Random-coil:α-helix equilibria as a reporter for the Lewis X-LewisX interaction

Article abstract of DOI:10.1002/anie.201101055

Probing weak interactions: A peptide random-coil:α-helix equilibrium has been used to identify a weak carbohydrate-carbohydrate interaction (CCI). Glucose and lactose destabilized the helical conformer while Lewis^X trisaccharide led to increased helicity. Though small, the trend observed indicates that this peptide reporter can detect a single CCI in isolation. Copyright

Full text of DOI:10.1002/anie.201101055

DOI: 10.1002/anie.201101055
Carbohydrates
Random-Coil:a-Helix Equilibria as a Reporter for the LewisX–LewisX
Interaction**
Timothy M. Altamore, Christian Fernꢀndez-Garcꢁa, Andrew H. Gordon, Tina Hꢂbscher,
Netnapa Promsawan, Maxim G. Ryadnov, Andrew J. Doig, Derek N. Woolfson,* and
Timothy Gallagher*
Weak multivalent interactions are now recognized as key to
many biological processes.[1] Since the discovery of carbohy-
drate–carbohydrate interactions (CCIs),[2] studies of this
phenomenon have now linked CCIs (both cis- and trans-
CCIs) to critical biological recognition events, such as cell
signaling and adhesion, fertilization, and metastasis.[3] CCIs
are intrinsically weak so their study and quantification at the
monovalent level is a significant challenge that represents the
focus of this Communication.
enon in isolation (i.e. outside of a multivalent environ-
ment),[10] we have evaluated the ability of a conformationally
dynamic system to report on a weak, attractive CCI based on
LeX–LeX. Random-coil:a-helix equilibria displayed by ala-
nine-rich peptides in aqueous solution, where helix content is
highly sensitive to small changes in the free energy of helix
formation, provide an attractive, effective and potentially
versatile vehicle for this purpose (Figure 1a). The requisite
Previous work has relied on macroscopic or multivalent
systems including synthetic polymers,[4] micelles and vesi-
cles,[5] glycosylated nanoparticles,[6] and Langmuir–Blodgett
monolayers.[7] These studies have generally (though not
exclusively) focused on the biologically important LewisX–
LewisX (LeX–LeX) interaction.[4–8] As a result, a number of
factors important in CCIs are now apparent, and these include
multivalent (Velcro-like) presentation of carbohydrates on a
surface; a requirement for polyamphiphilic surfaces associ-
ated with the hydraphobic effect;[9] and, in certain cases, roles
for both divalent metal cations (e.g. Ca2+) and ionic (charge)
effects. While multivalency effectively amplifies CCIs, the
complexity of such macroscopic systems makes mapping the
individual impact of component carbohydrate (CHO) units
and their associated molecular features difficult to define.
To achieve a more detailed picture of CCIs, while
recognizing the inherent challenge of studying this phenom-
[*] Dr. T. M. Altamore, C. Fernꢀndez-Garcꢁa, Dr. A. H. Gordon,
Dr. T. Hꢂbscher, Dr. N. Promsawan, Prof. Dr. D. N. Woolfson,
Prof. Dr. T. Gallagher
Figure 1. a) Schematic of random-coil:a-helix equilibrium for detection
of CCIs. b) Helical wheel diagram showing functionalized i, i+4, and
i+5 sites. c) General peptide sequences used in this study.
School of Chemistry, University of Bristol
Bristol BS8 1TS (UK)
E-mail: t.gallagher@bris.ac.uk
Prof. Dr. D. N. Woolfson
School of Biochemistry, University of Bristol
Bristol BS8 1TD (UK)
E-mail: d.n.woolfson@bris.ac.uk
peptides are readily accessible, and helix content can be
measured accurately by circular dichroism (CD) spectros-
copy. We posit that with two CHOs ligated at specified
positions (Figure 1b) on the peptide backbone, perturbation
of this highly sensitive equilibrium to a more helical state
would indicate the presence of an attractive (i.e. stabilizing)
CCI, thereby providing a means of studying this phenomenon
in comparative isolation, outside of a multivalent environ-
ment.
To validate the feasibility of a peptide-based reporter for
this purpose, a series of 19-residue host peptides was designed
(Figure 1c). These comprised mainly Ala and Lys residues,
incorporating Tyr (as a UV determinant of peptide concen-
Prof. Dr. A. J. Doig
Manchester Interdisciplinary Biocentre
Faculty of Life Sciences, University of Manchester
Manchester M1 7DN (UK)
Dr. M. G. Ryadnov
National Physical Laboratory, Teddington TW11 0LW (UK)
[**] EPSRC, the governments of Germany, Thailand, and Mexico
(CONACyT), and AstraZeneca are acknowledged for financial
support.
Supporting Information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201101055.
Angew. Chem. Int. Ed. 2011, 50, 11167 –11171
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
11167

Communications
Table 1: Helicities of peptides: controls and glycopeptides variants.
Control peptides (X=K)
% Helicity[a]
tration) and orthogonally protected Lys units provided the
two necessary glycosylation sites. The N- and C-termini were
capped with acetyl and amide groups, respectively, to remove
charges and stabilize the helix. Peptides were designed to be
close to 50% helical in order to be maximally sensitive (given
the weak nature of the interaction to side-chain interactions
being pursued) involving the CHO guests. Modified Lifson–
Roig helix–coil theory[11] was used to predict a-helix contents
for the isolated peptides in aqueous solution, using parame-
ters for interior and N-cap positions of helices.[12] Given the a-
helical repeat of 3.6 amino acids per turn (Figure 1b)
glycosylation sites were spaced i, i+4 to locate two CHO
moieties close in space, and to maximize the population of
(and preference for) the helical component in the presence of
a stabilizing CCI.
3a
3b
3c
3d
i, i+4
i, i+5
i, i+4
i, i+5
Ac-AKAAAAKAXAAAXAKAAGY-NH2
Ac-AKAAAAKAXAAAAXAKAGY-NH2
Ac-AKAAAAKAEAAAXAKAAGY-NH2
Ac-AKAAAAKAEAAAAXAKAGY-NH2
38
41
54
39
Acetylated control peptides (X=KAc)
4a
4b
4c
–
Ac-AKAAAAKAXAAAKAKAAGY-NH2
Ac-AKAAAAKAXAAAXAKAAGY-NH2
Ac-AKAAAAKAXAAAAXAKAGY-NH2
52
52
51
i, i+4
i, i+5
LeX glycopeptides (X=KCOCH2S(CH2)3OLeX)
5a
5b
5c
–
Ac-AKAAAAKAXAAAKAKAAGY-NH2
Ac-AKAAAAKAXAAAXAKAAGY-NH2
Ac-AKAAAAKAXAAAAXAKAGY-NH2
51
54
49
i, i+4
i, i+5
General controls were provided by i, i+5 variants since
this relationship provides no stabilizing interaction within an
a-helix (see Figure 1b); other controls/benchmarks were also
employed as discussed below. Specifically, we have used these
sequences to probe the LeX–LeX interaction, and to validate
the applicability and veracity of the coil:helix approach to
detecting and studying CCIs. The introduction of a function-
alized C3 linker based on LeX thioglycoside 1[13] was achieved
(see Supporting Information), and provided LeX thiol 2a and
disulfide 2b (Figure 2); both were used for peptide ligation.
The same C3 linker unit was common to all the CHO-based
controls employed in this study.
Other controls (X=KCOCH2S(CH2)3O-Glc 6a/b or O-Lac 7a/b)
6a
6b
7a
7b
i, i+4
i, i+5
i, i+4
i, i+5
Ac-AKAAAAKAXAAAXAKAAGY-NH2
Ac-AKAAAAKAXAAAAXAKAGY-NH2
Ac-AKAAAAKAXAAAXAKAAGY-NH2
Ac-AKAAAAKAXAAAAXAKAGY-NH2
40
48
37
49
[a] CD spectroscopy was performed at 58C (pH 7.0; 10 mm MOPS
buffer) with helicity values calculated using fh =(qobsÀqcoil)/(qhÀqcoil).[14]
Experimental errors, based on multiple scans and replicates (see
Supporting Information), were all within the range 0.4–1.4%.
(controls 3a and 3b) had similar and relatively low (38% and
41%, respectively) helical contents.[15] To demonstrate the
sensitivity of the peptide reporter to a stabilizing side-chain
interaction, we used a known[16] glutamate-to-lysine interac-
tion: the i, i+4 peptide 3c displayed a higher helix content
(54%) than the i, i+5 3d (39%), confirming the ability of a
stabilizing effect to enhance helix content in the peptide
reporter system used here.[17]
The N-e-Ac-K (mono- and two bisacetylated) variants
4a–c relate more closely to the LeX glycopeptides 5a–c in
structural and charge terms and offered an important control
set; we suggest that N-capped 4a–c are more relevant to this
study as controls than the free lysine 3a/b variants. Mono-
acetylated 4a, the i, i+4 and i, i+5 bisacetylated peptides 4b
and 4c, respectively, and the monoLeX glycopeptide 5a
displayed similar levels of helicity (51–52%, Table 1), indi-
cating that these substitutions (both NAc and monoglycosy-
lation) do not affect significantly the coil:helix distribution.
Comparison of the two key bisglycosylated LeX glyco-
peptides 5b/c showed that i, i+4 5b had a higher helical
content than i, i+5 isomer 5c (54% vs. 49%). Although small,
the increase in helicity associated with the i, i+4 isomer 5b
(5% vs. 5c compared against 1% for 4b vs. 4c), together with
the trend associated with two other glycopeptide controls 6a/
b and 7a/b (see below), indicates the presence of a stabilizing
carbohydrate–carbohydrate interaction.
Figure 2. LeX thioglycoside 1, LeX thiol 2a and disulfide 2b.
Solid-phase Fmoc protocols provided a series of un-
derivatized and N-e-acetyl peptides 3a–d and 4a–c, respec-
tively. In addition, and using the same C3 linker, monoLeX
peptide 5a, guest bisglycopeptides 5b and 5c incorporating
two LeX trisaccharide units, and the corresponding mono-
saccharide (glucose) 6a/b and disaccharide (lactose) 7a/b
variants were prepared. CD data were obtained as an average
of 30 spectra at five different concentrations and were
performed in triplicate to determine experimental repeat-
ability. The intensity of the CD signals at 222 nm indicated
that all peptides tested had a helical component in the range
of 37–54% (Table 1, Figure 3 and Figure 4, and Supporting
Information), consistent with the original peptide design.
Participation of higher-order aggregates was excluded as
no concentration dependencies were observed within the
range 12.5–200 mm; this is important as peptide aggregation
would interfere with the detection of a monovalent, isolated
CCI.
The differences in helical content observed are small,
though we suggest significant based on the effect associated
with other glycopeptides controls. Both i, i+4 and i, i+5
isomers of mono- and disaccharide bisglycopeptides 6a/b and
7a/b incorporating glucose (Glc) and lactose (Lac, Galb1-4-
Glc) moieties, respectively, were synthesized and coil:helix
distributions determined. In each case, and in marked
The control systems were further calibrated. Peptides
carrying free (i.e. positively charged) lysine at i, i+4 and i, i+5
11168 www.angewandte.org
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Angew. Chem. Int. Ed. 2011, 50, 11167 –11171

contrast to the bisLeX glycopeptides 5b/c, the i, i+4 variants
(6a and 7a) displayed a lower helical content than the
corresponding i, i+5 controls (6b and 7b); the i, i+4
monosaccharide 6a and disaccharide 7a controls were 8%
and 12% less helical than the i, i+5 variants 6b and 7b
respectively (Figure 3).[18]
responding to a weakly attractive, stabilizing and single
(monovalent) CCI.
Helix contents can be accurately predicted from helix–coil
theory, provided that parameters for the residues present are
known.[11b] This has been carried out for the glycopeptides
discussed here, to provide the free energies associated with
adding carbohydrate units to the helix and the free energies of
the side-chain interaction energies.[19] This indicates the LeX–
LeX interaction (associated with i, i+4 5b) stabilizes the helix
by approximately 0.5 kcalmolÀ1, compared to the other i, i+4
bisglycopeptides 6a and 7a.
Given the role reported for Ca2+ within LeX-based
carbohydrate interactions,[2,3] the effect of Ca2+ on the helix
preference of 5b was also examined. Using either a large
excess of Ca2+ (167 mm with 100 mm of 5b in 10 mm MOPS
buffer at pH 7.0) or under titration conditions (50 mm to
10 mm Ca2+), we observed no significant difference in helical
content of 5b.[20] This lack of a Ca2+ effect is interesting but
not without precedent[21] and, as articulated earlier by
Penadꢀs et al.,[8a] draws attention to the limited understand-
ing currently available as to the precise mechanism by which
Ca2+ mediates CCIs.
Figure 3. CD spectroscopy of glycopeptides 5b/c, 6a/b, and 7a/b
showing error to two standard deviations.
While reflecting the inherently weak nature of CCIs, the
differences in helical content and trend observed (Table 1 and
Figure 4) are consistent with observation of a monovalent and
favorable carbohydrate–carbohydrate interaction. This, in
turn, supports the ability of a dynamic random-coil:a-helical
peptide system to provide a qualitative read-out of CCIs.
Given that peptide synthesis and carbohydrate ligation are
straightforward, this reporter, validated here with LeX–LeX,
offers a new means of probing the molecular level details of a
weak but fundamentally important biological interaction.
This demonstrates that the introduction of a mono- then
disaccharide moiety (i.e. glyco-based controls possessing a
steric, and an escalating steric demand, but no associated
stabilizing interaction) results in an increasing destabilization
of the helical state. Also noteworthy is that the three
bisglycosylated i, i+5 (i.e. non-interacting, Figure 1b) pep-
tides 5c, 6b and 7b, all have closely similar helix contents at
approximately 49% suggesting that isolation of the CHO
units from each other has been achieved. These data, and in
particular the trend, shown schematically in Figure 4, of a
decrease in helical content associated with mono- and
disaccharide substrates (6a and 7a) compared to the increase
observed for the (trisaccharide) LeX–LeX glycopeptide 5b
supports the conclusion that this peptide reporter system is
Received: February 11, 2011
Revised: April 19, 2011
Published online: September 9, 2011
Keywords: carbohydrate–carbohydrate interaction · LewisX ·
.
peptide reporter
[1] M. Mammen, S. K. Choi, G. M. Whitesides, Angew. Chem. 1998,
110, 2908 – 2953; Angew. Chem. Int. Ed. 1998, 37, 2754 – 2794,
and references cited therein.
[2] For leading references to the discovery of CCIs, see a) S.
Hakomori, Glycoconjugate J. 2004, 21, 125 – 137; b) I. Bucior,
M. M. Burger, Glycoconjugate J. 2004, 21, 111 – 123. For a review
of the biological and functional role of cis- (intracellular) and
trans- (intercellular) CCIs, see S. Hakomori, Arch. Biochem.
Biophys. 2004, 426, 173 – 181.
[3] a) S. Hakomori, Glycoconjugate J. 2000, 17, 143 – 151; b) J. M.
Boggs, W. Gao, Y. Hirahara, J. Neurosci. Res. 2008, 86, 1448 –
1458; c) S. Yu, N. Kojima, S. Hakomori, S. Kudo, S. Inoue, Y.
Inoue, Proc. Natl. Acad. Sci. USA 2002, 99, 2854 – 2859; d) S.
Hakomori, K. Handa, FEBS Lett. 2002, 531, 88 – 92; e) Y. Song,
D. A. Withers, S. Hakomori, J. Biol. Chem. 1998, 273, 2517 –
2525.
[4] Q. Chen, Y. Cui, T. L. Zhang, J. Cao, B. H. Han, Biomacromol-
ecules 2010, 11, 13 – 19.
[5] a) A. Geyer, C. Gege, R. R. Schmidt, Angew. Chem. Int. Ed.
1999, 38, 1466 – 1468; b) P. V. Santacroce, A. Basu, Glycoconju-
gate J. 2004, 21, 89 – 95; c) C. Gourier, F. Pincet, E. Perez, Y. M.
Figure 4. Trends in helical content observed between free lysine 3a
and N-Ac 4b controls and mono-, di-, and trisaccharide-based glyco-
peptides (6a, 7a, and 5b, respectively). Ratios shown are for
helical:random coil in each of the relevant i, i+4 substrates.
Angew. Chem. Int. Ed. 2011, 50, 11167 –11171
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Communications
Zhang, J. M. Mallet, P. Sinaꢁ, Glycoconjugate J. 2004, 21, 165 –
discussed here, the relevant parameters are w(Ala) = 1.70,
w(Lys) = 1.00, w(Gly) = 0.048, w(Tyr) = 0.48, n(acetyl) = 5.9, n-
(Ala) = 1.0, n(Lys) = 0.72, n(Gly) = 3.9, c(All) = 1.0 and v =
0.048;[12b] Peptide 4c contains a KAc residue, with unknown
helix-coil parameters. We can safely assume that n = 1 for all
novel residues, as a helix is highly unlikely to initiate at a
substituted position in the middle of the sequence, so uncertainty
in this N-cap parameter makes very little difference to the
predicted helix contents. This leaves only one unknown in
peptide 4c, namely w(KAc). Fitting the experimental helix
content of 51% for 4c gives w(KAc) = 0.89 and hence the free
energy of transfer of KAc from the unfolded state to a helix
interior is ÀRTln(0.89) = 0.06 kcalmolÀ1. The error range in the
experimental helix content of 4c is 49% to 53%. Fitting to these
values gives a range of 0.83 to 0.95 for w(KAc). Similar analyses
gives the following helix interior preferences for the glycosylated
side-chains: w(KCOCH2S(CH2)3OLeX) = 0.83, range 0.78 to
174; d) C. Gourier, F. Pincet, E. Perez, Y. M. Zhang, Z. Zhu,
J. M. Mallet, P. Sinaꢁ, Angew. Chem. Int. Ed. 2005, 44, 1683 –
1687.
[6] a) J. M. de La Fuente, A. G. Barrientos, T. C. Rojas, J. Rojo, J.
CaÇada, A. Fernꢂndez, S. Penadꢀs, Angew. Chem. 2001, 113,
2317 – 2321; Angew. Chem. Int. Ed. 2001, 40, 2257 – 2261; b) J. M.
de La Fuente, P. Eaton, A. G. Barrientos, M. Menꢀndez, S.
Penadꢀs, J. Am. Chem. Soc. 2005, 127, 6192 – 6197; c) A. J.
Reynolds, A. H. Haines, D. A. Russell, Langmuir 2006, 22,
1156 – 1163; d) M. Fuss, M. Luna, D. Alcꢂntara, J. M. de La
Fuente, S. Penadꢀs, F. Briones, Langmuir 2008, 24, 5124 – 5128;
e) Z. C. Deng, S. Q. Li, X. Z. Jiang, R. Narain, Macromolecules
2009, 42, 6393 – 6405; f) N. Nagahori, M. Abe, S. I. Nishimura,
Biochemistry 2009, 48, 583 – 594; g) A. C. de Souza, D. N.
Ganchev, M. M. E. Snel, J. P. J. M. van der Eerden, J. F. G.
Vliegenthart, J. P. Kamerling, Glycoconjugate J. 2009, 26, 457 –
465.
[7] a) K. Matsuura, H. Kitakouji, N. Sawada, H. Ishida, M. Kiso, K.
Kitajima, K. Kobayashi, J. Am. Chem. Soc. 2000, 122, 7406 –
7407; b) K. Matsuura, R. Oda, H. Kitakouji, M. Kiso, K.
Kitajima, K. Kobayashi, Biomacromolecules 2004, 5, 937 – 941;
c) N. Seah, P. V. Santacroce, A. Basu, Org. Lett. 2009, 11, 559 –
562.
0.89, DG = ÀRTln(0.83)
=
0.10 kcalmol; w(KCOCH2S-
(CH2)3O-Glc) = 0.80; range 0.75 to 0.86, DG = ÀRTln(0.80) =
0.12 kcalmolÀ1; w(KCOCH2S(CH2)3O-Lac) = 0.83, range 0.78 to
0.89, DG = ÀRTln(0.83) = 0.10 kcalmolÀ1. All of these side-
chains are therefore weakly destabilizing to the helix. In peptides
with i, i+4 side-chain interactions, the only unknown is now the
side-chain interaction parameter, p. Fitting the experimental
helix content of 52% for peptide 4b, and using w(KAc), gives
p(KAc to KAc) = 1.18, range 1.04 to 1.34. The KAc to KAc
interaction thus weakly stabilizes the helix by ÀRTln(1.18) =
À0.09 kcalmolÀ1. Similarly, for 5b p(KCOCH2S(CH2)3OLeX to
KCOCH2S(CH2)3OLeX) = 1.54, range 1.35 to 1.77, DG =
ÀRTln(1.54) = À0.23 kcalmolÀ1; for 6a p(KCOCH2S(CH2)3O-
Glc to KCOCH2S(CH2)3O-Glc) = 0.69, range 0.61 to 0.78, DG =
[8] For other LeX-related work, see a) C. Tromas, J. Rojo, J. M.
de La Fuente, A. G. Barrientos, R. Garcꢃa, S. Penadꢀs, Angew.
Chem. Int. Ed. 2001, 40, 3052 – 3055; b) J. Rojo, V. Diaz, J. M.
de La Fuente, I. Segura, A. G. Barrientos, H. H. Riese, A.
Bernade, S. Penadꢀs, ChemBioChem 2004, 5, 291 – 297; c) G.
Nodet, L. Poggi, D. Abergel, C. Gourmala, D. X. Dong, Y. M.
Zhang, J. M. Mallet, G. Bodenhausen, J. Am. Chem. Soc. 2007,
129, 9080 – 9085.
[9] R. U. Lemieux, Acc. Chem. Res. 1996, 29, 373 – 380.
[10] a) T. Hasegawa, T. Sasaki, Chem. Commun. 2003, 978 – 979; b) T.
Hasegawa, M. Numata, M. Asai, M. Takeuchi, S. Shinkai,
Tetrahedron 2005, 61, 7783 – 7788; c) G. L. Simpson, A. H.
Gordon, D. M. Lindsay, N. Promsawan, M. P. Crump, K. Mulhol-
land, B. R. Hayter, T. Gallagher, J. Am. Chem. Soc. 2006, 128,
10638 – 10639; d) A. Otsuka, K. Sakurai, T. Hasegawa, Chem.
Commun. 2009, 5442 – 5444.
ÀRTln(0.69)
= ; and for 7a p(KCOCH2S-
0.20 kcalmolÀ1
(CH2)3O-Lac to KCOCH2S(CH2)3O-Lac) = 0.53, range 0.46 to
0.60, DG = ÀRTln(0.53) = 0.34 kcalmolÀ1
.
[20] Given the sensitivity of the conformational preferences of the
peptide backbone to cations, these titration experiments were
carried out under conditions of comparable ionic strength. In
addition, the effect of Na+ on the helix preference of 5b was
examined and no significant difference in helicity was observed
between Ca2+ (167 mm; I = 0.5) and Na+ (500 mm; I = 0.5). Full
details of these titration studies are provided in the Supporting
Information.
[11] a) B. J. Stapley, C. A. Rohl, A. J. Doig, Protein Sci. 1995, 4,
2383 – 2391; b) A. J. Doig, Biophys. Chem. 2002, 101 – 102, 281 –
293.
[12] a) C. A. Rohl, A. J. Doig, Protein Sci. 1996, 5, 1687 – 1696;
b) C. A. Rohl, A. Chakrabartty, R. L. Baldwin, Protein Sci. 1996,
5, 2623 – 2637.
[21] The interaction between LeX moieties with and without Ca2+ in a
variety of circumstances has been described and those regions of
LeX that interact with a divalent cation have been proposed.
Methods used include NMR spectroscopy,[8c,13,22] MS and
computational studies,[23] and models representing both trans-
and cis-CCIs have been reported.[24] However, no LeX–Ca2+
interactions have been detected by NMR spectroscopy in pure
water, only in aqueous MeOH, MeOH or DMSO or related
mixtures. A direct interaction between LeX units (in the absence
of Ca2+) has been reported,[13] but see also Ref. [22d]. Impor-
tantly, Penadꢀs et al. identified a Ca2+-independent LeX–LeX
interaction using atomic force microscopy (AFM)[8a] while
observing a Ca2+-dependent effect using analogously glycosy-
lated gold nanoparticles (AuNPs).[6a]
[22] a) M. R. Wormald, C. J. Edge, R. A. Dwek, Biochem. Biophys.
Res. Commun. 1991, 180, 1214 – 1221; b) B. Henry, H. Desvaux,
M. Pristchepa, P. Berthault, Y.-M. Zhang, J.-M. Mallet, J.
Esnault, P. Sinaꢁ, Carbohydr. Res. 1999, 315, 48 – 62; c) C.
Gege, A. Geyer, R. R. Schmidt, Eur. J. Org. Chem. 2002, 2475 –
2485; d) A. Wang, J. Hendel, F.-I. Auzanneau, Beilstein J. Org.
Chem. 2010, 6, DOI: 10.3762/bjoc.6.17; e) for NMR and
molecular dynamics studies of the Ca2+ complexes of a related,
pyruvated trisaccharide, see J. I. Santos, A. C. de Souza, F. J.
CaÇada, S. Martꢃn-Santamarꢃa, J. P. Kamerling, J. Jimꢀnez-
Barbero, ChemBioChem 2009, 10, 511 – 519.
[13] J. M. de La Fuente, S. Penadꢀs, Tetrahedron: Asymmetry 2002,
13, 1879 – 1888.
[14] P. Luo, R. L. Baldwin, Biochemistry 1997, 36, 8413 – 8421.
[15] Controls 3a/b are included here for completeness but the charge
(and derived effects) associated with the free lysine residues at i,
i+4 and i, i+5 is not mimicked in the glycopeptide substrates
used subsequently.
[16] J. S. Smith, J. M. Scholtz, Biochemistry 1998, 37, 33 – 40.
[17] Thermal denaturation of key peptides (3c/d and 5b/c) was also
performed. Analysis was complicated because the random
coil:helix equilibrium is not a two-state system, but a dynamic
ensemble of many conformations. Apparent TM values were
observed and the differences, though small, were consistent with
the equilibrium CD experiments (see Supporting Information).
[18] We suggest that, given the differences in structure of the side-
chain moieties, it is more meaningful to compare helicity values
within a series (i.e. i, i+4 vs. i, i+5) rather than rely solely on
absolute values between difference controls/glycopeptides.
[19] In modified Lifson–Roig theory, these parameters are prefer-
ences for helix interior (w), N-cap (n), C-cap (c) helix initiation
(v) and i, i+4 side-chain interactions (p).[11] In the peptides
11170 www.angewandte.org
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Angew. Chem. Int. Ed. 2011, 50, 11167 –11171

[23] a) G. Siuzdak, Y. Ichikawa, T. J. Caulfield, B. Munoz, C.-H.
Wong, K. C. Nicolaou, J. Am. Chem. Soc. 1993, 115, 2877 – 2881;
b) G. Siuzdak, Z.-L. Zheng, J. Y. Rhamphal, Y. Ichikawa, K. C.
Nicolaou, F. C. A. Gaeta, K. S. Chatman, C. H. Wong, Bioorg.
Med. Chem. Lett. 1994, 4, 2863 – 2866; c) S.-I. Nishimura, N.
Nagahori, K. Takyaya, Y. Tachibana, N. Miura, K. Monde,
Angew. Chem. Int. Ed. 2005, 44, 571 – 575.
[24] Models of trans-CCIs can be viewed as those associated with
intermolecular interactions, for example, glycosylated vesicles[5]
or AuNPs. a) Models representing a cis-CCI based on LeX have
been reported: A. Geyer, C. Gege, R. R. Schmidt, Angew. Chem.
Int. Ed. 2000, 39, 3246 – 3249.
Angew. Chem. Int. Ed. 2011, 50, 11167 –11171
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