Native chemical ligation, thiol-ene click: A methodology for the synthesis of functionalized peptides

Article abstract of DOI:10.1021/jo4001542

The sequential combination of native chemical ligation and thiol-ene radical chemistry (NCL-TEC) on the resulting cysteine thiol has been investigated as a methodology for rapidly accessing functionalized peptides. Three sequential cycles of native chemical ligation and subsequent thiyl radical reactions (including a free-radical-mediated desulfurization reaction) were carried out on a peptide backbone demonstrating the iterative nature of this process. The versatility of the thiyl radical reaction at cysteine was demonstrated through the introduction of a number of different side chains including an amino acid derivative, a carbohydrate group, and an alkyl azide. Conditions were developed that allowed the sequential NCL-TEC process to proceed in high yield.

Full text of DOI:10.1021/jo4001542

Article
pubs.acs.org/joc
Native Chemical Ligation,Thiol−Ene Click: A Methodology for the
Synthesis of Functionalized Peptides
Lyn Markey, Silvia Giordani,* and Eoin M. Scanlan*
Trinity Biomedical Sciences Institute, Trinity College, 152-160 Pearse Street, Dublin 2, Ireland
S
* Supporting Information
ABSTRACT: The sequential combination of native chemical ligation and thiol−ene radical chemistry
(NCL-TEC) on the resulting cysteine thiol has been investigated as a methodology for rapidly accessing
functionalized peptides. Three sequential cycles of native chemical ligation and subsequent thiyl radical
reactions (including a free-radical-mediated desulfurization reaction) were carried out on a peptide
backbone demonstrating the iterative nature of this process. The versatility of the thiyl radical reaction at
cysteine was demonstrated through the introduction of a number of different side chains including an
amino acid derivative, a carbohydrate group, and an alkyl azide. Conditions were developed that allowed
the sequential NCL-TEC process to proceed in high yield.
ligation and thiol−ene click chemistry could be applied to the
INTRODUCTION
■
general synthesis of highly functionalized S-linked peptides,
suitable for further conjugation onto nanomaterials. Simple
amino acid building blocks were employed for the initial model
studies, but we envisaged that SPPS could be used to access
more complex building blocks for further study.
Covalent modification of peptides and proteins is important for
understanding biological function and for the development of
new therapeutics. Free-radical-based methodologies at cysteine
are emerging as powerful tools for the chemoselective
modification of both peptides and proteins.1,2 The alkene
hydrothiolation reaction, also called the thiol−ene coupling
reaction (TEC), has allowed the site-specific elaboration of
peptides and proteins with a range of biomolecules including
carbohydrates3,4 and lipids.5 The methodology is high yielding
and is compatible with oxygen and an aqueous environment.
This methodology has been demonstrated to be efficient on
large peptides3 and can function as a synthetic equivalent to
post translational modification of proteins. The resulting
thioether linkage is robust and biologically stable.6
The ligation reaction between an unprotected peptide
thioester fragment and a second unprotected peptide
possessing an N-terminal cysteine that results in formation of
an amide bond is called native chemical ligation (NCL).7−10
This ligation methodology results in peptide fragments linked
by a cysteine residue and has allowed for the assembly of
peptides and proteins beyond the scope of current synthetic
SPPS methods.11,12 Because of their low abundance in nature,
the cysteine residues are often unwanted and are transformed
by desulfurization reactions to furnish the more abundant
alanine residue.13,14 However, the cysteine residue also provides
a synthetic handle for free-radical-based peptide functionaliza-
tion.3,5
Here we report the results of our investigation into
sequential NCL-thiyl radical reactions on a short peptide
backbone. The ultimate goal of this study was to realize an
iterative methodology that would allow continuous cycles of
NCL and thiyl radical chemistry to be carried out efficiently on
synthetic peptides. While there exists an abundance of
methodologies for the functionalization of peptides and
proteins at cysteine,17 the hydrothiolation reaction was chosen
due to its high yield and general applicability. Sequential NCL-
TEC, as a methodology for the synthesis of functionalized
peptides and proteins, is highly desirable in that both reactions
are high yielding and can be carried out in an aqueous
environment. Scheme 1 outlines the general iterative strategy
for the NCL-TEC process.
Following the NCL reaction between two peptides, the
resulting cysteine thiol is functionalized using a thiyl radical
reaction to introduce a robust thiother linkage. This can be
considered to be a thio-analogue of serine, a common
glycosylation site in glycoproteins. The carboxyl terminus on
the new glycopeptide is then converted to the reactive thioester
for the next sequential NCL step. Iterative cycles of NCL and
TEC can be repeated in order to furnish the extended
functionalized peptide or protein. Sequential NCL reactions
have previously been applied to the synthesis of long chain
As part of an ongoing research program into the synthesis of
glycosylated therapeutics15 and functionalized nanomaterials,16
we became interested in accessing functionalized peptides
containing both carbohydrate units and fluorescent probes. We
set out to investigate if a combination of native chemical
Received: January 28, 2013
© XXXX American Chemical Society
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Scheme 1. Strategy for Sequential NCL-TEC Methodology
Scheme 2. Proposed Mechanism of Native Chemical
Ligation
products.. We anticipated that any residual phosphine
compounds, phosphine oxides, free thiol byproducts, or
disulfides present following NCL could interfere with the
thiol−ene reaction outlined in Scheme 3.
Scheme 3. Mechanism of Thiol−Ene “Click” Reaction
peptides18−20 and methodology has been developed to
introduce thioesters onto the C-terminus of the growing
peptide chain in the presence of a range of functional groups.
To the best of our knowledge, sequential NCL-thiol−ene
coupling reactions remain unexplored. In this study, we aimed
to carry out three successive NCL-thiyl radical mediated
reactions on a peptide backbone in order to demonstrate the
iterative nature of this process.
The sequential NCL-desulfurization reaction to furnish
alanine linked peptide fragments has been reported by several
groups; indeed, Danishefsky and co-workers have reported a
mild desulfurization reaction that proceeds by way of a thiyl
radical intermedicate in the conversion of cysteine to alanine
following the NCL process.2,21,22 This strategy has been widely
adopted in peptide and protein synthesis.23−25 We reasoned
that the free-radical-based specific desulfurization of cysteine
was appropriate to include within this study since its
mechanism involves a thiyl radical intermediate, and this
reaction can occur readily in the presence of the phosphine
reagents used in NCL. We were also interested in
demonstrating that the conditions employed for the radical-
mediated desulfurization reaction would not affect the thioether
linkage formed in the thiol−ene reaction. It has previously been
demonstrated by Danishefsky and co-workers that the free-
radical desulfurization of cysteine is compatible with
thioethers.2 Initially, with a view toward developing a one-pot
procedure for the NCL-TEC sequence we first considered the
proposed mechanism of NCL,7,8 which is outlined in Scheme 2.
First, trans-thioesterification with the thiol side chain of an
N-terminal cysteine residue results in formation of a thioester-
linked intermediate. This thioester immediately undergoes a
spontaneous rearrangement to give the native peptide bond at
the ligation site. The reaction is normally carried out in aqueous
media and at physiological pH in the presence of a phosphine-
reducing reagent to avoid disulfide formation which would
inhibit the ligation methodology. Byproducts generated by the
NCL process include phosphine oxides and aromatic thiols.
Danishefsky2 and others26−28 have reported that phosphorus-
containing reagents react with thiyl radicals to give desulfurized
RESULTS AND DISCUSSION
■
We first investigated the sequential NCL-TEC process on a
model system (Scheme 4). The NCL reaction between the two
amino acids, Boc-protected alanine thioester 1 and cysteine
methyl ester 2, was first optimized. Native chemical ligation is
usually applied to long peptide fragments but is equally effective
for short peptides. Kent et al. determined that the rate of NCL
is dependent on both the identity of the C-terminal amino acid
derivatives and the thioester.7,8 Alanine was selected at the C-
terminal as it has a small side chain (Me) and should therefore
readily undergo ligation. Tributyl phosphine was employed as a
reducing agent. The NCL reaction proceeded in a yield of 96%
to furnish the dipeptide 3. In order to complete the sequential
NCL-TEC cycle, the dipeptide 3 was subjected to a thiol−ene
coupling reaction with a non-natural amino acid derivative
(fully protected L-homoallylglycine) 4. This residue had
previously been employed for thiol−ene coupling reactions to
furnish glycoproteins.4 The thiol−ene coupling reaction gave
the desired thioether-linked compound 5 in a yield of 63%
(unoptimized). This reaction completed the first sequential
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a
purification step was greatly simplified and the thiol−ene
coupling reaction between 7 and 8 was optimized to furnish
glycopeptide 9 in a 95% isolated yield (Table 1, entry 15).
Scheme 4. NCL-TEC Sequence on a Model System
Table 1. Optimization of TEC Conditions between Peptide
7 and Glycan 8 To Furnish Glycopeptide 9
a
b
entry peptide (equiv) sugar (equiv) initiator MAP conversion (%)
c
1
1.0
3.0
3.0
3.0
3.0
2.0
1.0
1.0
1.1
1.1
1.3
1.5
1.0
1.0
1.3
2
1
1
1
1
1
1
1
1
1
1
1
3
3
1
0.4
0.4
0.6
0.6
0.3
0.2
0.1
0.1
0.1
0.2
X
36
78
c
2
X
c
a
3
X
85
Initial NCL-TEC cycle: (i) MeOH, rt, 1 h, Bu3P, 96%; (ii) 4,4-
azobis(4-cyanovaleric acid) (ACVA) (0.6 equiv), DMF, hν, rt, 2 h,
63%.
c
4
0.6
X
87
d
d
d
d
d
d
5
>99
>99
70
6
X
7
X
NCL-TEC cycle for this system and demonstrated that the
methodology is viable for functionalized peptide synthesis.
Once the thiol−ene reaction between 3 and 4 had been
completed, we carefully studied the effect of NCL byproducts
on the thiol−ene reaction to see if the methodologies were
compatible for a one-pot procedure. First, tributylphosphine
was added to the TEC reaction, and as expected, the competing
desulfurization process was found to dominate. Addition of
tributylphosphine oxide also resulted in significantly diminished
yields of the thiol−ene product. Addition of thiophenol, the
byproduct from the trans-thioesterification step, also inhibited
the TEC reaction. An attempt to couple arylthiols to an alkene
using TEC was unsuccessful. It is likely that they form stable,
unreactive thiyl radicals that inhibit the radical chain process.
We concluded from this model study that a sequential NCL-
TEC process was feasible provided that all traces of NCL
byproducts had been removed since even trace quantities of
phosphorus reagents or thiol contaminants would inhibit the
radical reaction.
In order to further investigate the synthetic scope of this
methodology and to demonstrate its synthetic utility for the
preparation of functionalized peptides, we set out to complete
three sequential NCL-thiyl radical steps on a short peptide
backbone. For the iterative system, it was necessary to use
unprotected cysteine where the C-terminus could be converted
into a thioester following the NCL-thiyl radical sequence
(Scheme 1). For the initial NCL-TEC sequence, peptide
ligation followed by introduction of a carbohydrate group
bearing a terminal alkene at the anomeric position onto the
peptide backbone was investigated. Dondoni and co-workers
have previously demonstrated the thiol−ene coupling reaction
between glycans and cysteine-containing peptides and it has
been reported to proceed in good yield without racemization of
the cysteine.6 Following NCL between 1 and 6, purification of
dipeptide 7 proved difficult and the residual phosphine oxide
hindered the subsequent coupling reaction. Sodium borohy-
dride was investigated as an alternative reducing agent for the
NCL-TEC process since there would be no possibility of trace
phosphine inhibiting the subsequent radical ligation step. A
number of solvent systems were examined for their utility in the
NCL reaction. Sodium borohydride in MeOH was found to be
the most efficient system. When sodium borohydride was
employed as a reducing agent for the NCL reaction the
8
0.1
0.1
0.2
0.13
0.15
X
77
9
83
10
11
12
13
14
15
84
d
d
0.13
0.15
92
>95
84
d
0.1
d
0.1
0.1
93
d
e
0.13
0.13
95
a
b
4-Methoxyacetophenone (MAP). Conversions determined by 1H
NMR. 4,4-Azobis(4-cyanovaleric acid) (ACVA) as radical initiator.
2,2-Dimethoxy-2-phenyl-acetophenone (DPAP) as radical initiator.
c
d
e
Isolated yield.
The optimization study of this reaction addressed some of
the drawbacks associated with recent reports of TEC reactions
for glycopeptides synthesis. In particular, the reported require-
ment for a 3-fold excess of the thiol reagent6 was found to be
unnecessary for this system. Indeed, in our hands, it was
determined that 1.0:1.3 ratio of alkene 8 to thiol 7 furnished the
desired product in 95% yield (entry 15). Using an excess of the
sugar and reducing the equivalents of initiator allowed the
product to be formed in >90% conversion. This means that in
conjugation reactions where a large peptide or protein is
employed that an excess of carbohydrate can be used without
diminishing ligation yields. If the carbohydrate is the limiting
reagent then an excess or stoichiometric equivalent of the
peptide can be used. 4-Methoxyacetophenone (MAP) was
employed as a photosensitizer and found to generally improve
yields of the photochemical reaction. Following the successful
TEC reaction the glycopeptide 9 was converted into the
corresponding thioester 10 for the next NCL-Thiyl radical cycle
(Scheme 5).
The second NCL-thiyl radical process involved a chemical
ligation step between glycopeptide 10 and amino acid 6
followed by a thiyl-radical-mediated desulfurization of the
resulting cysteine linkage to alanine to furnish glycopeptide 12
(Scheme 6). As outlined in the Introduction, Danishefsky and
co-workers have previously reported a sequential NCL-radical
desulfurization reaction on a glycopeptide,2 and we felt this
reaction, which is becoming widely used in peptide synthesis
and which also involves formation a thiyl radical intermediate at
cysteine, was appropriate to include within this study. As was
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a
site. Mannose-containing glycopeptides labeled with fluorescein
have previously been used in cellular immunology screens for
the development of carbohydrate vaccines.29 The thermally
initiated thiol−ene reaction is inefficient for peptide function-
alization,3 and the conditions required for the photochemical
thiol−ene reaction would result in decomposition of the
photosensitive fluorescein. We therefore reasoned that a two-
step procedure would be the most efficient strategy for the
introduction of the fluorophore. In the first step an alkyl azide
group was introduced at the ligation site using the thiol−ene
reaction on 5-azidopent-1-ene to furnish the azide-function-
alized glycopeptide 15 (Scheme 7).
Scheme 5. NCL-TEC Cycle To Furnish Glycopeptide 10
a
Scheme 7. NCL-TEC Cycle To Introduce the Azido Group
a
Key: (i) MeOH, rt, 1 h, NaBH4, 96%; (ii) 4-methoxyacetophenone
(MAP) (0.1 equiv), 2,2-dimethoxy-2-phenylacetophenone (DPAP)
(0.1 equiv), DMF, hν, rt, 2 h, 87%; (iii) THF, 0 °C to rt, isobutyl
chloroformate (1.1 equiv), PhSH (1.2 equiv), N-methylmorpholine
(NMM) (1.2 equiv), 79%.
Scheme 6. NCL-Desulfurization Cycle To Furnish
a
Glycopeptides 12
a
Key: (i) MeOH, rt, 1 h, NaBH4, 85%; (ii) 4-methoxyacetophenone
(MAP) (0.1 equiv), 2,2-dimethoxy-2-phenylacetophenone (DPAP)
(0.1 equiv), DMF, hν, rt, 1 h, 83%.
Despite literature reports that thiyl radicals can add to the
central nitrogen of azides,30 this competing side reaction was
not observed and the desired thiol−ene coupling reaction
proceeded in good yield. The direct photochemical function-
alization of cysteine to furnish an azide-functionalized peptide
or protein has general applications for peptide and protein
modification reactions. The final thiol−ene reaction concluded
the third cycle of NCL-TEC process and demonstrated how
the iterative approach could be applied to functionalized
peptide synthesis. The resulting azide-functionalized glycopep-
tide 15 was then further modified via a copper-catalyzed azide−
alkyne cycloaddition reaction31 that could be carried out in the
absence of light to furnish the fluorescein-labeled glycopeptide
17 (Scheme 8)
a
Key: (i) MeOH, rt, 1 h, NaBH4, 87%; (ii) PBu3, 4,4-azobis(4-
cyanovaleric acid) (ACVA), DMF, 79%; (iii) THF, 0 °C to rt, isobutyl
chloroformate (1.1 equiv), PhSH (1.2 equiv), NMM (1.2 equiv), 60%.
CONCLUSION
■
previously reported by Danishefsky,2 the radical-mediated
desulfurization reaction did not affect the thioether bond,
therefore demonstrating that desulfurization reactions can be
carried out in the presence of this linkage. As part of the
iterative NCL-TEC pathway, conditions can therefore be
selected to allow for a functionalization reaction via the thiyl
radical or a desulfurization reaction to alanine to be carried out
at the point of ligation. The glycopeptide 12 was subsequently
converted to thioester 13 for the final NCL-thiyl radical cycle.
The first two NCL-thiyl radical cycles had demonstrated the
introduction of a a carbohydrate at the ligation site and a
desulfurization at the ligation site. For the final NCL-TEC
cycle, we considered how thiol−ene methodology could be
used to introduce a photosensitive fluorophore at the ligation
In summary, we have developed a methodology that
demonstrates the compatibility of native chemical ligation
and thiyl radical chemistry. We have demonstrated that thiyl
radical chemistry may be used to functionalize the ligation site
to access highly functionalized peptides. The versatility of the
thiyl radical click reaction was demonstrated through the
introduction of amino acid side chains, carbohydrate groups,
and alkyl azides. The thioether products were found to be
compatible with the well-established thiyl-radical-mediated
desulfurization chemistry. The presence of any trace phosphine
or phosphine oxide reagents was found to be detrimental to the
thiylene radical reaction. The use of sodium borohydride as a
reducing agent in the NCL step was found to have no adverse
effect on the subsequent radical reaction. A sequence of three
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chloroformate (1.1 equiv) was added, and the reaction mixture was
stirred for 15 min. Thiophenol (1.2 equiv) was added followed by N-
methylmorpholine (1.2 equiv). The reaction mixture was slowly
warmed to room temperature and stirred overnight. The reaction
mixture was filtered to remove molecular sieves, and the solvent was
removed in vacuo. The reaction mixture was redissolved in DCM and
washed with NaHCO3, H2O, and brine. The organic phase was dried
(MgSO4) and concentrated in vacuo and the product purified by
column chromatography.
Scheme 8. Synthesis of Fluorescein-Labeled Glycopeptide
17
a
General Procedure for Native Chemical Ligation (NCL).
Cysteine·HCl (1.0 equiv) was added to MeOH (0.03 mmol/mL).
NaBH4 (2.0 equiv) was added slowly, and the mixture was stirred for 1
h under N2. Thioester (1.0 equiv) was added in MeOH, and the
reaction mixture was stirred until TLC showed the disappearance of
starting materials (1−4 h). Silica gel was added to the reaction mixture
and solvent removed in vacuo. The product was purified by column
chromatography (60−100% EtOAc/hexane +1% AcOH).
(2S)-2-[(tert-Butyloxycarbonyl)amino]thiopropionic Acid S-
Phenyl Ester (1).33 The general procedure for the synthesis of
thioesters was carried out to prepare 1. The product was purified by
column chromatography (hexane/EtOAc, 9:1, Rf = 0.42) to yield a
white solid (21.22 g, 74%): 1H NMR (400 MHz, CDCl3) δ 7.41 (5H,
br s, CH Ar), 5.01 (1H, d, J = 7.5 Hz, NH), 4.52 (1H, app t, J = 7.5
Hz, CHCH3), 1.49 (9H, s, C(CH3)3), 1.44 (3H, d, J = 7.0 Hz, CH3);
HRMS (ES+) m/z calcd for C14H19NO3SNa [M + Na]+ 304.0983,
found 304.0976.
L-Cysteine Methyl Ester Hydrochloride (2).34 Methanol (150
mL) was added to a flame-dried round-bottom flask equipped with a
Teflon stir bar. The solvent was stirred and cooled to 0 °C, and acetyl
chloride (26.4 mL, 0.37 mol) was added dropwise. The solution was
stirred for an additional 10 min. L-Cysteine hydrochloride mono-
hydrate (4.35 g, 24.7 mmol) was added, the ice bath was removed, and
the reaction was stirred at room temperature for 24 h. The solvent was
removed in vacuo, and the resulting residue was dissolved in methanol
(40 mL) and water (8 mL). Tributylphosphine (3.0 mL, 12.4 mmol)
was added and the solution stirred at room temperature for 3.5 h. The
reaction mixture was diluted with water (50 mL) and the aqueous
phase washed with diethyl ether (4 × 25 mL). Solvent was removed in
vacuo and the product recrystallized in ethanol/ether and washed with
5% ethanol/ether to furnish 2 (2.89 g, 68%) as a white solid: 1H NMR
(400 MHz, MeOD) δ 4.36 (1H, t, J = 5.0 Hz, CH), 3.89 (3H, s,
OCH3), 3.11 (2H, d, J = 5.0 Hz, CH2); HRMS (ES+) m/z calcd for
C4H9NO2SNa [M + Na]+ 158.0252, found 158.0263.
a
Key: (i) sodium ascorbate, tris(3-hydroxypropyltriazolylmethyl)-
amine (THPTA), EtOH/H2O, Cu(MeCN)4PF6, 60 °C, 16 h, 65%.
iterative cycles has been demonstrated for the synthesis of a
fluorescein labeled glycopeptide 17. A solution to the problem
of introducing a photosensitive fluorophore is presented. In the
presence of phosphine reagents, the desulfurization reaction
dominates and the thiyl radical is inhibited by the presence of
aromatic thiols or disulfides. It is anticipated that this
methodology could be used in combination with solid phase
peptide synthesis (SPPS) methods to allow access longer chain
peptides of biological interest and that the methodology should
find general applications in functionalized peptide and protein
synthesis.
(R)-Methyl 2-[(S)-2-[(tert-Butoxycarbonyl)amino]-
propanamido]-3-mercaptopropanoate (3).35 Compounds 1
(0.166 g, 0.59 mmol) and 2 (0.101 g, 0.59 mmol) and
tributylphosphine (0.09 mL, 0.35 mmol) were stirred in MeOH (6
mL) under N2 at room temperature for 1 h. Solvent was removed in
vacuo, and purification by column chromatography (20−40% EtOAc/
EXPERIMENTAL SECTION
■
General Experimental Methods. For NMR spectra, a 400 MHz
spectrometer was employed for H (400.13 MHz) and 13C (100.61
1
MHz) spectra, a 600 MHz spectrometer was employed for 1H (600.13
MHz) and 13C (150.90 MHz) spectra. Resonances δ, are in ppm units
downfield from an internal reference in CDCl3 (δH = 7.26 ppm, δC =
1
hexane) furnished 3 as a white solid (0.17 g, 96%): H NMR (400
MHz, CDCl3) δ 6.99 (1H, br s, NHCHCH2), 5.06 (1H, br s, NHBoc),
4.88 (1H, m, CHCH2SH), 4.21 (1H, m, CHCH3), 3.82 (3H, s,
OCH3), 3.04 (2H, m, CH2SH), 1.48 (9H, s, (C(CH3)3)), 1.40 (3H, d,
J = 7.0 Hz, CH3CH); HRMS (ES+) m/z calcd for C12H22N2O5SNa [M
+ Na]+ 329.1147, found 329.1146.
77.0 ppm), or MeOH (δH = 3.31 ppm, δC = 49.0 ppm). H and 13C
1
NMR assignments were confirmed by 2D COSY, HSQC, HMBC, and
NOESY experiments. Mass spectrometry analysis was performed with
Maldi-quadrupole time-of-flight (Q-Tof) mass spectrometer equipped
with Z-spray electrospray ionization source (ESI). Silica gel (200
mesh) was used for column chromatography. Analytical thin-layer
chromatography was performed using silica gel (precoated sheets, 0.2
mm thick, 20 cm ×20 cm) and visualized by UV irradiation or
molybdenum staining (heating with a phosphomolybdic acid reagent).
DCM, MeOH, THF and toluene were dried over flame-dried 3 or 4 Å
sieves. Dimethylformamide (DMF), triethylamine (Et3N), and
trifluoroacetic acid (TFA) were used dry from Sure/Seal bottles.
Other reagents were purchased from an industrial supplier. All UV
reactions were carried out in a Luzchem photoreactor, LZC-EDU (110
V/60 Hz), containing 10 UVA lamps centered at 350 nm.
(S)-Methyl 2-[(tert-Butoxycarbonyl)amino]hex-5-enoate
(4).36,37 Compound 11 was synthesized according to the reported
literature procedure. Zinc dust (0.298 g, 4.56 mmol) was added to
iodine (0.012 g, 0.047 mmol) in a three-neck round-bottomed flask
and heated under vacuum for 10 min. The flask was flushed with
nitrogen and evacuated three times. N-(tert-Butoxycarbonyl)-3-iodo-L-
alanine methyl ester (0.50 g, 1.52 mmol) was dissolved in dry DMF (5
mL) and added to the zinc slurry at 0 °C. The reaction mixture was
stirred at room temperature for 1 h. While the zinc insertion reaction
was in progress, CuBr−DMS (0.041 g, 0.20 mmol) was placed in a
three-necked flask and gently dried under vacuum until a color change
from white to green was observed. Dry DMF (4 mL) and allyl chloride
(0.151 g, 1.98 mmol) were added, and the reaction was cooled to −15
°C. Once zinc insertion was complete (TLC), stirring of the reaction
mixture was ceased to allow the zinc to settle, and the supernatant was
General Procedure for the Synthesis of Thioesters.32 Peptide-
OH (1.0 equiv), N-methylmorpholine (1.2 equiv), and THF (0.5
mmol/mL) were added to a flame-dried flask and dried over 4 Å
molecular sieves. The solution was cooled to 0 °C, isobutyl
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removed and added dropwise to the electrophile and Cu catalyst. The
cold bath was removed, and the reaction mixture was stirred at room
temperature for 2 days. EtOAc (100 mL) was added, and the reaction
was stirred for 15 min. The reaction mixture was washed with 1 M
Na2S2O3 (100 mL), water (2 × 100 mL), and brine (100 mL) and
dried (MgSO4), and solvent was removed in vacuo. Purification by
column chromatography (95:5 hexane/ether) furnished 4 as a yellow
oil (0.143 g, 39%): 1H NMR (400 MHz, CDCl3) δ 5.84−5.68 (1H, m,
CH2CH), 5.10−4.93 (2H, m, CH2CH), 4.30 (1H, m, CHNHBoc),
3.72 (3H, s, OCH3), 2.10 (2H, m, CH2), 1.97−1.56 (4H, m, CH2),
1.42 (9H, s, C(CH3)3); HRMS (ES+) m/z calcd for C12H21NO4Na [M
+ Na]+ 266.1368, found 266.1371.
mmol/mL). The reaction was irradiated for 1 h at room temperature.
EtOAc (200 mL) was added, and the reaction was washed with brine
(6 × 100 mL). The organic phase was dried (MgSO4) and filtered, and
silica gel was added to the organic solution. Solvent was removed in
vacuo, and the product was purified by column chromatography (60−
80% EtOAc/hexane + 1% AcOH) to furnish 9 as a white solid (4.16 g,
95%): [α]23D = 13 (c = 0.1 g cm−1 in CHCl3); IR νmax (thin film) 3345
(NH), 2979 (OH), 1743 (CO), 1215 (CH), 1043 (COC) cm−1;
1H NMR (400 MHz, CDCl3) δ 7.06 (1H, d, J = 7.5 Hz, NH), 5.38
(1H, d, J = 3.0 Hz, H4), 5.18 (2H, dd, J = 11.0 Hz, J = 8.0 Hz, H2,
NH), 5.03 (1H, dd, J = 11.0 Hz, J = 3.5 Hz, H3), 4.78 (1H, m,
CHCH2S), 4.44 (1H, d, J = 8.0 Hz, H1), 4.30−4.06 (3H, m, CHCH3,
H6), 3.92 (2H, m, OCH2CH2, H5), 3.52 (1H, m, OCH2CH2), 3.05
(2H, m, CHCH2S), 2.70−2.43 (2H, m, SCH2CH2), 2.15, 2.11, 2.04,
1.98 (3H, s, OAc), 1.94−1.69 (2H, m, SCH2CH 2), 1.44 (9H, s,
(6S,9R,16S)-Methyl 16-[(tert-Butoxycarbonyl)amino]-9-(me-
thoxycarbonyl)-2,2,6-trimethyl-4,7-dioxo-3-oxa-11-thia-5,8-di-
azaheptadecan-17-oate (5). Compounds 3 (63.0 mg, 0.21 mmol)
and 4 (100.0 mg, 0.41 mmol) and 4,4-azobis(4-cyanovaleric acid)
(ACVA) (12.2 mg, 0.04 mmol) were dissolved in DMF (1 mL) and
irradiated in a UV reactor for 1 h (after this time, TLC showed
incomplete reaction). ACVA (12.2 mg, 0.04 mmol was added and the
reaction mixture placed in the UV reactor for 1 h. DMF was removed
in vacuo, and purification by column chromatography (40% EtOAc/
hexane) furnished 5 as a white solid (71 mg, 63%): [α]20D = −8.0 (c =
0.1 in CHCl3); IR νmax (thin film) 3320 (NH), 1742, 1672 (CO),
C(CH3)3), 1.38 (3H, d, J = 7.1 Hz, CHCH ); 13C NMR (100 MHz,
3
CDCl3) δ 172.7, 172.1, 171.2, 170.6, 170.3, 170.2, 155.6 (CO),
101.2 (C1), 80.2 (C(CH3)3), 70.7 (C-5), 70.6 (C3), 69.3 (C2), 68.4
(OCH2CH 2), 67.0 (C4), 61.2 (C6), 51.5 (CHCH2S), 50.0 (CHCH3),
33.6 (CHCH2S), 30.1 (SCH2CH 2), 30.0 (SCH2CH2), 28.2, 28.2, 28.2
(C(CH3)3), 21.0, 20.7, 20.7, 20.6 (C(O)CH3), 18.3 (CHCH3); HRMS
(ES−) m/z calcd for C28H44N2O15S [M − H]− 679.2383, found
679.2390.
1
1505 (CH),1161 (CO) cm−1; H NMR (400 MHz, CDCl3) δ 6.96
(2R,3S,4S,5R,6R)-2-(Acetoxymethyl)-6-[[(6S,9S)-2,2,6-tri-
methyl-4,7-dioxo-9-[(phenylthio)carbonyl]-3-oxa-11-thia-5,8-
diazatetradecan-14-yl]oxy]tetrahydro-2H-pyran-3,4,5-triyl Tri-
acetate (10). Following the general procedure for the synthesis of
thioester, 10 was purified by column chromatography (15−50%
(1H, m, NHCHCH2), 5.14 (2H, br s, NHBoc), 4.80 (1H, m,
CHCH2S), 4.31(1H, m, CHCH2CH2), 4.24 (1H, m, CHCH3), 3.79,
3.77 (3H, s, OCH3), 3.03−2.92 (2H, m, CHCH2S), 2.53 (2H, t, J =
7.0 Hz, SCH2CH2), 1.82 - 1.57 (4H, m, CH2), 1.52−1.36 (23H, m, 2
× C(CH3)3, CHCH3, CH2); 13C NMR (100 MHz, CDCl3) δ 173.0,
172.0, 170.6, 155.0, 154.9 (CO), 79.7, 79.5 (C(CH3)3), 52.8
(CHCH2), 52.2, 51.8 (OCH3), 51.3 (CHCH2CH2), 49.6 (CHCH3),
33.7, 31.8, 31.7, 28.3, 23.8 (CH2), 28.4, 28.4, 28.4, 28.4, 28.4, 28.4,
(C(CH3)3), 17.6 (CH3); HRMS (ES+) m/z calcd for C24H43N3O9SNa
[M + Na]+ 572.2618, found 572.2637.
EtOAc/Hexane) to furnish a white solid (3.41 g, 79%): [α]23 = −44
D
(c = 0.1 g cm−1 in CHCl3); IR νmax (thin film) 3316 (NH), 2935
(OH), 1745, 1654 (CO), 1513 (CC Ar), 1217 (CH), 1044
(COC), 753 (CH Ar) cm−1; 1H NMR (400 MHz, CDCl3) δ 7.41 (5H,
m, CH Ar), 7.12 (1H, br s, NH), 5.39 (1H, d, J = 3.1 Hz, H4), 5.19
(1H, dd, J = 11.0 Hz, J = 8.0 Hz, H2), 5.09 (1H, br s, NH), 5.00 (1H,
dd, J = 11.0 Hz, J = 3.1 Hz, H3), 4.95 (1H, m, CHCH2S), 4.45 (1H, d,
J = 8.0 Hz, H1), 4.30 (1H, br s, CHCH3), 4.22−4.07 (2H, m, H6),
3.91 (2H, m, OCH2CH2, H5), 3.60 (1H, m, OCH2CH2), 3.09−2.89
(2H, m, CHCH2S), 2.58 (2H, t, J = 7.2 Hz, SCH2CH2), 2.12, 2.05,
2.05, 1.98 (3H, s, OAc), 1.84 (2H, m, OCH2CH2), 1.46 (12H, br s,
CHCH3, C(CH3)3); 13C NMR (100 MHz, CDCl3) δ 197.8, 172.9,
170.5, 170.3, 170.2, 169.6, 155.6 (CO), 134.6, 134.6, 129.8, 129.3,
129.3, 126.9 (C Ar), 101.2 (C1), 80.6 (C(CH3)3), 70.9 (C3), 70.7
(C5), 68.8 (C2), 68.0 (OCH2CH2), 67.0 (C4), 61.3 (C6), 58.0
(CHCH3), 49.9 (CHCH3), 34.1 (CHCH2S), 29.2 (OCH2CH2), 28.9
(SCH2CH2), 28.4, 28.4, 28.4 (C(CH3)3), 20.8, 20.7, 20.7, 20.6 (C(O)
CH3), 17.8 (CHCH3); HRMS (ES+) m/z calcd for C34H48N2O14S2Na
[M + Na]+ 795.2447, found 795.2452.
N-(tert-Butoxycarbonyl)-L-alanyl-L-cysteine (7).38 Following
the general procedure for native chemical ligation furnished 7 as a
white solid (16.0 g, 96%): [α]23D = −29 (c = 0.1 g cm−1 in MeOH); IR
νmax (thin film) 3319 (NH), 2978 (OH), 1718, 1655 (CO), 1159
1
(CH) cm−1; H NMR (400 MHz, MeOD) δ 4.57 (1H, t, J = 4.7 Hz,
CHCH2), 4.10 (1H, q, J = 7.1 Hz, CHCH3), 3.00 (2H, m, CHCH2),
1.44 (9H, s, C(CH3)3), 1.33 (3H, d, J = 7.1 Hz, CHCH3); 13C NMR
(100 MHz, MeOD) δ 174.5, 171.4, 156.4 (CO), 79.3 (C(CH3)3),
54.1 (CHCH2), 50.2 (CHCH3), 27.2, 27.2, 27.2(C(CH3)3), 25.4
(CH2), 16.6 (CH3); HRMS (ES+) m/z calcd for C11H20N2O5SNa [M
+ Na]+ 315.0993, found 315.0995.
Allyl 2,3,4,6-Tetra-O-acetyl-β-D-galactopyranoside (8).39
Compound 8 was synthesized according to literature procedures. β-
D-Galactose pentaacetate (5.00 g, 12.80 mmol) and allyl alcohol (1.75
mL, 25.6 mmol) were dissolved in dry DCE (50 mL) and stirred over
4 Å molecular sieves for 2 h under N2. The reaction was cooled to −15
°C, and tin tetrachloride (1.60 mL, 13.35 mmol) was added and stirred
for 2 h. The reaction mixture was warmed to 5 °C, NaHCO3 (100
mL) was added, and the precipitate was filtered over Celite. DCM
(200 mL) was added to the filtrate, which was washed with NaHCO3
(200 mL), water (200 mL), and brine (200 mL), dried (MgSO4), and
concentrated in vacuo. Purification by column chromatography (35%
(6S,9R,12R)-12-(Mercaptomethyl)-2,2,6-trimethyl-4,7,10-tri-
oxo-9-[[[3-[[(2R,3R, 4S,5S, 6R)-3, 4,5-triacetoxy-6-
(acetoxymethyl)tetrahydro-2H-pyran-2-yl]oxy]propyl]thio]-
methyl]-3-oxa-5,8,11-triazatridecan-13-oic Acid (11). Com-
pound 11 was prepared according to the general procedure for native
chemical ligation to furnished 11 as a white solid (2.73 g, 87%): [α]23
D
= 6 (c = 0.1 g cm−1 in CHCl3); IR νmax (thin film) 3316 (NH), 2935
(OH), 1745, 1655 (CO), 1217 (CH), 1044 (COC) cm−1; 1H NMR
(600 MHz, CDCl3) δ 7.65 (1H, d, J = 7.3 Hz, NH), 7.37 (1H, br s,
NH), 5.41 (1H, d, J = 3.0 Hz, H4), 5.29−5.16 (2H, m, H2, NHBoc),
5.06 (1H, dd, J = 10.5 Hz, J = 3.0 Hz, H3), 4.86 (1H, br s,
CHCH2SH), 4.71 (1H, m, CHCH2S), 4.51 (1H, d, J = 8.0 Hz, H1),
4.32−4.09 (3H, m, CHCH3, H6), 3.96 (2H, br s, H5, OCH2CH2),
3.63 (1H, br s, OCH2CH2), 3.17−2.81 (4H, m, CHCH2S,
CHCH2SH), 2.65 (2H, m, SCH2CH2), 2.18, 2.09, 2.08, 2.01 (3H, s,
OAc), 1.88 (2H, m, SCH2CH2), 1.47 (9H, s, C(CH3)3), 1.41 (3H, d, J
= 6.7 Hz, CHCH3); 13C NMR (150 MHz, CDCl3) δ 173.1, 170.6,
170.2, 170.2, 170.1, 170.0, 169.7, 155.7 (CO), 101.2 (C1), 80.6
(C(CH3)3), 70.7 (C5), 70.5 (C3), 68.8 (C2), 68.0 (OCH2CH2), 66.9
(C4), 61.1 (C6), 54.4 (CHCH2SH), 52.4 (CHCH2S), 50.4 (CHCH3),
33.6 (CHCH2S), 29.1 (OCH2CH2), 28.7 (SCH2CH2), 28.2, 28.2, 28.2
(C(CH3)3), 26.1 (CHCH2SH), 20.8, 20.6, 20.6, 20.5 (C(O)CH3), 17.9
1
EtOAc/hexane) furnished 8 as a white solid (3.20 g, 64%): H NMR
(400 MHz, CDCl3) δ 5.90−5.77 (1H, m, CHCH2), 5.37 (1H, app
d, J = 3.0 Hz, H4), 5.30−5.15 (3H, m, H2, CHCH2), 5.00 (1H, dd,
J = 10.5 Hz, J = 3.5 Hz, H3) 4.50 (1H, d, J = 7.9 Hz, H1), 4.38−4.30
(1H, dd, J = 13.3 Hz, J = 5.0 Hz, H6′), 4.22−4.04 (3H, m, CH2CH
CH2, H6), 3.88 (1H, app t, J = 6.7 Hz, H5), 2.14, 2.05, 2.04, 1.96 (3H,
s, OAc); HRMS (ES+) m/z calcd for C17H24O10Na [M + Na]+
411.1267, found 411.1260.
(R)-2-[(S)-2-[(tert-Butoxycarbonyl)amino]propanamido]-3-
[[3-[[(2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-
tetrahydro-2H-pyran-2-yl]oxy]propyl]thio]propanoic Acid (9).
Compounds 7 (2.45 g, 8.37 mmol) and 8 (2.50 g, 6.44 mmol), 2,2-
dimethoxy-2-phenylacetophenone (DPAP) (0.215 g, 0.837 mmol),
and MAP (0.163 g, 0.837 mmol) were dissolved in DMF (17 mL, 0.5
F
dx.doi.org/10.1021/jo4001542 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(CHCH3); HRMS (ES−) m/z calcd for C31H49N3O16S2 [M − H]−
782.2475, found 782.2474.
8.1 Hz, H2), 5.04 (1H, dd, J = 11.0 Hz, J = 3.2 Hz, H3), 4.83−4.64
(2H, m, CHCH3, CHCH2SH), 4.55−4.45 (2H, m, H1, CHCH2S),
4.25 (1H, br s, CHCH3), 4.22−4.08 (2H, m, H6), 3.94 (2H, m,
OCH2CH2, H5), 3.60 (1H, m, OCH2CH2) 3.10 - 2.99 (3H, m, 1 ×
CHCH2S, 2 × CHCH2SH), 2.84 (1H, m, CHCH2S), 2.59 (2H, m,
SCH2CH2), 2.15, 2.07,2.05, 1.98 (3H, s, OAc),1.93−1.78 (2H, m,
(6S,9R,12S)-2,2,6,12-Tetramethyl-4,7,10-trioxo-9-[[[3-
[[(2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)-
tetrahydro-2H-pyran-2-yl]oxy]propyl]thio]methyl]-3-oxa-
5,8,11-triazatridecan-13-oic Acid (12). Compound 11 (1.32 g,
1.68 mmol) was dissolved in DMF (3.5 mL), and tributylphosphine
(0.42 mL, 1.68 mmol) was added. DPAP (0.043 g, 0.168 mmol) and
MAP (0.033 g, 0.168 mmol) were added, and the reaction was
irradiated for 1 h. EtOAc (100 mL) was added, the organic layer was
washed with brine (100 mL × 6) and dried (MgSO4), silica gel was
added, and the solvent was removed in vacuo. The product was
purified by column chromatography (75−100% EtOAc/hexane + 1%
AcOH) to furnish 12 as a white solid (1.00 g, 79%): [α]23D = −12 (c =
0.1 g cm−1 in CHCl3); IR νmax (thin film) 3317 (NH), 2933 (OH),
OCH2CH2), 1.50−1.35 (15H, br s, CHCH3, CHCH3, C(CH3)3); 13
C
NMR (150 MHz, CDCl3) δ 173.4, 172.6, 171.5, 170.5, 170.5, 170.2,
170.1, 169.7, 156.3 (CO), 101.2 (C1), 81.3 (C(CH3)3), 70.8 (C3),
70.6 (C5), 69.0 (C2), 68.0 (OCH2CH2), 67.0 (C4), 61.2 (C6), 54.8
(CHCH2SH), 52.9 (CHCH2S), 51.3 (CHCH3), 49.4 (CHCH3), 33.3
(SCH2CH), 29.3 (OCH2CH2), 28.6 (SCH2CH2), 28.4, 28.4, 28.4
(C(CH3)3), 26.2 (CHCH2SH), 20.8, 20.7, 20.6, 20.6 (C(O)CH3),
18.0, 17.9 (CHCH3); HRMS (ES−) m/z calcd for C34H54N4O17S2 [M
− H]− 853.2846, found 853.2856.
1
1745, 1654 (CO), 1217 (CH), 1044 (COC) cm−1; H NMR (400
(6S,9R,12S,15R)-15-[[(5-Azidopentyl)thio]methyl]-2,2,6,12-
tetramethyl-4,7,10,13-tetraoxo-9-[[[3-[[(2R,3R,4S,5S,6R)-3,4,5-
triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl]oxy]-
propyl]thio]methyl]-3-oxa-5,8,11,14-tetraazahexadecan-16-
oic Acid (15). Compound 14 (0.134 g, 0.157 mmol) was dissolved in
DMF (1 mL). MAP (2.83 μL, 15.7 μmol), DPAP (4.0 mg, 15.7 μmol),
and 5-azidopent-1-ene (52.0 mg, 0.471 mmol) were added. The
reaction mixture was irradiated for 1 h. EtOAc (50 mL) was added, the
reaction mixture was washed with brine (6 × 25 mL) and dried
(MgSO4), silica gel was added, and solvent was removed in vacuo. The
product purified by column chromatography (0−10% MeOH/EtOAc
MHz, CDCl3) δ 7.58 (1H, d, J = 7.0 Hz, NH), 7.39 (1H, d, J = 7.0 Hz,
NH), 5.38 (1H, d, J = 3.1 Hz, H4), 5.29 (1H, br s, NHBoc), 5.16 (1H,
dd, J = 10.8 Hz, J = 8.0 Hz, H2), 5.03 (1H, dd, J = 10.8 Hz, J = 3.5 Hz,
H3), 4.72−4.50 (2H, m, CHCH3, CHCH2S), 4.48 (1H, d, J = 8.0 Hz,
H1), 4.25−4.07 (3H, m, H6, CHCH3), 3.93 (2H, m, H5, OCH2), 3.60
(1H, m, OCH2), 3.13−2.74 (2H, m, CHCH2S), 2.60 (2H, m,
SCH2CH2), 2.15, 2.06, 2.05, 1.98 (3H, s, OAc), 1.86 (2H, m,
OCH2CH2), 1.50 (3H, d, J = 7.30 Hz, CHCH3), 1.45 (9H, s,
C(CH3)3), 1.39 (3H, d, J = 7.0 Hz, CHCH3); 13C NMR (100 MHz,
CDCl3) δ 170.3, 170.1, 169.9, 169.8, 169.6, 169.5, 169.4, 155.3 (C
O), 100.8 (C1), 80.4 (C(CH3), 70.4 (C3), 70.1 (C5), 68.5 (C2), 67.7
(OCH2CH2), 66.6 (C4), 60.8 (C6), 51.9 (CHCH2S), 51.5 (CHCH3),
48.2 (CHCH3), 33.1 (CHCH2S), 29.3 (OCH2CH2), 28.8 (SCH2CH2),
27.8, 27.8, 27.8 (C(CH3)3), 20.4, 20.3, 20.2, 20.2 (C(O)CH3), 17.6,
16.9 (CHCH3); HRMS (ES−) m/z calcd for C31H49N3O16S [M − H]−
750.2755, found 750.2748.
+ 1% AcOH) furnished 15 as a white solid (0.126 g, 83%): [α]24
=
D
−20 (c = 0.1 g cm−1 in CHCl3); IR νmax (thin film) 3301 (NH), 2925
(OH), 2097 (N3), 1747, 1640 (CO), 1218 (CH), 1045 (COC); 1H
NMR (600 MHz, CDCl3) δ 7.61, 7.54, 7.37 (1H, br s, NH), 5.42 (1H,
d, J = 3.0 Hz, H4), 5.28 (1H, br s, NHBoc), 5.20 (1H, m, H2), 5.07
(1H, dd, J = 10.8 Hz, J = 3.0 Hz, H3), 4.66−4.40 (4H, m, CHCH3,
CHCH2S, CHCH2S, H1), 4.23−4.15 (3H, m, CHCH3, H6), 4.03−
3.88 (2H, m, OCH2CH2, H5), 3.64 (1H, m, OCH2CH2), 3.29 (2H, t, J
= 6.8 Hz, CH2N3), 3.15−2.83 (4H, m, CHCH2S, CHCH2S), 2.72
(2R,3S,4S,5R,6R)-2-(Acetoxymethyl)-6-[[(6S,9R)-2,2,6-tri-
methyl-4,7-dioxo-9-[[(S)-1-oxo-1-(phenylthio)propan-2-yl]-
carbamoyl]-3-oxa-11-thia-5,8-diazatetradecan-14-yl]oxy]-
tetrahydro-2H-pyran-3,4,5-triyl Triacetate (13). Compound 13
was prepared according to the general procedure for the synthesis of
thioesters. Purification by column chromatography (60% EtOAc/
hexane) furnished 13 as a white solid (1.24 g, 65%): [α]23D = −8 (c =
0.1 g cm−1 in CHCl3); IR νmax (thin film) 3314 (NH), 2977 (OH),
1747, 1656 (CO), 1510 (CC Ar), 1217 (CH), 1044 (COC), 749
−2.51 (4H, m, SCH2CH2, SCH CH2), 2.18, 2.10, 2.08, 2.01 (3H, s,
2
OAc), 1.87 (2H, m, OCH2CH2), 1.62 (4H, m, SCH2CH2CH2C H2),
1.55−1.34 (15H, br s, C(C H3)3, CHCH3, CHCH3), 1.27 (2H, brs,
SCH2CH2CH2); 13C NMR (150 MHz, CDCl3) δ 173.5, 172.5, 171.2,
170.5, 170.2, 170.2, 170.1, 169.7, 156.3 (CO), 101.2 (C1), 80.9 (
C(CH3)3), 70.8 (C3), 70.5 (C5), 68.9 (C2), 68.1 (OCH2CH2), 67.0
(C4), 61.2 (C6), 52.8, 52.5 (CHCH2S), 51.2 (CHCH3), 50.9
1
(CH Ar) cm−1; H NMR (600 MHz, CDCl3) δ 7.49 (1H, br s, NH),
7.42−7.36 (5H, m, CH Ar), 6.93 (1H, d, J = 7.9 Hz, NH), 5.38 (1H, d,
J = 3.0 Hz, H4), 5.17 (1H, dd, J = 10.3 Hz, J = 8.0 Hz, H2), 5.01 (2H,
dd, J = 10.3 Hz, J = 3.0 Hz, H3, NHBoc), 4.77 (1H, q, J = 7.3 Hz,
CHCH3), 4.63 (1H, q, J = 6.4 Hz, CHCH2S), 4.44 (1H, d, J = 8.0 Hz,
H1), 4.20 - 4.06 (3H, m, H6, CHCH3), 3.98 - 3.85 (2H, m,
OCH2CH2, H5), 3.58 (1H, m, OCH2CH2), 3.05 (1H, br s, CHCH2S),
2.88 (1H, dd, J = 14.1 Hz, J = 6.4 Hz, CHCH2S), 2.63 (2H, t, J = 6.9
Hz, CH2CH2S), 2.13, 2.07, 2.05, 1.98 (3H, s, OAc), 1.87 (2H, m,
OCH2CH2), 1.50 (3H, d, J = 7.3 Hz, CHCH3), 1.42 (9H, s, C(CH3)3),
1.40 (3H, d, J = 7.2 Hz, CHCH3); 13C NMR (150 MHz, CDCl3) δ
198.6, 172.9, 170.4, 170.2, 170.1, 170.0, 169.8, 155.6 (CO), 134.7,
134.7 (CH Ar), 129.5 (C Ar), 129.2, 129.2, 127.2 (CH Ar), 101.5
(C1), 80.5 (C(CH3)3), 70.8 (C3), 70.7 (C5), 70.0 (C2), 68.1
(OCH2CH2), 67.1 (C4), 61.3 (C6), 55.4 (CHCH3), 52.1 (CHCH2S),
50.9 (CHCH3), 33.3 (CHCH2S), 29.4 (OCH2CH2), 28.5 (CH2CH2S),
28.3, 28.3, 28.3 (C(CH3)3), 20.8, 20.7, 20.6, 20.6 (C(O)CH3), 18.7,
17.7 (CHCH3); HRMS (ES+) m/z calcd for C37H53N3O15S2Na [M +
Na]+ 866.2818, found 866.2823.
(CH2N3), 49.4 (CHCH3), 33.6, 33.4 (CHCH2S), 32.2 (SCH CH2),
2
29.2 (OCH2CH 2), 28.9 (SCH2CH2), 28.6 (SCH2CH2), 28.4
(CH2CH2N3), 28.3, 28.3, 28.3 (C(CH3)3), 25.8 (CH2CH2CH2N3),
20.8, 20.6, 20.6, 20.6 (C(O)CH3), 18.2, 18.1 (CHCH3); HRMS (ES−)
m/z calcd forC39H63N7O17S [M − H]− 964.3643, found 964.3664.
2
Alkynyl Fluorescein Dye 16.40 Alkynyl fluoresceine dye was
prepared according to the literature. Briefly, fluoresceinamine (0.600 g,
1.73 mmol) was dissolved in pyridine (8 mL), and DCC (0.536 g, 2.60
mmol) and pentynoic acid (0.339 g, 3.46 mmol) were added. The
reaction mixture was stirred overnight, and DCU was filtered off. The
filtrate was poured into ice-cold water (30 mL). The pH was adjusted
to 2 with the addition of HCl (5 M) and the precipitate filtered off and
washed with water (3 mL). The product was purified by column
chromatography (0−6% MeOH/DCM) to furnish an orange solid
1
(0.465 g, 63%): H NMR (400 MHz, MeOD) δ 8.32 (1H, d, J = 1.8
Hz, CH Ar), 7.90 (1H, dd, J = 8.3, J = 1.8 Hz, CH Ar), 7.17 (1H, d, J =
1.8 Hz, CH Ar), 6.75−6.70 (4H, m, CH Ar), 6.60−6.56 (2H, m, CH
Ar), 2.67−2.60 (4H, m, CH2CH2), 2.33 (1H, t, J = 6.8 Hz, CCH);
HRMS (ES−) m/z calcd forC25H17NO6 [M − H]− 428.1134, found
428.1132.
Fluorescein-Functionalized Glycopeptide (17). Compound 15
(88.4 mg, 91.5 μmol), alkynylfluoresceine dye (86.0 mg, 201.0 μmol),
sodium ascorbate (36.2 mg, 182.4 μmol), and THPTA (39.7 mg, 91.5
μmol) were dissolved in EtOH (20 mL) and water (5 mL). Argon was
bubbled through the reaction mixture before the addition of
Cu(MeCN)4PF6 (6.8 mg, 18.2 μmol). The reaction was heated to
60 °C under argon for 16 h until TLC showed the consumption of
starting material (Rf = 0.7, 7:2:1 EtOAc/EtOH/H2O). The reaction
(6S,9R,12S,15R)-15-(Mercaptomethyl)-2,2,6,12-tetramethyl-
4,7,10,13-tetraoxo-9-[[[3-[[(2R,3R,4S,5S,6R)-3,4,5-triacetoxy-6-
(acetoxymethyl)tetrahydro-2H-pyran-2-yl]oxy]propyl]thio]-
methyl]-3-oxa-5,8,11,14-tetraazahexadecan-16-oic Acid (14).
Compound 14 was prepared according to the general procedure for
native chemical ligation to furnish 14 as a white solid (0.583 g, 85%):
[α]23 = −23 (c = 0.1 g cm−1 in CHCl3); IR νmax (thin film) 3303
D
(NH), 2927 (OH), 1745, 1641 (CO), 1218 (CH), 1044 (COC)
1
cm−1; H NMR (600 MHz, CDCl3) δ 7.63 (1H, d, J = 5.9 Hz, NH),
7.58 (1H, d, J = 6.7 Hz, NH), 7.36 (1H, d, J = 6.6 Hz, NH), 5.39 (1H,
d, J = 3.7 Hz, H4), 5.26 (1H, br s, NH), 5.17 (1H, dd, J = 11.0 Hz, J =
G
dx.doi.org/10.1021/jo4001542 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
mixture was concentrated in vacuo, and EtOAc (40 mL) was added.
This was washed with water (30 mL), and the aqueous phase extracted
with EtOAc (3 × 40 mL). The combined organics were dried
(MgSO4), and silica gel was added. Solvent was removed in vacuo and
the product purified by column chromatography (0−20% 2:1 EtOH/
H2O/EtOAc + 1% AcOH) to furnish a yellow solid (82.8 mg, 65%):
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3646−3649.
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1994, 266, 776−779.
[α]23 = −40 (c = 0.1 g cm−1 in MeOH); IR νmax (thin film) 3358
D
(NH), 2934 (OH), 1745, 1654 (CO), 1220 (CH Alkyl), 1048
(8) Tam, J. P.; Lu, Y. A.; Liu, C. F.; Shao, J. Proc. Natl. Acad. Sci.
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1
(COC), 850, 696 (CH-Ar) cm−1; H NMR (600 MHz, d-DMSO) δ
8.25 (1H, br s, NH), 7.97 (1H, s, NCH Ar), 7.92 (1H, d, J = 7.9 Hz,
NH), 7.59 (2H, d, J = 8.4 Hz, NH, CH Ar), 6.97−6.87 (4H, m, NH, 3
× CH Ar), 6.85 (1H, d, J = 8.8 Hz, CH Ar), 6.76 (1H, d, J = 8.4 Hz,
CH Ar), 6.66 (1H, dd, J = 8.8 Hz, J = 1.9 Hz, CH Ar), 6.55 (1H, m,
CH Ar), 6.29 (1H, br s, NH), 6.13 (1H, d, J = 1.6 Hz, CH Ar), 5.25
(1H, d, J = 3.7 Hz, H4), 5.16 (1H, dt, J = 10.4 Hz, J = 3.7 Hz, H3),
4.92 (1H, m, H2), 4.69 (1H, apt t, J = 7.4 Hz, H1), 4.45 (1H, br s,
CHCH2S), 4.34 (2H, t, J = 7.3 Hz, CH2N), 4.26 (1H, br s, CHCH3),
4.19 (1H, t, J = 6.5 Hz, H5), 4.05−3.95 (4H, m, H6, CHCH3,
CHCH2S), 3.78 (1H, m, OCH2), 3.56 (1H, m, OCH2), 3.04−2.89
(6H, m, CCH2CH2C(O), CH2S, CH2S), 2.83 (1H, m, CH2S), 2.66
(1H, m, CH2S), 2.56−2.49 (4H, br s, SCH2CH2, SCH2CH2), 2.11,
2.02, 2.00, 1.91 (3H, s, C(O)CH3), 1.83 (2H, quint, J = 7.3 Hz,
CH2CH2N), 1.74 (2H, t, J = 6.0 Hz, OCH2CH2), 1.58 (2H, t, J = 6.6
Hz, SCH2CH2), 1.38 (9H, s, C(CH3)3), 1.33 (2H, m,
CH2CH2CH2N), 1.23 (3H, d, J = 7.0 Hz, CHCH3), 1.19 (3H, d, J
= 7.1 Hz, CHCH3); 13C NMR (150 MHz, d-DMSO) δ 172.8 (C
ONH), 172.5 (COOH), 172.1, 172.5, 170.8 (CONH), 170.0,
169.9 (COCH3), 169.8 (COBoc), 169.5, 169.2 (COCH3),
168.9 (ArCCO), 160.3, 160.2 (ArCOCAr), 156.2 (ArCCO),
155.8 (Ar CNH), 151.2, 150.8 (Ar COH), 145.1 (Ar CN), 128.9,
128.6 (ArCH), 126.1 (Ar CH), 122.1 (Ar CHN), 117.8 (ArCHCOH),
115.6 (ArCHCNH), 113.4 (ArCHCOH), 111.6, 110.0, 109.9
(ArCCO), 105.5 (ArCHCNH), 102.4, 102.4 (ArCHCOH),100.0
(C1), 79.9 (CAr), 78.2 (C(CH3)3), 70.3 (C3), 69.8 (C5), 68.7
(C2), 67.8 (OCH2), 67.4 (C4), 61.2 (C6) 53.7 (CHCOOH), 52.2
(CHCH2S), 49.9 (CHNHBoc), 49.1 (CH2N), 48.6 (CHCH3), 33.8,
33.7 (CHCH2S), 33.2 (CH2CONH), 31.3 (SCH2), 29.4 (CH2CH2N),
29.0 (OCH2CH2), 28.3 (SCH2CH2), 28.2, 28.2, 28.2 (C(CH3)3), 27.7
(OCH2CH2CH2S), 24.8 (CH2CH2CH2N), 20.7 (CH2CH2CONH),
20.5, 20.5, 20.4, 20.3 (COCH3), 18.3, 18.2 (CHCH3); HRMS (ES−)
m/z calcd for C64H80N8O23S2 [M − H]− 1391.4700, found 1391.4741.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra for all compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: eoin.scanlan@tcd.ie, giordans@tcd.ie.
■
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Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by Science Foundation Ireland
(PIYRA 07/YI2/I1052). We are very grateful to Dr. Manuel
Ruether and Dr. John O’Brien for NMR, Dr. Martin Feeney
and Dr. Gary Hessman for MS, and Trinity College for support
(Trinity Award to L.M.). S.G. wishes to thank the L’Oreal
UNESCO For Women in Science Fellowship.
(37) Alcon
Asymmetry 1999, 10, 4639−4651.
(38) Ueki, M.; Shinozaki, K. Bull. Chem. Soc. Jpn. 1983, 56, 1187−
1191.
́ ̀
, M.; Moyano, A.; Pericas, M. A.; Riera, A. Tetrahedron:
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dx.doi.org/10.1021/jo4001542 | J. Org. Chem. XXXX, XXX, XXX−XXX