The importance of hydration for inhibiting ice recrystallization with C-linked antifreeze glycoproteins

Article abstract of DOI:10.1021/ja7103262

The role of hydration in modulating solution conformation, molecular recognition, and biological activity of oligosaccharides, proteins, and nucleotides is widely recognized but is often neglected when investigating many biological processes such as the mechanism by which biological antifreezes inhibit the growth of ice. We have investigated the relationship between carbohydrate configuration and recrystallization-inhibition (RI) activity in functional C-linked antifreeze glycoprotein (AFGP) analogues using a series of analogues 1-4. While analogues 1-4 did not show any thermal hysteresis (TH) activity, 1 did exhibit weak dynamic ice shaping indicating that this compound had the ability to interact with the ice lattice. The d-mannose and d-talose analogues (3 and 4, respectively) exhibited very weak RI activity with mean largest grain size values similar to phosphate buffered saline, the negative control. d-Glucose analogue 2 exhibited moderate RI activity while d-galactose analogue 1 was the most potent analogue with RI activity comparable to the native AFGP 8. These results suggest that the configuration of the carbohydrate moiety in C-linked AFGP analogues is extremely important and modulates recrystallization-inhibition activity. It seems likely that differences in hydration for each C-linked pyranose alter the compatibility of the carbohydrate moiety with the three-dimensional hydrogen-bonded network of supercooled bulk water. Consequently, the energy associated with transferring a water molecule to the ice lattice changes and can result in inhibition of ice growth. These results emphasize the importance of continued studies to further elucidate the role of hydration in antifreeze activity. Copyright

Full text of DOI:10.1021/ja7103262

S
1
The Importance of Hydration for Inhibiting Ice Recrystallization with
C-Linked Antifreeze Glycoproteins.
Pawel Czechura, Roger Y. Tam, Elena Dimitrijevic, Anastasia Murphy
and Robert N. Ben*
University of Ottawa, Department of Chemistry, D’Iorio Hall, 10 Marie Curie, Ottawa,
ON, Canada, K1N 6N5
*Rben@uottawa.ca
Supporting Information
Part A
I.
Synthesis of C-Linked Antifreeze Glycoprotein Analogues (1-4)
(i) General Experimental………………………………. S2
(ii) Synthesis of Carbohydrate Component…………….. S3
(iii) Synthesis of C-linked Glycoconjugate……………... S11
(iv) Solid Phase Synthesis of C-Linked Glycopeptides… S17
Assessing Antifreeze Activity……………………………………. S19
Conformational Analysis via Circular Dichroism………………... S19
II.
III.
Part B
IV.
NMR Spectra……………………………………………………... S21

S
2
I. Synthesis of C-Linked Antifreeze Glycoprotein Analogues (1-4)
(i) General Experimental
All anhydrous reactions were performed in flame-dried or oven-dried glassware under a
positive pressure of dry argon or nitrogen. 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 (230-400 mesh). All
solution phase reactions were monitored using analytical thin layer chromatography
(TLC) with 0.2mm pre-coated silica gel aluminum plates 60 F254 (E. Merck).
Components were visualized by illumination with a short-wavelength (254 nm) ultra-
violet light and/or staining (ceric ammonium molybdate, potassium permanganate, or
phosphomolybdate stain solution). All solvents used for anhydrous reactions were
distilled.
Tetrahydrofuran (THF) and diethyl ether were distilled from
sodium/benzophenone under nitrogen. Dichloromethane, acetonitrile, triethylamine,
benzene and diisopropylethylamine (DIPEA) were distilled from calcium hydride.
Methanol was distilled from calcium sulfate. N,N-dimethylformamide (DMF) was stored
over activated molecular sieves 4Å under argon.
13
1H (300, 360, 400, or 500 MHz) and C NMR (75, 90, 100 or 125 MHz) spectra were
recorded at ambient temperature on a Bruker Avance 300, Bruker AM 360, Bruker
Avance 400 or Bruker Avance 500 spectrometer. Deuterated chloroform (CDCl3),
methanol (CD3OD), or water (D2O) were used as NMR solvents, unless otherwise stated.
Chemical shifts are reported in ppm downfield from TMS and corrected using the solvent
residual peak or TMS 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 performed on a Micromass Quatro-LC Electrospray
spectrometer with a pump rate of 20µL/min using electrospray ionization (ESI) or a
Voyager DE-Pro matrix-assisted desorption ionization-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. High
resolution mass spectrometry (HRMS) data was acquired on Applied Biosystems/Sciex
QStar (Concord, ON). Samples in CH2Cl2/MeOH 1:1 were mixed with Agilent ES
tuning mix for internal calibration, and infused into the mass spectrometer at 5
µL/min. Infrared absorption spectra (IR) were recorded on a Shimadzu FTIR-8400S
spectrometer as a neat film from 4000 cm-1 to 650 cm-1. Analytical and preparatory scale
RP-HPLC were carried out with C-18 columns on a Varian Dynomax HPLC system
equipped with a variable wavelength detector (ProStar 330 PDA) or a Waters Delta 600E
HPLC system equipped with a variable wavelength detector. Automated solid phase
peptide synthesis (SPPS) was performed on APEX 396 (Advanced ChemTech) equipped
with a 40 well reaction vessel.

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3
(ii) Synthesis of Carbohydrate Component
Scheme 1
OAc
R1
OAc
R1
R3
R4
R3
R4
a
O
O
OAc
AcO
AcO
R2
R
2Br
6a: Gal 89%
6b: Glc 94%
6c: Man 92%
5a: Gal: R1,R4=H; R2,R3=OAc
5b: Glc: R1,R3=H; R2,R4=OAc
5c: Man: R1,R4=OAc; R2,R3=H
b
OAc
OAc
R3
R4
R3
R4
R1
R1
c
O
O
AcO
AcO
R2
R2
CO2H
8a: Gal 85%
8b: Glc 76%
8c: Man 82%
7a: Gal 88%
7b: Glc 77%
7c: Man 82%
Conditions: (a) HBr/HOAc; (b) (nBu)6Sn2, allylphenylsulfone, benzene, hν; (c)
.
RuCl3 H2O, NaIO4, CCl4/CH3CN/H2O
General protocol for Bromination of D-galactose pentaacetate, D-glucose
pentaacetate and D-mannose pentaacetate
To commercially available D-pyranose pentaacetate (5 g, 12.81 mmol), 30 mL of HBr in
AcOH (33% solution) was added at room temperature. The reaction was stirred for 40
minutes and then diluted with CH2Cl2. The solution was transferred to a separatory funnel
containing ice and the organic layer was washed with ice water until neutral pH. The
organic extract was dried over MgSO4, filtered and concentrated to afford product in
~90% yield. The crude product was crystallized from diethyl ether to form a white
powder.
OAc
AcO
O
AcO
AcO
Br
2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide (6a)
1H NMR (360 MHz, CDCl3) δ 6.56 (1H, d, J=3.9 Hz), 5.39 (1H, m), 5.22 (1H, dd, J=4.2,
8.2 Hz), 4.85 (1H, d, J=4.3 Hz), 4.82 (1H, d, J=4.2 Hz), 4.25 (1H, m), 3.97 (2H, m), 1.98
13
(3H, s), 1.98 (3H, s), 1.87 (3H, s), 1.82 (3H, s); C NMR (90 MHz, CDCl3) δ 169.98,
169.70, 169.64, 169.42, 88.19, 70.94, 67.73, 67.51, 66.78, 60.67, 20.47, 20.37, 20.31;
LRMS (ESI): Calcd for C14H23BrNO9 [M+NH4]+ 429.2, found 429.9.

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4
OAc
O
AcO
AcO
AcO
Br
2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (6b)
1H NMR (300 MHz, CDCl3) δ 6.16 (1H, d, J=1.3 Hz), 5.53 (1H, dd, J=3.6, 7.8 Hz), 5.27
(2H, m), 4.15 (1H, dd, J=9.6, 18 Hz), 4.07 (1H, m), 3.98 (1H, dd, J=2.1, 12 Hz), 2.01,
13
(3H, s), 1.98 (3H, s), 1.91 (3H, s), 1.84 (3H, s); C NMR (75 MHz, CDCl3) δ 170.28,
169.51, 169.39, 169.35, 83.20, 72.71, 71.90, 67.78, 65.05, 61.27, 20.60, 20.49, 20.41;
LRMS (ESI): Calcd for C14H23BrNO9 [M+NH4]+ 429.2, found 429.9.
OAc
OAc
O
AcO
AcO
Br
2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl bromide (6c)
1H NMR (360 MHz, CDCl3) δ 6.45 (1H, d, J=3.9 Hz), 5.39 (1H, dd, J=9.6, 10 Hz), 5.01
(1H, dd, J=9.6, 9.9Hz), 4.67, (1H, ddd, J=1.5, 3.9, 5.4Hz), 4.16 (2H, m), 3.95 (1H, d,
13
J=12.0 Hz), 1.92 (3H, s), 1.91 (3H, s), 1.91 (3H, s), 1.90 (3H, s); C NMR (90 MHz,
CDCl3) δ 169.98, 169.70, 169.64, 169.42, 88.19, 70.94, 76.73, 67.57, 66.78, 60.67, 20.47,
20.37, 20.31; LRMS (ESI): Calcd for C14H23BrNO9 [M+NH4]+ 429.2, found 429.9.
General protocol for photochemical allylation of 2,3,4,6-tetra-O-acetyl-α-D-
galactopyranosyl bromide, 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide,
2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl bromide1
To a solution of 2,3,4,6-tetra-O-acetyl-α-D-pyranosyl bromide (1.24 g, 3.01 mmol) in
benzene (15 mL), allyl phenyl sulfone (1.418 mL, 7.52 mmol), and bis-tributyl tin (2.19
mL, 4.21 mmol) were added. The solution was degassed and sonicated for 30 minutes
under argon atmosphere; the sealed flask was irradiated for 9 hours under a 450W
mercury arc lamp. The reaction was monitored using TLC at time intervals of 2, 6 and 8
hours. The reaction mixture was loaded directly onto a silica gel column packed with
hexanes. The organostannanes were flushed with 3 void volumes of hexanes, after which
the solvent system was changed to 5:1 hexanes/EtOAc to remove the unreacted allyl
phenyl sulfone. The product was eluted with 3:1 hexanes/EtOAc and concentrated to
afford the allylated derivative as colorless oil (~95% yield).
1 Pontén, F.; Magnusson, G. J. Org. Chem. 1996, 61, 7463.

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5
OAc
O
AcO
AcO
AcO
Allyl 2,3,4,6-tetra-O-acetyl-α-D-galactopyranoside (7a)
1H NMR (360 MHz, CDCl3) δ 5.76 (1H, dddd, J=6.5, 7.1, 10.0, 16.6 Hz), 5.42 (1H, dd,
J=2.4, 3.1 Hz), 5.28 (1H, dd, J=4.8, 9.3 Hz), 5.22 (1H, dd, J=3.1, 9.3 Hz), 5.13 (1H, ddd,
J=1.6, 3.0, 16.6 Hz), 5.12 (1H, ddd, J=1.4, 3.0, 10.0 Hz), 4.30 (1H, ddd, J=4.7, 5.2, 10.3
Hz), 4.21 (1H, dd, J=8.9, 12.5 Hz), 4.09 (2H, m), 2.46 (1H, m), 2.29 (1H, m), 2.12 (1H,
13
s), 2.07 (1H, s), 2.04 (1H, s), 2.03 (1H, s); C NMR (90 MHz, CDCl3) δ 170.5, 170.1,
169.9, 169.8, 133.3, 117.6, 71.4, 68.2, 67.9, 61.4, 30.9, 20.8, 20.7, 20.6; LRMS (ESI):
Calcd for C17H25O9 [M+H]+ 373.4, found 373.1.
OAc
O
AcO
AcO
AcO
Allyl 2,3,4,6-tetra-O-acetyl-α-D-glucopyranoside (7b)
The product was co-eluted with starting material with 3:1 hexanes to EtOAc and
concentrated. The mixture was re-dissolved in a 1:1 mixture of acetone and water, to
which silver carbonate was added. The reaction was allowed to stir for 30 minutes, after
which it was filtered. The product was extracted with CH2Cl2, washed with water, brine,
dried over MgSO4 and concentrated. The colorless oil was purified with flash column
chromatography in 3:1 hexanes/EtOAc, and afforded 7b as a colorless oil in ~70% yield
over two steps.
1H NMR (360 MHz, CDCl3) δ 5.76 (1H, dddd, J=6.6, 8.0, 16.0Hz), 5.41 (1H, dd, J=6.8,
13.2Hz), 5.18 (2H, m), 5.01 (1H, dd, J=6.5, 12.1Hz), 4.35 (1H, m), 4.23 (1H, dd, J=5.9,
12.8Hz), 4.10 (1H, dd, J=3.0, 13.2Hz), 3.89 (1H, m), 2.59 (1H, m), 2.38 (1H, m), 2.19
13
(3H, s), 2.01 (3H, s), 1.95 (3H, s), 1.91 (3H, s); C NMR (90 MHz, CDCl3) δ 177.30,
174.48, 170.58, 169.55, 169.43, 168.66, 163.66, 140.11, 115.29, 76.97, 76.61, 69.31,
68.76, 67.71, 67.47, 32.83, 26.55, 20.51, 20.48, 20.36, 20.22, 20.09, 20.06, 19.86; HRMS
(ESI): Calcd for C17H25O9 [M+H]+ 373.1498, found 373.1461.

S
6
OAc
OAc
O
AcO
AcO
Allyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (7c)
1H NMR (360 MHz, CDCl3) δ 5.77 (1H, dddd, J=6.8, 10.0, 10.4Hz), 5.23 (1H, dd,
J=3.24, 6.8Hz), 5.15 (1H, m), 5.07 (1H, m), 4.29 (1H, dd, J=6.4, 12.2Hz), 4.06 (1H, dd,
J=2.9, 8.2 Hz), 4.03 (1H, m), 3.86 (1H, ddd, J=2.9, 6.8, 8.6Hz), 2.52 (1H, m), 2.29 (1H,
13
m), 2.07 (3H, s), 2.02 (3H, s), 1.99 (3H, s), 1.94 (3H, s); C NMR (90 MHz, CDCl3) δ
174.30, 171.58, 170.38, 169.98, 169.13, 168.16, 163.11, 140.21, 114.21, 73.91, 68.81,
68.36, 67.69, 61.49, 35.81, 26.95, 22.53, 22.49, 22.31, 20.20, 20.01, 19.98, 19.45; LRMS
(ESI): Calcd for C17H25O9 [M+H]+ 373.4, found 373.1.
General protocol for oxidation of allyl 2,3,4,6-tetra-O-acetyl-α-D-pyranoside to
2,3,4,6-tetra-O-acetyl-α-D-pyranosyl acid
Allylated pyranose tetraacetate (0.83 g, 2.23 mmol) was dissolved in 14 mL of a solution
of (2:2:3) acetonitrile: carbon tetrachloride: water, followed by the addition of sodium
periodate (1.90 g. 8.92 mmol). A catalytic amount of ruthenium trichloride trihydrate was
then added, and the reaction was allowed to stir at room temperature for 2-3 hours. The
solution was filtered through celite and transferred to a separatory funnel with
dichlroromethane. The organic layer was washed successively with saturated ammonium
chloride solution, saturated brine solution, dried over MgSO4, filtered and concentrated to
afford the carboxylic acid derivative in >75 % yield.
OAc
AcO
O
AcO
AcO
O
OH
2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl acid (8a)
1H NMR (400 MHz, CDCl3) δ 5.43 (1H, t, J=2.8 Hz), 5.33 (1H, dd, J=8.9, 5.0 Hz,), 5.17
(1H, dd, J =8.9, 3.3 Hz), 4.70 (1H, ddd, J=9.3, 8.3, 5.3 Hz), 4.30-4.08 (3H, m), 2.73 (1H,
dd, J=15.6, 8.7 Hz), 2.63 (1H, dd, J=15.6, 5.7 Hz), 2.13 (3H, s), 2.07 (3H, s), 2.04 (6H,
s); 13C NMR (90 MHz, CDCl3) δ: 175.5, 170.8, 170.1, 170.0, 169.7, 69.4, 68.9, 67.8,
67.6, 67.1, 61.2, 33.15, 20.7; IR (thin film): 3706-2355, 1748 cm-1.

S
7
OAc
O
AcO
AcO
AcO
O
OH
2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl acid (8b)
1H NMR (360 MHz, CDCl3) δ 5.28 (1H, ddd, J=4.6, 9.7Hz), 5.17 (1H, ddd, J=6.7,
9.9Hz), 5.03 (1H, q, J=8.9Hz), 4.84 (1H, m), 4.68 (1H, m), 4.26 (1H, ddd, J=6.2, 9.9Hz),
4.06 (1H, m), 3.94 (1H, m), 3.86 (1H, m), 2.81 (1H, m), 2.04 (3H, s), 2.02 (3H, s), 2.00
(3H, s), 1.99 (3H, s); 13C NMR (90 MHz, CDCl3) δ 177.28, 174.50, 170.62, 169.84,
101.95, 69.31, 68.76, 67.71, 67.50, 67.47, 67.01, 66.73, 66.43, 61.00, 32.92, 20.89, 20.77,
20.72, 20.67, 20.65, 20.60; HRMS (ESI): Calcd for C16H23O11 [M+H]+ 391.1240, found
391.1216.
OAc
OAc
O
AcO
AcO
O
OH
2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl acid (8c)
1H NMR (300 MHz, CDCl3) δ 5.33 (1H, dd, J=5.4, 7.8 Hz), 5.14 (1H, m), 4.49 (1H, m),
4.21 (1H, dd J=5.7, 13.5 Hz), 4.01 (1H, m), 2.63 (1H, ddd, J=6.4, 7.9, 9.3 1H), 2.10 (3H,
13
s), 1.99 (3H, s), 1.89 (3H, s), 1.75 (3H, s), 1.44 (4H, m); C NMR (75 MHz, CDCl3) δ
174.53, 171.15, 170.30, 170.10, 169.99, 133.96, 116.38, 73.19, 68.81, 68.34, 67.63,
63.13, 61.79, 35.76, 35.13, 26.93, 24.79, 21.17, 21.09, 21.06, 20.56, 19.23, 14.22; IR
(thin film): 3300-2350, 1750 cm-1; HRMS (ESI): Calcd for C16H23O11 [M+H]+ 391.1240,
found 391.1228.

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8
Scheme 2
OH
O
OTBS
O
OAc
O
O
AcO
AcO
c
a,b
O
O
O
69%
67%
HO
HO
AcO
9
10
7a
d
75%
OTBS
OH
O
OAc
OAc
O
OAc
AcO
AcO
AcO
AcO
O
OAc
g
e, f
O
O
82%
75%
X
O
14
OH
11
12 X=CH2
13 X=O
Conditions: (a) NaOMe, MeOH; (b) (CH3)2C(OCH3)2, p-TsOH, CH3CN; (c) TBDMSCl, DMAP, py;
(d) (CO)2Cl2, DMSO, Et3N, then NaBH4, CH3OH; (e) 80% HOAc-H2O, then Ac2O, py-CH2Cl2;
(f) O3, PPh3, CH2Cl2, -78 ºC; (g) NaClO2, KH2PO4, 2-methyl-2-butene, tBuOH-iPrOH-H2O.
OH
O
O
O
HO
3,4-O-isopropylidene-1-allyl-α-D-galactopyranoside (9)
To a solution of allylglycoside (0.81 g, 3.97 mmol) and dimethoxypropane (2.44 mL,
19.85 mmol) in dry acetonitrile (15 mL), p-TsOH (40 mg) was added and the reaction
mixture was stirred for 2h. Water (3 mL) was then added and after 30 min the reaction
was neutralized with triethylamine and evaporated. Flash column chromatography (10%
CH2Cl2/EtOAc) afforded 9 (0.69 g, 70%) as white crystalline solid.
1H NMR (400 MHz, CDCl3) δ 5.86 (1H, dddd, J=7.1, 10.2, 17.2 Hz), 5.15 (1H, dd,
J=1.8, 17.2 Hz), 5.09 (1H, dddd, J=1.5, 17.2 Hz), 4.33-4.25 (2H, m), 4.08-4.02 (2H, m),
3.87-3.77 (2H, m), 2.45-2.30 (2H, m), 2.20 (1H, dd, J=3.2, 9.3 Hz), 2.00 (1H, d, J=4.5
Hz), 1.47 (3H, s), 1.32 (3H, s); 13C NMR (100 MHz, CDCl3) δ 134.3, 117.7, 109.7, 74.8,
73.1, 70.6, 69.6, 69.1, 63.6, 34.9, 26.8, 24.6; HRMS (ESI): Calcd for C12H21O5 [M+H]+
245.1388, found 245.1377.

S
9
OTBS
O
O
O
HO
3,4-O-isopropylidene-6-O-tert-butyldimethylsilyl-1-allyl-α-D-galactopyranoside
(10)
To a solution of diol 9 (0.30 g, 1.21 mmol) in dry pyridine:CH2Cl2 (5:5 mL), tert-
butyldimethylsilyl chloride (0.22 g, 1.45 mmol) and DMAP (7 mg) were added
sequentially. The reaction mixture was stirred overnight, diluted with CH2Cl2 and washed
successively with 0.5 N HCl, water and brine. The organic extract was dried over MgSO4
and concentrated in vacuo. Flash column chromatography (25% EtOAc/hexanes) gave
the product in quantitative yield (0.43 g, >99%).
1H NMR (400 MHz, CDCl3) δ 5.82 (1H, dddd, J=7.0, 7.0, 10.2, 17.1 Hz), 5.16-5.10 (1H,
m), 5.08-5.04 (1H, m), 4.34 (1H, dd, J=1.8, 7.1 Hz), 4.17 (1H, dd, J=3.4, 7.1 Hz), 3.99-
3.93 (2H, m), 3.76-3.64 (3H, m), 2.45 (1H, br s), 2.35-2.28 (2H, m), 1.43 (3H, s), 1.28
13
(3H, s), 0.84 (9H, s), 0.02 (3H, s), 0.02 (3H, s); C NMR (100 MHz, CDCl3) δ 134.4,
117.5, 109.0, 74.6, 71.7, 70.5, 70.0, 69.7, 62.6, 35.0, 27.0, 25.9, 24.5, -5.3 ,-5.4; HRMS
(ESI): Calcd for C18H35O5Si [M+H]+ 359.2253, found 359.2257.
OTBS
O
OH
O
O
3,4-O-isopropylidene-6-O-tert-butyldimethylsilyl-1-allyl-α-D-talopyranoside (11)
To a solution of oxalyl chloride (0.13 mL, 1.51 mmol) in CH2Cl2 (10 mL) at -78 °C,
DMSO (0.23 mL, 3.31 mmol) was added dropwise and the reaction mixture was stirred
for 30 min. Then, 10 (0.27 g, 0.75 mmol) in CH2Cl2 (10 mL) was added over 10 min.
After an additional 30 min of stirring, triethylamine (0.73 mL, 5.27 mmol) was added at -
60°C and the reaction mixture was brought to room temperature. After 1h, the reaction
mixture was diluted with CH2Cl2 and water was added. The layers were separated and the
organic extract was washed with water, brine, dried over MgSO4, and concentrated. The
crude product was immediately dissolved in MeOH (20 mL) and NaBH4 (0.06 g, 1.51
mmol) was added. After stirring for 30 min, the reaction mixture was quenched with
acetic acid and the solvent was evaporated. The residue was taken up in CH2Cl2, washed
with water, brine and then dried over MgSO4. The organic phase was concentrated in
vacuo and after flash column chromatography (25% EtOAc/hexanes), compound 11 was
obtained as an oil (0.41 g, 75%).
1H NMR (400 MHz, CDCl3) δ 5.93 (1H, dddd, J=6.7, 7.4, 10.2, 17.1 Hz), 5.19-5.07 (2H,
m), 4.50 (1H, dd, J=3.6, 8.0 Hz), 4.42 (1H, dd, J=1.5, 8.0 Hz), 3.77 (1H, ddd, J=4.24,
6.50, 9.65 Hz), 3.74-3.57 (4H, m), 2.52-2.27 (2H, m), 1.99 (1H, br s), 1.51 (3H, s), 1.36
13
(3H, s), 0.89 (9H, s), 0.06 (3H, s), 0.06 (3H,s); C NMR (100 MHz, CDCl3) δ 134.4,

S
117.6, 109.8, 73.4, 73.1, 72.5, 70.5, 68.2, 62.3, 36.9, 26.2, 25.9, 24.7, -5.3, -5.5;
HRMS (ESI): Calcd for C18H38NO5Si [M+NH4]+ 376.2519, found 376.2508.
10
OAc
AcO
AcO
OAc
O
Allyl 2,3,4,6-tetra-O-acetyl-α-D-talopyranoside (12)
A solution of acetal 11 (0.22 g, 0.61 mmol) in 80% acetic acid (20 mL) was heated for 1h
at 80 °C. The reaction mixture was cooled and the solvent was evaporated. The residue
was taken up in pyridine (20 mL), followed by addition of acetic anhydride (10 mL) and
DMAP (10 mg). The reaction mixture was stirred overnight, then diluted with CH2Cl2
and washed successively with saturated CuSO4 solution, water and brine. The organic
extract was dried over MgSO4 and concentrated. Flash column chromatography afforded
compound 12 as a white crystalline solid (0.17 g, 75%).
1H NMR (500 MHz, CDCl3) δ 5.77 (1H, dddd, J=6.9, 6.9, 10.7, 16.6 Hz), 5.44 (1H, t,
J=3.2 Hz), 5.18 (1H, dd, J=3.2, 5.1 Hz), 5.11-5.08 (1H, m), 5.08-5.06 (1H, m), 4.83 (1H,
dd, J=3.2, 7.3 Hz), 4.62 (1H, dd, J=8.9, 12.1 Hz), 4.18 (1H, ddd, J=3.8, 4.8, 8.8 Hz), 4.09
(1H, dd, J=3.6, 12.2 Hz), 4.01 (1H, ddd, J=4.8, 7.5, 7.5 Hz), 2.39-2.23 (2H, m), 2.08 (3H,
13
s), 2.05 (3H, s), 2.04 (3H, s), 2.02 (3H, s); C NMR (125 MHz, CDCl3) δ 170.6, 169.6,
169.4, 169.3, 132.8, 117.7, 70.5, 69.2, 68.7, 67.1, 66.4, 60.3, 34.6, 20.7, 20.6, 20.5;
HRMS (ESI): Calcd for C17H28NO9 [M+NH4]+ 390.1764, found 390.1777.
OAc
AcO
AcO
OAc
O
O
2,3,4,6-tetra-O-acetyl-α-D-talopyranosyl aldehyde (13)
Ozone gas was bubbled through a solution of 12a (0.86 g, 2.33 mmol) in 15 mL of dry
o
CH2Cl2 at -78 C until the solution turned blue in color. Nitrogen was then bubbled
through the solution until it turned colorless. Triphenylphosphine (1.5 g, 5.7 mmol) was
then added, the solution was allowed to warm up to room temperature and stirred
overnight. Dichloromethane was removed under reduced pressure and the residue was
purified by flash column chromatography (toluene/acetone 4:1) to afford 13 in 85% yield.
1H NMR (500 MHz, CDCl3) δ 9.70 (1H, t, J=2.1 Hz), 5.56 (1H, t, J=3.0 Hz), 5.18 (1H,
dd, J=3.0, 6.3 Hz), 4.90 (1H, dd, J=9.8, 12.6 Hz), 4.77 (1H, dd, J=3.0, 9.3 Hz), 4.53 (1H,
ddd, J=5.2, 7.4, 9.2 Hz), 4.23 (1H, ddd, J=2.7, 6.3, 9.6 Hz), 4.07 (1H, dd, J=2.8, 12.7
13
Hz), 2.57-2.54 (2H, m), 2.15 (3H, s), 2.07 (3H, s), 2.05 (3H, s), 2.00 (3H, s); C NMR
(125 MHz, CDCl3) δ 199.1, 171.0, 169.6, 169.3, 169.1, 71.8, 69.1, 67.4, 66.5, 63.3, 59.5,

S
44.9, 20.8, 20.8, 20.6, 20.5; HRMS (ESI): Calcd for C16H26NO10 [M+NH4]+
392.1556, found 392.1568.
11
OAc
AcO
AcO
OAc
O
O
OH
2,3,4,6-tetra-O-acetyl-α-D-talopyranosyl acid (14)
To a solution of 13 (2.0 g, 5.4 mmol) in 1:1 (v:v) mixture of tert-butyl alcohol and 2-
methyl-2-butene (20 mL), an aqueous solution of potassium dihydrogen phosphate (3.7
g) and sodium chlorite (2.4 g) in 10 mL distilled water was added. The mixture was
stirred vigorously at 0 °C for 6 hours. The product was extracted with EtOAc and washed
with 100 mL water and 50 mL brine solution. The organic layer was then dried over
MgSO4 and concentrated to afford 14 in 96% yield.
1H NMR (400 MHz, CDCl3) δ 5.57 (1H, t, J=3.0 Hz), 5.20 (1H, dd, J=3.1, 6.2 Hz), 4.83
(1H, dd, J=3.0, 9.2 Hz), 4.76 (1H, dd, J=9.5,12.5 Hz), 4.38 (1H, ddd, J=3.7, 8.9, 8.9 Hz),
4.29 (1H, ddd, J=3.0, 6.2, 9.3 Hz), 4.17 (1H, dd, 3.0, 12.6 Hz), 2.62 (1H, dd, J=3.7, 15.8
Hz), 2.53 (1H, dd, J=8.8, 15.8 Hz), 2.16 (3H, s), 2.08 (3H, s), 2.07 (3H, s), 2.03 (3H, s);
13C NMR (100 MHz, CDCl3) δ 173.9, 171.0, 169.7, 169.3, 169.2, 71.6, 68.9, 67.4, 66.5,
65.0, 59.8, 36.1, 20.9, 20.7, 20.6, 20.6; LRMS (ESI): Calcd for C16H23O11 [M+H]+ 391.3,
found 391.4; HRMS (ESI): Calcd for C16H26NO11 [M+NH4]+ 408.1505, found 408.1512.
(iii) Synthesis of C-linked glycoconjugate
Scheme 3
OAc
R1
OAc
R1
R3
R3
O
O
R4
AcO
R4
AcO
R2
R2
BocHN
O
O
c
X
, b
a
HN
HN
X = 8a, 8b,
8c, or 14
FmocHN
CO2Bn
FmocHN
CO2Bn
FmocHN
CO2H
15
20: Gal: 84%
21: Glc: 87%
22: Man: 82%
23: Tal: 85%
16: Gal: 83%
17: Glc: 85%
18: Man: 93%
19: Tal: 87%
Conditions: (a) TFA/CH2Cl2; (b) HBTU, DIPEA, CH2Cl2; (c) 10% Pd/C, H2, EtOH-EtOAc.

S
12
General Protocol for amide coupling of C-linked glycoconjugate
To a solution of fully protected amino acid derivative 15 (0.21 g, 0.38 mmol) in 10 mL of
CH2Cl2, 2 mL of TFA was added. The reaction was stirred for 40 minutes and then
concentrated under reduced pressure. The syrup was re-dissolved in a 1:1 mixture of
toluene and CH2Cl2, concentrated, then re-dissolved in diethyl ether and concentrated to
afford a white solid. To a solution of carboxylic acid carbohydrate derivative (0.15 g,
0.38 mmol), HBTU (0.17 g, 0.46 mmol) in 15 mL of CH2Cl2, and 0.20 mL (0.15 g, 1.14
mmol) of DIPEA were added. The reaction was allowed to stir for 20 minutes and then
the deprotected amino acid derivative was added. After stirring overnight, the reaction
mixture was washed successively with saturated ammonium chloride, water, brine, dried
over MgSO4 and concentrated. The product was purified by flash column
chromatography in 50:1 CH2Cl2 and MeOH to afford a C-linked glycosyl amide building
block in ~80% yield.
OAc
O
AcO
AcO
AcO
O
HN
OBn
FmocHN
O
Benzyl
Fluorenylmethoxycarbonyl-L-ornithine-1-(2,3,4,6-tetra-O-acetyl-α-D-
galactopyranoside) (16)
1H NMR (300 MHz, CDCl3) δ 7.76 (2H, d, J=7.5 Hz), 7.59 (2H, d, J=7.21 Hz), 7.42-7.28
(9H, m), 6.12 (1H, br s), 5.55 (1H, d, J=8.4 Hz), 5.4 (1H, t, J=3.3 Hz), 5.29-5.23 (1H, m),
5.17-5.12 (3H, m), 4.67-4.61 (1H, m), 4.46-4.34 (3H, m), 4.25-4.19 (2H, m), 4.15-4.09
(2H, m), 3.29-3.23 (2H, m), 2.57-2.47 (1H, m), 2.41-2.34 (1H, m), 2.10 (3H, s), 2.05 (3H,
s), 2.04 (3H, s), 2.00 (3H, s), 1.93-1.82 (1H, m), 1.73-1.47 (3H, m); 13C NMR (75 MHz,
CDCl3) δ 171.9, 170.5, 169.9, 169.7, 169.5, 169.2, 156.0, 143.6, 141.2, 135.0, 128.6,
128.5, 127.7, 127.0, 125.0, 119.9, 69.4, 68.7, 67.8, 67.7, 67.2, 67.0, 66.8, 61.0, 53.5, 47.0,
38.6, 34.4, 30.0, 25.3, 20.7, 20.6; LRMS (ESI): Calcd for C43H49N2O14 [M+H]+ 817.8,
found 817.4.

S
13
OAc
O
AcO
AcO
AcO
O
HN
OBn
FmocHN
O
Benzyl
Fluorenylmethoxycarbonyl-L-ornithine-1-(2,3,4,6-tetra-O-acetyl-α-D-
glucopyranoside) (17)
1H NMR (300 MHz, CDCl3) δ 7.76 (2H, d, J=7.4 Hz), 7.60 (2H, d, J=7.1 Hz), 7.45-7.25
(9H, m), 6.17 (1H, br s), 5.64 (1H, d, J=7.9 Hz), 5.23 (1H, t, J=8.4 Hz), 5.20-5.04 (3H,
m), 4.97 (1H, t, J=8.3 Hz), 4.25-4.12 (3H, m), 3.97-3.90 (1H, m), 3.35-3.17 (2H, m), 2.60
(1H, dd, J=9.6, 12.4 Hz), 2.45 (1H, dd, J=4.4, 15.4 Hz), 2.07-1.98 (12H, m), 1.95-1.77
(1H, m), 1.76-1.60 (1H, m), 1.60-1.46 (2H, m); 13C NMR (75 MHz, CDCl3) δ 171.9,
170.6, 169.9, 169.4, 169.0, 156.0, 143.8, 143.6, 141.2, 135.1, 128.6, 128.5, 128.5, 128.3,
127.7, 127.0, 125.0, 119.9, 69.9, 69.7, 69.4, 69.3, 68.2, 67.3, 67.0, 62.0, 53.5, 47.1, 38.9,
38.6, 34.2, 30.0, 25.3, 20.7, 20.6, 20.6; HRMS (ESI): Calcd for C43H49N2O14 [M+H]+
817.3183, found 817.3192.
OAc
OAc
O
AcO
AcO
O
HN
OBn
FmocHN
O
Benzyl
Fluorenylmethoxycarbonyl-L-ornithine-1-(2,3,4,6-tetra-O-acetyl-α-D-
mannopyranoside) (18)
1H NMR (300 MHz, CDCl3) δ 7.76 (2H, d, J=7.4 Hz), 7.60 (2H, d, J=7.1 Hz), 7.44-7.25
(9H, m), 6.18 (1H, br s), 5.71 (1H, d, J=8.1 Hz), 5.25 (1H, dd, J=3.2, 7.2 Hz), 5.20-5.07
(3H, m), 4.47-4.30 (5H, m), 4.25-4.15 (2H, m), 4.02-3.94 (1H, m), 3.40-3.20 (2H, m),
2.52 (2H, d, J=6.87 Hz), 2.10-2.06 (12H, m), 2.00-1.82 (1H, m), 1.80-1.65 (1H, m), 1.64-
13
1.50 (2H, m); C NMR (75 MHz, CDCl3) δ 172.0, 170.6, 169.9, 169.8, 169.5, 168.8,
156.1, 143.8, 143.6, 141.2, 135.1, 128.6, 128.6, 128.5, 128.5, 128.3, 127.7, 127.6, 127.0,
126.9. 125.0, 119.9, 72.0, 69.9, 69.1, 68.0, 67.3, 67.2, 67.0, 65.2, 61.7, 53.6, 47.1, 38.9,
38.6, 37.3, 29.9, 25.5, 20.7, 20.7, 20.6; HRMS (ESI): Calcd for C43H49N2O14 [M+H]+
817.3183, found 817.3198.

S
14
OAc
OAc
O
AcO
AcO
O
HN
OBn
FmocHN
O
Benzyl
Fluorenylmethoxycarbonyl-L-ornithine-1-(2,3,4,6-tetra-O-acetyl-α-D-
talopyranoside) (19)
1H NMR (500 MHz, CDCl3) δ 7.69 (2H, d, J=7.3 Hz), 7.52 (2H, d, J=7.8 Hz), 7.35-7.17
(9H, m), 6.18-6.11 (1H, m), 5.54 (1H, d, J=7.6 Hz), 5.46 (1H, t, J=3.3 Hz), 5.14-5.05
(3H, m), 4.70 (1H, dd, J=2.6, 9.0 Hz), 4.55 (1H, dd, J=9.8, 12.6 Hz), 4.40-4.00 (7H, m),
3.23-3.10 (2H, m), 2.37 (1H, dd, J=2.7, 15.1 Hz), 2.23 (1H, dd, J=9.0, 14.8 Hz), 2.06
(3H, s), 1.99 (3H, s), 1.96 (3H, s), 1.93 (3H, s), 1.87-1.77 (1H, m), 1.67-1.57 (1H, m),
1.53-1.42 (2H, m); 13C NMR (125 MHz, CDCl3) δ 172.0, 170.7, 169.6, 169.4, 169.2,
169.2, 156.0, 143.8, 143.6, 141.2, 135.1, 128.6, 128.5, 128.2, 127.6, 127.0, 125.0, 125.0,
119.9, 71.7, 69.0, 67.4, 67.2, 67.0, 66.4, 65.6, 60.1, 53.6, 47.0, 38.9, 38.5, 38.2, 29.9,
25.4, 20.8, 20.8, 20.6; HRMS (ESI): Calcd for C43H49N2O14 [M+H]+ 817.3183, found
817.3149.
General protocol for debenzylation of C-linked glycoconjugate
To a solution of benzyl ester in 1:1 ethanol/ethyl acetate, a catalytic amount (8% w/w) of
palladium on charcoal (10% w/w) was added. The reaction mixture was stirred under H2
atmosphere and monitored via TLC. After the reaction was completed, the catalyst was
gravity filtered through Whatman No.2 filter paper. The combined filtrates were
concentrated and the compound was purified by column chromatography (5%
MeOH/CH2Cl2) to afford a crystalline solid in >80% yield.

S
15
OAc
O
AcO
AcO
AcO
O
HN
OH
FmocHN
O
Fluorenylmethoxycarbonyl-L-ornithine-1-(2,3,4,6-tetra-O-acetyl-α-D-
galactopyranoside) (20)
1H NMR (300 MHz, CDCl3) δ 7.76 (2H, d, J=7.2 Hz), 7.60 (2H, d, J=6 Hz), 7.42-7.37
(2H, m), 7.33-7.28 (2H, m) 6.25 (1H, br s), 5.6 (1H, d, J=7.5 Hz), 5.39 (1H, t, J=3.3 Hz),
5.27-5.23 (1H, m), 5.16-5.12 (1H, m), 4.68-4.63 (1H, m), 4.39 (2H, d, J=6.6), 4.33-4.05
(5H, m), 3.37-3.25 (2H, m), 2.60 (1H, dd, J=15.5, 9.8 Hz), 2.44 (1H, dd, J=15.5, 4.2 Hz),
2.11 (3H, s), 2.05 (3H, s), 2.04 (3H, s), 2.02 (3H, s), 1.98-1.86 (1H, m), 1.82-1.69 (1H,
m), 1.70-1.54 (2H, m); 13C NMR (100 MHz, CDCl3) δ 174.4, 172.4, 170.9, 170.4, 170.0,
169.8, 156.1, 143.6, 141.2, 127.7, 127.0, 125.0, 119.9, 69.1, 68.9, 67.8, 67.7, 66.9, 61.2,
53.4, 50.6, 47.0, 39.1, 34.1, 31.7, 28.6, 22.1, 20.7; IR (thin film): 3367, 1748 cm-1; LRMS
(ESI): Calcd for C36H43N2O14 [M+H]+ 726.3, found 727.0.
OAc
O
AcO
AcO
AcO
O
HN
OH
FmocHN
O
Fluorenylmethoxycarbonyl-L-ornithine-1-(2,3,4,6-tetra-O-acetyl-α-D-
glucopyranoside) (21)
1H NMR (300 MHz, CDCl3) δ 7.75 (2H, d, J=7.6 Hz), 7.63-7.55 (2H, m), 7.38 (2H, t,
J=7.4 Hz), 7.33-7.25 (2H, m), 6.64-6.53 (1H, m), 5.83 (1H, d, J=7.4 Hz), 5.24 (1H, t,
J=8.3 Hz), 5.07 (1H, dd, J=5.2, 8.6 Hz), 4.98 (1H, t, J=8.3 Hz), 4.70-4.60 (1H, m), 4.45-
4.30 (3H, m), 4.25-4.12 (2H, m), 4.05-3.90 (3H, m), 3.40-3.20 (2H, m), 2.66 (2H, dd,
J=9.9, 15.2 Hz), 2.49 (2H, dd, J=4.3, 15.3 Hz), 2.05 (3H, s), 2.03 (3H, s), 2.02 (3H, s),
2.01 (3H, s), 1.97-1.82 (1H, m), 1.81-1.67 (1H, m), 1.67-1.51 (2H, m); 13C NMR (75
MHz, CDCl3) δ 171.0, 170.0, 169.6, 169.5, 156.3, 143.8, 143.7, 141.3, 127.7, 127.1,
125.1, 120.0, 77.2, 69.9, 69.7, 69.5, 69.4, 68.2, 67.1, 62.1, 53.5, 47.1, 39.1, 34.2, 29.8,
25.2, 20.7, 20.7, 20.6, 20.6; HRMS (ESI): Calcd for C36H43N2O14 [M+H]+ 727.2714,
found 727.2739.

S
16
OAc
OAc
O
AcO
AcO
O
HN
OH
FmocHN
O
Fluorenylmethoxycarbonyl-L-ornithine-1-(2,3,4,6-tetra-O-acetyl-α-D-
mannopyranoside) (22)
1H NMR (300 MHz, CDCl3) δ 7.74 (2H, d, J=7.4 Hz), 7.58 (2H, dd, J=4.8, 7.1 Hz), 7.37
(2H, dd, 7.2, 7.9 Hz), 7.27 (2H, dt, J=1.1, 7.7, 7.8 Hz), 6.72 (1H, br m), 5.94 (1H, d,
J=7.5 Hz), 5.24 (1H, dd, J=3.1, 7.4 Hz), 5.15-5.08 (2H, m), 4.45-4.30 (4H, m), 4.22-4.12
(2H, m), 4.02-3.92 (1H, m), 3.38-3.20 (2H, m), 2.65-2.45 (2H, m), 2.05 (3H, s), 2.04 (3H,
13
s), 2.03 (6H, s), 1.97-1.82 (1H, m), 1.80-1.65 (1H, m), 1.65-1.52 (2H, m); C NMR (75
MHz, CDCl3) δ 174.5, 170.9, 170.2, 169.9, 169.8, 169.5, 156.3, 143.7, 143.6, 141.2,
127.7, 127.0, 125.0, 119.9, 77.2, 71.7, 70.1, 69.3, 68.0, 67.2, 67.0, 61.8, 53.4, 47.0, 39.1,
36.9, 29.6, 25.2, 20.7, 20.7, 20.7, 20.6; HRMS (ESI): Calcd for C36H43N2O14 [M+H]+
727.2714, found 727.2715.
OAc
OAc
O
AcO
AcO
O
HN
OH
FmocHN
O
Fluorenylmethoxycarbonyl-L-ornithine-1-(2,3,4,6-tetra-O-acetyl-α-D-
talopyranoside) (23)
1H NMR (400 MHz, CDCl3) δ 9.3-8.2 (1H, br s), 7.74 (2H, d, J=7.4 Hz), 7.59 (2H, d,
J=6.9 Hz), 7.33 (4H, dt, J=7.1, 28.6 Hz), 6.73 (1H, br s), 5.99 (1H, br d, J=5.6 Hz), 5.52
(1H, br s), 5.16 (1H, br dd, J=3.0, 5.2), 4.80 (1H, br dd, J=2.2, 8.3 Hz), 4.62 (1H, br dd,
J=9.8, 12.6 Hz), 4.50-4.11 (6H, m), 3.4-3.15 (2H, br m), 2.58-2.30 (2H, br m), 2.12 (3H,
s), 2.06 (3H, s), 2.03 (3H, s), 2.00 (3H, s), 1.96-1.83 (1H, m), 1.80-1.66 (1H, m), 1.66-
1.52 (2H, m); 13C NMR (100 MHz, CDCl3) δ 171.0, 170.2, 169.7, 169.5, 156.3, 143.8,
143.6, 141.2, 127.7, 127.0, 125.1, 120.0, 71.5, 68.9, 67.3, 66.9, 66.4, 66.0, 60.3, 47.0,
39.0, 37.9, 29.7, 25.3, 20.8, 20.8, 20.6; HRMS (ESI): Calcd for C36H43N2O14 [M+H]+
727.2714, found 727.2701.

S
17
(iv) Solid Phase Synthesis of C-Linked Glycopeptides
General protocol for manual and automated solid phase synthesis of C-linked
analogues
All polypeptides were prepared by linear solid-phase synthesis using standard Fmoc
chemistry.2 A typical procedure started from Fmoc-glycine Wang resin with loading
capacities of ~0.066 mmol/g. The resin was swollen in DMF for 30 minutes. The solvent
was then drained and 20% piperidine solution in DMF was added. The solution was
allowed to stir for 1 hour, then it was drained, and the resin was washed with three
aliquots of DMF. Kaiser and TNBS tests for free amine were then performed. Building
block (1.5 equivalents) was premixed with 1.5 equivalents of HBTU in DMF, followed
by the addition of 1.5 equivalents of DIPEA in DMF. The reaction was stirred for 30
minutes, transferred to the SPS flask and stirred for 24 hours. The flask was drained and
the resin was rinsed three times with DMF. Kaiser and TNBS tests were performed to
verify the coupling had reached completion (negative test outcome). The resin was
treated with 20% piperdine in DMF solution for 1 hour, the flask was drained and the
resin was washed thoroughly with DMF. Kaiser and TNBS tests were performed to
ensure the presence of free amine. Then the next successive building block (5
equivalents) to be coupled (commercially available amino acid) was premixed with 5
equivalents of HBTU and 5 equivalents of DIPEA in DMF for 20 minutes. The reaction
solution was then transferred to the resin, and the reaction mixture was allowed to stir for
4 hours. The Fmoc deprotection and coupling steps were repeated until twelve amino acid
residues were coupled to the resin. To cleave the glycopeptide from the resin, the resin
was successively rinsed with DMF, MeOH, and CH2Cl2, and stirred for 2 hours in 1:1
(v:v) trifluoroacetic acid and CH2Cl2. The solution was filtered and concentrated, and the
product crystallized from diethyl ether to produce a white powder. The glycopolymer was
dissolved in a sodium methoxide (pH=10) solution (4:1 sodium methoxide/ water) and
stirred for 5 hours. The solution was neutralized with IR-120 ion exchange resin, filtered,
concentrated to remove MeOH and lyophilized to give the product as a white powder.
Purification via HPLC using reverse phase chromatography (C-18 column, liquid phase:
0.1% TFA in acetonitrile/water) was then performed to obtain the desired glyopeptide.
[L-Ornithine(galactose)-glycine-glycine]4 -glycine (1)
1H NMR (300 MHz, D2O) δ 4.42-4.32 (4H, m), 4.28-4.18 (4H, m), 4.00-3.80 (29H, m),
3.75-3.67 (4H, m), 3.67-3.47 (14H, m), 3.20-3.05 (8H, m), 2.62-2.42 (8H, m), 1.85-1.70
(5H, m), 1.70-1.57 (4H, m), 1.57-1.37 (9H, m); 13C NMR (75 MHz, D2O) δ 175.0, 174.9,
174.1, 173.9, 173.6, 172.3, 172.3, 172.2, 172.0, 171.9, 171.8, 170.7, 163.6, 163.1, 73.3,
72.8, 70.0, 69.1, 67.9, 61.2, 54.1, 53.2, 42.9, 42.7, 41.5, 41.4, 39.1, 32.7, 28.4, 25.2;
LRMS (MALDI-TOF): Calcd for C70H117N17O38 [M+H]+ 1805.8, found 1805.3.
2 Fields, G. B.; Fields, C. G. J. Am. Chem. Soc. 1991, 113, 4202.

S
18
[L-Ornithine(glucose)-glycine-glycine]4 -glycine (2)
1H NMR (360 MHz, D2O) δ 4.47 (32H, s), 4.08 (2H, br s), 3.73 (6H, m), 3.32 (10H, m),
3.01 (6H, m), 2.45 (2H, m), 2.19 (18H, s), 1.98 (8H, s), 1.68 (8H, m), 1.30 (4H, m), 0.87
(2H, m); 13C NMR (90 MHz, D2O) δ 187.76, 186.77, 185.19, 184.83, 184.24, 180.00,
159.30, 143.77, 142.41, 142.34, 139.011, 138.55, 97.19, 86.21, 85.68, 82.81, 81.66,
80.76, 80.63, 74.08, 71.31, 66.13, 60.96, 55.79, 55.60, 51.98, 45.61, 45.43, 38.05, 36.468,
19.55; LRMS (ESI): Calcd for C70H117N17O38 [M+H]+ 1805.8, found 1805.2
[L-Ornithine(mannose)-glycine-glycine]4 -glycine (3)
1H NMR (500 MHz, D2O) δ 4.83 (32H, m), 4.45 (4H, m), 4.07 (6H, m), 3.93 (8H, m),
3.65 (4H, m), 3.46 (4H, m), 3.21 (3H, m), 2.98 (1H, m), 2.80 (4H, m), 2.68 (1H, m), 2.58
(1H, m), 2.25 (4H, m), 2.07 (16H, m), 1.79 (4H, m), 1.60 (8H, m), 1.33 (8H, m), 1.06
(4H, m); 13C NMR (125 MHz, D2O) δ 181.01, 170.59, 117.47, 117.05, 74.60, 74.33,
74.07, 70.34, 70.06, 66.70, 62.04, 60.51, 42.73, 42.07, 41.95, 38.29, 35.01, 34.79, 29.77,
27.69, 24.29, 22.81; LRMS (ESI): Calcd for C70H117N17O38 [M+H]+ 1805.8, found
1804.1.
[L-Ornithine(2,3,4,6-peracetylated talose)-glycine-glycine]4 -glycine
1H NMR (300 MHz, MeOD) δ 5.51 (1H, br s), 5.21 (1H, br s), 4.65-4.53 (1H, m), 4.46-
3.82 (10H, m), 3.24 (3H, m), 2.60-2.45 (2H, m), 2.11 (3H, s), 2.08 (3H, s), 2.06 (6H, s),
1.97-1.83 (1H, m), 1.83-1.72 (1H, m), 1.72-1.52 (2H, m); 13C NMR (75 MHz, MeOD) δ
174.3, 174.0, 171.9, 171.5, 171.4, 171.1, 170.8, 170.5, 170.5, 170.4, 71.1, 69.4, 68.1,
67.5, 67.0, 60.9, 53.9, 53.9, 53.2, 42.7, 40.9, 39.2, 38.9, 37.6, 29.8, 28.7, 25.9, 25.0, 24.9,
22.8, 20.0, 19.8, 19.7, 19.7; LRMS (MALDI-TOF): Calcd for C102H149N17O54 [M+H]+
2478.4, found 2476.8.
[L-Ornithine(talose)-glycine-glycine]4 -glycine (4)
1H NMR (500 MHz, D2O) δ 4.27-4.17 (5H, m), 3.92-3.72 (20H, m), 3.67-3.55 (7H, m),
3.45-3.40 (1H, m), 3.14-3.07 (6H, m), 2.54-2.40 (6H, m), 1.80-1.70 (2H, m), 1.70-1.55
13
(3H, m), 1.55-1.37 (6H, m); C NMR (125 MHz, D2O) δ 174.4, 172.6, 171.8, 171.5,
170.9, 99.9, 73.9, 72.9, 72.8, 70.6, 68.8, 66.8, 60.0, 53.8, 53.5, 43.0, 42.4, 42.3, 42.2,
38.8, 38.6, 36.0, 28.0, 24.7, 24.6, 24.4; LRMS (MALDI-TOF): Calcd for C70H117N17O38
[M+H]+ 1805.8, found 1804.8.

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19
II. Assessing Antifreeze Activity:
Antifreeze activity of analogues 1-4 was evaluated using two standard methods. Thermal
hysteresis (TH) was measured using a Clifton Nanoliter Osmometer3
and
recrystallization-inhibition (RI) activity was assessed using the splat cooling technique
previously described by Knight and co-workers.4 Briefly, the latter technique involves
generating a frozen wafer from the analyte which is dissolved in phosphate buffered
saline (PBS) solution; a 10 uL drop of this solution is then dropped from a pipette
through a two metre high plastic tube (10 cm in diameter) onto a block of polished
o
aluminum pre-cooled to approximately -80 C. The droplet freezes instantly on the
polished aluminum block and is approximately 1 cm in diameter and 20 um thick. This
wafer is then carefully removed from the surface of the block and transferred to a
o
cryostage (refrigerated microscope stage) held at -6.4 C. The wafer is left to anneal at
this temperature for a period of 30 mins and then photographed between crossed
polarizing filters using a digital camera (Nikon CoolPix 5000) fitted to the microscrope.
A total of six different images are taken from each wafer; three from the middle and three
from the edge. The images are then analyzed using the mean elliptical method. In this
method, the ten largest ice crystals were chosen from the field of view (FOV) in each
image.5,6 Selection of these crystals was arbitrary in that they were chosen after a visual
inspection of the image. The two dimensional surface area of each of these ten crystals
was then calculated via approximation of the crystal as an elliptical area. The major and
minor elliptical axes were defined by the two largest orthogonal dimensions across the
ice grain surface. The surface area of each ice grain was then calculated based on the
formula: A = πab, in which A represented area; a and b represented the length of the
major and minor elliptical axes. Totalling all individual measurements for each FOV
produces a value for the average grain surface area referred to as the mean largest grain
size (MLGS). Error was calculated using standard error of the mean (SEM). T-tests were
performed to a 95% confidence level.
III. Conformational Analysis via Circular Dichroism
Circular Dichroism was performed on JASCO Model J-810 automatic spectrophotometer
interfaced with Acer computer. Spectral data were obtained with the following
parameters: 1nm bandwidth; four scans at a scan speed of 50nm/min; 190-350nm
o
wavelength range. Samples were measured at 22 C in a quartz cell (Hellma QS) with a
1cm path length. A molar concentration of 43 µM was used for each sample, diluted in
doubly distilled water. Data obtained from CD spectroscopy was converted into molar
ellipticity (deg cm2 dmol-1). Glycopeptide secondary structures were estimated using the
deconvolution software CD Pro (SELCON 3 program and IBASIS 4 protein dataset).7
3 Chakrabartty, A.; Hew, C.L.; Eur. J. Biochem. 1991, 202, 1057.
4 Knight, C. A.; Hallett, J.; DeVries, A.L. Cryobiology 1988, 25, 55.
5 Eniade, A.; Purushotham, M.; Ben, R.N.; Wang, J.B.; Horwath, K. Cell Biochem. Biophys. 2003, 38, 115.
6 Liu, S.; Ben, R.N. Org. Lett. 2005, 7, 2385.
7
(a) Sreerama, N.; Venyaminov, S.Y.; Woody, R.W. Anal. Biochem. 2000, 287, 243. (b) Sreerama, N.; Woody, R.W.
Anal. Biochem. 2000, 287, 252-260. (c) Greenfield, N. J. Anal. Biochem. 1996, 35, 1-10.

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20
IBASIS 4
Conformation
Regular Distorted Regular Distorted
α-Helix α-Helix β-Strand β-Strand
Random
coil
Turn
Sum NRMSD
OGG Gal (1) -0.011 0.160
OGG Glc (2) -0.012 0.172
0.031
-0.004
0.008
0.084
0.164
0.092
0.073
0.007
0.101
0.094
0.256 0.504 1.032 0.543
0.232 0.568 1.029 0.425
0.225 0.540 0.954 0.440
0.248 0.529 1.031 0.407
0.237 0.378 1.036 0.833
OGG Man (3) 0.010
OGG Tal (4) 0.020
0.164
0.049
0.114
AFGP-8
0.049
Table 1. Deconvolution data for AFGP-8, C-linked analogues 1-4, using SELCON 3 and IBASIS 4.

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21
OAc
O
AcO
AcO
AcO
Br
6a
OAc
O
AcO
AcO
AcO
Br
6a

S
22
OAc
O
AcO
AcO
AcO
7a
OAc
O
AcO
AcO
AcO
7a

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23
OAc
O
AcO
AcO
AcO
O
OH
8a
OAc
O
AcO
AcO
AcO
O
OH
8a

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24
OAc
O
AcO
AcO
AcO
O
HN
OBn
FmocHN
O
16
OAc
AcO
AcO
O
AcO
O
HN
OBn
FmocHN
O
16

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25
OAc
O
AcO
AcO
AcO
O
HN
OH
FmocHN
O
20
OAc
AcO
AcO
O
AcO
O
HN
OH
FmocHN
O
20

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26
[L-Ornithine(galactose)-glycine-glycine]4 -glycine (1)
[L-Ornithine(galactose)-glycine-glycine]4 -glycine (1)

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27
OAc
OAc
O
AcO
AcO
Br
6c
OAc
OAc
O
AcO
AcO
Br
6c

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28
OAc
O
AcO
AcO
AcO
Br
6b
OAc
O
AcO
AcO
AcO
Br
6b

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29
OAc
OAc
O
AcO
AcO
7c
OAc
OAc
O
AcO
AcO
7c

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30
OAc
OAc
O
AcO
AcO
O
OH
8c
OAc
OAc
O
AcO
AcO
O
OH
8c