An insight into the radical thiol/yne coupling: The emergence of arylalkyne-tagged sugars for the direct photoinduced glycosylation of cysteine-containing peptides

Article abstract of DOI:10.1021/jo101906j

An explorative study of the Thiol-Yne Coupling (TYC) reaction has been carried out using an aliphatic (1-octyne) and an aromatic alkyne (phenylacetylene) and two alkanethiols (methyl thioglycolate and N-acetyl-L-cysteine methyl ester). The outcomes of the TYC reactions strongly depend on the experimental conditions (e.g., temperature, solvent, and alkyne/thiol ratio), but these can be properly adjusted to achieve selective production of either mono-or bis-coupling products. With respect to 1-octyne, phenylacetylene undergoes notably easier radical hydrothiolation, further showing a notably higher aptitude for monohydrothiolation exclusive of bis-hydrothiolation. The overall findings were exploited in glycosylation of cysteine derivatives as well as of cysteine-containing peptides. A sugar featuring an arylacetylene moiety gave rise to a true click-reaction, that is, glycosylation of the tripeptide glutathione in its native form, by means of virtually equimolar amounts of reagents. This reaction was successfully applied, under physiological conditions, to a cysteine-containing nonapeptide with marked advantages over the analogous Thiol-Ene Coupling (TEC) derivatization. A TYC/TEC sequence affording bis-armed cysteine derivatives through dual functionalization of an alkynyl sugar was additionally devised.

Full text of DOI:10.1021/jo101906j

pubs.acs.org/joc
An Insight into the Radical Thiol/Yne Coupling: The Emergence
of Arylalkyne-Tagged Sugars for the Direct Photoinduced Glycosylation
of Cysteine-Containing Peptides
Matteo Minozzi,§ Alessandro Monesi,§ Daniele Nanni,*,§ Piero Spagnolo,§
Nicola Marchetti,† and Alessandro Massi*,‡
§
ꢀ
Dipartimento di Chimica Organica “A. Mangini”, Universita di Bologna, Viale Risorgimento 4,
ꢀ
†
I-40136 Bologna, Italy, Dipartimento di Chimica, Laboratorio di Chimica Analitica, Universita di Ferrara,
Via L. Borsari 46, I-44100 Ferrara, Italy, and ‡Dipartimento di Chimica, Laboratorio di Chimica Organica,
ꢀ
Universita di Ferrara, Via L. Borsari 46, I-44100 Ferrara, Italy
daniele.nanni@unibo.it; msslsn@unife.it
Received September 27, 2010
An explorative study of the Thiol-Yne Coupling (TYC) reaction has been carried out using an
aliphatic (1-octyne) and an aromatic alkyne (phenylacetylene) and two alkanethiols (methyl
thioglycolate and N-acetyl-L-cysteine methyl ester). The outcomes of the TYC reactions strongly
depend on the experimental conditions (e.g., temperature, solvent, and alkyne/thiol ratio), but these
can be properly adjusted to achieve selective production of either mono- or bis-coupling products.
With respect to 1-octyne, phenylacetylene undergoes notably easier radical hydrothiolation, further
showing a notably higher aptitude for monohydrothiolation exclusive of bis-hydrothiolation. The
overall findings were exploited in glycosylation of cysteine derivatives as well as of cysteine-
containing peptides. A sugar featuring an arylacetylene moiety gave rise to a true click-reaction, that
is, glycosylation of the tripeptide glutathione in its native form, by means of virtually equimolar
amounts of reagents. This reaction was successfully applied, under physiological conditions, to a
cysteine-containing nonapeptide with marked advantages over the analogous Thiol-Ene Coupling
(TEC) derivatization. A TYC/TEC sequence affording bis-armed cysteine derivatives through dual
functionalization of an alkynyl sugar was additionally devised.
Introduction
functionalization of both molecules of biological interest3
and many assorted materials,4 owing to its special features
such aschemoselectivity, versatility, andtheabsenceofanyneed
for metal catalysts.3,5 Recently, it has been shown that
thiol-ene couplings can be also accomplished in green
solvents such as ionic liquids.6 The main drawback of TEC
is that, on many occasions, an excess of either reagents is
In recent years, organic synthesis has witnessed a dramatic
improvement of click-chemistry methods both in the biochem-
istry field (in particular for bioconjugation)1 and in material
science (for material derivatization).2 Among the most pop-
ular ‘click’-procedures, the thermally or photochemically
induced radical addition of thiols to alkenes (the Thiol-Ene
Coupling, TEC) has become an outstanding tool for
(3) van Dijk, M.; Rijkers, D. T. S.; Liskamp, R. M. J.; van Nostrum,
C. F.; Hennink, W. E. Bioconjugate Chem. 2009, 20, 2001–2016.
(4) Hoyle, C. E.; Bowman, C. N. Angew. Chem., Int. Ed. 2010, 49, 1540–1573.
(5) Dondoni, A. Angew. Chem., Int. Ed. 2008, 47, 8995–8997.
(6) Lanza, T.; Minozzi, M.; Monesi, A.; Nanni, D.; Spagnolo, P.;
Chiappe, C. Curr. Org. Chem. 2009, 13, 1726–1732.
(1) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004–2021. (b) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol.
2005, 1, 13–21.
(2) Moses, J. E.; Moorhouse, A. D. Chem. Soc. Rev. 2007, 36, 1249–1262.
450 J. Org. Chem. 2011, 76, 450–459
Published on Web 12/22/2010
DOI: 10.1021/jo101906j
r
2010 American Chemical Society

Minozzi et al.
JOCArticle
required in order to obtain complete conversions, a problem
due to the reversibility of the attack of sulfur-centered radicals to
C-C double bonds.7 In those cases, additional workup is
necessary to get rid of the excess of alkene/thiol and one of the
main advantages of click-reactions is therefore lost. In prin-
ciple, a possible way of overcoming this drawback could be
to perform the reactions in the presence of alkynes instead
of alkenes, that is, to carry out Thiol-Yne Couplings (TYCs).
Indeed, although reported as a slower process, sulfanyl radical
addition to C-C triple bonds is known to be virtually irrevers-
ible, at least with alkanesulfanyl radicals.8 This breakthrough
has actually been developed in a group of very recent papers
where the authors exploited this idea in the field of polymer
chemistry.9 It should, however, be emphasized that those
authors aimed at creating very highly functionalized materials
(polymers and dendrimers) by means of a one-pot reaction and
under very mild conditions. Hence, they drove the reaction to
full completion by letting the initially formed vinyl sulfide
adducts to react with additional thiol through a consecutive
thiol-ene coupling. On the other hand, the issue of the selective
formation of the primary vinyl sulfide TYC products could be
crucial in the field of bioconjugation, where molecular complex-
ity might be a secondary concern with respect to finding a
reliable, ‘clickable’ way of derivatization of valuable biological
substrates. As far as we know, only two papers have so far
appeared focusing somewhat on this matter: the more recent
one10a deals with dual glycosylation of peptides, thus, concen-
trating again on bis-addition coupling products, whereas the
other one10b actually focuses on vinyl sulfide adducts, which
SCHEME 1. Mechanisms of TYC and TEC Radical Chains
are nevertheless attained through an ionic addition of thiols
to suitable electron-deficient alkynes.
Scheme 1 shows the radical chain reactions that can operate
under TYC conditions. Addition of a sulfanyl radical to the
terminal carbon of the alkyne C-C triple bond gives a
β-sulfanylvinyl radical that readily abstracts hydrogen from
the thiol to regenerate the chain-carrier, sulfur-centered radical
intermediate with concomitant formation of the vinyl sulfide
monoadduct (TYC chain). Under certain circumstances, the
monoadduct can undergo further addition of another sulfa-
nyl radical to give an R,β-disulfanyl-disubstituted alkyl radical
that can subsequently abstract hydrogen from the thiol to
give the final bis-sulfide bis-adduct (TEC chain). Unlike the
primary vinyl radical, the successive alkyl-counterpart oc-
curs in a reversible fashion: this allows for properly tuning
reaction conditions in order to favor formation of the bis-
adduct (TYC-TEC sequence) or to stop the reaction at the
vinyl sulfide stage (TYC).
In our opinion, the latter outcome is that deserving
thorough attention. Indeed, the (virtually) irreversible oc-
currence of vinyl sulfide adduct from the radical TYC
process could be exploited in a more ‘clickable’ reaction than
the TEC one, since it could provide easier access to assorted
sulfur-tethered products employing equivalent amounts of
starting materials. This latter point could be of extreme
importance in the field of bioconjugation, where the isola-
tion of target molecules from complex reaction mixtures con-
taining excess substrates is often a difficult task.10a Further-
more, synthesis and activity of bioconjugates containing the
vinyl sulfide linkage are highly worthy of being explored and,
to date, only a unique example dealing with this issue has been
reported.10b Finally, the feasibility of selective syntheses of
monoadducts can open the door to TYC-TEC sequences lead-
ing to nonsymmetric bis-functionalizations, hence, paving
the way to very attractive dual-labeling investigations.11
Here, we report an explorative study on model substrates
that aimed at getting more insight into the conditions affect-
ing the coupling reaction outcome, particularly the ensuing
mono- and bis-adduct ratio. Both classical organic solvents
and water (or water/solvent mixtures) were taken into
account for carrying out the reactions, since the aqueous
medium would be crucial for applying Thiol-Yne Couplings
as a ligation strategy, for instance, for peptide glycosylation,
(7) (a) Benati, L.; Montevecchi, P. C.; Spagnolo, P. J. Chem. Soc., Perkin
Trans. 1 1991, 2103–2109. The reversibility of the addition of sulfanyl
radicals to C-C double bonds precludes, for example, the radical addition
of disulfides to C-C double bonds, which is inefficient unless either by
employing alkynes instead of alkenes (see refs 7a above and 13a below) or in
the presence of a very good radical trap such as a diselenide, see: (b) Ogawa,
A.; Tanaka, H.; Yokoyama, H.; Obayashi, R.; Yokoyama, K.; Sonoda, N.
J. Org. Chem. 1992, 57, 111-115 or a very efficient H-donor, see: (c) Taniguchi,
T.; Fujii, T.; Idota, A.; Ishibashi, H. Org. Lett. 2009, 11, 3298–3301.
(8) (a) Heiba, E. I.; Dessau, R. M. J. Org. Chem. 1967, 32, 3837–3840.
(b) Montevecchi, P. C.; Navacchia, M. L. J. Org. Chem. 1997, 62, 5600–5607.
(c) Melandri, D.; Montevecchi, P. C.; Navacchia, M. L. Tetrahedron 1999,
55, 12227–12236. For a kinetic study on the addition of benzenesulfanyl
radicals to alkynes, see: (d) Ito, O.; Omori, R.; Matsuda, M. J. Am. Chem.
Soc. 1982, 104, 3934-3937. where that addition has been suggested to be
slightly reversible. It is worth noting that addition of sulfur-centered radicals
to alkynes is not always necessarily an irreversible process, provided that the
radicals possess additional stabilization with respect to sulfanyls: sulfonyl
radicals, for example, have been reported to add reversibly to C-C triple
bonds, see: (e) Rosenstein, I, J. In Radicals in Organic Synthesis; Renaud, P.,
Sibi, M. P., Eds.: Wiley-VCH: Weinheim, Germany, 2001; Vol. 1, Chapter
1.4, pp 50-71. (f) Bertrand, M. P.; Ferreri, C. In Radicals in Organic
Synthesis; Renaud, P., Sibi, M. P., Eds.: Wiley-VCH: Weinheim, Germany,
Vol. 2, Chapter 5.5, pp 485-504. (g) Chatgilialoglu, C.; Ferreri, C. In The
Chemistry of Triple-Bonded Functional Groups; Patai, S., Ed.; Wiley: Chichester,
U.K., 1994; Vol. 2, pp 917-944. (h) Chatgilialoglu, C.; Bertrand, M. P.;
Ferreri, C. In S-Centered Radicals; Alfassi, Z. B., Ed.; Wiley: Chichester,
U.K., 1999; pp 311-354.
(9) (a) Fairbanks, B. D.; Scott, T. F.; Kloxin, C. J.; Anseth, K. S.;
Bowman, C. N. Macromolecules 2009, 42, 211–217. (b) Chen, G.; Kumar,
J.; Gregory, A.; Stenzel, M. H. Chem. Commun. 2009, 6291–6293. (c) Chan,
J. W.; Hoyle, C. E.; Lowe, A. B. J. Am. Chem. Soc. 2009, 131, 5751–5753.
(d) Hensarling, R. M.; Doughty, V. A.; Chan, J. W.; Patton, D. L. J. Am.
Chem. Soc. 2009, 131, 14673–14675. (e) Konkolewicz, D.; Gray-Weale, A.;
Perrier, S. J. Am. Chem. Soc. 2009, 131, 18075–18077. (f) Lowe, A. B.; Hoyleb,
C. E.; Bowman, C. N. J. Mater. Chem. 2010, 20, 4745–4750. (g) Fairbanks, B. D.;
Sims, E. A.; Anseth, K. S.; Bowman, C. N. Macromolecules 2010, 43, 4113–4119.
(h) Chan, J. W.; Shin, J.; Hoyle, C. E.; Bowman, C. N.; Lowe, A. B. Macro-
molecules 2010, 43, 4937–4942. (i) Hoogenboom, R. Angew. Chem., Int. Ed. 2010,
49, 3415–3417.
(10) (a) Lo Conte, M.; Pacifico, S.; Chambery, A.; Marra, A.; Dondoni,
A. J. Org. Chem. 201075, 4644–4647. (b) Shiu, H.-Y.; Chan, T.-C.; Ho, C.-M.;
Liu, Y.; Wong, M.-K.; Che, C.-M. Chem.—Eur. J. 2009, 15, 3839–3850.
(11) For an outstanding example, see: 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.
J. Org. Chem. Vol. 76, No. 2, 2011 451

Minozzi et al.
JOCArticle
similarly to what has been done with TEC procedures.3 In
addition, we are going to confirm that, compared to alkyla-
cetylenes, C-C triple bonds linked to aromatic substrates
(arylacetylenes) work as a better trap toward sulfanyl radicals:12
to the best of our knowledge, this enhanced behavior has been
exploited neither in the domain of bioconjugation nor in that
of material derivatization and could entail interesting con-
sequences in both fields. Finally, some preliminary results
will be reported showing that the data obtained from the
explorative study could be successfully applied to some
bioconjugation examples including glycosylation of the native
form of glutathione and of an unmodified nonapeptide con-
taining a cysteine residue.
TABLE 1. Thiol-Yne Couplings of Methyl Thioglycolate 1 with
1-Octyne 2a
Results and Discussion
Our long, earlier interest in the radical reactions of thiols
with alkynes13 led us to ascertain that, under thermal condi-
tions (80-100 °C) and in hydrocarbon solvents (e.g., ben-
zene or toluene), derived sulfanyl radicals usually lead to
vinyl sulfide adducts only. Consistent with our original evidence,
methyl thioglycolate 1 was presently found to react with
equimolar amounts of 1-octyne 2 in toluene solution at 80 °C
in the presence of AIBN as a radical initiator, affording vinyl
sulfide 3 (62%) and disulfide 5 (28%) as the only identifiable
reaction products (Table 1, entry 1). Nevertheless, the reac-
tion outcomes alter to a considerable extent by changing
conditions (i.e., solvent, temperature, concentration, and
initiation method), as proved by a series of experiments
(Tables 1-4) carried out between two thiols (methyl thiogly-
colate 1 and N-acetyl-L-cysteine methyl ester 10) and two
alkynes (1-octyne 2 and phenylacetylene 6). The parallel beha-
vior of thiols with aromatic and aliphatic alkynes has never
been addressed in the recently reported TYC studies, but in
light of our previous investigations, we thought that this
point should deserve adequate consideration. Different
reaction solvents were chosen on the basis of their potential
employment in bioconjugation procedures (water and 95:5
water/DMSO mixtures) or material derivatization (DMSO,
DMF). All the reactions were carried out at room temperature
entry
1/2
solvent (c [M])
3/4 [%]b,c
yield 3 þ 4 [%]d
1
2
3
4
5
6
7
8
9
1:1
1:1
1:1
1:1
1:1
1:1
2:1
2:1
2:1
Toluene (0.5)e
Toluene (0.5)f
DMSO (0.02)
H2O-DMSO (0.5)g
H2O (0.5)
100/-
45/55
80/20
45/55
11/89
25/75
-/100
-/100
-/100
62
76
55
90
85
52
75
90
89
[bmim][PF6]
DMSO (0.5)
H2O-DMSO (0.5)g
H2O (0.5)
aPhotoinduced reactions were carried out at r.t. with a household
UVA lamp apparatus at λmax 365 nm (see the Experimental Section for
equipment setup). Reactions performed with 1 normally gave the
corresponding disulfide 5 in <5% yield; only the reaction of entry 3
b
afforded 5 in ca. 10% yield. Values are relative percentages and were
determined by GC-MS and 1H NMR analysis. cAdduct 3 was a 1.2:1
mixture of E and Z isomers. dIsolated yield calculated on the basis of the
starting alkyne. eReaction performed under thermal conditions (80 °C)
f
with AIBN as the radical initiator. Similar results were obtained in
DMSO or DMF. gH2O/DMSO 95:5.
(r.t.)14 for 1-2 h by irradiation with UV-lamp (λmax 365 nm)
using 2,2-dimethoxy-2-phenylacetophenone (DMPA, 10 mol %)
as a radical initiator, that is, the conditions normally em-
ployed in this kind of radical couplings.9 Thus, the model
reaction of thiol 1 with alkyne 2 (1 equiv) in toluene (0.5 M)
solution afforded, under these conditions, both mono- (3)
and bis-adduct (4) in comparable amounts (45:55 ratio, 76%
overall yield), together with very minor amounts (<5%) of
disulfide 5 (Table 1, entry 2).
(12) For comparisons between the reactivities of alkyl- and arylacety-
lenes, see refs 8d above, 13a, 13b, below, and (a) Ogawa, A.; Obayashi, R.;
Ine, H.; Tsuboi, Y.; Sonoda, N.; Toshikazu, H. J. Org. Chem. 1998, 63, 881–
884. For another recent example of radical monothiolation of arylacetylenes,
see: (b) Beauchemin, A.; Gareau, Y. Phosphorus, Sulfur, Silicon Relat. Elem.
1998, 139, 187–192. It is worth emphasizing that a higher reactivity (kinetic
constant of more than one order of magnitude higher) of phenylacetylene
with respect to an alkyl congener (1-propyne) has been also reported for
addition of alkyl (methyl) radicals, see: (c) Fischer, H.; Radom, L. Angew.
Chem., Int. Ed. 2001, 40, 1340–1371.
(13) (a) Benati, L.; Montevecchi, P. C.; Spagnolo, P. J. Chem. Soc., Perkin
Trans. 1 1991, 2103–2109. and references therein. (b) Benati, L.; Montevecchi,
P. C.; Spagnolo, P. J. Chem. Soc., Perkin Trans. 1 1992, 1659–1664. (c) Benati, L.;
Capella, L.; Montevecchi, P. C.; Spagnolo, P. J. Chem. Soc., Perkin Trans. 1 1995,
1035–1038. (d) Benati, L.; Capella, L.; Montevecchi, P. C.; Spagnolo, P. J. Org.
Chem. 1995, 60, 7941–7946. (e) Montevecchi, P. C.; Navacchia, M. L. J. Org.
Chem. 1997, 62, 5600–5607. (f) Montevecchi, P. C.; Navacchia, M. L. J. Org.
Chem. 1998, 63, 537–542. (g) Montevecchi, P. C.; Navacchia, M. L.; Spagnolo, P.
Tetrahedron 1998, 54, 8207–8216. (h) Montevecchi, P. C.; Navacchia, M. L.;
Spagnolo, P. Eur. J. Org. Chem. 1998, 1219–1226. (i) Leardini, R.; Nanni, D.;
Zanardi, G. J. Org. Chem. 2000, 65, 2763–2772. We recently exploited addition of
sulfanyl radicals to alkynes as a novel tin-/metal-free method to generate assorted
kinds of radicals, see: (j) Benati, L.; Calestani, G.; Leardini, R.; Minozzi, M.;
Nanni, D.; Spagnolo, P.; Strazzari, S. Org. Lett. 2003, 5, 1313–1316. (k) Benati,
L.; Leardini, R.; Minozzi, M.; Nanni, D.; Scialpi, R.; Spagnolo, P.; Zanardi, G.
Synlett 2004, 987–990. (l) Benati, L.; Bencivenni, G.; Leardini, R.; Minozzi, M.;
Nanni, D.; Scialpi, R.; Spagnolo, P.; Zanardi, G. J. Org. Chem. 2006, 71, 3192–
3197. (m) Bencivenni, G.; Lanza, T.; Leardini, R.; Minozzi, M.; Nanni, D.;
Spagnolo, P.; Zanardi, G. Org. Lett. 2008, 10, 1127–1130.
As far as the 3/4 ratio is concerned, almost identical results
were obtained by substituting toluene with DMSO or DMF,
albeit with lower overall yields. It would therefore seem that
temperature plays a pivotal role in affecting the outcoming
sulfide-3/bis-sulfide-4ratio (by comparison of entries 1 and 2), as
an expected result of a faster or slower back fragmentation of
the dithioalkyl radical precursor of the bis-adduct. Interest-
ingly, unlike the aliphatic acetylene 2, underthesameconditions
phenylacetylene 6 gave only vinyl sulfide 7 in 90% yield
(Table 2, entry 1).15
(14) Under these conditions, the reaction vessel actually warmed up to
35-40 °C. Anyway, strictly identical results were obtained with reaction
mixtures kept at 25 °C by simultaneous air-cooling with compressed air.
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TABLE 2. Thiol-Yne Couplings of Methyl Thioglycolate 1 with
SCHEME 2. Reaction Mechanism of Formation of Bis-Adducts
4, 8, and 9
Phenylacetylene 6a
afforded small amounts (10%) of bis-adduct as a mixture
of regioisomers 8 and 9 in a 2:1 ratio (Table 2, entry 4).
The outcoming difference in the regiochemistry of the bis-
sulfide adducts arising from octyne 2 and phenylacetylene 6
probably reflects the relative stability of the alkyl radicals R
and β that could in principle arise from further addition of
sulfanyl radical to the respective double bond of vinyl sulfides 3
and 7 (Scheme 2). With sulfide 3, the sulfanyl would strictly
prefer to form the more stable radical β, R = C6H13, owing
to back-donation stabilization provided by the attached
sulfur atom; with sulfide 7, the formation of the correspond-
ing radical β, R = Ph, would be discouraged to some extent
in favor of the resonance-stabilized benzylic radical R, R =
Ph (Scheme 2).
entry 1/6
solvent (c [M])
7/8 þ 9 [%]b,c yield 7 þ 8 þ 9 [%]d
1
2
3
4
5
6
7
8
1:1 Toluene (0.5)e
1:1 DMSO (0.02)
1:1 H2O-DMSO (0.5)f
1:1 H2O (0.5)
1:1 [bmim][PF6]
2:1 DMSO (0.5)
2:1 H2O-DMSO (0.5)f
2:1 H2O (0.5)
100/-
100/-
100/-
90/10g
90/10g
40/60g
22/78g
5/95g
90
65
87
91
58
69
88
91
It is worth noting that an analogous effect in favor of bis-
hydrothiolation was observed by changing water with an
ionic liquid ([bmim][PF6]): with this solvent, 1-octyne gave
adducts 4 and 3 in a 3:1 ratio (Table 1, entry 6) and phenyl-
acetylene afforded again monoadduct 7 accompanied with
minor amounts of bis-adducts 8 and 917 (Table 2, entry 5).
Preferential occurrence of bis-sulfide 4 at the expense of vinyl
sulfide 3 would possibly entail especially fast H-transfer from
thiol 1 to the intermediate dithioalkyl radical. Under these
circumstances, in fact, that radical intermediate could be
(seriously) discouraged to suffer usual β-elimination of
sulfanyl radical yielding back vinyl sulfide 3 (see Scheme 1).
Thus, our present findings with octyne 2 in the above ionic
liquid seem to substantiate previous chemical evidence that
thiols in ionic liquid solvents could act as very strong H-donors.6
Moreover, the corresponding findings achieved in pure
water first suggest that in such medium thiols should inter-
estingly become even stronger H-donors. Whether the en-
hanced H-donor properties of thiol in ionic liquid and,
especially, water would result from some solvent stabilization
of the H-transfer transition state, as previously suggested,6
or would just be a consequence of ‘neat’ reactions occurring
inside organic droplets18 is a debated question that will be
dealt with in future studies.
aPhotoinduced reactions were carried out at r.t. with a household
UVA lamp apparatus at λmax 365 nm (see the Experimental Section for
equipment setup). Reactions performed with 1 normally gave the
corresponding disulfide 5 in <5% yield; only the reaction of entry
2 afforded 5 in ca. 10% yield. bValues are relative percentages and were
c
determined by GC-MS and 1H NMR analysis. Adduct 7 was a 2:1
mixture of Z and E isomers. dIsolated yield calculated on the basis of the
starting alkyne. eSimilar results were obtained in DMSO or DMF. fH2O/
DMSO 95:5. g8/9 ∼2:1.
Dilution of the reaction mixture from 0.5 to 0.02 M led,
with 1-octyne in DMSO, to preferential formation of mono-
adduct 3 (3/4 ratio ca. 4:1; Table 1, entry 3), whereas the
reaction with alkyne 6 was not substantially affected, yield-
ing again the monoadduct 7 (65%) as the only coupling
product (Table 2, entry 2); both reactions also afforded disulfide
5 as a byproduct (ca. 10%). Since, in general, dilution was
found to affect negatively the reaction outcome in terms of
lower overall yields, formation of byproducts, and lower con-
versions, all the subsequent reactions were performed in
0.5 M solutions. When the reaction of 1 with 2 was carried
out in water/DMSO 95:5, no change in product distribution
was observed (Table 1, entry 4), whereas pure water16 strongly
favored occurrence of bis-adduct 4 over the monoadduct 3
(3/4 ratio ca. 1:8; Table 1, entry 5). So strong seems this effect
of water that with this solvent also phenylacetylene 6
Taking into account the overall results obtained with thiol
1 (Tables 1 and 2), we can infer that it is possible to modify
properly the reaction outcome by tuning the reaction condi-
tions. Starting from equivalent amounts of thiol and alkyne,
(15) All the vinyl sulfides were formed as mixtures of E- and Z-isomers:
see the Experimental Section for details. Studies on the stereoselective formation
of the kinetic (Z) or thermodynamic (E) product are currently underway.
(16) Reactions carried out in water were heterogeneous mixtures, whereas
those carried out in water/DMSO 95:5 were normally homogeneous.
(17) Both in water and in the ionic liquid the 8/9 ratio is ca. 2:1.
(18) For a recent discussion about organic synthesis “on water”, see:
Chanda, A.; Fokin, V. V. Chem. Rev. 2009, 109, 725–748.
J. Org. Chem. Vol. 76, No. 2, 2011 453

Minozzi et al.
JOCArticle
with 1-octyne 2 (Table 1), formation of monoadduct 3 is
favored at higher temperatures (AIBN-initiated reaction in
toluene) or diluted mixtures (0.02 M in DMSO), whereas
formation of bis-adduct 4 is strongly favored in an ionic liquid
such as [bmim][PF6] and, particularly, in water. With phenyl-
acetylene 6 (Table 2), the monoadduct 7 is always the exclusive
product, with the exception of the small amounts of the
regioisomeric bis-adducts 8 and 9 isolated in water or
[bmim][PF6]. If total production of bis-sulfide is desired, this
can be readily achieved by using a 2-fold excess of thiol
reagent. Indeed, the photolytically initiated reaction of alkyne 2
in the presence of 2 equiv of thiol 1 was found to afford virtually
quantitative amounts of bis-sulfide 4 irrespective of the solvent
employed (DMSO, H2O/DMSO, or H2O) (Table 1, entries
7-9). Even in the presence of the same excess of thiol 1, the
aromatic alkyne 6 behaved in a different fashion, since it could
still afford significant amounts of monoadduct 7 both in DMSO
and H2O/DMSO (Table 2, entries 6 and 7). In pure water,
however, 6 succeeded in forming virtually exclusive amounts of
the bis-sulfides 8 and 9, in line with the discovered effect of water
solvent (Table 2, entry 8).
TABLE 3. Thiol-Yne Couplings of N-Acetyl-L-cysteine Methyl Ester
10 with 1-Octyne 2a
entry 10/2
solvent (c [M])
11/12 [%]b,c yield 11 þ 12 [%]d
As far as the cysteine thiol 10 is concerned (Tables 3 and 4),
its reactions appear slower than those of the congener 1 as a
possible result of a higher steric hindrance.19 This probably
justifies the preferential formation of monoadduct 11,9d which is
the major product both in DMSO, water/DMSO, and pure
water (Table 3, entries 1-3).
1
2
3
4
5
6
1:1 DMSO (0.5)
83/17
67/33
62
85
82
81
91
92
1:1 H2O-DMSO (0.5)e
1:1 H2O (0.5)
1:2 H2O (0.5)
55/45
95/5
2:1 H2O-DMSO (0.5)
<5/>95
<5/>95
2:1 H2O (0.5)e
aPhotoinduced reactions were carried out at r.t. with a household
UVA lamp apparatus at λmax 365 nm (see the Experimental Section for
equipment setup). Reactions performed with 10 normally gave the
corresponding cystine 13 in <5% yield; only the reaction of entry 1
afforded 13 in ca. 20% yield. bValues are relative percentages and were
determined by GC-MS and 1H NMR analysis. cAdduct 11 was a 1.5:1
mixture of Z and E isomers. dIsolated yield calculated on the basis of the
starting alkyne (entries 1-3 and 5-6) or the starting thiol (entry 4).
eH2O/DMSO 95:5.
It is worth noting that highly selective production of the
monoadduct 11 could be achieved by carrying out the
reaction under diluted (0.02 M DMSO) conditions (35%
yield of 11, with only 50% conversion; not reported) or using
a 2-fold excess of the alkyne 2 (11/12 ratio ∼19:1; Table 3,
entry 4). Conversely, the use of a 2-fold excess of cysteine 10
allowed highly selective occurrence of the bis-sulfide 12
(11/12 ratio >1:19 both in water/DMSO and water) in very
good yield (>90%) (Table 3, entries 5 and 6). In parallel, the
TYC of the same cysteine 10 with equimolar phenylacetylene
6 afforded the monoadduct 14 in a very selective fashion
under all conditions employed (Table 4, entries 1-3). No
evidence of formation of a bis-adduct was observed either
with 2 equiv of thiol (entries 4 and 5).
peptides. The usefulness of this radical reaction in glycopep-
tide chemistry has been recently validated by the synthesis of
dually glycosylated peptides by a two-step strategy, which
involved first the S-propargylation of a cysteine containing
peptide, and then the photoinduced coupling with excess of
glycosyl thiol.10a Undoubtedly, if peptide mono glycosyla-
tion is the target, the direct TYC of sugar alkynes with
peptides bearing a free cysteine residue appears as a more
straightforward strategy. Our investigation on this comple-
mentary approach took advantage of the information gained
from the above explorative study and involved peptide portions
of increasing complexity. Accordingly, the coupling between
the peracetylated O-propargyl β-glycoside 15 with a single
cysteine residue, that is, the N-Fmoc cysteine tert-butyl ester
16, was initially considered to establish optimal conditions
for the selective monohydrothiolation pathway of such more
complex substrates (Table 5).
The photoinduced TYC (λmax 365 nm, DMPA 10 mol %)
of equimolar 15 and 16 proceeded smoothly under homo-
geneous conditions with DMF (0.5 M) as the solvent to give
exclusively the monoadduct 17 (25%) as a 1.5:1 mixture of
E/Z isomers (Table 5, entry 1), albeit in low yields. Complete
conversion of cysteine 16 was conveniently achieved by using
a 3-fold excess of sugar alkyne 15, thus, obtaining 17 in 88%
isolated yield (entry 2). It is worth noting that, owing to the
set of orthogonal protective groups, the glycosyl amino acid
It is noteworthy that our explorative study with octyne 2
and phenylacetylene 6 clearly revealed a deeply different
behavior of these two alkynes toward their radical coupling
reactions with thiols 1, 10. Indeed, phenylacetylene 6 usually
gave mono/bis-sulfide adducts in notably higher (overall)
yields and, furthermore, showed a distinct propensity to form
monosulfide rather than bis-sulfide product. It is therefore
plausible that the aromatic alkyne 6 could act as a much
stronger trap for sulfur-centered radicals than the aliphatic
congener 2. This point was actually substantiated by our
additional finding that the usual reaction of cysteine 10 with
equimolar amounts of both alkynes 2 and 6 in DMSO could
basically afford the phenylvinyl sulfide 14 at the expense of
the alkylvinyl one 11 (11/14 ∼ 1:15, Scheme 3).
As anticipated, the ultimate goal of this study was to
establish the potential role of photoinduced TYC as a click
ligation tool for the direct monoglycosylation of unmodified
(19) We cannot exclude that polar factors may also play an additional role
in the overall thiolation reaction, see: Escoubet, S.; Gastaldi, S.; Vanthuyne,
N.; Gil, G.; Siri, D.; Bertrand, M. P. J. Org. Chem. 2006, 71, 7288–7292.
454 J. Org. Chem. Vol. 76, No. 2, 2011

Minozzi et al.
JOCArticle
TABLE 4. Thiol-Yne Couplings of N-Acetyl-L-cysteine Methyl
TABLE 5. Synthesis of Glycosyl Cysteine 17 and Bis-Armed
Cysteine 18a
Ester 10 with Phenylacetylene 6a
entry
10/6
solvent (c [M])
yield 14 [%]b,c
1
2
3
4
5
1:1
1:1
1:1
2:1
2:1
DMSO (0.5)
60
78
88
86
91
H2O-DMSO (0.5)d
H2O (0.5)
DMSO (0.5)
H2O-DMSO (0.5)d
aPhotoinduced reactions were carried out at r.t. with a household
UVA lamp apparatus at λmax 365 nm (see the Experimental Section for
equipment setup). Reactions performed with 10 normally gave the
corresponding cystine 13 in <5% yield; only the reaction of entry 1
afforded 13 in ca. 20% yield. bAdduct 14 was a ∼1:1 mixture of Z and E
c
entry
15/16
solvent (c [M])
time (h)
yield 17/18 [%]b
isomers. Isolated yield calculated on the basis of the starting alkyne.
dH2O/DMSO 95:5.
1
2
3
4
5
1:1
3:1
1:1
1:1
1:2
DMF (0.5)
DMF (0.5)
1
1
2
2
2
25c/-
88c/-
-/-d
6c/81f
-/85f
H2O (0.5)
H2O-PhMe (0.01)e
SCHEME 3. Competitive Reaction of N-Acetyl-L-cysteine
Methyl Ester 10 with 1-Octyne 2 and Phenylacetylene 6
(1:1:1 Ratio)
H2O-PhMe (0.01)e
aPhotoinduced reactions were carried out at r.t. with a household
UVA lamp apparatus at λmax 365 nm (see the Experimental Section for
equipment setup). bIsolated yield calculated on the basis of the starting
alkyne (entries 1, 3-5) or the starting thiol (entry 2). cAdduct 17 was a
1.5:1 mixture of E and Z isomers. dCysteine 16 stuck on reaction vessel
walls. eToluene (10% v/v) was used to disperse reagents and catalyst in
water. fd.r. ∼1:1.
conditions in the model couplings of 1-octyne (Table 1, entry
5 and Table 3, entry 3), the photoinduced coupling (λmax 365 nm,
DMPA 10 mol %) of sugar alkyne 15 and cysteine 16 was
initially performed in H2O (0.5 M). Unfortunately, these
conditions did not produce any results, very likely because of
the ‘sticky’ nature of 16, which resulted in agglomeration and
hence precluded the intimate contact between the reaction
partners (entry 3). On the other hand, when equimolar 15, 16,
and the sensitizer DMPA (10 mol %) were previously dissolved
with minimal toluene, the subsequent addition of H2O (0.01 M)
resulted in the formation of organic droplets which dispersed
under vigorous magnetic stirring. Irradiation of that mixture
for 2 h afforded, after concentration and column chroma-
tography, the bis-glycosylated cysteine derivative 18 as the
main product (81%, d.r. ∼1:1) along with small amounts of
the monoadduct 17 (6%; entry 4). The selective formation of
the bis-adduct 18 was finally achieved by simply using 2
equiv of cysteine 16 under the same conditions (entry 5).
Although a detailed analysis of this reaction outcome goes
beyond the object of this research, it can be speculated that
reactants concentration into organic droplets by means of
hydrophobic interactions is responsible for the observed rate
acceleration of the double hydrothiolation reaction.18,22
17 appeared to be a suitable substrate for a co-translational
approach20 to glycopeptides through N-Fmoc-based peptide
synthesis.
From a mechanistic point of view, possible formation of
the bis-adduct 18 seemed to be strongly inhibited by steric
factors in agreement with the results reported above and
previous observations on photoinduced TYC of bulky thiols.9d
Nevertheless, due to the relevance of ‘bis-armed’ amino acids
of type 18 in peptide chemistry,21 the double hydrothiolation
of sugar alkyne 15 with cysteine 16 was actively pursued.
Thus, bearing in mind the beneficial effect of heterogeneous
(20) McGarvey, G. J.; Benedum, T. E.; Schmidtmann, F. W. Org. Lett.
2002, 4, 3591–3594.
(21) For leading references on the synthesis of bis-amino acids, see: (a) Li,
C.; Tang, J.; Xie, J. Tetrahedron 2009, 65, 7935–7941. For an interesting
application, see: (b) Schafmeister, C. E.; Brown, Z. Z.; Gupta, S. Acc. Chem.
Res. 2008, 41, 1387–1398.
J. Org. Chem. Vol. 76, No. 2, 2011 455

Minozzi et al.
SCHEME 5. Optimized Conditions for the Complete
Glycosylation of Glutathione 22 by Sugar Alkynes 21 and 24
JOCArticle
SCHEME 4. Synthesis of Orthogonally Protected Bis-Armed
Cysteine 20
The discovery of a reaction window for both mono- and
double-hydrothiolation of sugar alkyne 15 prompted us to
investigate the sequential hydrothiolation of 15 using the two
different cysteine derivatives 16 and 19 (Scheme 4). Indeed,
we thought this proof-of-principle experiment might be of
interest to establish the potential of photoinduced TYC/
TEC sequences in dual-labeling investigations.11 Thus, the vinyl
thioether intermediate 17 was first prepared by TYC of alkyne
15 with cysteine 16 under optimized conditions (Table 5, entry 2),
and then subjected to the photoinduced TEC (λmax 365 nm, 2 h,
DMPA 10 mol %) with cysteine 19 (2 equiv) in diluted water/
toluene (10:1 v/v) (0.01 M) to give the target bis-adduct 20
(d.r. ∼1:1) in 66% overall yield (Scheme 4). Noteworthy,
the orthogonal protection of the bis-amino acid 20 allows
for differential peptide chain elongation via Boc- and Fmoc-
based peptide synthesis.21
The investigation of ‘click’ equimolar glycosylation of
cysteine-containing peptides via photoinduced TYC was
the next step in our program. To this aim, the readily available
glycosyl alkyne 21 and the natural tripeptide glutathione 22 (γ-L-
Glu-L-Cys-Gly, GSH) in its native form were considered suitable
substrates for testing the efficiency of this approach (Scheme 5).
After some experimentation, full conversion of GSH 22 could be
achieved under irradiation (λmax 365 nm, DMPA 10 mol %, 1 h)
in a 19:1 H2O-MeOH mixture23 when using at least a 5-fold
excess of sugar alkyne 21, as it was established by LC-MS
analyses (see Supporting Information). This rather disappoint-
ing, even though successful, result prompted us to synthesize an
aromatic counterpart of the alkyne 21 (Scheme 5) in view of our
previous discovery that an aromatic alkyne should be more
effective than an aliphatic one in photoinduced TYC (Scheme 3).
Accordingly, the unknown ethynylbenzyl β-D-glucopyranoside
24 was obtained by quantitative hydroxyl groups deprotection
(NaOMe/MeOH) of the corresponding peracetylated deriva-
tive, which in turn was prepared, under nonoptimized conditions
of a mixture of glutathione 22, DMPA (10 mol %), and sugar
alkyne 24 (1.1 equiv) in H2O-MeOH 3:1 (v/v) (0.1 M)23
resulted in quantitative formation of the glycoconjugate 25 as
judged by 1H NMR and LC-MS analyses of the crude reaction
mixture (see Supporting Information). These optimal coupling
conditions did not involve any significant excess of either
reagents as required by a true ‘click’ reaction, and then allowed
for a simple, rapid purification process of 25. This compound
was readily isolated by short-column chromatography with
Sephadex LH20 in 82% yield as a 6:1 mixture of E/Z isomers
(Scheme 5). With the pure glycopeptide 25 in hand, the
stability of the vinyl thioether linkage in aqueous medium was
1
next investigated by recording H NMR spectra of a D2O
solution of 25 (0.03 M) over the time. Rewardingly, no degrada-
tion occurred during a week, as it was also confirmed by a final
LC-MS analysis of the same solution.
Paralleling the previous study on peptide glycosylation via
photoinduced thiol-ene coupling,24 the efficacy of the opti-
mized thiol-yne coupling was evaluated in aqueous solu-
tions at physiological pH with the higher synthetic nonapeptide
TALNCNDSL 26,25 which displays a single cysteine residue as
well (Scheme 6). Hence, the coupling of 24 with 26 was optimized
by adding a solution of DMPA (50 mol %) and 24 (5 equiv) in
DMSO (5% final volume content) to a solution of 26 in 20 mM
phosphate buffer (pH 7.4) and maintaining irradiation (λmax
365 nm) for 1 h. Under these conditions, peptide 26 was
quantitatively converted into the glycoconjugate 27, which was
obtained in 68% isolated yield (Sephadex LH20) as a 2:1
mixture of E/Z isomers. The selective attachment of 24 to the
sulfhydryl group of 26 was duly confirmed by LC-MS/MS
(45% yield), by BF3 OEt2-promoted glycosylation of β-D-
glucose pentaacetate with ethynylbenzyl alcohol (Scheme S1,
3
Supporting Information). Gratifyingly, irradiation for 1 h
(22) Pirrung, M. C.; Das Sarma, K.; Wang, J. J. Org. Chem. 2008, 73,
8723–8730.
(24) Dondoni, A.; Massi, A.; Nanni, P.; Roda, A. Chem.—Eur. J. 2009,
15, 11444–11449.
(23) Homogeneous conditions were required to achieve good conversions
of GSH 22.
(25) This compound was kindly provided as trifluoroacetic salt by
UFPeptides S.r.L. (University of Ferrara).
456 J. Org. Chem. Vol. 76, No. 2, 2011

Minozzi et al.
JOCArticle
SCHEME 6. Preparation of Glycopeptide 27
of a cysteine-containing nonapeptide under physiological con-
ditions. Aromatic alkynes have hence become a new, attractive
tag for bioconjugation studies based on the TYC ligation
strategy.
Experimental Section
Photoinduced reactions were carried out in a glass vial
(diameter, 1 cm; wall thickness, 0.65 mm), sealed with a natural
rubber septum, located 2.5 cm away from the UVA lamp
(irradiation on sample: 365 nm, 1.04 W/m2).
General Procedure for the Thiol/Yne Radical Couplings Re-
ported in Tables 1-4. A mixture of alkyne (0.50 mmol), thiol
(0.50 mmol unless otherwise stated), 2,2-dimethoxy-2-phenyla-
cetophenone (13 mg, 0.05 mmol), and the stated solvent (1 mL
unless otherwise stated) was irradiated at room temperature for
1-2 h under magnetic stirring. The crude mixtures of reactions
performed in DMSO, H2O/DMSO, or DMF were diluted with
H2O (5 mL) and extracted with AcOEt (3 ꢀ 5 mL). The com-
bined organic phases were washed several times with H2O, dried
(Na2SO4), and concentrated. The crude mixtures of the reac-
tions carried out in [bmim][PF6] were extracted with AcOEt (3 ꢀ
5 mL). The combined organic phases were dried (Na2SO4) and
concentrated. For the reactions performed in toluene, the solvent
was simply evaporated off. The resulting residues were eluted from a
column of silica gel with the suitable elution system. Yields are
reported in Tables 1-4.
(E/Z)-Methyl 2-(Oct-1-enylthio)acetate (3). Column chroma-
tography with 5:1 hexanes-AcOEt afforded 3 as a 1.2:1 mixture
of E and Z isomers. GC-MS: Z-3, r.t. 8.18, m/z 216 (Mþ, 25),
145 (59), 143 (39), 109 (59), 85 (47), 71 (100), 55 (44); E-3, r.t.
8.23, m/z 216 (Mþ, 39), 145 (74), 143 (40), 109 (50), 85 (42), 71
analyses of both (E) and (Z) isomers of glycopeptide 27
(Supporting Information). Inevitably, the use of a 5-fold excess
of sugar alkyne 24 was required to drive the coupling to com-
pletion (LC-MS analysis). This result, however, appears quite
satisfactory if compared to that obtained in the parallel TEC
study.24 In that occasion, even a 30-fold excess of a peracetylated
allyl C-glycoside was required to achieve complete conversion of
the same peptide 26. It should be noted, however, that a detailed
comparison between the two methodologies is complicated at
this stage by too many variables, and therefore, it will be the
object of a dedicated study.
1
(100), 55 (37). H NMR (400 MHz): δ = 6.02-5.94 (m, 1 H,
H-10), 5.77 (ddd, 0.55 H, J2 ,3 a = 7.0 Hz, J2 ,3 b = 7.1 Hz, J1 ,2
0
=
=
0
0
0
0
0
14.9 Hz, H-20(E)), 5.65 (ddd, 0.45 H, J2 ,3 a = 7.3 Hz, J2 ,3 b
0
0
0
0
0
0
0
7.4 Hz, J1 ,2 = 9.5 Hz, H-2 (Z)), 3.74 (s, 3 H, OMe), 3.35 (s, 2 H,
2H-2), 2.17-2.04 (m, 2 H, 2H-30), 1.42-1.20 and 0.92-0.80
(2 m, 11 H, 2H-40, 2H-50, 2H-60, 2H-70, 3H-80). 13C NMR (100
MHz): δ = 170.3, 134.3, 131.9, 122.7, 120.7, 52.5, 52.4, 35.1,
35.0, 33.0, 31.9, 31.7, 31.6, 29.7, 29.3, 29.0, 28.9, 28.7, 22.7, 22.6,
14.1, 14.0. ESI MS (216): 239 (M þ Naþ). HRMS (ESI/Q-TOF):
calcd m/z for C11H20NaO2S [M þ Na]þ, 239.1082; found,
239.1087.
Conclusions
(R/S)-Methyl 2-[(1-[(2-Methoxy-2-oxoethyl)sulfanyl]methyl-
heptyl)sulfanyl]acetate (4). Column chromatography with 5:1 hex-
anes-AcOEt afforded 4 as a yellow oil. GC-MS: r.t. 11.52, m/z322
(Mþ, <1%), 263 (7), 216 (40), 203 (47), 146 (19), 143 (73), 110 (26),
107 (29), 97 (35), 87 (27), 69 (46), 55 (100). 1H NMR (600 MHz):
δ = 3.71 (s, 3 H, OMe), 3.70 (s, 3 H, OMe), 3.32 and 3.21 (2 d, 2 H,
J = 14.5 Hz, CH2CO), 3.25 and 3.21 (2 d, 2 H, J = 14.7 Hz,
CH2CO), 3.00-2.91 (m, 2 H, H-1a, H-2), 2.81-2.75 (m, 1 H, H-1b),
1.80-1.70, 1.50-1.40, 1.38-1.10 (3 m, 10 H, 2H-3, 2H-4, 2H-5, 2H-
6, 2H-7), and 0.85 (t, 3 H, J = 7.0, 3H-8). 13C NMR (150 MHz):
δ = 171.2, 171.0, 51.5, 51.4, 44.8 (C-2), 37.1 (C-1), 32.7, 32.2, 31.5,
30.6, 28.0, 25.5, 21.5, 13.0. ESI MS (322): 345 (M þ Naþ). HRMS
(ESI/Q-TOF): calcd m/z for C14H26NaO4S2 [M þ Na]þ, 345.1170;
found, 345.1173.
(R/S)-Methyl 2-(2-[(2-Methoxy-2-oxoethyl)sulfanyl]-1-phenyl-
ethylsulfanyl)acetate (8). Column chromatography with 5:1
hexanes-AcOEt afforded 8 as a yellow oil. GC-MS: r.t. 12.05, m/z
314 (Mþ, <1), 208 (55), 195 (100), 177 (18), 149 (38), 135 (29), 121
(97), 115 (20), 104 (27), 91 (25), 77 (18). 1H NMR (400 MHz): δ =
7.36-7.24(m,5H, Ph),4.26(t, 1H,J1,2 =7.8Hz, H-1),3.71(s,3H,
OMe), 3.67 (s, 3 H, OMe), 3.15 (d, 2 H, 2H-2), 3.14 and 3.08 (2 d,
2 H, J = 14.7 Hz, CH2CO), 3.13 and 2.98 (2 d, 2 H, J = 15.1 Hz,
CH2CO). 13C NMR (100 MHz): δ = 170.4 (2C), 139.4, 129.1,
128.5, 128.2, 128.0, 127.8, 52.2 (2C), 49.3, 37.7, 33.4, 32.6. ESI MS
Our explorative study showed that the radical alkanethiol/
terminal alkyne coupling reactions are strongly affected by
the adopted experimental conditions, including thiol/alkyne
molar ratio, temperature and, above all, solvent. A proper
choice of the reaction conditions can hence favor highly selective
occurrence of either mono- or bis-sulfide coupling product. This
study also showed that an aromatic alkyne, besides being much
more reactive than an aliphatic congener, has a notably en-
hanced propensity to form the monocoupling product exclusive
of the double-coupling one. Our present observations, which are
unprecedented in the reported studies of TYC reactions in the
fields of bioconjugation and/or material derivatization, may
possibly pave the way to new important applications of
the TYC strategy in those fields. The findings were preliminarily
exploited in successful glycosylation of cysteine derivatives as
well as in the production of a bis-armed cysteine through dual
labeling of an alkynyl sugar in a TYC/TEC sequence. Further,
the appealing behavior of aromatic alkynes in TYC was reward-
ingly applied to glycosylation of the native form of GSH just
using virtually equimolar amounts of a sugar bearing an aryla-
cetylene moiety and, more importantly, to a similar glycosylation
J. Org. Chem. Vol. 76, No. 2, 2011 457

Minozzi et al.
JOCArticle
(314): 337 (M þ Naþ). HRMS (ESI/Q-TOF): calcd m/z for
0.26 mmol), 2,2-dimethoxy-2-phenyl-acetophenone (7 mg,
0.026 mmol), and DMF (1.5 mL) was irradiated at room tempera-
ture for 1 h under magnetic stirring, and then concentrated. The
resulting residue was eluted from a column of silica gel with (i-Pr)2O
and then with 9:1 (i-Pr)2O/AcOEt to give 17 (179 mg, 88%) as 1.5:1
C14H18NaO4S2 [M þ Na]þ, 337.0544; found, 337.0548.
Methyl 2-(1-[(2-Methoxy-2-oxoethyl)sulfanyl]-2-phenylethyl-
sulfanyl)acetate (9). Column chromatography with 5:1 hexanes-
AcOEt afforded 9 as a yellow oil. GC-MS: r.t. 11.80, m/z 314 (Mþ,
<1), 241 (27), 223 (50), 209 (26), 177 (48), 149 (100), 135 (50),
115(38), 103(14), 91(51). 1HNMR(400 MHz):δ=7.35-7.22(m,
5 H, Ph), 4.38 (t, 1 H, J1,2 = 7.1 Hz, H-1), 3.70 (s, 6 H, OMe), 3.45
1
mixture of E and Z isomers. H NMR (300 MHz, DMSO-d6,
120 °C, mixture of E and Z isomers): δ = 7.86 (d, 2 H, J = 7.4 Hz,
Ar), 7.68 (d, 2 H, J = 7.4 Hz, Ar), 7.46-7.39 and 7.38-7.30 (2 m,
and 3.22 (2 d, 4 H, J = 15.2 Hz, 2 CH2CO), 3.13 (d, 2 H, 2H-2). 13
C
4 H, Ar), 7.14 (bs, 1 H, NH), 6.360(ddd, 0.6 H, J1 ,3a = 0.5 Hz,
0
0
0
J1 ,3b =0.6Hz, J1 ,2 = 15.0 Hz, H-1 (E)), 6.32(ddd, 0.4H, J1 ,3a
0
0
0
0
0
NMR (100 MHz): δ = 170.6 (2C), 137.5, 129.3 (2C), 128.4 (2C),
127.0, 53.8, 52.4 (2C), 42.2, 32.4 (2C). ESI MS (314): 337 (M þ
Naþ). HRMS (ESI/Q-TOF): calcd m/z for C14H18NaO4S2 [M þ
Na]þ, 337.0544; found, 337.0549.
=
0
0
0
0
0
0.5 Hz, J1 ,3b =0.6Hz,J1 ,2 =9.5Hz,H-1 (Z)),5.75-5.60 (m, 1 H,
H-20), 5.28-5.15 (m, 1 H, H-300), 4.94 (dd, 0.4 H, J3 ,4 = 9.0 Hz,
00 00
00
00 00
00 00
00 00
J4 ,5 = 9.5 Hz, H-4 (Z)), 4.92 (dd, 0.6 H, J3 ,4 = 9.0 Hz, J4 ,5
=
(2R,E/Z)-Methyl 2-Acetamido-3-(oct-1-enylthio)propanoate
(11). Column chromatography with 2:1 hexanes-AcOEt afforded
11 as a 1.5:1 mixture of Zand Eisomers. GC-MS: Z-11, r.t. 9.41, m/
z287 (Mþ,1),228(22),177(8),144(16),111(13),87(26),81(11),69
(38), 55 (32), 43 (100); E-11, r.t. 9.56, m/z 287 (Mþ, 2), 228 (18), 177
(5), 144 (14), 111 (13), 87 (14), 81 (15), 69 (33), 55 (34), 43 (100). 1H
NMR(400 MHz): δ = 6.33 (bd, 1 H, J = 7.0 Hz, NH), 5.90-5.70
9.5 Hz, H-400(Z)), 4.85-4.70 (m, 1 H, H-100, H-200), 4.40-4.32 (m,
2 H, FmocCH2), 4.30-4.06 and 4.00-3.88 (2 m, 7 H, FmocCH,
H-2, 2 H-30, H-500, 2 H-600), 3.20-2.80 (m, 2 H, 2 H-3), 2.05-1.92
(8 s, 12 H, CH3), 1.45 (s, 9 H, t-Bu). 13C NMR (75 MHz, mixture of
E and Z isomers and conformers, selected data): δ = 170.3, 169.3,
115.6, 143.8, 141.3,128.5, 127.7, 125.1, 123.7, 120.0, 99.2, 83.2, 72.8,
71.7, 713, 69.3, 68.4, 67.2, 65.5, 61.9, 54.8, 54.1, 47.1, 37.0, 35.2, 27.9,
20.7. ESI MS (785): 803 (M þ NH4þ). HRMS (ESI/Q-TOF): calcd
m/z for C39H51N2O14S [M þ NH4]þ, 803.3050; found, 803.3061.
(2R,20R/S,200R)-tert-Butyl 2-(((9H-Fluoren-9-yl)methoxy)car-
bonylamino)-3-(10-(200-((9H-fluoren-9-yl)methoxy)carbonylamino)-
300-tert-butoxy-300-oxopropylthio)-30-((2000,3000,4000,6000-tetra-O-acetyl-
β-D-glucopyranosyl)oxypropan-20-ylthio)propanoate (18). To a
mixture of cysteine derivative 16 (155 mg, 0.39 mmol), propar-
gyl glycoside 15 (75 mg, 0.20 mmol), and 2,2-dimethoxy-2-phenyl-
acetophenone (10 mg, 0.039 mmol) in toluene (2 mL) was added
H2O (19 mL). The resulting dispersion was irradiated at room
temperature for 2 h under vigorous magnetic stirring, then concen-
trated, and eluted from a column of silica gel with (i-Pr)2O and then
with 9:1 (i-Pr)2O/AcOEt to give 18 (206 mg, 85%) as a ∼1:1 mixture
of C20-diastereoisomers. 1H NMR (300 MHz, DMSO-d6, 120 °C,
mixture of diastereoisomers): δ=7.86(d, 4H, J= 7.4 Hz, Ar), 7.68
(d, 4 H, J = 7.4 Hz, Ar), 7.45-7.36 and 7.35-7.28 (2 m, 8 H, Ar),
(m, 1.4 H, H-10(E), H-10(Z), H-20(E)), 5.58 (ddd, 0.6 H, J2 ,3a
0
0
=
0
0
0
0
0
7.2 Hz, J2 ,3 b = 7.3 Hz, J1 ,2 = 9.3 Hz, H-2 (Z), 4.91-4.83 (m, 1 H,
H-2), 3.75 (s, 3 H, OMe), 3.23-3.04 (m, 2 H, 2H-3), 2.17-2.00 (m,
2 H, 2H-30), 1.42-1.20 and 0.98-0.84 (2 m, 11 H, 2H-40, 2H-50,
2-H60, 2H-70, 3-H80). 13C NMR (100 MHz, selected data): δ =
170.8, 169.7, 134.2, 131.4, 123.8, 121.5, 52.6, 52.5, 52.1, 36.0, 35.3,
33.1, 31.6, 31.5, 29.0, 28.9, 28.8, 28.7, 23.1, 22.6, 22.5, 14.0. ESI MS
(287): 310 (M þ Naþ). HRMS (ESI/Q-TOF): calcd m/z for
C14H25NNaO3S [M þ Na]þ, 310.1453; found, 310.1457.
Dimethyl (4R,11R)-7-Hexyl-2,13-dioxo-6,9-dithia-3,12-dia-
zatetradecane-4,11-dicarboxylate (12). Column chromatography
with 1:2 hexanes-AcOEt afforded 12 as a 1:1 mixture of C2
diastereoisomers. GC-MS: r.t. ∼25 (very broad peak), m/z 320
(Mþ-N-Ac-Cys, 2%), 288 (18), 287 (13), 246 (14), 232 (17), 228
(28), 176 (34), 144 (9), 43 (100). 1H NMR (400 MHz): δ = 6.64 (t,
1 H, J = 7.2 Hz, NH), 6.54 (t, 1 H, J = 7.9 Hz, NH), 4.89-4.79 (m,
2H, H-20, H-200), 3.78 (s, 6H, OMe), 3.10-2.91 and 2.84-2.65 (2 m,
7 H, 2H-1, H-2, 2H-30, 2H-300), 2.07 and 2.06 (2 s, 6 H, C(O)Me),
1.78-1.60, 1.52-1.40, and 1.39-1.20 (3 m, 10 H, 2H-3, 2H-4, 2H-5,
2H-6, 2H-7), 0.88(t, 3 H, J = 6.7 Hz, 3H-8). 13C NMR (100 MHz,
selected data): δ = 171.3, 171.2 (2C), 170.0, 169.9, 52.7, 52.6, 52.3,
52.1, 52.0, 46.0, 38.7 (2C), 35.1, 35.0, 34.0, 33.9, 32.9, 32.7, 31.6, 29.0,
26.7, 26.6, 23.0, 22.5, 14.0. ESI MS (464): 487 (M þ Naþ). HRMS
(ESI/Q-TOF): calcd m/z for C20H36N2NaO6S2 [M þ Na]þ,
487.1912; found, 487.1921.
000,4000
000,3000
= 9.0 Hz, J2
7.06 (bs, 2 H, NH), 5.22 (dd, 0.5 H, J3
=
=
9.5 Hz, H-3000(diast. I)), 5.20 (dd, 0.5 H, J3
000,4000
000,3000
= 9.0 Hz, J2
9.5 Hz, H-3000(diast. II)), 4.93 (dd, 0.5 H, J4
= 9.0 Hz, H-4000-
000,5000
000,5000
(diast. I)), 4.92 (dd, 0.5 H, J4
= 9.0 Hz, H-4000(diast. II)),
4.86-4.74 (m, 2 H, H-1000, H-2000), 4.44-3.89 (m, 12 H, 2 FmocCH,
2 FmocCH2, H-2, H-200, H-30a, H-5000, 2 H-6000), 3.82 (dd, 0.5 H,
J
J
2 ,3 b = 5.0 Hz, J3 a,3 b = 10.5 Hz, H-30b (diast. I), 3.70 (dd, 0.5 H,
0 0 0 0
2 ,3 b = 5.5 Hz, J3 a,3 b = 10.5 Hz, H-30b (diast. II), 3.20-2.70 (m,
0
0
0
0
5 H, 2 H-3, 2 H-100, H-20), 2.02-1.92 (8 s, 12 H, CH3), 1.46-1.42
(4 s, 18 H, t-Bu). 13C NMR (75 MHz, mixture of diastereoisomers
and conformers, selected data):δ= 170.6, 169.4, 155.8, 143.8, 141.3,
127.7, 127.0, 125.1, 120.0, 101.2, 100.8, 83.0, 82.9, 72.7, 72.6, 71.8,
71.0, 68.3, 67.2, 61.8, 54.6, 53.4, 47.0, 34.4, 28.0, 20.7, 20.6. ESI MS
(1184): 1203 (M þ NH4þ). HRMS (ESI/Q-TOF): calcd m/z for
C61H76N3O18S2 [M þ NH4]þ, 1202.4560; found, 1202.4551.
(2R,E/Z) Methyl 2-Acetamido-3-(styrylthio)propanoate (14).
Column chromatography with AcOEt afforded 14 as a ∼1:1
mixture of E and Z isomers (reported NMR spectra are for a 4:1
Z/E mixture). GC-MS: Z-14, r.t. 10.27, m/z 279 (Mþ, 14), 220
(45), 161 (23), 144 (63), 134 (43), 116 (28), 115 (54), 98 (72), 91
(46), 84 (17), 77 (23), 43 (100); E-14, r.t. 10.52, m/z 279 (Mþ, 14),
220 (49), 161 (25), 144 (62), 134 (47), 116 (28), 115 (61), 98 (74),
(2R,20R/S,200R)-Methyl 2-((tert-Butoxycarbonylamino)-3-(10-
(200-((9H-fluoren-9-yl)methoxy)carbonylamino)-300-tert-butoxy-
300-oxopropylthio)-30-(2000,3000,4000,6000-tetra-O-acetyl-β-D-gluco-
pyranosyl)oxypropan-20-ylthio)propanoate (20). To a mixture of
alkene 17 (150 mg, 0.19 mmol), cysteine derivative 19 (90 mg, 0.38
mmol), 2,2-dimethoxy-2-phenyl-acetophenone (10 mg, 0.038 mmol)
in toluene (2.0 mL) was added H2O (19 mL). The resulting
dispersion was irradiated at room temperature for 2 h under
magnetic stirring, and then concentrated. The resulting residue was
eluted from a column of silica gel with 8:1 (i-Pr)2O/AcOEt to give 20
(146 mg, 75%) as a ∼1:1 mixture of C20-diastereoisomers. 1H NMR
(300 MHz, DMSO-d6, 120 °C, mixture of diastereoisomers): δ =
7.86 (d, 2 H, J = 7.4 Hz, Ar), 7.70 (d, 2 H, J = 7.4 Hz, Ar),
7.45-7.38 and 7.37-7.30 (2 m, 4 H, Ar), 7.14 (bs, 1 H, NH), 6.60
1
91 (51), 84 (17), 77 (25), 43 (100). H NMR (400 MHz): δ =
0
0
7.46-7.16 (m, 5 H, Ph), 6.64 and 6.56 (2 d, 1 H, J1 ,2 = 15.6 Hz,
H-10(E), H-20(E), 6.42 and 6.12 (2 d, 1 H, J1 ,2 = 10.7 Hz,
H-10(Z), H-20(Z), 6.38 (d, 1 H, J = 7.1 Hz, NH), 4.97-4.88 (m,
1 H, H-2), 3.77 (s, 1.5 H, OMe), 3.72 (s, 1.5 H, OMe), 3.38-3.21
(m, 2 H, 2H-3), 2.03 (s, 3 H, C(O)Me), 2.00 (s, 3 H, C(O)Me). 13C
NMR (100 MHz, selected data): δ = 171.0 (E), 170.7 (Z), 170.5
(E), 169.8 (Z), 136.3, 129.4, 128.6, 128.5, 128.1, 127.3, 126.9,
126.5, 126.2, 125.5, 123.7, 60.3, 52.6, 52.4, 52.2, 37.7, 35.0, 22.9,
20.9, 14.0. ESI MS (279): 302 (M þ Naþ). HRMS (ESI/Q-TOF):
calcd m/z for C14H17NNaO3S [M þ Na]þ, 302.0827; found,
302.0830.
0
0
(2R,E/Z)-tert-Butyl 2-(((9H-Fluoren-9-yl)methoxy)carbonyl-
amino)-3-(30-(200,300,400,600-tetra-O-acetyl-β-D-glucopyranosyl)-
oxyprop-1-enylthio)propanoate (17). A mixture of propargyl glyco-
side 15 (300 mg, 0.78 mmol), cysteine derivative 16 (103 mg,
000,4000
000,3000
= 9.5 Hz,
(bs, 1 H, NH), 5.35 (dd, 0.5 H, J3
= 9.0 Hz, J2
H-3000(diast. I)), 5.90(d, 0.5H,J1
=7.5Hz, H-1000 (diast. I),5.24
= 9.5 Hz, H-3000(diast. II)),
000,2000
000,4000
(dd, 0.5 H, J3
000,3000
= 9.0 Hz, J2
458 J. Org. Chem. Vol. 76, No. 2, 2011

Minozzi et al.
JOCArticle
5.00-4.75 (m, 2.5 H, H-2000, H-4000, H-1000 (diast. II), 4.40-4.32,
4.28-4.06, 4.00-3.90, and 3.80-3.64 (4 m, 13 H, FmocCH,
FmocCH2, H-2, H-200, 2 H-30, H-5000, 2 H-6000, OCH3), 3.10-2.80
(m, 5 H, H-3, H-100), 2.05-1.94 (8 s, 12 H, CH3), 1.46 (s, 9 H, t-Bu),
1.43 and 1.42 (2 s, 9 H, t-Bu). 13C NMR (75 MHz, mixture of
diastereoisomers and conformers, selected data): δ = 170.7, 169.4,
155.8, 155.2, 143.8, 141.3, 127.7, 127.1, 125.2, 120.0, 101.2, 100.8,
91.7, 83.0, 72.8, 72.6, 71.9, 71.0, 68.3, 68.2, 67.2, 61.8, 61.4, 60.4, 54.7,
53.4, 52.6, 47.0, 46.8, 45.8, 40.3, 35.9. 29.7, 28.3, 28.0, 21.0, 20.9. ESI
MS (1020): 1038 (M þ NH4þ). HRMS (ESI/Q-TOF): calcdm/z for
C48H68N3O18S2 [M þ NH4]þ, 1038.3934; found, 1038.3922.
(dd, 1 H, J5
b = 5.5 Hz, H-6000b), 3.57 (t, 1 H, J = 7.0 Hz, H-10),
000,6000
3.40-3.10 (m, 5 H, H-2000, H-3000, H-4000, H-5000, H-10a), 3.03 (dd,
1 H, J1 b,5 = 8.0 Hz, J1 a,1 b = 14.5 Hz, H-10b), 2.36-2.28 (m, 2 H,
0
0
0
1
2 H-8), 2.05-1.90 (m, 2 H, 2 H-9). H NMR (400 MHz, D2O,
(Z)-isomer; selected data): δ= 6.49 and 6.22 (2 d, 2 H, J = 10.8 Hz,
000,2000
CHdCH), 4.37 (d, 1 H, J1
= 8.0 Hz, H-1000). 13C NMR
(75 MHz, D2O, (E/Z)-isomers; selected data): δ = 174.9, 173.8,
172.4, 116.7, 135.9, 129.5, 129.0, 127.3, 126.0, 124.3, 101.3, 76.1, 76.0,
73.3, 71.3, 69.9, 61.0, 54.0, 53.7, 41.8, 33.8, 31.4, 26.1. ESI MS (601):
619 (M
þ
NH4þ). HRMS (ESI/Q-TOF): calcd m/z for
C25H39N4O12S [M þ NH4]þ, 619.2280; found, 619.2275. The
LC-MS analysis of the reaction mixture was performed to establish
the full conversion of glutathione and confirm the E/Z ratio of 25
(see Figure S2). The LC MS/MS analysis of 25 was also performed
to confirm the selective glycosylation of the cysteine residue of
glutathione (see Figure S3).
Glycopeptide 23. A mixture of sugar alkyne 21 (272 mg,
1.25 mmol), reduced glutathione 22 (77 mg, 0.25 mmol), 2,2-
dimethoxy-2-phenyl-acetophenone (6 mg, 0.025 mmol), MeOH
(0.1 mL), and H2O (1.9 mL) was irradiated at room temperature
for 1 h under magnetic stirring, and then concentrated. The
resulting white solid residue was suspended in MeOH (2 mL)
and triturated with portions of MeOH (3 ꢀ 2 mL) which were
pipetted off. The residue left after trituration was eluted from a
column of Sephadex LH20 with 3:1 H2O-MeOH to give 23 (102
mg, 78%) as a 8:1 mixture of (E)- and (Z)-isomers (reported
NMR spectra are for a 2:1 E/Z mixture). The occurrence of the
E-isomer as the major compound was confirmed by analysis of
the vinyl protons region of the 1H NMR spectrum of the crude
Glycopeptide 27. A solution of sugar alkyne 24 (13.8 mg,
0.047 mmol) and DMPA (1.2 mg, 4.71 μmol) in DMSO (0.25 mL)
was added to a solution of peptide 26 (10.0 mg, 9.41 μmol) in 20 mM
phosphate buffer (pH 7.4, 5.0 mL). The resulting solution was
irradiated at room temperature for 1 h under magnetic stirring, and
then lyophilized. The resulting residue was eluted from a column of
Sephadex LH20 with H2O to give 27 (8.0 mg, 68%) as a 2:1 mixture
of (E)- and (Z)-isomers but slightly contaminated by excess sugar
alkyne (reported 1H NMR spectrum is for a ∼1:2 E/Zmixture). The
occurrence of the E-isomer as the major compound was confirmed
by analysis of the vinyl protons region of the 1H NMR spectrum of
1
reaction mixture. H NMR (300 MHz, D2O, (E/Z)-isomers):
δ = 6.25 (d, 0.66 H, J = 15.0 Hz, CHdCH (E)), 6.19 (d, 0.33 H,
J = 10.5 Hz, CHdCH (Z)), 5.70-5.60 (m, 1 H, CHdCH),
1
000,2000
4.48-4.40 (m, 1 H, H-5), 4.30 (d, 0.66 H, J1
= 8.0 Hz,
the crude reaction mixture. H NMR (400 MHz, D2O, (E/Z)-
H-1000(E)), 4.28 (d, 0.33 H, J1
= 8.5 Hz, H-1000(Z)),
isomers, selected data): δ = 6.64 and 6.58 (2 d, 066 H, J = 13.5 Hz,
CHdCH(E)), 6.44 and 6.165 (2 d, 1.32 H, J = 9.5 Hz, CHdCH-
(Z)). The LC-MS analysis of the reaction mixture was performed
to establish the full conversion of peptide 26 and confirm the E/Z
ratio of 27 (see Figure S4). The LC MS/MS analyses of peptide 26
and glycopeptide 27 were performed to confirm the selective
glycosylation of the cysteine residue of 26 (see Figures S5 and S6).
000,2000
4.22-4.00 (m, 2 H, 2 H-300), 3.80-3.50 (m, 6 H, 2 H-2, H-10,
H-5000, 2 H-6000), 3.34-3.04 (m, 4 H, H-10a, H-2000, H-3000, H-4000),
2.94-2.82 (m, 1 H, H-10b), 2.40-2.30 (m, 2 H, 2 H-8), 2.02-1.94
(m, 2 H, 2 H-9).13C NMR (75 MHz, D2O, (E/Z)-isomers;
selected data): δ = 174.7, 174.6, 173.8, 173.6, 172.1, 172.0,
129.7, 129.0, 128.3, 124.8, 124.4, 101.1, 100.7, 75.9, 75.8, 75.7,
72.9, 69.5, 69.4, 65.6, 60.6, 53.7, 52.9, 41.7 31.1, 25.9. ESI MS
(525): 526 (M þ Hþ). HRMS (ESI/Q-TOF): calcd m/z for
C19H32N3O12S [M þ H]þ, 526.1707; found, 526.1701. The LC-
MS analysis of the reaction mixture was performed to establish
the full conversion of glutathione and confirm the E/Z ratio of
23 (see Figure S1).
Acknowledgment. A.Ma. and N.M. gratefully acknowl-
edge the University of Ferrara (Progetto FAR 2009) and the
Italian Ministry of University and Scientific Research (Progetto
FIRB Chem-Profarma-Net Grant RBPR05NWWC 008) for
financial supports. A.Mo., M.M., D.N., and P.S. gratefully
acknowledge financial support from the Italian Ministry of
University and Scientific Research (2008 PRIN funds for “Prop-
erties and reactivity of free radicals in complex environments and
their role in oxidative processes and in organic synthesis”).
Thanks are also given to Mr. Paolo Formaglio for NMR
experiments.
Glycopeptide 25. A mixture of sugar alkyne 24 (79 mg, 0.27
mmol), reduced glutathione 22 (75 mg, 0.24 mmol), 2,2-
dimethoxy-2-phenyl-acetophenone (6 mg, 0.024 mmol), and
MeOH (0.5 mL), and H2O (1.5 mL) was irradiated at room tem-
perature for 1 h under magnetic stirring, and then concentrated.
The resulting residue was eluted from a column of Sephadex
LH20 with 1:1 H2O-MeOH to give 25 (120 mg, 82%) as a 6:1
mixture of (E)- and (Z)-isomers. The occurrence of the E-isomer
as the major compound was confirmed by analysis of the vinyl
protons region of the 1H NMR spectrum of the crude reaction
mixture. 1H NMR (400 MHz, D2O, (E)-isomer): δ = 7.40-7.20
(m, 4, H, Ar), 6.72 and 6.54 (2 d, 2 H, J = 15.6 Hz, CHdCH),
4.74 and 4.57 (2 d, 2 H, J = 11.7 Hz, 2 H-20), 4.56-4.50 (m, 1 H,
Supporting Information Available: General experimental
methods, synthesis of 24, LC ESI-IT MS analyses of glycopep-
tides 23, 25, and 27; copies of 1H and 13C NMR spectra of 3, 4,
7-9, 11, 12, 14, 17, 18, 20, 23, peracetylated-24, 24, 25, and
27. This material is available free of charge via the Internet at
http://pubs.acs.org.
H-5), 4.36 (d, 1 H, J1
=8.0Hz, H-1000), 3.77(dd, 1H, J5
000,2000
000,6000
a
=
= 12.5 Hz, H-6000a), 3.74 (s, 2 H, 2H-2), 3.58
000a,6000
2.0 Hz, J6
b
J. Org. Chem. Vol. 76, No. 2, 2011 459