl)ꢀαꢀ
Dꢀgalactopyranosyl)mercaptopropyl)ꢀ
L
ꢀserine
benzyl
ester (3)
A solution of NꢀBocꢀLꢀSer(Oꢀallyl)ꢀOBn (2bꢀSer) (90 mg, 0.27 mmol), 3,4,6ꢀtriꢀOꢀacetylꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀ1ꢀthioꢀαꢀ
ꢀgalactopyranose (1) (166 mg, 0.46 mmol) and AIBN (27 mg, 0.16 mmol) in CHCl3 (3 mL) was heated at reflux. After
D
stirring for 3 h, another portion of AIBN (27 mg, 0.16 mmol) was added and the reaction was stirred for 4 h more. The
reaction mixture was cooled, concentrated, and then chromatographed on silica gel using EtOAc/CH2Cl2 (8:2) as eluent to
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give compound 3 (104 mg, 56%) as a white foam. [α]D (c=1.00, CHCl3): +71.4. HRMS (ESI+) (m/z) 699.2791
[M+H]+; calculated C32H47N2O13S+: 699.2793. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.39–7.29 (m, 5H, arom), 5.58 (d, J
= 8.6 Hz, 1H, NH sugar), 5.45 (d, J = 5.1 Hz, 1H, H1), 5.41–5.34 (m, 2H, NH, H4), 5.25 (d, J = 12.4 Hz, 1H, CH2Bn), 5.13
(d, J = 12.4 Hz, 1H, CH2Bn), 5.03 (dd, J = 11.6, 2.7 Hz, 1H, H3), 4.81–4.71 (m, 1H, H2), 4.51 (t, J = 6.4 Hz, 1H, H5),
4.49–4.43 (m, 1H, Hα), 4.16–4.03 (m, 2H, 2H6), 3.84 (dd, J = 9.4, 2.6 Hz, 1H, Hβ), 3.64 (dd, J = 9.4, 3.2 Hz, 1H, Hβ),
3.51–3.35 (m, 2H, 2Hδ), 2.67–2.47 (m, 2H, 2Hζ), 2.15 (s, 3H, COCH3), 2.03–1.95 (m, 9H, COCH3, COCH3, NHCOCH3),
1.87–1.71 (m, 2H, 2Hε), 1.44 (s, 9H, C(CH3)3). 13C NMR (100 MHz, CDCl3) δ (ppm): 171.1, 170.7, 170.5, 170.4, 170.2,
155.6 (CO), 135.6, 128.7, 128.5, 128.3 (arom), 85.3 (C1), 80.2 (C(CH3)3), 71.0 (Cβ), 69.7 (Cδ), 68.6 (C3), 67.5 (C4, C5),
67.2 (CH2Bn), 62.0 (C6), 54.3 (Cα), 48.5 (C2), 29.8 (Cε), 28.5 (C(CH3)3), 28.0 (Cζ), 23.5 (NHCOCH3), 20.8 (3 COCH3).
NꢀAcetylꢀOꢀ(Sꢀ((2ꢀacetamidoꢀ2ꢀdeoxy)ꢀ(3,4,6ꢀtriꢀOꢀacetyl)ꢀαꢀ
Dꢀgalactopyranosyl)mercaptopropyl)ꢀ
L
ꢀserine benzyl
ester (4)
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Trifluoroacetic acid (378 ꢀL, 4.91 mmol) was added over a solution of compound 3 (104 mg, 0.15 mmol) in CH2Cl2 (8
mL) and the resulting solution was stirred for 2 h at room temperature and the solvent was removed under reduced
pressure. The residue was dissolved in Et2O (20 mL) and then evaporated. This operation was repeated several times to
obtain the corresponding compound, which was used directly in the next reaction. Acetic anhydride (2.0 mL) and pyridine
(4 mL) were added to previous compound, and the solution was stirred for 12 h at room temperature. The solvent was
removed under reduced pressure and the residue was chromatographed on silica gel using CH2Cl2/MeOH (9:1) as eluent
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to give compound 4 (67 mg, 70%) as a white foam. [α]D (c=1.01, CHCl3): +81.9. HRMS (ESI+) (m/z) 641.2382
[M+H]+; calculated C29H41N2O12S+: 641.2375. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.43–7.22 (m, 5H, arom), 6.42 (d, J
= 8.0 Hz, 1H, NH), 5.57 (d, J = 8.7 Hz, 1H, NH sugar), 5.48 (d, J = 5.2 Hz, 1H, H1), 5.38 (s, 1H, H4), 5.25 (d, J = 12.3
Hz, 1H, CH2Bn), 5.16 (d, J = 12.3 Hz, 1H, CH2Bn), 5.04 (dd, J = 11.7, 2.5 Hz, 1H, H3), 4.83–4.71 (m, 2H, Hα, H2), 4.51
(t, J = 6.4 Hz, 1H, H5), 4.17–4.05 (m, 2H, 2H6), 3.86 (dd, J = 9.3, 1.5 Hz, 1H, Hβ), 3.69–3.63 (m, 1H, Hβ), 3.52–3.46 (m,
1H, Hδ), 3.43–3.35 (m, 1H, Hδ), 2.66–2.58 (m, 1H, Hζ), 2.55–2.48 (m, 1H, Hζ), 2.15 (s, 3H, COCH3), 2.08–1.95 (m,
12H, COCH3, COCH3, NHCOCH3, NHCOCH3), 1.86–1.72 (m, 2H, 2Hε). 13C NMR (100 MHz, CDCl3) δ (ppm): 171.1,
170.5, 170.1, 170.2, 170.3 (6 CO), 135.5, 128.7, 128.5, 128.2 (arom), 85.1 (C1), 70.7 (Cβ), 69.4 (Cδ), 68.5 (C3), 67.4
(C4, C5), 67.3 (CH2Bn), 61.8 (C6), 52.8 (Cα), 48.5 (C2), 29.7 (Cε), 27.6 (Cζ), 23.3 (NHCOCH3), 23.2 (NHCOCH3), 20.8,
20.8 (3 COCH3).
NꢀAcetylꢀOꢀ(Sꢀ((2ꢀacetamidoꢀ2ꢀdeoxy)ꢀ(3,4,6ꢀtriꢀOꢀacetyl)ꢀαꢀDꢀgalactopyranosyl)mercaptopropyl)ꢀLꢀserine methyl
amide (5)
A solution of compound 4 (67 mg, 0.10 mmol) in MeOH (10 mL) was treated with hydrogen under atmospheric pressure
at room temperature, using Pd/C 10% as a catalyst (67 mg). This reaction mixture was stirred for 24 h. The solution was
then filtered over celite, evaporated and this crude was used directly in the next reaction. It was dissolved in dry CH3CN
(20 mL) under an inert atmosphere. Methylamine hydrochloride (21 mg, 0.31 mmol), TBTU (44 mg, 0.14 mmol) and
DIEA (91 ꢀL, 0.52 mmol) were then added and the reaction was stirred at room temperature for 24 h. After evaporation,
the residue was purified by a silica gel column chromatography, eluting with CH2Cl2/MeOH (9:1), to give compound 5
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(27 mg, 46%) as a yellow oil. [α]D (c=1.00, CHCl3): +101.4. HRMS (ESI+) (m/z) 564.2234 [M+H]+; calculated
1
C23H38N3O11S+: 564.2222. H NMR (400 MHz, CDCl3) δ (ppm): 6.71–6.60 (m, 2H, NHCOCH3, NHCH3), 6.00 (t, J =
8.4 Hz, 1H, NH sugar), 5.53 (dd, J = 8.0, 5.4 Hz, 1H, H1), 5.38–5.36 (m, 1H, H4), 5.04 (dt, J = 11.8, 3.1 Hz, 1H, H3),
4.75–4.66 (m, 1H, H2), 4.55–4.47 (m, 2H, H5, Hα), 4.14–4.07 (m, 2H, 2H6), 3.77 (ddd, J = 14.4, 9.4, 4.6 Hz, 1H, Hβ),
3.61–3.43 (m, 3H, Hβ, 2Hδ), 2.81 (d, J = 4.8 Hz, 3H, NHCH3), 2.73–2.58 (m, 2H, 2Hζ), 2.14 (s, 3H, COCH3), 2.06–1.95
(m, 12H, COCH3, COCH3, NHCOCH3, NHCOCH3), 1.92–1.77 (m, 2H, 2Hε). 13C NMR (100 MHz, CDCl3) δ (ppm)
(Duplication of signals are observed, probably due to the coexistence of various conformers, since when the spectra were
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registered at higher temperature some of them collapse in one signal): 171.2, 170.6, 170.6, 170.5, 170.3, 170.3 (CO),
85.4, 85.4 (C1), 70.1, 70.1 (Cβ), 69.3, 69.2 (Cδ), 68.5 (C3), 67.6, 67.5, 67.4, 67.4 (C4, C5), 61.9, 61.9 (C6), 52.8, 52.8
(Cα), 48.8, 48.7 (C2), 29.7, 29.6 (Cε), 28.0, 27.9 (Cζ), 26.5 (NHCH3), 23.4, 23.4 (NHCOCH3), 23.3, 23.3 (NHCOCH3),
20.9, 20.8, 20.8 (3 COCH3).
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NꢀAcetylꢀOꢀ(Sꢀ((2ꢀacetamidoꢀ2ꢀdeoxy)ꢀαꢀDꢀgalactopyranosyl)mercaptopropyl)ꢀLꢀserine methyl amide (6)
To a solution of compound 5 (27 mg, 0.05 mmol) in MeOH (2 mL), MeONa (0.5 M, 150 ꢀL) was added and the reaction
was stirred at room temperature for 2 h. Then, Dowex 50WꢀX8 (40 mg) was added to the reaction and it was stirred for 10
min. After that, the mixture was filtered and evaporated obtaining compound 6 in a 90% yield. In order to perform ELLA
test, the residue was dissolved in H2O (0.5 mL) and compound 6 was purified by semipreparative HPLC analysis using
the following procedure: 0.5 mL injection loop was conducted on a Waters Delta Prep 4000 reverse phase HPLC and
Waters 2987 Dual Absorbance Detector, using a Phenomenex Luna C18(2) column (10 ꢁ, 250 mm × 21.2 mm), 5% (v/v)
CH3CN in H2O (containing 0.1% v/v TFA) gradient to 30% CH3CN (t = 30 min), 10 mL/min, λ = 212 nm to furnish 6 (10
mg, 50%, tR 25.3 min) as a white foam after lyophilization. [α]D20 (c=1.00, CHCl3): +85.0. HRMS (ESI+) (m/z) 438.1911
[M+H]+; calculated C17H32N3O8S+: 438.1905. 1H NMR (400 MHz, H2O/D2O 9:1) δ (ppm) amide protons: 8.20 (d, J =
5.5 Hz, 1H, NHCOCH3), 8.13 (d, J = 7.6 Hz, 1H, NH sugar), 7.91 (s, 1H, NHCH3). 1H NMR (400 MHz, D2O) δ (ppm):
5.50 (d, J = 5.5 Hz, 1H, H1), 4.46 (t, J = 4.9 Hz, 1H, Hα), 4.35 (dd, J = 11.4, 5.5 Hz, 1H, H2), 4.28 (t, J = 6.1 Hz, 1H,
H5), 4.00 (d, J = 2.4 Hz, 1H, H4), 3.84 (dd, J = 11.4, 3.1 Hz, 1H, H3), 3.80–3.71 (m, 4H, 2H6, 2Hβ), 3.69–3.55 (m, 2H,
2Hδ), 2.75 (s, 3H, NHCH3), 2.72–2.61 (m, 2H, 2Hζ), 2.07 (s, 3H, NHCOCH3), 2.05 (s, 3H, COCH3), 1.94–1.81 (m, 2H,
2Hε). 13C NMR (100 MHz, D2O) δ (ppm): 174.6, 174.6, 172.2 (CO), 83.9 (C1), 71.7 (C5), 69.2 (Cβ, Cδ), 68.6 (C4), 67.8
(C3), 61.3 (C6), 54.0 (Cα), 50.3 (C2), 28.5 (Cε), 26.8 (Cζ), 26.1 (NHCH3), 22.0 (NHCOCH3), 21.9 (NHCOCH3).
NꢀFmocꢀOꢀ(allyl)ꢀLꢀserine tertꢀbutyl ester (9ꢀSer)
To a solution of compound 2aꢀSer (1.02 g, 4.16 mmol) in CH2Cl2 (5 mL), trifluoroacetic acid (5 mL) was added. The
resulting solution was stirred for 2 h at room temperature and the solvent was removed under reduced pressure. The
residue was dissolved in Et2O (10 mL) and then evaporated. This operation was repeated several times to obtain
compound 7ꢀSer, which was used directly in the next reaction. A solution of acetone/H2O (1:1) (30 mL) was added to the
previous compound and it was then treated with Na2CO3 (882 mg, 8.32 mmol) and FmocOSu (1.4 g, 4.16 mmol). The
mixture was stirred for 18 h at room temperature and concentrated. The crude mixture was diluted with H2O and extracted
with Et2O. The aqueous layer was then acidified to pH 3ꢀ4 with a 10% KHSO4 solution. This aqueous solution was
extracted with CH2Cl2 (3 x 50 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered,
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and concentrated to give compound 8ꢀSer (1.50 g, 98%) as a white solid, which was used directly in the next reaction.
tButyl alcohol (1.6 mL, 16.73 mmol) was added over DCC (2.72 g, 13.07 mmol) and CuCl (40 mg, 0.41 mmol) under
inert atmosphere, light protected, and the resulting mixture was stirred for 5 days and dry CH2Cl2 (6 mL) was then added.
After that, compound 8ꢀSer (1.50 g, 4.08 mmol) dissolved in dry CH2Cl2 (6 mL) was added over the previous solution
and the mixture was stirred for 4 h. The dicyclohexylurea byproduct was removed by filtration over celite, and the
solution was washed with saturated NaHCO3 (2 x 50 mL). The organic layer was dried, filtered and concentrated, and the
residue was purified by silica gel column chromatography using hexane/EtOAc (8:2) as eluent, to give compound 9ꢀSer
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(1.0 g, 58%) as a colorless oil. [α]D (c=1.00, CHCl3): +16.5. HRMS (ESI+) (m/z) 446.1943 [M+Na]+; calculated
+
1
C25H29NNaO5 : 446.1938. H NMR (400 MHz, CDCl3) δ (ppm): 7.80–7.73 (m, 2H, arom), 7.65–7.59 (m, 2H, arom),
7.44–7.36 (m, 2H, arom), 7.34–7.28 (m, 2H, arom), 5.86 (m, 1H, Hε), 5.67 (d, J = 8.3 Hz, 1H, NH), 5.27 (d, J = 17.3 Hz,
1H, Hζtrans), 5.20 (d, J = 10.4 Hz, 1H, Hζcis), 4.47–4.33 (m, 3H, Hα, CH2Fmoc), 4.24 (t, J = 7.2 Hz, 1H, CHFmoc), 4.04
(dd, J = 12.8, 5.3 Hz, 1H, Hδ), 3.96 (dd, J = 12.8, 5.4 Hz, 1H, Hδ), 3.86 (dd, J = 9.4, 2.5 Hz, 1H, Hβ), 3.69 (dd, J = 9.3,
2.2 Hz, 1H, Hβ), 1.49 (s, 9H, C(CH3)3). 13C NMR (100 MHz, CDCl3) δ (ppm): 169.5 (CO), 156.2 (CO), 144.1, 144.0,
141.4 (arom), 134.2 (Cε), 127.8, 127.2, 125.3, 120.1 (arom), 117.5 (Cζ), 82.4 (C(CH3)3), 72.4 (Cδ), 70.4 (Cβ), 67.3
(CH2Fmoc), 55.1 (Cα), 47.3 (CHFmoc), 28.2 (C(CH3)3).
NꢀFmocꢀSꢀ(allyl)ꢀLꢀcysteine tertꢀbutyl ester (9ꢀCys)
Following the same procedure above described for compound 9ꢀSer and starting from 2aꢀCys (0.76 g, 2.91 mmol),
compound 9ꢀCys (679 mg) was obtained as a white solid with a 54% overall yield.
Mp: 61–63 ºC. [α]D20 (c=1.00, CHCl3): ꢀ6.0. HRMS (ESI+) (m/z) 440.1896 [M+H]+; calculated C25H30NO4S+: 440.1890.
1H NMR (400 MHz, CDCl3) δ (ppm): 7.78–7.76 (m, 2H, arom), 7.64–7.60 (m, 2H, arom), 7.43–7.38 (m, 2H, arom),
7.35–7.29 (m, 2H, arom), 5.82–5.71 (m, 1H, Hε), 5.62 (d, J = 7.7 Hz, 1H, NH), 5.16–5.08 (m, 2H, 2Hζ), 4.50 (m, 1H,
Hα), 4.40 (d, J = 7.1 Hz, 1H, CH2Fmoc), 4.25 (t, J = 7.1 Hz, 1H, CHFmoc), 3.18–3.15 (m, 2H, 2Hδ), 2.97 (dd, J = 13.8, 4.5
Hz, 1H, Hβ), 2.87 (dd, J = 13.8, 5.6 Hz, 1H, Hβ), 1.50 (s, 9H, C(CH3)3). 13C NMR (100 MHz, CDCl3) δ (ppm): 169.9
(CO), 155.8 (CO), 144.0, 143.9, 141.4 (arom), 133.9 (Cε), 127.8, 127.2, 125.2, 120.1 (arom), 117.9 (Cζ), 82.9 (C(CH3)3),
67.2 (CH2Fmoc), 54.2 (Cα), 47.2 (CHFmoc), 35.5 (Cδ), 33.3 (Cβ), 28.1 (C(CH3)3).
NꢀFmocꢀOꢀ(allyl)ꢀLꢀthreonine tertꢀbutyl ester (9ꢀThr)
Following the same procedure above described for compound 9ꢀSer and starting from 2aꢀThr (2.24 g, 8.64 mmol),
compound 9ꢀThr (2.03 g) was obtained as a white solid with a 59% overall yield.
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Mp: 58–60 ºC. [α]D20 (c=1.00, CHCl3): ꢀ0.3. HRMS (ESI+) (m/z) 438.2292 [M+H]+; calculated C26H32NO5 : 438.2275.
1H NMR (400 MHz, CDCl3) δ (ppm): 7.78–7.74 (m, 2H, arom), 7.68–7.63 (m, 2H, arom), 7.43–7.38 (m, 2H, arom),
7.35–7.30 (m, 2H, arom), 5.94–5.82 (m, 1H, Hε), 5.60 (d, J = 9.6 Hz, 1H, NH), 5.29 (d, J = 17.0 Hz, 1H, Hζtrans), 5.19
(d, J = 10.4 Hz, 1H, Hζcis), 4.48–4.38 (m, 2H, CH2Fmoc), 4.34 (dd, J = 9.6, 1.3 Hz, 1H, Hα), 4.26 (t, J = 7.2 Hz, 1H,
CHFmoc), 4.14–4.06 (m, 2H, Hβ, Hδ), 3.91 (dd, J = 12.6, 5.5 Hz, 1H, Hδ), 1.51 (s, 9H, C(CH3)3), 1.25 (d, J = 6.2 Hz, 3H,
CH3β). 13C NMR (100 MHz, CDCl3) δ (ppm): 169.6 (CO), 156.7 (CO), 144.0, 143.8, 141.2 (arom), 134.5 (Cε), 127.6,
127.0, 127.0, 125.1, 125.1, 119.9 (arom), 116.9 (Cζ), 81.8 (C(CH3)3), 74.7 (Cβ), 70.0 (Cδ), 67.1 (CH2Fmoc), 59.1 (Cα),
47.1 (CHFmoc), 28.0 (C(CH3)3), 16.2 (CH3β).
+
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NꢀFmocꢀOꢀ(Sꢀ((2ꢀacetamidoꢀ2ꢀdeoxy)ꢀ(3,4,6ꢀtriꢀOꢀacetyl)ꢀαꢀ
Dꢀgalactopyranosyl)mercaptopropyl)ꢀ
L
ꢀserine
tertꢀ
butyl ester (10ꢀSer)
THERMAL WAY: A solution of 9ꢀSer (195 mg, 0.46 mmol), compound 1 (284 mg, 0.78 mmol) and AIBN (46 mg, 0.28
mmol) in CHCl3 (5 mL) was heated at reflux. After stirring for 3 h, another portion of AIBN (46 mg, 0.28 mmol) was
added and the reaction was stirred for 4 h more. The reaction mixture was cooled, concentrated and then chromatographed
on silica gel using EtOAc/CH2Cl2 (8:2) as eluent to give protected glycosyl amino acid 10ꢀSer (253 mg, 70%) as a white
solid.
PHOTOCHEMICAL WAY: A solution of compound 9ꢀSer (232 m, 0.55 mmol), compound 1 (240 mg, 0.66 mmol) and
DPAP (30 mg, 0.11 mmol) in CH2Cl2 (2.5 mL) was irradiated at λmax 365 nm. After stirring for 3 h, another portion of
DPAP (30 mg, 0.11 mmol) was added and the reaction was stirred for 4 h more. The reaction mixture was then
concentrated and the residue was chromatographed on silica gel using EtOAc/CH2Cl2 (8:2) as eluent to give protected
glycosyl amino acid 10ꢀSer (227 mg, 53%) as a white solid.
REPAIRING WAY: A solution of compound 9ꢀSer (205 mg, 0.48 mmol), compound 1 (352 mg, 0.97 mmol), catechol
(64 mg, 0.58 mmol) and BEt3 (84 ꢀL, 0.58 mmol) in CH2Cl2 (0.65 mL) was stirred at room temperature. After stirring for
1 h, another portion of BEt3 (7 ꢀL) was added (until complete consumption of compound 9ꢀSer). The reaction mixture
was then concentrated and the residue chromatographed on silica gel using EtOAc/CH2Cl2 (8:2) as eluent, to give
protected glycosyl amino acid 10ꢀSer (265 mg, 70%) as a white solid.
Physical data: mp: 63–65 ºC, [α]D (c=1.00, CHCl3): +95.5. HRMS (ESI+) (m/z) 787.3119 [M+H]+; calculated
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C39H51N2O13S+: 787.3106. H NMR (400 MHz, CDCl3) δ (ppm): 7.78–7.74 (m, 2H, arom), 7.65–7.59 (m, 2H, arom),
1
7.42–7.37 (m, 2H, arom), 7.34–7.29 (m, 2H, arom), 5.70–5.60 (m, 2H, NHAc, NHFmoc), 5.49 (d, J = 5.2 Hz, 1H, H1),
5.38 (s, 1H, H4), 5.04 (dd, J = 11.6, 2.6 Hz, 1H, H3), 4.82–4.72 (m, 1H, H2), 4.53 (t, J = 6.3 Hz, 1H, H5), 4.46–4.33 (m,
3H, CH2Fmoc, Hα), 4.25 (t, J = 7.1 Hz, 1H, CHFmoc), 4.17–4.03 (m, 2H, 2H6), 3.82 (dd, J = 9.4, 2.8 Hz, 1H, Hβ), 3.67 (dd,
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J = 9.3, 2.3 Hz, 1H, Hβ), 3.60–3.43 (m, 2H, 2Hδ), 2.75–2.57 (m, 2H, 2Hζ), 2.14 (s, 3H, COCH3), 2.06–1.95 (m, 9H, 2
COCH3, NHCOCH3), 1.91–1.83 (m, 2H, 2Hε), 1.48 (s, 9H, C(CH3)3). 13C NMR (100 MHz, CDCl3) δ (ppm): 171.1,
170.4, 170.4, 170.2, 169.4, 156.1 (6 CO), 144.0, 144.0, 141.4, 127.8, 127.2, 125.3, 120.0 (arom), 85.2 (C1), 82.4
(C(CH3)3), 71.2 (Cβ), 69.7 (Cδ), 68.6 (C3), 67.5, 67.4 (C4, C5), 67.2 (CH2Fmoc), 61.9 (C6), 55.0 (Cα), 48.5 (C2), 47.3
(CHFmoc), 29.8 (Cε), 28.1 (C(CH3)3), 28.0 (Cζ), 23.4 (NHCOCH3), 20.8, 20.8 (3 COCH3).
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NꢀFmocꢀSꢀ(Sꢀ((2ꢀacetamidoꢀ2ꢀdeoxy)ꢀ(3,4,6ꢀtriꢀOꢀacetyl)ꢀαꢀ
D
ꢀgalactopyranosyl)mercaptopropyl)ꢀ
L
ꢀcysteine
tertꢀ
butyl ester (10ꢀCys)
THERMAL WAY: Following the same procedure above described for compound 10ꢀSer and starting from 9ꢀCys (191
mg, 0.43 mmol), compound 10ꢀCys (70 mg, 20%) was obtained as a white solid.
PHOTOCHEMICAL WAY: Following the same procedure above described for compound 10ꢀSer and starting from 9ꢀ
Cys (203 mg, 0.46 mmol), compound 10ꢀCys (111 mg, 30%) was obtained as a white solid.
REPAIRING WAY: Following the same procedure above described for compound 10ꢀSer and starting from 9ꢀCys (310
mg, 0.71 mmol), compound 10ꢀCys (201 mg, 36%) was obtained as a white solid.
Physical data: mp: 69–71 ºC, [α]D (c=1.00, CHCl3): +82.9. HRMS (ESI+) (m/z) 803.2878 [M+H]+; calculated
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C39H51N2O12S2 : 803.2878. H NMR (400 MHz, CDCl3) δ (ppm): 7.77–7.74 (m, 2H, arom), 7.62–7.58 (m, 2H, arom),
7.41–7.36 (m, 2H, arom), 7.33–7.28 (m, 2H, arom), 5.69 (d, J = 8.0 Hz, 2H, NHAc, NHFmoc), 5.49 (d, J = 5.3 Hz, 1H,
H1), 5.36 (d, J = 2.4 Hz, 1H, H4), 5.02 (dd, J = 11.7, 3.1 Hz, 1H, H3), 4.79–4.71 (m, 1H, H2), 4.54–4.44 (m, 2H, H5,
Hα), 4.43–4.34 (m, 2H, CH2Fmoc), 4.22 (t, J = 7.0 Hz, 1H, CHFmoc), 4.15–4.03 (m, 2H, 2H6), 2.99 (dd, J = 13.7, 4.8 Hz,
1H, Hβ), 2.92 (dd, J = 13.7, 5.1 Hz, 1H, Hβ), 2.75–2.13 (m, 4H, 2Hζ, 2Hδ), 2.13 (s, 3H, COCH3), 2.02–1.93 (m, 9H, 2
COCH3, NHCOCH3), 1.91–1.82 (m, 2H, 2Hε), 1.48 (s, 9H, C(CH3)3). 13C NMR (100 MHz, CDCl3) δ (ppm): 171.0,
170.4, 170.3, 170.2, 169.8, 155.8 (6 CO), 143.9, 143.9, 141.4, 127.8, 127.2, 125.2, 120.1 (arom), 85.2 (C1), 83.0
(C(CH3)3), 68.6 (C3), 67.5 (C5), 67.4 (C4), 67.2 (CH2Fmoc ), 61.9 (C6), 54.4 (Cα), 48.4 (C2), 47.2 (CHFmoc), 34.8 (Cβ),
31.7 (Cδ), 29.9 (Cζ), 29.3 (Cε), 28.1 (C(CH3)3), 23.4 (NHCOCH3), 20.8, 20.8 (3 COCH3).
The MS spectrum of the crude reaction mixture, before to be purified by column chromatography, showed signals
corresponding to the expected product 10ꢀCys and sideꢀproducts named 24, 26, 27, 28 and 29 that probably are formed by
radical couplings as shown in the scheme shown in the Supporting Information. This feature may explain the low yield
obtained in this reaction, following the tree methodologies, when it is compared with the formation of 10ꢀSer, 10ꢀThr or
12.
NꢀFmocꢀOꢀ(Sꢀ((2ꢀacetamidoꢀ2ꢀdeoxy)ꢀ(3,4,6ꢀtriꢀOꢀacetyl)ꢀαꢀ
D
ꢀgalactopyranosyl)mercaptopropyl)ꢀ
L
ꢀthreonine tertꢀ
butyl ester (10ꢀThr)
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THERMAL WAY: Following the same procedure above described for compound 10ꢀSer and starting from 9ꢀThr (206
mg, 0.47 mmol), compound 10ꢀThr (268 mg, 71%) was obtained as a white solid.
6
7
8
9
PHOTOCHEMICAL WAY: Following the same procedure above described for compound 10ꢀSer and starting from 9ꢀ
Thr (160 mg, 0.37 mmol), compound 10ꢀThr (181 mg, 62%) was obtained as a white solid.
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REPAIRING WAY: Following the same procedure above described for compound 10ꢀSer and starting from 9ꢀThr (242
mg, 0.55 mmol), compound 10ꢀThr (322 mg, 73%) was obtained as a white solid.
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Pysical data: mp: 76–78 ºC, [α]D (c=1.00, CHCl3): +78.3. HRMS (ESI+) (m/z) 801.3271 [M+H]+; calculated
1
C40H53N2O13S+: 801.3263. H NMR (400 MHz, CDCl3) δ (ppm): 7.76–7.73 (m, 2H, arom), 7.64–7.59 (m, 2H, arom),
7.40–7.35 (m, 2H, arom), 7.33–7.27 (m, 2H, arom), 5.75 (d, J = 8.7 Hz, 1H, NHAc), 5.54–5.46 (m, 2H, H1, NHFmoc),
5.37 (s, 1H, H4), 5.04 (dd, J = 11.7, 2.7 Hz, 1H, H3), 4.71–4.82 (m, 1H, H2), 4.53 (t, J = 6.4 Hz, 1H, H5), 4.43–4.34 (m,
2H, CH2Fmoc), 4.20–4.19 (m, 2H, Hα, CHFmoc), 4.14–4.04 (m, 2H, 2H6), 3.98 (d, J = 6.1 Hz, 1H, Hβ), 3.62–3.54 (m, 1H,
Hδ), 3.41–3.30 (m, 1H, Hδ), 2.74–2.55 (m, 2H, 2Hζ), 2.12 (s, 3H, COCH3), 2.02–1.95 (m, 9H, 3 COCH3, NHCOCH3),
1.89–1.78 (m, 2H, 2Hε), 1.46 (s, 9H, C(CH3)3), 1.18 (d, J = 6.2 Hz, 3H, 3Hγ). 13C NMR (100 MHz, CDCl3) δ (ppm):
171.0, 170.4, 170.3, 170.2, 169.8, 156.8 (6 CO), 144.0, 143.9, 141.4, 127.8, 127.1, 125.2, 120.0 (arom), 85.1 (C1), 82.1
(C(CH3)3), 75.6 (Cβ), 68.6 (C3), 67.4 (C4, C5, Cδ), 67.2 (CH2Fmoc), 61.9 (C6), 59.1 (Cα), 48.4 (C2), 47.3 (CHFmoc), 30.2
(Cε), 28.2 (C(CH3)3), 28.0 (Cζ), 23.3 (NHCOCH3), 20.8, 20.8 (3 COCH3), 16.4 (Cγ).
NꢀFmocꢀSꢀ((2ꢀacetamidoꢀ2ꢀdeoxy)ꢀ(3,4,6ꢀtriꢀOꢀacetyl)ꢀαꢀDꢀgalactopyranosyl)ꢀLꢀhomohomocysteine tertꢀbutyl ester
(12)
THERMAL WAY: A solution of NꢀFmocꢀLꢀallylglycine tertꢀbutyl ester 11 (100 mg, 0.25 mmol), compound 1 (157 mg,
0.43 mmol) and AIBN (25 mg, 0.15 mmol) in CHCl3 (2.5 mL) was heated at reflux. After stirring for 3 h, another portion
of AIBN (25 mg, 0.15 mmol) was added and the reaction was stirred for 4 h more. The reaction mixture was cooled,
concentrated and then chromatographed on silica gel using EtOAc/CH2Cl2 (8:2) as eluent to give protected glycosyl
amino acid 12 (140 mg, 70%) as a white solid.
PHOTOCHEMICAL WAY: A solution of compound 11 (142 mg, 0.36 mmol), compound 1 (156 mg, 0.43 mmol) and
DPAP (19 mg, 0.07 mmol) in CH2Cl2 (2.5 mL) was irradiated at λmax 365 nm. After stirring for 3 h, another portion of
DPAP (19 mg, 0.07 mmol) was added and the reaction was stirred for an additional time (4 h). The reaction mixture was
concentrated and the residue was chromatographed on silica gel using EtOAc/CH2Cl2 (8:2) as eluent to give protected
glycosyl amino acid 12 (137 mg, 50%) as a white solid.
REPAIRING TEC WAY: A solution of compound 11 (214 mg, 0.54 mmol), compound 1 (395 mg, 1.09 mmol),
catechol (72 mg, 0.65 mmol) and BEt3 (95 ꢀL, 0.65 mmol) in CH2Cl2 (0.72 mL) was stirred at room temperature. After
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stirring for 1 h, another portion of BEt3 (8 ꢀL, 0.05 mmol) was added (until complete consumption of compound 11). The
reaction mixture was concentrated and the residue chromatographed on silica gel using EtOAc/CH2Cl2 (7:3) as eluent to
give protected glycosyl amino acid 12 (288 mg, 70%) as a white solid.
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Physical data: mp: 59–61 ºC, [α]D (c=1.00, CHCl3): +85.5. HRMS (ESI+) (m/z) 757.3012 [M+H]+; calculated
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C38H49N2O12S+: 757.3001. H NMR (400 MHz, CDCl3) δ (ppm): 7.76–7.71 (m, 2H, arom), 7.61–7.56 (m, 2H, arom),
7.40–7.30 (m, 2H, arom), 7.33–7.27 (m, 2H, arom), 5.80 (d, J = 8.7 Hz, 1H, NHAc), 5.54–5.48 (m, 2H, NHFmoc, H1),
5.35 (s, 1H, H4), 5.03 (dd, J = 11.6, 2.3 Hz, 1H, H3), 4.79–4.71 (m, 1H, H2), 4.51 (t, J = 6.4 Hz, 1H, H5), 4.37 (d, J = 6.9
Hz, 2H, CH2Fmoc), 4.29–4.18 (m, 2H, CHFmoc, Hα), 4.13–4.03 (m, 2H, H6), 2.66–2.54 (m, 2H, 2Hδ), 2.12 (s, 3H, COCH3),
2.01–1.85 (m, 10H, 2 COCH3, NHCOCH3, Hβ), 1.78–1.59 (m, 3H, Hβ, 2Hγ), 1.46 (s, 9H, C(CH3)3). 13C NMR (100
MHz, CDCl3) δ (ppm): 171.4, 171.2, 170.5, 170.4, 170.2, 156.1 (6 CO), 144.0, 141.4, 127.9, 127.2, 125.2, 120.1 (arom),
85.3 (C1), 82.6 (C(CH3)3), 68.7 (C3), 67.4, 67.4 (C4, C5), 67.1 (CH2Fmoc), 61.8 (C6), 53.6 (Cα), 48.5 (C2), 47.3 (CHFmoc),
32.2 (Cβ), 30.9 (Cδ), 28.7 (C(CH3)3), 25.7 (Cγ), 23.5 (NHCOCH3), 20.9, 20.8 (3 COCH3).
NꢀFmocꢀSꢀ((2ꢀacetamidoꢀ2ꢀdeoxy)ꢀ(3,4,6ꢀtriꢀOꢀacetyl)ꢀαꢀDꢀgalactopyranosyl)ꢀLꢀhomocysteine tertꢀbutyl ester (14)
Under an inert atmosphere, a solution of compound 1 (1.21 g, 3.36 mmol) in dry DMF (6 mL) was transferred into a
Schlenk, which contains 4 Å molecular sieves, previously activated at 280 °C for 4 h under vacuum. After stirring for 5
min, a solution of compound 13 (500 mg, 1.12 mmol) in dry DMF (12 mL) was dropped into the Schlenk. The reaction
was stirred for 24 h at room temperature, filtered over celite (to discard the molecular sieves) and the solvent was
concentrated. The residue was purified by gradient column chromatography on silica gel using the following set of
proportions: EtOAc/hexane, (1:1), (6:4), (7:3) to give protected glycosyl amino acid 14 (416 mg, 50%) as a white solid.
Mp: 54–56 ºC, [α]D (c=1.00, CHCl3): +68.2. HRMS (ESI+) (m/z) 743.2844 [M+H]+; calculated C37H47N2O12S+:
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743.2846. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.77–7.75 (m, 2H, arom), 7.60–7.58 (m, 2H, arom), 7.42–7.38 (m, 2H,
arom), 7.33–7.30 (m, 2H, arom), 5.56 (d, J = 8.7 Hz, 1H, NHAc), 5.48 (d, J = 5.0 Hz, 1H, H1), 5.43 (d, J = 7.9 Hz, 1H,
NHFmoc), 5.39–5.38 (m, 1H, H4), 5.06–5.02 (m, 1H, H3), 4.82–4.75 (m, 1H, H2), 4.56–4.53 (m, 1H, H5), 4.40 (d, J =
6.7, 2H, CH2Fmoc), 4.35–4.31 (m, 1H, Hα), 4.22 (t, J = 6.2 Hz, 1H, CHFmoc), 4.17–4.04 (m, 2H, 2H6), 2.74–2.55 (m, 2H,
2Hγ), 2.16–1.97 (m, 14H, 3 COCH3, NHCOCH3, 2Hβ), 1.48 (s, 9H, C(CH3)3). 13C NMR (100 MHz, CDCl3) δ (ppm):
171.0, 170.4, 170.3, 170.2, 167.8, 155.9 (6 CO), 143.9, 143.8, 141.3, 127.8, 127.1, 125.0, 120.0 (arom), 85.7 (C1), 82.9
(C(CH3)3), 68.5 (C3), 67.5 (C5), 67.3 (C4), 67.0 (CH2Fmoc), 61.7 (C6), 53.6 (Cα), 48.3 (C2), 47.2 (CHFmoc), 33.5 (Cβ),
28.0 (C(CH3)3), 27.5 (Cγ), 23.3 (NHCOCH3), 20.7, 20.7, 20.7 (3 COCH3).
NꢀFmocꢀSꢀ((2ꢀacetamidoꢀ2ꢀdeoxy)ꢀ(3,4,6ꢀtriꢀOꢀacetyl)ꢀαꢀDꢀgalactopyranosyl)ꢀLꢀcysteine tertꢀbutyl ester (16)
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Under an inert atmosphere, a solution of compound 1 (1.01 g, 2.784 mmol) in dry DMF (6 mL) was transferred into a
Schlenk, which contains the 4 Å molecular sieves, previously activated at 280 °C for 4 h under vacuum. After stirring for
5 min, a solution of compound 15 (400 mg, 0.928 mmol) in dry DMF (12 mL) was dropped into the Schlenk. The reaction
was stirred for 24 h at room temperature, filtered over celite (to discard the molecular sieves) and the solvent was
concentrated. The residue was purified by gradient column chromatography on silica gel using the following set of
proportions: EtOAc/hexane, (1:1), (6:4), (7:3) to give protected glycosyl amino acid 16 (330 mg, 51%) as a white solid.
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Mp: 59–61 ºC, [α]D (c=1.00, CHCl3): +72.8. HRMS (ESI+) (m/z) 729.2686 [M+H]+; calculated C36H45N2O12S+:
729.2688. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.77–7.75 (m, 2H, arom), 7.64–7.62 (m, 2H, arom), 7.42–7.29 (m, 4H,
arom), 6.10 (d, J = 8.5 Hz, 1H, NHAc), 5.65 (d, J = 8.9 Hz, 1H, NHFmoc), 5.38–5.37 (m, 2H, H1, H4), 4.98–4.94 (m, 1H,
H3), 4.85–4.78 (m, 1H, H2), 4.59–4.55 (m, 1H, Hα), 4.48–4.38 (m, 3H, H5, CH2Fmoc), 4.23–4.19 (m, 2H, H6, CHFmoc),
4.02–3.98 (m, 1H, H6), 3.29–3.20 (m, 1H, Hβ), 3.05−2.98 (m, 1H, Hβ), 2.18 (s, 3H, COCH3), 2.01 (s, 3H, NHCOCH3),
1.99 (s, 3H, COCH3), 1.97 (s, 3H, COCH3), 1.47 (s, 9H, C(CH3)3). 13C NMR (100 MHz, CDCl3) δ (ppm): 171.0, 170.5,
170.2, 170.0, 168.8, 155.8 (6 CO), 143.9, 143.8, 141.4, 127.7, 127.1, 125.1, 120.1 (arom), 87.3 (C1), 82.9 (C(CH3)3), 68.3
(C3), 68.2 (C5), 67.4 (C4), 66.9 (CH2Fmoc), 62.0 (C6), 54.5 (Cα), 48.4 (C2), 47.2 (CHFmoc), 36.2 (Cβ), 28.0 (C(CH3)3),
23.3 (NHCOCH3), 20.7, 20.7, 20.6 (3 COCH3).
AlaꢀProꢀAspꢀCys(αꢀSꢀGalNAc)ꢀArgꢀProꢀNH2 (17)
Following SPPS methodology with FmocꢀProꢀOH (337 mg, 1 mmol), FmocꢀArg(Pbf)ꢀOH (649 mg, 1 mmol), Fmocꢀ
Cys(αꢀSꢀ(AcO)3GalNAc)ꢀOH (137 mg, 0.2 mmol), FmocꢀAsp(OtBu)ꢀOH (412 mg, 1 mmol), FmocꢀProꢀOH (337 mg, 1
mmol), FmocꢀAlaꢀOH (311 mg, 1 mmol), peptide 17 was obtained with a 58% yield after purification by reversed phase
HPLC and lyophilization (tR 20.7 min). HRMS (ESI+) (m/z) 860.3936 [M+H]+; calculated C34H58N11O13S+: 860.3938.
1H NMR (400 MHz, H2O/D2O 9:1) δ (ppm) amide protons: 8.67 (t, J = 6.9 Hz, 1H, NHAsp), 8.40 (d, J = 7.5 Hz, 1H,
NHCys), 8.36 (d, J = 7.4 Hz, 1H, NHArg), 8.20 (d, J = 8.2 Hz, 1H, NHCOCH3), 7.70 (s, 1H, CONH2), 7.20 (t, J = 5.3 Hz,
1H, NHεArg), 7.00 (s, 1H, CONH2), 6.67 (br s, 2H, NH2ηArg). 1H NMR (400 MHz, D2O) δ (ppm): 5.58 (d, J = 5.4 Hz,
1H, H1), 4.73 (t, J = 6.7 Hz, 1H, HαAsp),4.68 (d, J = 3.9 Hz, 1H, HαArg), 4.65–4.57 (m, 1H, HαCys), 4.54–4.46 (m, 1H,
HαPro), 4.44–4.35 (m, 3H, H2, HαPro, HαAla), 4.27–4.17 (m, 1H, H5), 4.00 (d, J = 2.9 Hz, 1H, H4), 3.85–3.59 (m, 7H,
2HδPro, 2HδPro, 2H6, H3), 3.30–3.18 (m, 2H, 2HδArg), 3.16–2.83 (m, 4H, 2HβAsp, 2HβCys), 2.45–2.25 (m, 2H,
HβPro, HβPro), 2.14–1.91 (m, 9H, NHCOCH3, 2HβArg, HβPro, HβPro, HγPro, HγPro), 1.82–1.60 (m, 4H, HγPro,
HγPro, 2HγArg), 1.55 (d, J = 7.0 Hz, 3H, 3HβAla). 13C NMR (100 MHz, CDCl3) δ (ppm): 176.8, 174.6, 173.9, 173.6,
171.9, 171.4, 171.1, 169.2 (8 CO), 156.8 (CζArg), 85.4 (C1), 71.9 (C5), 68.4 (C4), 67.6 (C3), 61.1 (C6), 60.3 (CαPro ,
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CαPro), 53.7 (CαCys), 51.1 (CαArg), 50.1 (CαAsp), 50.0 (C2), 48.0 (CαAla), 47.9 (CδPro), 47.7 (CδPro), 40.6 (CδArg),
35.2 (CβAsp), 32.6 (CβCys), 29.6 (CβPro), 29.3 (CβPro), 27.7 (CγPro), 24.7(CγPro), 24.6 (CβArg), 24.1 (CγArg), 21.9
(NHCOCH3), 15.1 (CβAla).
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AlaꢀProꢀAspꢀhCys(αꢀSꢀGalNAc)ꢀArgꢀProꢀNH2 (18)
Following SPPS methodology with FmocꢀProꢀOH (337 mg, 1 mmol), FmocꢀArg(Pbf)ꢀOH (649 mg, 1 mmol), Fmocꢀ
hCys(αꢀSꢀ(AcO)3GalNAc)ꢀOH (135 mg, 0.2 mmol), FmocꢀAsp(OtBu)ꢀOH (412 mg, 1 mmol), FmocꢀProꢀOH (337 mg, 1
mmol), FmocꢀAlaꢀOH (311 mg, 1 mmol), peptide 18 was obtained with a 67% yield after purification by reversed phase
HPLC and lyophilization (tR 20.8 min). HRMS (ESI+) (m/z) 874.4084 [M+H]+; calculated C35H60N11O13S+: 874.4087.
1H NMR (400 MHz, H2O/D2O 9:1) δ (ppm) amide protons: 8.63 (d, J = 7.0 Hz, 1H, NHAsp), 8.39 (d, J = 7.6 Hz, 1H,
NHArg), 8.27 (d, J = 7.2 Hz, 1H, NHhCys), 8.21 (d, J = 8.2 Hz, 1H, NHCOCH3), 7.71 (s, 1H, CONH2), 7.21 (t, J = 5.2
Hz, 1H, NHεArg), 7.00 (s, 1H, CONH2), 6.67 (br s, 2H, NH2ηArg). 1H NMR (400 MHz, D2O) δ (ppm): 5.50 (d, J = 5.4
Hz, 1H, H1), 4.68–4.63 (m, 2H, HαAsp, HαhCys), 4.54–4.47 (m, 2H, HαPro, HαArg), 4.42–4.34 (m, 3H, H2, HαPro,
HαAla), 4.30–4.27 (m, 1H, H5), 4.00 (s, 1H, H4), 3.89–3.64 (m, 7H, 2HδPro, 2HδPro, 2H6, H3), 3.23 (t, J = 6.7 Hz, 2H,
2HδArg), 3.01–2.85 (m, 2H, 2HβAsp), 2.67 (t, J = 6.9 Hz, 2H, 2HγhCys), 2.39–2.28 (m, 2H, HβPro, HβPro), 2.23–1.67
(m, 15H, HβPro, HβPro, 2HγPro, 2HγPro, 2HβhCys, 2HβArg, 2HγArg, NHCOCH3), 1.55 (d, J = 6.9 Hz, 3H, 3HβAla).
13C NMR (100 MHz, CDCl3) δ (ppm): 175.8, 174.5, 173.8, 173.5, 172.8, 172.1, 171.3, 169.3 (8 CO), 156.8 (CζArg),
84.8 (C1), 71.8 (C5), 68.5 (C4), 67.7 (C3), 61.2 (C6), 60.3 (CαPro, CαPro), 52.4 (CαArg), 51.2 (CαhCys), 50.1 (CαAsp),
50.0 (C2), 48.0 (CαAla), 47.9 (CδPro), 47.7 (CδPro), 40.5 (CδArg), 35.0 (CβAsp), 31.3 (CγPro), 29.6 (CβPro), 29.4
(CβPro), 27.4 (CγPro), 27.1 (CγhCys), 24.7 (CβArg), 24.7 (CβhCys), 24.1 (CγArg), 21.9 (NHCOCH3), 15.1 (CβAla).
AlaꢀProꢀAspꢀhhCys(αꢀSꢀGalNAc)ꢀArgꢀProꢀNH2 (19)
Following SPPS methodology with FmocꢀProꢀOH (337 mg, 1 mmol), FmocꢀArg(Pbf)ꢀOH (649 mg, 1 mmol), Fmocꢀ
hhCys(αꢀSꢀ(AcO)3GalNAc)ꢀOH (140 mg, 0.2 mmol), FmocꢀAsp(OtBu)ꢀOH (412 mg, 1 mmol), FmocꢀProꢀOH (337 mg, 1
mmol), FmocꢀAlaꢀOH (311 mg, 1 mmol), peptide 19 was obtained with a 59% yield after purification by reversed phase
HPLC and lyophilization (tR 24.4 min). HRMS (ESI+) (m/z) 888.4251 [M+H]+; calculated C36H62N11O13S+: 888.4249.
1H NMR (400 MHz, H2O/D2O 9:1) δ (ppm) amide protons: 8.63 (d, J = 7.0 Hz, 1H, NHAsp), 8.32 (d, J = 7.3 Hz, 1H,
NHArg), 8.27 (d, J = 7.1 Hz, 1H, NHhhCys), 8.20 (d, J = 8.1 Hz, 1H, NHCOCH3), 7.69 (s, 1H, CONH2), 7.20 (t, J = 5.2
Hz, 1H, NHεArg), 7.00 (s, 1H, CONH2), 6.67 (br s, 2H, NH2ηArg). 1H NMR (400 MHz, D2O) δ (ppm): 5.49 (d, J = 5.4
Hz, 1H, H1), 4.68–4.64 (m, 2H, HαAsp, HαArg), 4.50–4.46 (m, 1H, HαPro), 4.41–4.30 (m, 4H, H2, HαPro, HαAla,
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HαhhCys), 4.25 (t, J = 5.9 Hz, 1H, H5), 3.98 (d, J = 2.8 Hz, 1H, H4), 3.83 (dd, J = 11.4, 3.1 Hz, 1H, H3), 3.81–3.58 (m,
6H, 2H6, 2HδPro, 2HδPro), 3.27–3.16 (m, 2H, 2HδArg), 2.95 (dd, J = 17.0, 6.5 Hz, 1H, HβAsp), 2.85 (dd, J = 17.0, 7.1
Hz, 1H, HβAsp), 2.70–2.55 (m, 2H, 2HδhhCys), 2.38–2.27 (m, 2H, HβPro, HβPro), 2.10–1.81 (m, 11H, NHCOCH3,
2HβhhCys, 2HγhhCys, HβPro, HβPro, HγPro, HγPro), 1.80–1.58 (m, 6H, 2HγArg, 2HβArg, HγPro, HγPro), 1.54 (d, J =
7.0 Hz, 3H, 3HβAla). 13C NMR (100 MHz, D2O) δ (ppm): 176.8, 174.5, 173.8, 173.5, 173.1, 172.0, 171.3, 169.2 (8 CO),
156.7 (CζArg), 83.6 (C1), 71.7 (C5), 68.5 (C4), 67.7 (C3), 61.2 (C6), 60.3 (CαPro), 60.3 (CαPro), 53.2 (CαhhCys), 51.0
(CαArg), 50.1 (CαAsp), 50.0 (C2), 48.0 (CαAla), 47.9 (CδPro), 47.7 (CδPro), 40.6 (CδArg), 35.1 (CβAsp), 30.0 (CγPro),
29.6 (CβPro, CβhhCys), 29.4 (CβPro), 29.4 (CδhhCys), 27.4 (CγPro), 25.0 (CβArg), 25.0 (CγhhCys), 24.1 (CγArg), 21.9
(NHCOCH3), 15.1 (CβAla).
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AlaꢀProꢀAspꢀSer(OꢀmercaptopropylꢀαꢀSꢀGalNAc)ꢀArgꢀProꢀNH2 (20)
Following SPPS methodology with FmocꢀProꢀOH (337 mg, 1 mmol), FmocꢀArg(Pbf)ꢀOH (649 mg, 1 mmol), Fmocꢀ
Ser(mercaptopropylꢀαꢀSꢀ(AcO)3GalNAc)ꢀOH (146 mg, 0.2 mmol), FmocꢀAsp(OtBu)ꢀOH (412 mg, 1 mmol), FmocꢀProꢀ
OH (337 mg, 1 mmol), FmocꢀAlaꢀOH (311 mg, 1 mmol), peptide 20 was obtained with a 67% yield after purification by
reversed phase HPLC and lyophilization (tR 25.7 min). HRMS (ESI+) (m/z) 459.7210 [M+2H]+2; calculated
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C37H65N11O14S+2: 459.7211. H NMR (400 MHz, H2O/D2O 9:1) δ (ppm) amide protons: 8.60 (d, J = 6.8 Hz, 1H,
NHAsp), 8.31–8.20 (m, 3H, NHSer, NHArg, NHCOCH3), 7.69 (s, 1H, CONH2), 7.25 (d, J = 6.8 Hz, 1H, NHεArg), 7.01
(s, 1H, CONH2), 6.68 (br s, 2H, NH2ηArg). 1H NMR (400 MHz, D2O) δ (ppm): 5.50 (d, J = 5.4 Hz, 1H, H1), 4.76–4.68
(m, 2H, HαAsp, HαArg), 4.53 (t, J = 5.0 Hz, 1H, HαSer), 4.51–4.47 (m, 1H, HαPro), 4.42–4.32 (m, 3H, H2, HαPro,
HαAla), 4.26 (t, J = 6.0 Hz, 1H, H5), 3.99 (d, J = 2.8 Hz, 1H, H4), 3.83 (dd, J = 11.4, 3.0 Hz, 1H, H3), 3.81–3.70 (m, 6H,
2H6, HδPro, HδPro, 2HβSer), 3.69–3.55 (m, 4H, HδPro, HδPro, 2HδSer), 3.27–3.17 (m, 2H, 2HδArg), 2.98 (dd, J = 17.1,
6.4 Hz, 1H, HβAsp), 2.89 (dd, J = 17.1, 6.9 Hz, 1H, HβAsp), 2.72–2.56 (m, 2H, HζSer), 2.39–2.26 (m, 2H, HβPro,
HβPro), 2.11–1.80 (m, 12H, NHCOCH3, 2HβArg, 2HεSer, HβPro, HβPro, 2HγPro, HγPro), 1.79–1.59 (m, 3H, HγPro,
2HγArg), 1.55 (d, J = 7.0 Hz, 3H, 3HβAla). 13C NMR (100 MHz, D2O) δ (ppm): 176.8, 174.5, 173.8, 173.6, 172.2,
171.1, 170.0, 169.3 (8 CO), 156.7 (CζArg), 83.9 (C1), 71.7 (C5), 69.5 (CδSer), 69.1 (CβSer), 68.5 (C4), 67.7 (C3), 61.2
(C6), 60.4 (CαPro), 60.4 (CαPro), 53.6 (CαSer), 51.0 (CαArg), 50.2 (C2), 50.0 (CαAsp), 48.1 (CαAla), 47.9 (CδPro),
47.8 (CδPro), 40.6 (CδArg), 35.2 (CβAsp), 29.6 (CβPro), 29.4 (CβPro), 28.7 (CεSer), 27.7 (CγPro), 26.9 (CζSer), 24.7,
24.6 (CβArg, CγPro), 24.0 (CγArg), 21.9 (NHCOCH3), 15.1 (CβAla).
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Page 29 of 38
The Journal of Organic Chemistry
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AlaꢀProꢀAspꢀCys(SꢀmercaptopropylꢀαꢀSꢀGalNAc)ꢀArgꢀProꢀNH2 (21)
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Following SPPS methodology with FmocꢀProꢀOH (337 mg, 1 mmol), FmocꢀArg(Pbf)ꢀOH (649 mg, 1 mmol), Fmocꢀ
Cys(mercaptopropylꢀαꢀSꢀ(AcO)3GalNAc)ꢀOH (149 mg, 0.2 mmol), FmocꢀAsp(OtBu)ꢀOH (412 mg, 1 mmol), FmocꢀProꢀ
OH (337 mg, 1 mmol), FmocꢀAlaꢀOH (311 mg, 1 mmol), peptide 21 was obtained with a 63% yield after purification by
reversed phase HPLC and lyophilization (tR 30.3 min). HRMS (ESI+) (m/z) 934.4123 [M+H]+; calculated
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C37H64N11O13S2 : 934.4121. H NMR (400 MHz, H2O/D2O 9:1) δ (ppm) amide protons: 8.56 (d, J = 7.3 Hz, 1H,
NHAsp), 8.31 (d, J = 7.5 Hz, 1H, NHArg), 8.18 (d, J = 7.4 Hz, 1H, NHCys), 8.13 (d, J = 8.0 Hz, 1H, NHCOCH3), 7.59
(s, 1H, CONH2), 7.09 (t, J = 5.4 Hz, 1H, NHεArg), 6.91 (s, 1H, CONH2), 6.57 (br s, 1H, NH2ηArg). 1H NMR (400 MHz,
D2O) δ (ppm): 5.51 (d, J = 5.4 Hz, 1H, H1), 4.74–4.66 (m, 2H, HαAsp, HαArg), 4.55–4.47 (m, 2H, HαCys, HαPro),
4.41–4.31 (m, 3H, H2, HαPro, HαAla), 4.26 (t, J = 6.0 Hz, 1H, H5), 3.99 (d, J = 2.4 Hz, 1H, H4), 3.86–3.79 (m, 2H, H3,
HδPro), 3.77 (d, J = 6.2 Hz, 2H, 2H6), 3.75–3.69 (m, 1H, HδPro), 3.69–3.60 (m, 2H, 2HδPro), 3.26–3.18 (m, 2H,
2HδArg), 3.02–2.94 (m, 2H, HβCys, HβAsp), 2.91–2.84 (m, 2H, HβAsp, HβCys), 2.77–2.63 (m, 4H, 2HζCys, 2HδCys),
2.39–2.27 (m, 2H, HβPro, HβPro), 2.10–1.92 (m, 9H, NHCOCH3, 2HβArg, 2HγPro, 2HβPro), 1.91–1.82 (m, 3H,
2HεCys, HγPro), 1.80–1.61 (m, 3H, HγPro, 2HγArg), 1.54 (d, J = 7.0 Hz, 3H, 3HβAla). 13C NMR (100 MHz, D2O) δ
(ppm): 176.8, 174.5, 173.8, 173.5, 171.9, 171.5, 171.1, 169.3 (8 CO), 156.7 (CεArg), 83.9 (C1), 71.7 (C5), 68.5 (C4),
67.6 (C3), 61.2 (C6), 60.4 (CαPro), 60.3 (CαPro), 52.9 (CαCys), 51.1 (CαArg), 50.2 (C2), 49.9 (CαAsp), 48.0 (CαAla),
47.9 (CδPro), 47.7 (CδPro), 40.6 (CδArg), 35.1 (CβAsp), 32.5 (CβCys), 30.3 (CδCys), 29.6 (CβPro), 29.4 (CβPro), 29.1
(CζCys), 28.6 (CεCys), 27.6 (CγPro), 24.7, 24.6 (CβArg, CγPro), 24.1 (CγArg), 21.9 (NHCOCH3), 15.1 (CβAla).
AlaꢀProꢀAspꢀThr(OꢀmercaptopropylꢀαꢀSꢀGalNAc)ꢀArgꢀProꢀNH2 (22)
Following SPPS methodology with FmocꢀProꢀOH (337 mg, 1 mmol), FmocꢀArg(Pbf)ꢀOH (649 mg, 1 mmol), Fmocꢀ
Thr(mercaptopropylꢀαꢀSꢀ(AcO)3GalNAc)ꢀOH (149 mg, 0.2 mmol), FmocꢀAsp(OtBu)ꢀOH (412 mg, 1 mmol), FmocꢀProꢀ
OH (337 mg, 1 mmol), FmocꢀAlaꢀOH (311 mg, 1 mmol), glycopeptide 22 was obtained with a 62% yield after
purification by reversed phase HPLC and lyophilization (tR 29.7 min). HRMS (ESI+) (m/z) 932.4506 [M+H]+; calculated
C38H66N11O14S+: 932.4506. H NMR (400 MHz, H2O/D2O 9:1) δ (ppm) amide protons: 8.71 (d, J = 7.0 Hz, 1H,
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NHAsp), 8.23 (d, J = 7.9 Hz, 1H, NHCOCH3), 8.18 (d, J = 7.4 Hz, 1H, NHArg), 8.06 (d, J = 8.2 Hz, 1H, NHThr), 7.68 (s,
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1H, CONH2), 7.21 (t, J = 4.5 Hz, 1H, NHεArg), 7.01 (s, 1H, CONH2), 6.67 (br s, 2H, NH2ηArg). H NMR (400 MHz,
D2O) δ (ppm): 5.45 (d, J = 5.5 Hz, 1H, H1), 4.78–4.74 (m, 1H, HαAsp), 4.65 (dd, J = 8.1, 5.3 Hz, 1H, HαArg), 4.46 (dd,
J = 8.1, 6.3 Hz, 1H, HαPro), 4.40–4.28 (m, 4H, H2, HαThr, HαPro, HαAla), 4.22 (t, J = 6.0 Hz, 1H, H5), 4.01–3.94 (m,
2H, HβThr, H4), 3.79 (dd, J = 11.4, 3.1 Hz, 1H, H3), 3.76–3.55 (m, 7H, 2H6, 2HδPro, 2HδPro, HδThr), 3.46–3.38 (m,
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