Photoinduced addition of glycosyl thiols to alkynyl peptides: Use of free-radical thiol-yne coupling for post-translational double-glycosylation of peptides

Article abstract of DOI:10.1021/jo1008178

(Figure presented) Double glycosylation of cysteine-containing peptides has been carried out by a one-pot two-step sequence comprising selective S-propargylation followed by photoinduced (λ _max 365 nm) free-radical hydrothiolation with glycosyl thiols. Conditions were established for the sequential introduction of two different thiol residues such as a glycosyl and a biotinyl derivative.

Full text of DOI:10.1021/jo1008178

pubs.acs.org/joc
hydrate-based anticancer vaccines.3,4 Hence, the develop-
Photoinduced Addition of Glycosyl Thiols to Alkynyl
Peptides: Use of Free-Radical Thiol-Yne Coupling
for Post-Translational Double-Glycosylation
of Peptides
ment of methods for peptide and protein glycosylation by
efficient and site-specific ligation tools is at the forefront in
biotechnology and proteomics. Synthetic glycopeptides and
glycoproteins containing unnatural linkages between the
carbohydrate and aglycone moieties have been reported in
recent years.5 One of the most used of these unnatural
linkages is the 1,4-disubstituted triazole ring3b,6,7 due to
its robusteness8 and ease of formation by the Huisgen
Cu(I)-catalyzed azide-alkyne cycloaddition.9 Furthermore,
S-linked glycoproteins, i.e. compounds featuring an S-
glycosidic bond, have been prepared by Michael addition
of glycosyl thiols to synthetic dehydroalanine-containing
proteins10 and by phosphine-mediated dechalcogenation
of a disulfide protein.11 Very recently Davis and co-workers
reported on free-radical addition of glycosyl thiols to geneti-
cally modified proteins in which an homoallylglycine tag was
introduced.12 Almost at the same time we reported a com-
plementary method leading to C-linked glycopeptides and a
glycoprotein, i.e. compounds featuring a C-glycosidic bond.
To this end, we employed the photoinduced coupling be-
tween allyl C-glycosides and cysteine-containing peptides
and the natural protein bovine serum albumine (BSA).13
Both Davis and our method demonstrated how the free-
radical thiol-ene coupling can be exploited as a click liga-
tion tool for bioconjugation. Indeed, while the potential of
thiol-ene reaction is amply documented in polymer and
material synthesis,14,15 its use as a metal-free ligation process
in bioorganic chemistry is relatively scanty.16 Nevertheless,
the assembly of biomolecules under mild and neutral reac-
tion conditions through the specific formation of robust
sulfide bridges is an attractive target due to convenient
features of the C-S bond.17 In this context we would like
to report here on the application of a sister reaction to the
thiol-ene, that is the radical-mediated hydrothiolation of
terminal alkyne (thiol-yne process). This reaction, whose
Mauro Lo Conte,† Salvatore Pacifico,† Angela Chambery,‡
Alberto Marra,† and Alessandro Dondoni*,†
†Dipartmento di Chimica, Laboratorio di Chimica Organica,
ꢀ
Universita di Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy,
and Dipartmento di Scienze della Vita, II Universitaꢀ
di Napoli, Via Vivaldi 43, I-81100 Caserta, Italy
‡
adn@unife.it
Received April 26, 2010
Double glycosylation of cysteine-containing peptides has
been carried out by a one-pot two-step sequence compris-
ing selective S-propargylation followed by photoinduced
(λ max 365 nm) free-radical hydrothiolation with glycosyl
thiols. Conditions were established for the sequential
introduction of two different thiol residues such as a
glycosyl and a biotinyl derivative.
It is well established that protein glycosylation is a post-
translational modification that profoundly affects protein
folding, stability, immunogenicity, and biological properties
and activities.1 As native glycoproteins are isolated as mix-
tures of glycoforms, there is a pressing need for synthetic
glycopeptides and glycoproteins with a well-defined struc-
ture and composition.2 These compounds may serve as
probes for studies in glycobiology as well as leads toward
the development of pharmaceutical agents, such as carbo-
(5) (a) Specker, D.; Wittmann, V. Top. Curr. Chem. 2007, 267, 65–107.
(b) Nicotra, F.; Cipolla, L.; Peri, F.; La Ferla, B.; Redaelli, C. Adv.
Carbohydr. Chem. Biochem. 2007, 61, 353–398.
(6) (a) van Kasteren, S. I.; Kramer, H. B.; Jensen, H. H.; Campbell, S. J.;
Kirkpatrick, J.; Oldham, N. J.; Anthony, D. C.; Davis, B. G. Nature 2007,
446, 1105–1109. (b) Gamblin, D. P.; van Kasteren, S. I.; Chalker, J. M.;
Davis, B. G. FEBS J. 2008, 275, 1949–1959.
(7) Lee, D. J.; Mandal, K.; Harris, P. W. R.; Brimble, M. A.; Kent,
S. B. H. Org. Lett. 2009, 11, 5270-5273 and references cited therein.
(8) Dondoni, A. Chem. Asian J. 2007, 2, 700–708.
(9) Meldal, M.; Tornøe, C. W. Chem. Rev. 2008, 108, 2952–3015.
(10) (a) Wang, J.; Schiller, S. M.; Schultz, P. G. Angew. Chem., Int. Ed.
2007, 46, 6849–6851. (b) Bernardes, G. J. L.; Calker, J. M.; Errey, J. C.;
Davis, B. G. J. Am. Chem. Soc. 2008, 130, 5052–5053.
(11) Bernardes, G. J. L.; Grayson, E. J.; Thompson, S.; Chalker, J. M.;
Errey, J. C.; El Oualid, F.; Claridge, T. D. W.; Davis, B. G. Angew. Chem.,
Int. Ed. 2008, 47, 2244–2247.
(12) Floyd, N.; Vijayakrishnan, B.; Koeppe, J. R.; Davis, B. G. Angew.
Chem., Int. Ed. 2009, 48, 7798–7802.
(13) Dondoni, A.; Massi, A.; Nanni, P.; Roda, A. Chem.;Eur. J. 2009,
15, 11444–11449.
(14) (a) Lowe, A. B. Polym. Chem. 2010, 1, 17–36. (b) Kade, M. J.; Burke,
D. J.; Hawker, C. J. J. Polym. Sci., Part A: Polym. Chem. 2010, 48, 743–750.
(15) Hoyle, C. E.; Bowman, C. N. Angew. Chem., Int. Ed. 2010, 49, 1540–
1573.
(16) Dondoni, A. Angew. Chem., Int. Ed. 2008, 47, 8995–8997.
(17) Fiore, M.; Marra, A.; Dondoni, A. J. Org. Chem. 2009, 74, 4422–
4425.
(1) (a) Varki, A. Glycobiology 1993, 3, 97–130. (b) Dwek, R. A. Chem.
Rev. 1996, 96, 683–720. (c) Seitz, O. ChemBioChem 2000, 1, 214–246.
(d) Essentials of Glycobiology; Varki, A., Cummings, R. D., Esko, J. D., Freeze,
H. H., Hart, G. W., Marth, J., Eds.; Cold Spring Harbor Laboratory Press: Cold
Spring Harbor, NY, 1999.
(2) (a) Hang, H. C.; Bertozzi, C. R. Acc. Chem. Res. 2001, 34, 727–736.
(b) Davis, B. G. Chem. Rev. 2002, 102, 579–601. (c) Wong, C.-H. J. Org.
Chem. 2005, 70, 4219–4225. (d) Hackenberger, C. P. R.; Schwarzer, D.
Angew. Chem., Int. Ed. 2008, 47, 10030–10074. (e) Gamblin, D. P.; Scanlan,
E. M.; Davis, B. G. Chem. Rev. 2009, 109, 131–163. (f) Kent, S. B. H. Chem.
Soc. Rev. 2009, 38, 338–351. (g) Davis, B. G. Angew. Chem., Int. Ed. 2009, 48,
4674–4678. (h) Payne, R. J.; Wong, C.-H. Chem. Commun. 2010, 46, 21–43.
(3) (a) Danishefsky, S. J.; Allen, J. R. Angew. Chem., Int. Ed. 2000, 39,
836–863. (b) Wan, Q.; Chen, J.; Chen, G.; Danishefsky, S. J. J. Org. Chem.
2006, 71, 8244–8249.
(4) Buskas, T.; Thompson, P.; Boons, G.-J. Chem. Commun. 2009, 5335–
5349.
4644 J. Org. Chem. 2010, 75, 4644–4647
Published on Web 06/09/2010
DOI: 10.1021/jo1008178
r
2010 American Chemical Society

Lo Conte et al.
JOCNote
SCHEME 1
SCHEME 2
FIGURE 1. Partial 1H NMR spectra of the reaction mixtures
between S-propargyl cysteine 2 and glucosyl thiol 3 (4 equiv): initial
reaction mixture (spectrum A); after 20 min irradiation at λmax
365 nm (spectrum B); after 45 min irradiation at λmax 365 nm
(spectrum C).
bearing a propargylic tag was established by exploiting the
unique reactivity of the cysteine side chain thiol.23 As shown
in Scheme 2 the model reaction of protected cysteine 1 with a
slight excess of propargyl bromide (1.2 equiv) and triethyl-
amine (2 equiv) in CH2Cl2 at room temperature afforded the
S-propargyl derivative 2 in 86% yield.
discovery dates back to the mid-1900s,18 serves to introduce
two thiol fragments across a carbon-carbon triple bond via
a multistep mechanism as depicted in Scheme 1.
Then, the photoinduced reaction of 2 with the peracety-
lated glucosyl thiol 3 was examined. Taking advantage of the
expertise acquired from our recent work on thiol-ene
coupling,13 the reaction was carried out in methanol as the
solvent by irradiation at λmax 365 nm in the presence (10 mol
%) of 2,2-dimethoxy-2-phenylacetophenone (DPAP) as the
sensitizer. No effort was made to exclude air and moisture.
After some experimentation, the use of excess thiol 3
(4 equiv) with respect to alkyne 2 was optimized. Within
45 min at room temperature, NMR analysis of the reac-
tion mixture showed the complete consumption of starting
alkyne and vinyl thioether intermediate as judged by the
absence of signals at 2.26 (CtC;H) and 5.75-6.30 ppm
(HCdCH) (Figure 1). Also the doublet at 2.4 ppm corre-
sponding to the thiol proton was not any more detectable as
the excess of this reagent was transformed into the corre-
sponding disulfide by homocoupling. The glycosylated
bisthioether 4 was isolated by chromatography (53%) as a
ca. 1:1 mixture of diastereomers.
The glycosylation of small peptides by the strategy out-
lined above was examined by using the natural tripeptide
Glu-Cys-Glc (glutathione, GSH, 5) and the synthetic tetra-
peptide Arg-Gly-Asp-Cys (RGDC, 6) as substrates. The
main difference between 5 and 6 was that the former con-
tained an internal cysteine residue while the latter was a
C-terminal cysteine derivative. A one-pot two-step sequence
comprising peptide alkynylation and thiol-yne coupling
was planned in order to avoid intermediate isolation. No-
tably, the use of unprotected peptides GSH 5 and RGDC 6
allowed the alkynylation to be performed under very mild
conditions, that is with propargyl bromide (1.1 equiv) and
The first, slower, step involves the anti-Markovnikov-like
addition of a thiyl radical to the CtC bond to yield an
intermediate vinyl thioether that is capable of undergoing
a second, faster, thiyl radical addition, a formal thiol-ene
reaction, leading to the dithioether with exclusive 1,2-addi-
tion mode.19 Notably, the thiol-yne reaction can be photo-
initiated in the UV-visible range (254-470 nm) and pro-
ceeds at room temperature with high efficiency in the
presence of oxygen/water. Finally, the tolerance to a wide
range of functional groups and orthogonality to various
types of chemistry make this reaction an ideal and broadly
applicable tool for multiple bioconjugation. However, while
recent work highlighted thiol-yne chemistry as a valuable
tool in polymer and material science,20 its use in organic
synthesis is limited to a few cases such as for example the
synthesis of small vinyl sulfides and dithioether adducts.21
Thus, the potential of this process as a chemoselective and
bioorthogonal ligation tool22 for multiple hydrothiolation of
biologically relevant molecules remains to be established.
Considering the above attributes, we envisioned the fabrica-
tion of dually glycosylated peptides using the photoinduced
thiol-yne reaction between glycosyl thiols and alkynyl pep-
tides. It is conceivable that the peptide double glycosylation
can affect much more substantially than monoglycosylation
the peptide structure and biological activity. Thus, at first a
method for introducing into peptides a chemical handle
(18) Bader, H.; Cross, L. C.; Heilbron, I.; Jones, E. R. H. J. Chem. Soc.
1949, 619–623.
(19) Yu, B.; Chan, J. W.; Hoyle, C. E.; Lowe, A. B. J. Polym. Sci., Part A:
Polym. Chem. 2009, 47, 3544–3557.
(20) Lowe, A. B.; Hoyle, C. E.; Bowman, C. N. J. Mater. Chem. 2010, 20,
4745-4750.
(21) Benati, L.; Capella, L.; Montevecchi, P. C.; Spagnolo, P. J. Org.
Chem. 1995, 60, 7941–7946.
(23) (a) Chalker, J. M.; Lin, Y. A.; Boutureira, O.; Davis, B. G. Chem.
Commun. 2009, 3714–3716. (b) Chalker, J. M.; Bernardes, G. J. L.; Lin,
Y. A.; Davis, B. G. Chem. Asian J. 2009, 4, 630–640. (c) Hong, V.; Kislukhin,
A. A.; Finn, M. G. J. Am. Chem. Soc. 2009, 131, 9986–9994.
(22) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. 2009, 48, 6974–
6998.
J. Org. Chem. Vol. 75, No. 13, 2010 4645

Lo Conte et al.
JOCNote
TABLE 1. Synthesis of Bis-Glycosylated Peptides via One-Pot Propargylation and Photoinduced Thiol-Yne Coupling
aIsolated yields after chromatography on Sephadex LH20 column. bConversion determined from the reacted alkyne as estimated by 1H NMR
analysis of the crude reaction mixture.
NH3 in water at 0 °C for 1 h and then at room temperature
(ca. 22 °C) for 2 h. From the NMR and MS spectra of the
crude reaction mixtures the purity of the alkynyl peptides
thus formed was estimated to be 80-85%. Hence, the
photoinduced double hydrothiolation was carried out by
using 4 equiv of the unprotected glucosyl thiol12 9 under the
conditions outlined above with the only change being that
the solvent was a 1:1 H2O/MeOH mixture. We were pleased
to observe that thiol-yne reactions took place with great
efficiency with conversion >95% into the corresponding
13, 14, 16, and 17. All glycopeptides 12-17 were isolated by
chromatography on a Sephadex LH20 column as ca. 1:1
mixtures of diastereomers as established by 13C NMR ana-
lysis (multiple signals for anomeric carbons at ca. δ 85 ppm).
Unfortunately, attempts to separate the isomers were un-
successful. It has to be noted that isolated yields quoted
in Table 1 largely reflect difficulties handling amphiphilic
products.
The scope of the one-pot two-step glycosylation strategy
was broadened by using higher peptides such as the synthetic
cysteine-containing octapeptides 7 and 8 (Table 1). Adopting
the above procedure and conditions employed for propar-
gylation of GSH 5 and RGDC 6 followed by thiol-yne
coupling with thiol 9, these peptides were transformed into
1
S-glucosyl peptides 12 and 15 as determined by H NMR
analysis of the crude reaction mixtures (Table 1). The same
approach was carried out with galactosyl thiol12 10 and
lactosyl thiol12 11 to give the corresponding S-glycopeptides
4646 J. Org. Chem. Vol. 75, No. 13, 2010

Lo Conte et al.
JOCNote
SCHEME 3
allowed the dithioether 21 to be isolated in 34% yield. The
same procedure was employed for the preparation of the
regioisomer of 21 with use of a reversal order of hydrothiola-
tion, i.e. biotinylation first and then glycosylation (see the
Supporting Information). As biotin is an important biologi-
cal tag24 that is typically conjugated to peptides and proteins
via primary amines (e.g., lysines), this method constitutes an
alternative approach that can be employed for the selective
biotin labeling of cysteine-containing compounds.
In summary, we have demonstrated the selective propar-
gylation of cysteine-containing peptides followed by cou-
pling with glycosyl thiols as a one-pot two-step platform for
post-translational dual glycosylation of peptides. Single
glycosylation and conjugation with biotin also have been
performed. Although demonstrated here for peptides, this
approach is certainly extendable to proteins exposing a cys-
teine SH. As a support to this expectation are the mild reac-
tion conditions under which the photoinduced thiol-yne
coupling takes place (water as a solvent, room temperature,
no metal-based catalyst) as well as irradiation at wavelengths
compatible with protein stability. The lack of diastereoselec-
tivity observed with peptides can be inconsequential when
only one protein diastereomer interacts stereospecifically
with enzyme or receptors.10b Furthermore, the relatively
low abundance of natural proteins displaying cysteine resi-
dues should be compensated by the approach to proteins
incorporating a single cysteine via site-synthetic mutagene-
sis. Research on these topics is underway in our group.
the corresponding S-glycosides 18 and 19. In each case
almost total conversion was registered by NMR analysis
while chromatography over the Sephadex LH20 column
allowed for isolating small samples of pure compounds for
analytical purposes. That the propargylation and subse-
quent glycosylation involved only the peptide cysteine resi-
due was confirmed by ESI-QTOF MS/MS analysis of 15 and
19. The clear fragmentation pattern revealed both the y and b
fragment ions, easily allowing the sequence deconvolution
(see Figures S1 and S2 in the Supporting Information).
Within the derived sequence, the Cys* assignment corre-
sponded to the cysteine residue derivatized with the glyco-
sylated thioether linker. This is noteworthy because the
peptides 6 and 8 contained a basic arginine and lysine
residue, respectively, and therefore there was the possibility
of nonselective propargylation. Evidently this event did not
occur due to the superior nucleophilicity of the cysteine
sulfhydryl group with respect to the basic nitrogen atoms
of other amino acid residues.
Having observed in earlier coupling experiments of alky-
nyl cysteine 2 with sugar thiol 3 that the use of 2-2.5 equiv of
the latter afforded a considerable amount of vinyl thioether
after short irradiation time, it appeared that the thiyl radical
addition to the internal alkene was slow presumably due to
steric considerations. In contrast to the mechanism estab-
lished for the thiol-yne coupling in model systems,19 the
kinetics of the above reaction suggested the possibility to
perform the sequential hydrothiolation of alkynyl peptides
by two thiyl radicals generated from different thiols. The
feasibility of this approach was demonstrated by using GSH
5 as the starting material. Thus, a solution of crude S-
propargyl GSH in MeOH was treated with glucosyl thiol 9
(1.1 equiv) and sensitizer DPAP (0.1 equiv), and the mixture
was irradiated at λmax 365 nm for 15 min (Scheme 3). The 1H
NMR spectrum showed the total consumption of the alkynyl
derivative. Then, a solution of biotin thiol 20 (2 equiv) in
MeOH was added and the mixture was again irradiated at
λmax 365 nm for 45 min. The NMR spectrum of the crude
reaction mixture showed the presence of vinyl proton signals
at ca. 6.3 and ca. 5.6 ppm, which corresponded to 5-10% of
unreacted glycosylated vinylthioether intermediate. Never-
theless, chromatography over the Sephadex LH20 column
Experimental Section
Glycopeptide 12. To a cooled (0 °C), stirred solution of
glutathione 5 (13 mg, 0.042 mmol) in MeOH (0.25 mL) were
added NH4OH (0.25 mL of a 28% solution in H2O) and
propargyl bromide (3.3 μL, 0.046 mmol). The solution was
stirred at 0 °C for 1 h, then warmed to rt, stirred for an additional
2 h, and then concentrated. A stirred solution of the crude
product, glucosyl thiol 9 (33 mg, 0.168 mmol), and DPAP (4 mg,
0.017 mmol) in 2:1 MeOH-H2O (1.2 mL) was irradiated (λmax
365 nm) at rt for 45 min and then concentrated. The residue was
eluted from a column of Sephadex LH20 with MeOH to give 12
(24 mg, 77%) as an amorphous solid. 1H NMR (300 MHz, D2O)
selected data: δ 4.46 (d, 1H, J1,2 = 9.5 Hz, H-1 Glc), 3.70 (s, 2H),
2.79 (ddd, 1H, J = 5.0, 8.6, 14.2 Hz), 2.44 and 2.38 (2 ddd, 2H,
J = 7.5, 7.5, 15.0 Hz), 2.03 (ddd, 2H, J = 7.0, 7.5, 7.5 Hz). 13
C
NMR (75 MHz, D2O): δ 175.4 (C), 174.8 (C), 173.8 (C), 171.9
(C), 86.4 (CH), 85.1 (CH), 85.0 (CH), 84.6 (CH), 79.8 (CH), 77.1
(CH), 72.4 (CH), 72.2 (CH), 69.4 (CH), 60.8 (CH2), 54.0 (CH),
53.1 (CH), 48.8 (CH), 45.6 (CH), 44.9 (CH), 42.8 (CH2), 36.0
(CH2), 35.8 (CH2), 35.2 (CH2), 34.1 (CH2), 33.5 (CH2), 31.3
(CH2), 26.1(CH2). HRMS (ESI/Q-TOF) m/z calcd for C25H44-
N3O16S3 (M þ H)þ 738.1884, found 738.1874.
Acknowledgment. We thank Dr. Claudio Trapella (UFPep-
tides s.r.l., Ferrara, Italy) for a generous gift of peptides 7 and 8.
Supporting Information Available: Experimental proce-
dures, physical data, ESI-QTOF MS/MS analyses, and copies
of the NMR spectra of the new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
(24) (a) Chapman-Smith, A.; Cronan, J. E. Trends Biochem. Sci. 1999, 24,
359–363. (b) Yeo, D. S. Y.; Srinivasan, R.; Chen, G. Y. J.; Yao, S. Q.
Chem.;Eur. J. 2004, 10, 4664–4672.
J. Org. Chem. Vol. 75, No. 13, 2010 4647