Synthesis of C -linked triazole-containing afgp analogues and their ability to inhibit ice recrystallization

Article abstract of DOI:10.1021/bc100394k

C-Linked antifreeze glycoprotein (C-AFGP) analogues have been shown to have potent ice recrystallization inhibition (IRI) activity. However, the lengthy synthesis of these compounds is not amenable to large-scale preparation for the many commercial, industrial, and medical applications that exist. This paper describes the synthesis of triazole-containing AFGPs using a convergent solid-phase synthesis (SPS) approach in which multiple carbohydrate derivatives are coupled to a resin-bound synthetic peptide in a single step. Modified "Click" conditions using dry DMF as solvent with catalytic Cu(II), sodium ascorbate, and microwave radiation afforded the synthesis of AFGP analogues 9-12 in 16-54% isolated yield. Compound 9 demonstrated no IRI activity, while compounds 10, 11, and 12 were moderate inhibitors of ice recrystallization. These results suggest that, while the triazole group is a structural mimetic of an amide bond, the amide bond in C-AFGP analogue 3 is an essential structural feature necessary for potent IRI activity.

Full text of DOI:10.1021/bc100394k

ARTICLE
pubs.acs.org/bc
Synthesis of C-Linked Triazole-Containing AFGP Analogues and Their
Ability to Inhibit Ice Recrystallization
Chantelle J. Capicciotti, John F. Trant, Mathieu Leclꢀere, and Robert N. Ben*
Department of Chemistry, D’Iorio Hall, 10 Marie Curie, University of Ottawa, Ottawa, Ontario, Canada, K1N 6N5.
S Supporting Information
b
ABSTRACT: C-Linked antifreeze glycoprotein (C-AFGP)
analogues have been shown to have potent ice recrystallization
inhibition (IRI) activity. However, the lengthy synthesis of
these compounds is not amenable to large-scale preparation for
the many commercial, industrial, and medical applications that
exist. This paper describes the synthesis of triazole-containing
AFGPs using a convergent solid-phase synthesis (SPS) ap-
proach in which multiple carbohydrate derivatives are coupled
to a resin-bound synthetic peptide in a single step. Modified
“Click” conditions using dry DMF as solvent with catalytic
Cu(II), sodium ascorbate, and microwave radiation afforded the synthesis of AFGP analogues 9ꢀ12 in 16ꢀ54% isolated yield.
Compound 9 demonstrated no IRI activity, while compounds 10, 11, and 12 were moderate inhibitors of ice recrystallization. These
results suggest that, while the triazole group is a structural mimetic of an amide bond, the amide bond in C-AFGP analogue 3 is an
essential structural feature necessary for potent IRI activity.
’ INTRODUCTION
bond-containing side chain in 3 was either lengthened or
shortened (Figure 1).6 Molecular dynamics (MD) simulations
and variable temperature (VT) NMR experiments suggest that
the IRI activity of 3 is attributed to an unusual conformation in
which the carbohydrate moiety folds back over the peptide chain
creating a hydrophobic pocket. It was hypothesized that this
“pocket” disrupts the ordering of water molecules in the primary
hydration shell of the galactose moiety, resulting in IRI activity.6
However, it is not known whether the amide bond in these
analogues is an important structural feature necessary for this
activity. One manner in which to probe the importance of this
linkage is to utilize a suitable isostere. The 1,4-disubstituted
triazole has been shown by Wong and co-workers to be an
isostere of a Z-amide bond (Figure 2).7,8 Furthermore, the mild,
highly efficient copper-catalyzed Huisgen cycloaddition for the
preparation of triazoles has facilitated their use in many biologi-
cally significant molecules.9ꢀ19
Antifreeze glycoproteins (AFGPs) are found in many deep sea
Teleost fish and serve to protect these organisms against cryo-
injury and death. This is accomplished by inhibiting the growth
of ice crystals in vivo—a phenomenon referred to as thermal
hysteresis (TH). AFGPs are also potent inhibitors of ice recrys-
tallization, and it has been recently hypothesized that this
property would be beneficial for a cryoprotectant.1 Cryopreser-
vation experiments have been performed with AFGPs, but
thermal hysteresis activity has been shown to cause significant
cellular damage at temperatures below the thermal hysteretic
gap,2,3 thus ensuring that these compounds will not be further
developed as cryoprotectants. However, AFGP analogues
possessing “custom-tailored antifreeze activity” (i.e., are potent
inhibitors of ice recrystallization but do not possess TH
activity) have great potential as cryoprotectants that is pre-
sently unrealized.
Prior syntheses of C-AFGPs by our laboratory have utilized a
traditional linear synthesis requiring the preparation of a glyco-
sylated amino acid prior to solid-phase synthesis (SPS). This
approach can be lengthy and expensive.20,21 Alternatively, con-
vergent methodologies have been reported for the synthesis of
glycoconjugates in which several carbohydrates are simulta-
neously coupled to the resin-bound peptide.22ꢀ24 Two conver-
gent syntheses of triazole-containing AFGP analogues have been
reported. A triazole-containing proline AFGP analogue was
During the past decade, our lab has been actively design-
ing and synthesizing C-linked analogues of the native AFGP
(C-AFGPs) in an effort to better understand their interactions at
the iceꢀbulk water interface. Towards this end, we have reported
the synthesis of several C-AFGP analogues (e.g., 3 and 5) that
exhibit potent ice recrystallization inhibition (IRI) activity but
display no TH activity and have tremendous potential as novel
cryoprotectants (Figure 1).4,5 Structureꢀfunction studies on
these C-AFGPs have questioned the functional importance of
the amide bond in 3 for IRI activity. For instance, when the amide
bond is removed as in C-serine analogue 5, IRI activity is
retained.5 In contrast, other studies have shown that acti-
vity is significantly decreased when the length of the amide
Received: September 2, 2010
Revised:
March 14, 2011
Published: April 01, 2011
r
2011 American Chemical Society
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Figure 2. Electronic similarities between a Z-amide bond and a 1,4-
disubstituted triazole ring.
and diisopropylethylamine (DIPEA) were distilled from calcium
hydride. N,N-Dimethylformamide (DMF) was stored over acti-
vated 4 Å molecular sieves under argon. 1H (400 or 500 MHz)
and 13C NMR (100 or 125 MHz) spectra were recorded at
ambient temperature on a Bruker Avance 400, Bruker Avance
500, or Varian Inova 500 spectrometer. Deuterated chloroform
(CDCl3), methanol (CD3OD), or water (D2O) were used as
NMR solvents. Chemical shifts are reported in ppm downfield
from trimethylsilane (TMS) or the solvent residual peak as an
internal standard. Splitting patterns are designated as follows: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br,
broad. Low resolution mass spectrometry (LRMS) was per-
formed on a Micromass Quatro-LC Electrospray spectrometer
with a pump rate of 20 μL/min using electrospray ionization
(ESI), a Voyager DE-Pro matrix-assisted laser desorption/ioni-
zation-time-of-flight (MALDI-TOF) (Applied Biosystem, Foster
City, CA) mass spectrometer operated in the reflectron/positive-
ion mode with DHB in 20% EtOH/H2O as the MALDI matrix,
or a Bruker Microflex MALDI-TOF mass spectrometer with
DHB in 20% acetonitrile/H2O as the MALDI matrix. Analy-
tical and preparatory scale RP-HPLC were carried out with
Varian Polaris C-18 columns on a Varian Prostar HPLC system
equipped with a variable wavelength detector (ProStar 330
PDA). Desalting was accomplished using C18 solid-phase ex-
traction cartridges (Discovery, Supelco) using 40% acetonitrile
in water for elution of glycopeptides. All yields are unoptimized.
Circular Dichroism. CD spectra of the glycopolymers 9ꢀ12
were obtained using a Jasco model J-810 automatic recorder
spectropolarimeter interfaced with a Dell computer. All of the
measurements were performed in quartz cells with 1.0 cm path
length. Spectra were obtained with a 1.0 nm bandwidth, a time
constant of 2 s, and a scan speed of 50 nm/min. Eight scans were
added to improve the signal-to-noise ratio, and baseline correc-
tions were made against each sample. All of the spectra were
recorded between 190 and 300 nm, and all of the CD experi-
ments were performed in doubly distilled H2O at pH 7.4. Data
obtained from CD spectroscopy were converted into molar
ellipticities (deg cm2 dmolꢀ1). Glycopeptide secondary struc-
tures were estimated using the deconvolution software CD Pro.
The data from each spectrum were analyzed using three different
deconvolution programs: SELCON3, CDSSTR, and CON-
TINLL. Of these three programs, SELCON3 and CONTINLL
gave the most consistent results. IBASIS 5 was used as the set of
reference proteins; it contains 37 proteins with R-helix, β-
structure, polyproline II, and unordered conformations with
optimal wavelengths of 185ꢀ240 nm.6
Figure 1. Previously prepared C-linked C-AFGP analogues.
synthesized in a solution-phase approach through coupling of an
acetylenic carbohydrate derivative and an azido peptide using
10ꢀ20% CuSO4 and 25ꢀ50% sodium ascorbate at 80 °C in the
presence of microwaves.25 In another approach, three AFGP
peptoids (6, 7, and 8, shown in Figure 3) were prepared by
coupling azido sugars with a resin-bound acetylenic peptoid
using excess CuI and sodium ascorbate in anhydrous DMF and
DIPEA.26 On the basis of this precedent, we sought to prepare
triazole-containing AFGP analogues of 3 using “Click” chemistry
in a convergent solid-phase approach. The structures of these
analogues (9ꢀ12) are shown in Figure 4. The goal of this study is
to understand the functional importance of the amide bond in
the side chain of C-AFGP analogue 3.
’ EXPERIMENTAL PROCEDURES
General Methods and Materials. All anhydrous reactions
were performed in flame-dried glassware under a positive
pressure of dry argon. Air- or moisture-sensitive reagents and
anhydrous solvents were transferred with oven-dried syringes or
cannulae. All flash chromatography was performed with E. Merck
silica gel 60 (230ꢀ400 mesh). All solution-phase reactions were
monitored using analytical thin layer chromatography (TLC)
with 0.2 mm precoated silica gel aluminum plates 60 F254 (E.
Merck). Components were visualized by illumination with a
short-wavelength (254 nm) ultraviolet light and/or staining
(ceric ammonium molybdate, potassium permanganate, or
orcinol stain solution). Dry-vacuum chromatography was carri-
ed out according to the protocol outlined by Pedersen and
Rosenbohm.27 All solvents used for anhydrous reactions were
distilled. Tetrahydrofuran (THF) was distilled from sodium/benzo-
phenone under nitrogen. Dichloromethane (DCM), triethylamine,
Thermal Hysteresis (TH) Assay. Nanoliter osmometry was
performed using a Clifton nanoliter osmometer (Clifton Tech-
nical Physics, Hartford, NY), as described by Chakrabartty and
Hew.28 All of the measurements were performed in doubly
distilled water. Ice crystal morphology was observed through a
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Figure 3. Triazole-containing peptoid analogues synthesized by Norgren et al.
Figure 4. Proposed structures of triazole-containing AFGP analogues.
Leitz compound microscope equipped with an Olympus 20ꢁ
(infinity-corrected) objective, a Leitz Periplan 32ꢁ photo eye-
piece, and a Hitachi KPM2U CCD camera connected to a
Toshiba MV13K1 TV/VCR system. Still images were captured
directly using a Nikon CoolPix digital camera.
Ice Recrystallization Inhibition (IRI) Assay. Sample analysis
for IRI activity was performed using the “splat cooling” method
as previously described.1 In this method, the analyte was
dissolved in phosphate buffered saline (PBS) solution and a 10
μL droplet of this solution was dropped from a micropipet
through a 2-m-high plastic tube (10 cm in diameter) onto a block
of polished aluminum precooled to approximately ꢀ80 °C. The
droplet froze instantly on the polished aluminum block and was
approximately 1 cm in diameter and 20 μm thick. This wafer was
then carefully removed from the surface of the block and
transferred to a cryostage held at ꢀ6.4 °C for annealing. After
a period of 30 min, the wafer was photographed between crossed
polarizing filters using a digital camera (Nikon CoolPix 5000)
fitted to the microscope. A total of three images were taken from
each wafer. During flash freezing, ice crystals spontaneously
nucleated from the supercooled solution. These initial crystals
were relatively homogeneous in size and quite small. During the
annealing cycle, recrystallization occurred, resulting in a dramatic
increase in ice crystal size. A quantitative measure of the
difference in recrystallization inhibition of two compounds X
and Y is the difference in the dynamics of the ice crystal size
distribution. Image analysis of the ice wafers was performed
using a novel domain recognition software (DRS) program.29
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This processing employed the Microsoft Windows Graphical
User Interface to allow a user to visually demarcate and store
the vertices of ice domains in a digital micrograph. The data
was then used to calculate the domain areas. All data was
plotted and analyzed using Microsoft Excel. IRI activity is
reported as a ratio of the mean grain size (MGS) of ice crystals
in the presence of the solute divided by the mean grain size
(MGS) of ice crystals in a control solution of phosphate
buffered saline (PBS). Small ratios indicate high levels of ice
recrystallization inhibition.
N-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-L-propar-
gylglycine allyl ester (13). In a flame-dried round-bottom flask
with 4 Å molecular sieves, Fmoc-L-Propargylglycine (338 mg,
1.01 mmol) was dissolved in anhydrous DCM (7 mL) under
argon atmosphere. HBTU (460 mg, 1.2 mmol) was added and
the mixture was stirred for 20 min. Allyl alcohol (347 μL, 5.1
mmol) was added, followed by a dropwise addition of DIPEA
(355.5 μL, 2.04 mmol). The reaction was stirred at room
temperature for 16 h. Upon consumption of starting material,
the reaction was diluted with DCM, filtered through a Celite pad,
and washed with 10% HCl, NaHCO3, and brine successively.
The final product was purified by dry vacuum chromatography
(DVC) to yield 326 mg of a white powder (86% yield). 1H NMR
(400 MHz, CDCl3): δppm 7.77 (d, 2H, J = 7.5 Hz), 7.61 (d, 2H
J = 7.4 Hz), 7.40 (t, 2H, J = 7.5 Hz), 7.32 (ddd, 2H, J = 7.5, 4.3, 1.1
Hz), 5.92 (ddd, 1H, J = 16.4, 10.8, 5.6 Hz), 5.66 (d, 1H, J =
8.1 Hz), 5.36 (dd, 1H, J = 17.2, 0.8 Hz), 5.28 (dd, 1H, J = 10.4, 0.9
Hz), 4.70 (dd, 2H, J = 2.7, 1.3 Hz), 4.61ꢀ4.54 (m, 1H), 4.40
(d, 2H, J = 7.2 Hz), 4.25 (t, 1H, J = 7.2 Hz), 2.82 (dd, 2H, J = 4.5,
2.6 Hz), 2.08 (t, 1H, J = 2.4 Hz). 13C NMR (100 MHz, CDCl3):
δppm 170.0, 155.6, 143.8, 143.7, 141.3, 131.3, 127.7, 127.1, 125.1,
120.0, 119.0, 72.0, 67.3, 66.4, 52.4, 47.1, 22.8. ESI-MS m/z calcd
for C23H21NO4 [MþH]þ: 376.154; [MþNa]þ: 398.137.
Found: 376.14, 398.12.
2,3,4,6-Tetra-O-acetyl-r-D-galactopyranosyl Azide (14).
2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl chloride30 (4.68 g,
12.8 mmol) was dissolved in 15 mL of HMPA. Sodium azide (2.5 g,
38.4 mmol) was added and the reaction was stirred vigorously for
16 h at ambient temperature. The mixture was poured into an
iceꢀwater mixture and the solid was collected by filtration and
washed with water. The product was recrystallized from ether
and petroleum ether to yield 4.3 g of white crystals (89% yield).
Characterization data are consistent with that reported pre-
viously in the literature.31 1H NMR (400 MHz, CDCl3): δppm 5.67
(d, 1H, J = 3.9 Hz), 5.46 (dd, 1H J = 2.9, 1.2 Hz), 5.25 (dd, 1H,
J = 10.8, 3.0 Hz), 5.20 (dd, 1H, J = 10.8, 3.9 Hz), 4.37 (dd, 1H,
J = 6.5, 6.5 Hz), 4.12 (dd, 2H, J = 6.5, 4.4 Hz), 2.15 (s, 3H), 2.11
(s, 3H), 2.06 (s, 3H), 2.00 (s, 3H). 13C NMR (100 MHz,
CDCl3): δppm 170.4, 170.1, 170.0, 169.8, 86.7, 68.6, 67.6, 67.4,
67.2, 61.5, 20.67, 20.64, 20.60, 20.5. ESI-MS m/z calcd for
C14H20N3O9 [MþH]þ: 374.12; [MþNa]þ: 396.10. Found:
374.09, 396.02.
1-Azido-2-(2,3,4,6-tetra-O-acetyl-r-D-galactopyranosyl)
ethane (16). 2-(2,3,4,6-Tetra-O-acetyl-R-D-galactopyranosyl)
acetaldehyde5 (2.5 g, 6.7 mmol) was added to a round-bottom
flask purged with argon and cooled to 0 °C. 70 mL of MeOH
was then added followed by sodium borohydride (983 mg, 26
mmol) and the reaction was stirred for 40 min when TLC (1:1
hexanes:ethyl acetate) analysis showed consumption of start-
ing material. The reaction was then quenched with 4 mL of
acetic acid and the solvent was removed in vacuo. Following
evaporation, the product was dissolved in ethyl acetate and
washed successively with 10% HCl, sodium bicarbonate, and
brine to yield 2.6 g of crude oil. The product was sufficiently
pure to be used directly in the next transformation.
A round-bottom flask was cooled to 0 °C in an ice bath. Crude
2-(2,3,4,6-tetra-O-acetyl-R-D-galactopyranosyl)ethanol (2.6 g,
6.7 mmol) and methanesulfonylchloride (2.1 mL, 26.8 mmol)
were then added to the flask and dissolved in 33 mL of pyridine
(0.2 M). The reaction was stirred at 0 °C for 1.5 h. Once
complete, the reaction was diluted with ethyl acetate and was
washed successively with saturated aqueous copper(II) acetate,
10% HCl, saturated sodium bicarbonate, and brine. The organic
layer was then dried with magnesium sulfate and the sol-
vent removed in vacuo to yield a slightly brown solid. This
crude product (2.2 g) was used directly without any further
purification.
The crude mesylate (2.2 g, 4.8 mmol) was dissolved in 20 mL
of DMF under argon. Sodium azide (760 mg, 11.7 mmol) was
added and the reaction was stirred vigorously at room tempera-
ture overnight. The mixture was then poured into an iceꢀwater
mixture and the solid was filtered out and washed with water.
Flash chromatography (7:3 ethyl acetate:hexanes) followed by
recrystallization from ether and hexanes yielded 1.6 g (68.2%
over three steps) of 16 as a pure white powder. Characterization
data are consistent with that reported previously in the
literature.32 1H NMR (400 MHz, CDCl3): δppm 5.42 (dd, 1H,
J = 3.1, 3.1 Hz), 5.25 (dd, 1H, J = 8.7, 4.7 Hz), 5.18 (dd, 1H,
J = 8.7, 3.3 Hz), 4.34ꢀ4.32 (m, 2H), 4.09 (ddd, 2H, J = 7.8, 5.9,
3.4 Hz), 3.43ꢀ3.41 (m, 2H), 2.12 (s, 3H), 2.10 (s, 3H), 2.07
(s, 3H), 2.05 (s, 3H), 1.99ꢀ1.87 (m, 1H), 1.75ꢀ1.65 (m, 1H).
13C NMR (100 MHz, CDCl3): δppm 170.6, 170.0, 169.8, 169.7,
69.0, 68.9, 68.2, 67.9, 67.2, 61.2, 47.7, 26.0, 20.8, 20.8, 20.7. ESI-
MS m/z calcd for C16H23N3O9 [MþH]þ: 402.151; [MþNa]þ:
424.133. Found: 402.13, 424.11.
(2,3,4,6-Tetra-O-acetyl-r-D-galactopyranosyl)methyl Azide
(24). Azido sugar 24 was synthesized according to a modified
procedure33 as indicated in Scheme 1 in the Supporting Informa-
tion. Spectral data are consistent with previously published data.
1H NMR (400 MHz, CDCl3): δppm 5.44 (dd, 1H, J = 3.3,
3.3 Hz), 5.27 (dd, 1H, J = 8.4, 4.6 Hz), 5.22 (dd, 1H, J = 8.4, 3.2
Hz), 4.39 (m, 2H), 4.25 (ddd, 1H, J = 8.0, 4.5, 3.4 Hz), 4.11 (dd,
1H, J = 11.7, 4.7 Hz), 3.63 (dd, 1H, J = 13.5, 8.8 Hz), 3.24 (dd,
1H, J = 13.5, 3.7 Hz), 2.12 (s, 3H), 2.11 (s, 3H), 2.07 (s, 3H), 2.06
(s, 3H). 13C NMR (100 MHz, CDCl3): δppm 170.7, 169.8, 169.6,
169.5, 70.8, 69.8, 67.7, 67.5, 66.9, 60.7, 48.3, 20.67, 20.66, 20.59.
ESI-MS m/z calcd for C15H21N3O9 [M þ K]þ: 426.091. Found:
426.12.
(2S)-1-Allyl-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-
3-(4-(2,3,4,6-tetra-O-acetyl-r-D-galactopyranosyl)-2,3,4-triazol-
1-yl)-propanoate (15). Compounds 13 (10 mg, 0.027 mmol) and
14 (10 mg, 0.027 mmol) were dissolved in 200 μL of the organic
solvent and 150 μL of water in a 3 mL culture tube. A 0.2 M cupric
acetate solution (25 μL, 0.005 mmol) was then added and the
reaction was sealed with a septum. The tube was flushed with argon
for 10 min and then placed in a 40 °C oil bath. A 0.4 M sodium
ascorbate solution (25 μL, 0.01 mmol) was added and the reaction
was stirred until completion (for a maximum of 6 h). The reaction
mixture was then cooled to room temperature, diluted with ethyl
acetate, and washed with water and brine. Solvent was removed in
vacuo and the crude product was purified by preparative TLC (6:4
1
ethyl acetate:hexanes). H NMR (400 MHz, CDCl3): δppm 7.76
(d, 2H, J = 7.5 Hz), 7.61 (t, 2H, J = 7.3 Hz), 7.40 (ddd, 2H, J = 7.4,
7.4, 3.3 Hz), 7.37ꢀ7.29 (m, 3H), 6.38 (d, 1H, J = 6.1 Hz), 6.16 (dd,
608
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1H, J = 10.7, 3.3 Hz), 5.97ꢀ5.86 (m, 1H), 5.85 (d, 1H, J = 8.2 Hz),
5.65 (d, 1H, J = 3.1 Hz), 5.46 (dd, 1H, J = 10.8, 6.0 Hz), 5.35 (d, 1H,
J = 17.1 Hz), 5.27 (d, 1H, J = 10.4 Hz), 4.73 (ddd, 1H, J = 8.2, 5.2,
5.2 Hz), 4.64 (d, 2H, J = 5.6 Hz), 4.60 (dd, 1H, J = 6.4, 6.4 Hz), 4.39
(d, 2H, J = 7.1 Hz), 4.24 (dd, 1H, J = 7.0, 7.0 Hz), 4.10 (dd, 1H,
J = 11.3, 6.6 Hz), 4.00 (dd, 1H, J = 11.3, 6.5 Hz), 3.32 (d, 2H,
J= 5.1 Hz), 2.19 (s, 3H), 2.01 (s, 3H), 1.95 (s, 3H), 1.88 (s, 3H). 13C
NMR (100 MHz, CDCl3): δppm 170.6, 170.6, 170.3, 170.0, 169.4,
155.9, 143.8, 143.7, 142.0, 141.3, 141.2, 131.4, 127.8, 127.1, 127.1,
125.1, 124.5, 120.0, 119.2, 81.7, 70.5, 67.7, 67.5, 67.2, 67.2, 66.2, 61.2,
53.4, 47.1, 28.0, 20.61, 20.61, 20.6, 20.3. ESI-MS m/z calcd
for C37H40N4O13 [MþH]þ: 749.2665; [MþNa]þ: 771.2490.
Found: 749.22, 771.20.
(2S)-1-Allyl-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-
3-(4-(2,3,4,6-tetra-O-acetyl-r-D-galactopyranosylethyl)-2,3,
4-triazol-1-yl)-propanoate (17). Compounds 13 (10 mg,
0.027 mmol) and 16 (11 mg, 0.027 mmol) were treated with
the same conditions as the preparation of 15. The crude
product 17 was purified by preparative TLC (7:3 ethyl acetate:
hexanes). 1H NMR (400 MHz, CDCl3): δppm 7.76 (d, 2H, J =
7.5 Hz), 7.59 (t, 2H, J = 7.4 Hz), 7.40 (t, 2H, J = 7.4 Hz), 7.37
(s, 1H), 7.31 (t, 2H, J = 7.5, 7.5 Hz), 5.94 (d, 1H, J = 8.9 Hz),
5.89 (m, 1H), 5.41 (t, 1H, J = 2.8, 2.8 Hz), 5.32 (d, 1H, J = 17.1
Hz), 5.25 (dd, 1H, J = 10.4, 1.1 Hz), 5.20 (d, 1H, J = 4.5 Hz),
5.17 (dd, 1H, J = 8.7, 3.1 Hz), 4.73 (dd, 1H, J = 13.3, 5.4 Hz),
4.66 (d, 2H, J = 5.7 Hz), 4.46 (ddd, 1H, J = 12.9, 7.9, 4.8 Hz),
4.41ꢀ4.27 (m, 4H), 4.23 (dd, 1H, J = 7.2, 7.2 Hz), 4.19ꢀ4.13 (m,
1H), 4.11ꢀ4.02 (m, 2H), 3.31 (d, 2H, J = 4.9 Hz), 2.31ꢀ2.17 (m,
1H), 2.10 (s, 3H), 2.07 (s, 6H), 2.03 (s, 3H). 13C NMR (100
MHz, CDCl3): δppm 170.8, 170.6, 169.9, 169.7, 169.7, 156.0,
143.8, 143.8, 142.7, 141.2, 141.2, 131.6, 127.7, 127.1, 125.2,
125.2, 122.8, 120.0, 118.7, 77.5, 77.2, 76.9, 69.2, 68.6, 68.0,
67.7, 67.1, 66.1, 61.1, 53.5, 47.1, 46.4, 28.1, 27.1, 20.77, 20.74,
20.71, 20.63. ESI-MS m/z calcd for C39H44N4O13 [MþH]þ:
777.2978. Found: 777.21.
protocols.34 5 mg of resin was directly cleaved and the crude
peptide was purified through preparatory TLC (30% water in
methanol) to yield 0.8 mg of 18 as a white powder (91% based on
resin loading). H NMR (400 MHz, D2O): δppm 3.94ꢀ3.78
1
(m, 5H), 3.59 (d, 2H, J = 1.35 Hz), 2.64 (d, 2H, J = 3.23 Hz, 2H),
2.36 (t, 1H, J = 2.21). 13C NMR (100 MHz, D2O): δppm 171.4,
171.32, 171.31, 168.9, 76.2, 74.4, 51.4, 42.6, 42.4, 42.3, 21.0. ESI-MS
m/z calcd for C11H16N4O5 [MþH]þ: 285.1199. Found: 285.072.
Resin-Supported Peptide 20. Peptide 20 was prepared using
115 mg of Wang resin using standard Fmoc-based SPS
protocols.34 18 mg of resin was cleaved directly and purified by
reversed-phase HPLC to yield 3.0 mg of 20 as a white powder in
78% yield (based on resin loading). 1H NMR (400 MHz, D2O):
δppm 7.40ꢀ7.15 (m, 5H), 4.60 (dd, 1H, J = 8.2, 6.6 Hz), 4.30
(q, 1H, J = 7.3 Hz), 4.19 (t, 1H, J = 6.1 Hz), 3.91 (q, 2H, J =
16.9 Hz), 3.11 (dd, 1H, J = 13.9, 6.5 Hz), 2.96 (dd, 1H, J = 13.9,
8.3 Hz), 2.84 (td, 2H, J = 3.3, 2.9 Hz), 2.52 (t, 1H, J = 2.7 Hz),
1.33 (d, 3H, J = 7.3 Hz). 13C NMR (125 MHz, D2O): δppm
176.7, 173.2, 171.0, 169.3, 136.8, 129.9, 129.4, 127.8, 83.2, 75.1,
55.4, 52.0, 49.3, 42.869, 42.861, 37.9, 21.7. ESI-MS m/z calcd for
C19H24N4O5 [MþH]þ: 389.182; [MþNa]þ: 411.164. Found:
389.19, 411.17.
Resin-Supported Peptide 23. Peptide 23 was prepared using
115 mg of Wang resin according to standard Fmoc-based SPS
protocols.34 10 mg of resin was cleaved directly and purified by
reversed-phase HPLC to yield 1.8 mg of 23 as a white powder in
70% yield (based on resin loading). 1H NMR (400 MHz, D2O):
δppm 4.58 (m, 3H), 4.29 (t, 1H, J = 5.9 Hz), 4.19ꢀ3.79 (m, 18H),
2.93 (dd, 2H, J = 5.9, 2.8 Hz), 2.76 (bd, 6H, J = 6.1 Hz), 2.60 (t,
1H, J = 2.5 Hz), 2.46 (t, 3H, J = 2.4 Hz). 13C NMR (125 MHz,
D2O): δppm 172.7, 171.8, 171.5, 171.3, 168.8, 79.2, 76.2, 74.3,
72.2, 52.4, 52.3, 51.3, 42.5, 42.4, 42.2, 20.8. ESI-MS m/z calcd for
C38H49N13O14 [MþH]þ: 912.3595; [MþNa]þ: 934.3420.
Found: 912.34, 934.32.
Resin-Supported Peptide 25. Peptide 25 was prepared using
150 mg of Wang resin using standard Fmoc-based SPS protocols
with amino acid 26 (Scheme 3b).34 20 mg of resin was directly
cleaved and the crude peptide was purified by reversed-phase
HPLC (isocratic flow with 10% acetonitrile in water for 5 min
followed by a linear gradient from 10% to 60% acetonitrile in
water) to yield 4.1 mg of 25 as a white powder in 48% yield
(based on resin loading). 1H NMR (500 MHz, D2O, presatura-
tion on HOD): δppm 4.36 (m, 3H), 3.90ꢀ3.75 (m, 17H), 3.60
(s, 2H), 2.28ꢀ2.09 (m, 12H), 1.91 (m, 4H), 1.80 (m, 4H). 13C
NMR (125 MHz, D2O): δppm 176.4, 174.2, 174.2, 174.1, 171.9,
171.8, 171.7, 171.6, 171.5, 170.9, 83.3, 82.6, 70.8, 70.3, 52.9, 52.8,
52.8, 52.6, 43.1, 42.5, 42.4, 42.3, 29.3, 29.2, 14.4, 13.9. ESI-MS
m/z calcd for C42H58N13O14 [MþH]þ: 968.42. Found: 968.76.
(S)-2-(9-Fluorenylmethoxycarbonylamino)-hex-5-ynoic
Acid (26). Amino acid 26 was synthesized as indicated in Scheme
2 in the Supporting Information. 1H NMR (CDCl3/CD3OD 2/
1, 400 MHz): δppm 7.71 (d, 2H, J = 7.6 Hz), 7.58 (dd, 2H, J = 7.1,
3.9 Hz), 7.35 (t, 2H, J = 7.6 Hz), 7.27 (t, 2H, J = 7.6 Hz), 4.35
(m, 3H), 4.18 (t, 1H, J = 6.8 Hz), 2.24 (m, 2H), 2.09 (m, 1H),
2.01 (t, 1H, J = 2.6 Hz), 1.86 (m, 1H). 13C NMR (CDCl3/
CD3OD 2/1, 100 MHz): δppm 173.9, 156.7, 143.8, 143.7, 141.2,
141.2, 127.7, 127.0, 125.0, 119.8, 82.6, 77.5, 69.3, 66.9, 53.0, 47.1,
30.9, 14.9. ESI-MS m/z calcd for C21H19NO4 [M þ Na]þ:
372.12. Found: 372.24.
General Method for Peptide Synthesis. The syntheses of
the peptides were carried out using an Advanced Chem Tech
Apex 396 automated peptide synthesizer (40 wells), equipped
with a dual-arm system and argon atmosphere controlled from an
IBM PC using Advanced Chem Tech v 1.6 software. Preloaded
Fmoc-Gly-Wang or Fmoc-Ala-Wang resins were swollen in
DMF for 1 h followed by filtration, and then were subjected to
20% piperidine in DMF twice successively for 30 min. Peptide
couplings were performed using standard conditions for Fmoc
solid-phase synthesis using HCTU as coupling agent.34 Follow-
ing the coupling of each residue, deprotection of the Fmoc
moiety was accomplished by treatment with 20% piperidine in
DMF. Between each deprotection and coupling, and after every
coupling, the beads were shaken 4 times with 4 mL of DMF
followed by filtration. Upon completion of the synthesis, the
beads were washed extensively with DMF (6 ꢁ 4 mL), MeOH
(6 ꢁ 4 mL), DCM (6 ꢁ 4 mL), hexanes (6 ꢁ 4 mL), and
ethanol (3 ꢁ 6 mL) and then removed from the synthesizer
and stored in a desiccator under vacuum in the presence of
P2O5 until required. A small amount of the peptide was
cleaved from the resin for characterization using 92.5:5:2.5
(v/v/v) TFA:triisopropylsilane:water. The peptides were
purified using reversed-phase HPLC (ramp from 0% to 18%
acetonitrile in water over 5 min followed by isocratic flow for
25 min) unless stated otherwise.
General Protocol for the Solid-Phase Organic Synthesis
Microwave “Click” Reaction. A CEM Mars X Microwave
System with 300 W maximum power and quartz reaction vessels
Resin-Supported Peptide 18. Peptide 18 was prepared
using 100 mg of Wang resin using standard Fmoc-based SPS
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Bioconjugate Chemistry
ARTICLE
was used for these reactions. The resin supported peptide was
initially stirred in DMF for 30 min under argon. Five equivalents
of azide per mole of alkyne and 0.2 equiv of cupric acetate per mol
of alkyne were added and the solution was degassed under argon.
0.4 equivalents of sodium ascorbate per mole of alkyne was then
added and the stirbar was removed. The flask was sealed under
argon and placed in the microwave and run under the following
conditions: 2 min ramp to 90 °C then held at 15% power for 1.5 h.
The solvent was then drained, and the beads were washed
extensively with 1:1 water:isopropanol followed by 4 ꢁ 4 mL
DMF, 4 ꢁ 4 mL MeOH, 4 ꢁ 4 mL DCM, and finally 4 ꢁ 4 mL
hexanes. The beads were then stirred in 4 mL of 92.5:5:2.5
TFA:triisopropylsilane:water cleavage cocktail to remove the
product off the resin. Solvent was evaporated at ambient
temperature. The residue was washed with ether (4 ꢁ 5 mL),
then dissolved in a solution of 0.01 M sodium methoxide in
methanol with a drop of water and stirred for 1.5 h at room
temperature. TFA was added until the pH of the solution
was adjusted to 6. The mixture was evaporated to dryness,
desalted, and resuspended in water and purified by reversed-
phase HPLC.
Glycopeptide 21. Resin-supported 20 (80 mg, 0.046 mmol)
was treated as per the general procedure described above with 14
(85.2 mg, 0.23 mmol), cupric acetate (1.7 mg, 0.0093 mmol),
and sodium ascorbate (3.4 mg, 0.017 mmol). Purification by
reversed-phase HPLC (1% acetonitrile in water for 12 min
followed by a linear gradient from 1% acetonitrile in water to
10% acetonitrile in water over 28 min followed by a linear
gradient from 10% acetonitrile in water to 98% acetonitrile in
water over 40 min) yielded 16.7 mg of 21 as a white powder (61%
based on resin-loading). 1H NMR (500 MHz, D2O): δppm 8.01
(s, 1H), 7.38ꢀ7.15 (m, 5H), 6.26 (s, 1H), 4.69ꢀ4.56 (m, 1H),
4.51ꢀ4.42 (m, 1H), 4.35ꢀ4.15 (m, 2H), 4.13ꢀ3.96 (m, 3H),
3.89ꢀ3.75 (m, 2H), 3.70ꢀ3.56 (m, 2H), 3.33ꢀ3.09 (m, 3H),
3.04ꢀ2.95 (m, 1H), 1.33ꢀ1.18 (m, 3H). 13C NMR (125 MHz,
D2O): δppm 179.4, 171.8, 170.860, 170.7, 136.6, 129.1, 128.6,
1267.0, 74.9, 69.2, 68.7, 66.8, 61.1, 54.8, 54.2, 46.5, 42.4, 36.7,
17.5. ESI-MS m/z calcd for C25H35N7O10 [M þ H]þ: 594.25.
Found: 594.42.
reversed-phase HPLC (1% acetonitrile in water for 12 min
followed by a linear gradient from 1% acetonitrile in water to
10% acetonitrile in water over 28 min followed by a linear
gradient from 10% acetonitrile in water to 98% acetonitrile in
water over 40 min) yielded 9 mg of 9 as a white powder (20%
based on resin loading). 1H NMR (500 MHz, D2O): δppm 7.83
(s, 4H), 6.12 (d, 4H, J = 5.9 Hz), 4.33 (d, 4H, J = 10.1 Hz), 4.17
(dd, 4H, J = 10.0, 6.3 Hz), 3.93 (m, 5H), 3.86ꢀ3.71 (m, 17H),
3.60ꢀ3.47 (m, 12H), 3.18ꢀ2.98 (m, 12H). 13C NMR (125
MHz, D2O): δppm 173.2, 173.0, 172.0, 171.9, 171.83, 171.76,
171.3, 171.28, 171.22, 171.1, 148.8, 125.3, 85.2, 74.8, 69.3, 68.7,
67.0, 61.1, 53.2, 53.16, 53.14, 46.57, 42.59, 42.2, 27.1, 26.9, 26.8,
26.7, 26.65, 26.59, 25.1, 23.7. MALDI-TOF MS m/z calcd for
C62H93N25O34 [M þ Na þ H]2þ: 877.814. Found: 877.8.
Glycopeptide 10. Resin-supported 23 (70 mg 0.030 mmol)
was treated as per the general procedure described above with 24
(94 mg, 0.24 mmol), cupric acetate (6.5 mg, 0.036 mmol), and
sodium ascorbate (13 mg, 0.072 mmol). Purification by reversed-
phase HPLC (1% acetonitrile in water for 12 min followed by a
linear gradient from 1% acetonitrile in water to 10% acetonitrile
in water over 28 min followed by a linear gradient from 10%
acetonitrile in water to 98% acetonitrile in water over 40 min)
yielded 25 mg of 10 as a white powder (45% based on resin
1
loading). H NMR (500 MHz, D2O): δppm 7.73 (s, 4H), 4.53
(m, 4H), 4.24 (s, 4H), 3.93 (m, 5H), 3.88ꢀ3.63 (m, 28H),
3.59ꢀ3.56 (m, 5H), 3.54ꢀ3.38 (m, 12H), 3.13ꢀ2.97 (m, 8H).
13C NMR (125 MHz, D2O): δppm 171.4, 171.3, 171.2, 171.1,
171.1, 171.0, 170.9, 129.8, 122.6, 74.7, 74.6, 74.5, 72.5, 69.5, 69.4,
69.2, 68.7, 68.6, 68.4, 67.1, 60.8, 45.74, 45.70, 45.6, 42.6, 42.4,
42.3, 42.2, 27.2, 27.13, 27.09, 27.02. MALDI-TOF MS m/z calcd
for C66H101N25O34 [M þ H]þ: 1788.69. Found: 1788.6.
Glycopeptide 11. Resin-supported 23 (40 mg 0.017 mmol)
was treated as per the general procedure described above with 16
(136 mg, 0.34 mmol), cupric acetate (2.5 mg, 0.0136 mmol), and
sodium ascorbate (5.5 mg, 0.0275 mmol). Purification by
reversed-phase HPLC (2% acetonitrile in water for 9 min
followed by a linear gradient from 2% acetonitrile in water to
40% acetonitrile in water over 30 min) yielded 17 mg of 11 as a
white powder (54% based on resin loading). 1H NMR (500 MHz,
D2O): δppm 7.84 (s, 1H), 7.76 (s, 3H), 4.62ꢀ4.51 (m, 4H),
4.40ꢀ4.32 (m, 9H), 4.26 (t, 1H, J = 6.40 Hz), 3.92ꢀ3.70 (m,
24H), 3.63 (dd, 12H, J = 10.1, 3.0 Hz), 3.58ꢀ3.53 (m, 8H), 3.25
(d, 2H, J = 6.2 Hz), 3.21ꢀ2.96 (m, 6H), 2.24ꢀ2.10 (m, 4H),
1.95ꢀ2.10 (m, 4H). 13C NMR (125 MHz, D2O): δppm 173.0,
172.0, 171.87, 171.80, 171.69, 171.42, 171.4, 171.36, 171.32,
171.19, 169.8, 169.3, 126.2, 126.1, 72.2, 72.1, 72.0, 69.6, 68.8,
67.7, 65.8, 61.0, 54.4, 53.8, 53.3, 52.7, 47.0, 46.95, 46.91, 42.6,
42.6, 42.3, 42.2, 27.9, 27.2, 27.0, 26.9, 26.8, 26.6, 24.6, 22.2.
MALDI-TOF MS m/z calcd for C70H109N25O34 [M]þ: 1843.76.
Found: 1843.8.
Glycopeptide 22. Resin-supported 20 (24 mg, 0.0137 mmol)
was treated as per the general procedure described above with 16
(27.5 mg, 0.0685 mmol), cupric acetate (0.5 mg, 0.0028 mmol),
and sodium ascorbate (1 mg, 0.0051 mmol). Purification by
reversed-phase HPLC (2% acetonitrile in water for 9 min
followed by a linear gradient from 2% acetonitrile in water to
40% acetonitrile in water over 30 min) yielded 6 mg of 22 as a
1
white powder (70% based on resin-loading). H NMR (500
MHz, D2O): δppm 7.76 (s, 1H), 7.27ꢀ7.09 (m, 5H), 4.37 (dd,
2H, J = 14.0, 6.3 Hz), 4.18 (t, 1H, J = 6.3 Hz), 4.02 (dd, 1H,
J = 14.3, 7.0 Hz), 3.76ꢀ3.85 (m, 3H), 3.74 (d, 1H, J = 1.6 Hz),
3.66ꢀ3.58 (m, 2H), 3.55 (d, 2H, J = 5.8 Hz), 3.18 (d, 1H,
J = 6.3), 3.07 (dd, 1H, J = 13.9, 5.4 Hz), 2.87 (dd, 1H, J = 13.9, 9.3
Hz), 2.30ꢀ1.99 (m, 2H), 1.19 (d, 3H, J = 7.2 Hz). 13C NMR
(125 MHz, D2O): δppm 171.9, 170.4, 169.0, 162.8, 136.4, 129.2,
128.6, 127.0, 72.1, 69.5, 68.7, 67.6, 60.9, 54.8, 52.6, 47.0, 42.2,
36.9, 26.6, 24.6, 17.2. ESI-MS m/z calcd for C27H39N7O10
[MþH]þ: 622.284; [MþNa]þ: 644.266. Found: 622.15,
644.18.
Glycopeptide 12. Resin-supported 25 (25 mg, 0.011 mmol)
was treated as per the general procedure described above with 24
(85 mg, 0.22 mmol), cupric acetate (1.6 mg, 0.009 mmol), and
sodium ascorbate (3.6 mg, 0.018 mmol). Purification by re-
versed-phase HPLC (isocratic 1% acetonitrile in water for 10 min
followed by a linear gradient from 1% acetonitrile in water to 30%
acetonitrile in water over 30 min) yielded 3.3 mg of 12 as a white
1
powder (16% based on resin loading). H NMR (500 MHz,
Glycopeptide 9. Resin-supported 23 (60 mg 0.026 mmol)
was treated as per the general procedure described above with 14
(190 mg, 0.51 mmol), cupric acetate (3.8 mg, 0.020 mmol),
and sodium ascorbate (8.2 mg, 0.041 mmol). Purification by
D2O, presaturation on HOD): δppm 7.71 (br s, 4H), 4.31 (s, 4H),
4.17 (s, 3H), 4.08ꢀ3.43 (m, 51H), 2.65 (m, 8H), 2.05ꢀ1.77
(m, 8H).13C NMR (125 MHz, D2O): δppm 174.2, 171.8, 171.0,
124.0, 96.4, 74.8, 72.5, 69.6, 68.7, 67.1, 60.8, 52.8, 45.5, 43.1, 42.5,
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ARTICLE
Table 1. Optimization of Solution-Phase “Click” Coupling
organic solvent
solvent composition (% organic)
reaction time (hrs)
% conversiona
1
2
3
4
5
6
7
8
9b
t-BuOH
DMF
NMP
Dioxane
THF
50
50
50
50
50
75
80
90
90
5
81
76
4
3
>98
95
1
0.5
0.75
0.25
0.5
0.25
>98
>98
>98
>98
>98
THF
THF
THF
THF
a Conversions calculated based upon analysis of crude reaction mixture using 1H NMR. b Reaction carried out with 5 equiv of azide.
Table 2. Optimization of Solvent Conditions Using a Primary Azido Sugar
organic solvent
solvent composition (% organic)
reaction time (hrs)
conversiona
1
2
3
4
DMF
50
50
50
50
0.25
0.5
78
>98
>98
>98
NMP
Dioxane
THF
0.5
0.5
a Conversions calculated based upon analysis of crude reaction mixture using 1H NMR.
42.3, 30.1, 20.9. MALDI-TOF MS m/z calcd for C70H109N25O34
[M þ Na]þ: 1866.7. Found: 1867.5.
organic solvents capable of swelling Wang resin were examined
for the solution-phase reaction between alkyne 13 and azido
sugar 14 before applying the conditions to the solid-phase
synthesis of triazole-containing C-AFGP analogues.40 The reac-
tion between 13 and 14 was investigated using aqueous mixtures
(50%) of N-methyl morpholine (NMP), tetrahydrofuran
(THF), N,N-dimethyl formamide (DMF), dioxane and tert-
butanol (Table 1). All of these solvents have excellent swelling
properties for Wang resin except for tert-butanol, which swells
resins poorly.40 Under typical Sharpless reaction conditions
’ RESULTS AND DISCUSSION
Several modifications to the original “Click” reaction condi-
tions for solid-phase applications have been reported.16,17,35ꢀ39
We sought to utilize solvents that would permit reaction condi-
tions with sub-stoichiometric amounts of copper and also facil-
itate swelling of the solid-phase resin. As a result, a number of
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ARTICLE
Scheme 1. Solid-Phase Synthesis of a Triazole-Linked Glycopeptide Using Optimized Conditions
Scheme 2. Microwave Assisted Solid-Phase Synthesis of Triazole-Linked Glycopeptides
using tert-butanol,11 15 was formed, but reaction conversion was
only 81% after 5 h (Table 1, entry 1). DMF resulted in only 76%
conversion after 4 h (entry 2), while NMP resulted in complete
conversion after 3 h (entry 3). In contrast, dioxane afforded 95%
conversion after only 1 h (entry 4). However, the best results
were obtained when THF was employed, with complete con-
version after only 30 min (entry 5).
conversion by using a 10% water/THF solvent system in the
presence of 5 equiv of azide (entry 9).
With optimized solution-phase results in hand, the solid-phase
synthesis of a triazole-linked glycopeptide was attempted
(Scheme 1). Initial attempts to couple resin-supported peptide
18 using the optimized conditions (10% water in THF, 0.2 equiv
of Cu(OAc)2, 0.4 equiv of sodium ascorbate) with azido sugar 14
(5 equiv) failed. Even upon heating the reaction for 96 h at 80 °C
or adding 10 equiv of azido sugar 14, the reaction still failed to
yield glycopeptide 19. Given the precedent for microwave
accelerated Click couplings on solid-phase resins,42 we investi-
gated whether glycopeptide formation would occur in the
presence of microwaves. Similar conditions were then attempted
for the microwave accelerated “Click” coupling, but peptide 20
(Scheme 2) was utilized instead of 18. Peptide 20 bearing a
phenylalanine residue was selected, as the strong UV absorbance
of this residue would facilitate purification by HPLC and
anhydrous THF was used as the solvent. Consequently, resin-
supported peptide 20 was reacted with azido sugar 14 in
anhydrous THF, utilizing 0.2 equiv of Cu(OAc)2, 0.4 equiv of
sodium ascorbate, and microwave heating (300 W) for 2 h.
Analysis by MALDI mass spectrometry of the crude reaction
mixture failed to show a trace of product. However, when the
solvent was changed to anhydrous DMF trace quantities of
glycoconjugate 21 were detected after 2 h (the product was
cleaved from the resin prior to analysis). By decreasing the power
to 45 W, the reaction went to completion after only 1.5 h and
glycoconjugate 21 was obtained in 61% yield after cleavage from
the resin and subsequent deprotection and purification by
reversed-phase HPLC (Scheme 2). Similarly, when resin-sup-
ported peptide 20 was reacted with azido sugar 16 under these
These solvent mixtures were also applicable with other azide
substrates (Table 2). For instance, reaction of alkyne 13 with
azido sugar 16 yielded similar results to those described above,
but reaction times were generally shorter. When DMF was used
as a cosolvent, glycoconjugate 17 was obtained in 78% conver-
sion after only 15 min (Table 2, entry 1), as opposed to 76%
conversion in 4 h for glycoconjugate 15 prepared from azido
sugar 14 (Table 1, entry 2). Quantitative conversions for 17 were
also obtained within 30 min when NMP, dioxane, and THF were
employed (Table 2, entries 2ꢀ4). Overall, THF yielded the best
results, showing it to be an ideal solvent for the solution-phase
coupling regardless of the structure of the azide substrate.
As stated previously, a solvent system with minimal water
content is desirable for solid-phase applications to ensure an
efficient swelling of the resin. However, an aqueous component is
necessary to dissolve the sodium ascorbate and facilitate reduc-
tion of Cu(II) to Cu(I).9,41 Hence, the efficiency of the solution-
phase Click reaction of alkyne 13 with azido sugar 14 was
repeated using a decreased percentage of water in THF
(Table 1, entries 6ꢀ9). Surprisingly, decreasing the water
content had no effect upon conversions and reaction times were
still less than one hour (entries 6ꢀ8). However, the reaction time
could further be reduced to 15 min without decreasing reaction
612
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Bioconjugate Chemistry
ARTICLE
Scheme 3. Microwave Assisted Solid-Phase Synthesis of Glycopeptides 9ꢀ12
Table 3. Analysis of Secondary Structure for Glycopeptides
9ꢀ12
R helix
β sheet
β turn
poly proline type 2
random coil
9
0.011
0.022
0.0135
0
0.2975
0.2505
0.2855
0.358
0.1435
0.1445
0.1345
0.093
0.1225
0.1125
0.107
0.4275
0.469
10
11
12
0.4555
0.356
0.193
reacted with resin-supported peptide 25 (prepared under standard
Fmoc solid-phase coupling conditions utilizing amino acid 26)
under the microwave “Click” conditions described above, and
glycopeptide 12 was obtained in 16% yield (Scheme 3b).
Triazole-containing AFGP analogues 9ꢀ12 were first exam-
ined for TH activity using a nanoliter osmometer.28 This
instrument is regarded as the “gold standard” for assessing
thermal hysteresis. All four C-AFGP analogues were found to
exhibit no thermal hysteresis activity. Furthermore, no dynamic
ice shaping was evident indicating there was no interaction with
the ice surface, a result consistent with the lack of thermal
hysteresis activity in previously reported ornithine analogues
1ꢀ4.4,6 Analysis of solution conformation using circular dichro-
ism (CD) spectroscopy (Figure 5 and Table 3) suggests that the
predominant secondary structure in solution is random coil with
β-sheet being the next likely solution structure for glycopeptides
9ꢀ12. This is consistent with the solution conformation re-
ported for AFGP-8 and other C-AFGP analogues studied by our
laboratory2,4,43 and suggests that the lack of TH activity is not
due to the fact that AFGP analogues 9ꢀ12 adopt different
solution conformations compared to the native AFGP.
Figure 5. CD spectrum of glycopeptides 9ꢀ12. All spectra were
acquired at 22 °C, with a concentration of 43 μM. CD data has been
normalized for concentration and number of chromophores.
reaction conditions, glycopeptide 22 was obtained in 70%
isolated yield after cleavage from the resin, deprotection, and
purification by reversed-phase HPLC.
With this promising result, the convergent syntheses of
triazole-containing C-AFGP glycopeptides 9, 10, 11, and 12
were attempted. Glycosyl azide 14 and resin-supported peptide
23 (prepared under standard Fmoc solid-phase coupling con-
ditions) were subjected to microwave heating in DMF with 0.2
equiv of Cu(OAc)2 and 0.4 equiv of sodium ascorbate
(Scheme 3a). Glycoconjugate 9 was obtained in 20% isolated
yield following cleavage from the Wang resin, deprotection, and
purification by reversed-phase HPLC. Similarly, reaction of azido
sugar 24 with resin-supported peptide 23 yielded glycopeptide
10 (45% yield), and when azido sugar 16 was utilized, glycopep-
tide 11 was obtained in 54% yield. Glycosyl azide 24 was also
The triazole-containing C-AFGP analogues 9ꢀ12 were al-
so screened for their ability to inhibit ice recrystallization (IRI
613
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Chart 1. IRI Activity of C-Linked AFGP Analoguesa
activity) using a splat cooling assay.1,29 Chart 1 shows the IRI
activity of triazole-containing C-AFGP analogues 9ꢀ12, native
AFGP-8, and previously synthesized C-AFGP analogues 1ꢀ4
(the latter contain an amide bond in the side chain).6,44 In this
assay, a phosphate buffered saline (PBS) solution is utilized as a
positive control for ice recrystallization; thus, bars smaller than
PBS indicate the ability to inhibit ice recrystallization. All samples
have been normalized to PBS. As evident from Chart 1, triazole-
containing AFGP analogue 9 failed to exhibit IRI activity. AFGP
analogue 9 is closely related to peptoid AFGP analogue 6
(published by Norgren et al.) which also failed to exhibit IRI
activity.26 AFGP analogue 6 contains N-methyl glycine instead of
glycine in the polypeptide backbone; however, the nature of the
anomeric linkage, total length of the side chain, and the position
of the triazole within the side chain are identical to those of AFGP
analogue 9. While these analogues did not inhibit the recrystalli-
zation of ice, the results are consistent with the suggestion that
IRI activity is sensitive to the length of the side chain bearing the
carbohydrate moiety. Similar results have been reported by our
laboratory in analogues that contain an amide bond in the side
aPBS represents a positive control for ice recrystallization.
Figure 6. The presence of a hydrophobic methylene spacer and “reversed” directionality of the triazole moiety in AFGP analogues 9ꢀ12.
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chain instead of a triazole.6 For instance, as the side chain of IRI
active C-AFGP analogue 3 is shortened (compounds 1 and 2) or
lengthened (compound 4) IRI activity is lost.6
presence of microwaves were utilized. Triazole-containing AFGP
analogue 9 failed to exhibit IRI activity, while analogues 10, 11,
and 12 exhibited moderate IRI activity. The results of this study
suggest that substitution of the amide bond in C-AFGP analogue
3 with a triazole moiety is detrimental to IRI activity. In addition,
a hydrophobic component (i.e., methylene spacer) close to the
carbohydrate residue as well as the length of the side chain
bearing the triazole (or amide) linkage is also important. This
result is consistent with earlier findings by our laboratory.
Molecular dynamics simulations and variable temperature
NMR studies are currently being performed on analogues 10,
11, and 12 in an effort to elucidate how the methylene spacer
and position of the triazole linker are influencing orientation of
the carbohydrate residue. The results of these studies will be
reported in due course.
Triazole-containing AFGP analogue 9 is also a close analogue
of C-AFGP analogue 1, which did not possess IRI activity. There
are two key structural changes in AFGP analogue 9 relative to 1.
First, the triazole moiety has been employed to mimic the amide
bond in 1. Second, the orientation of the triazole in the side chain
mimics an amide bond that is “reversed” in its orientation
compared to 1. These structural modifications in 9 do not restore
IRI activity since this analogue failed to inhibit ice recrystalliza-
tion. However, the fact that the lengths of the side chains in 9 and
1 are the same supports the hypothesis that the distance between
the carbohydrate moiety and the peptide backbone is important
for IRI activity.
Some of the most IRI active C-AFGP analogues prepared by
our laboratory possess a methylene group between the carbohy-
drate moiety and the amide bond in the side chain. It has been
proposed that the hydrophobic nature of this group is an
important structural feature in C-AFGP analogue 3. This methy-
lene group is absent in triazole-containing AFGP analogue 9.
Interestingly, when this group is present as in triazole-containing
C-AFGP analogue 10 (Figure 6a), the glycoconjugate is a
moderate inhibitor of ice recrystallization, validating its
importance.6 Triazole-containing C-AFGP analogue 11 has
two methylene groups incorporated between the carbohydrate
moiety and the triazole linkage. This glycoconjugate has similar
IRI activity as triazole-containing analogue 10, further validating
the importance of a hydrophobic group in the spacer close to the
carbohydrate.6 C-AFGP derivative 11 is also a close analogue of
potent ice recrystallization inhibitor C-AFGP analogue 3. How-
ever, 11 is not as active as 3. The number of atoms between the
carbohydrate moiety and the polypeptide backbone in analogues
3 and 11 is the same. However, the triazole moiety in 11 mimics
an amide bond that has the directionality “reversed” relative to 3.
Furthermore, the position of the triazole linker in the side chain
of 11 is different than the position of the amide bond in 3
(Figure 6b). The triazole linker in C-linked AFGP 11 is actually
one atom closer to the polypeptide backbone than the amide
bond in 3. This structural modification is detrimental to IRI
activity as analogue 11 is much less active than analogue 3.
Triazole-containing C-AFGP analogue 12 is also a close
analogue of 3 with the exception of the orientation of the triazole
moiety. The orientation of the triazole mimics an amide bond
that is “reversed” relative to 3 (Figure 6b). However, the length of
the side chain between the carbohydrate and the peptide back-
bone, as well as the position of the triazole linker within this side
chain, is the same (shown in Figure 6b). While triazole-contain-
ing analogue 12 exhibited moderate IRI activity, it is much less
active than C-AFGP analogue 3. These results indicate that while
the presence of a hydrophobic methylene spacer and the length
of the side chain bearing the triazole (or amide) linkage directly
affect IRI activity, the amide bond in 3 is an essential structural
feature necessary for potent IRI activity.
’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic procedures for azido
b
sugar 24, amino acid 26, and 1H and 13C spectra are available for
all characterized compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*rben@uottawa.ca.
’ ACKNOWLEDGMENT
R.N.B. acknowledges NSERC, CBS, and CFI for financial
support. J.F.T. holds an NSERC PGS-D.
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