Influence of glucose-templated proline mimetics on the β-turn conformation of the peptide fragment Ac-Leu-d-Phe-Pro-Val-NMe2 found in Gramicidin S

Article abstract of DOI:10.1016/j.carres.2010.03.037

The synthesis of tetrapeptide-based β-turn mimetics containing spirocyclic glucose-templated 3-hydroxyproline hybrids Glc3′(S)-5′(R)(CH₂OH)HypH and Glc3′(S)-5′(S)(CH₂OH)HypH as proline mimetics is presented. NMR-based conformational analysis of Ac-Leu-d-Phe-[Glc3′(S)-5′(R)(CH₂OH)HypH]-Val-NMe₂ and Ac-Leu-d-Phe-[Glc3′(S)-5′(S)(CH₂OH)HypH]-Val-NMe₂ demonstrates the presence of β-turn conformations. Different turn structures were observed by changing the stereochemistry at 5′-position of Glc3′(S)-5′(R)(CH₂OH)HypH. The major prolyl amide cis isomer of glucose-protected tetrapeptide Ac-Leu-d-Phe-[Glc(MOM)₄3′(S)-5′(R)(CH₂OMOM)HypH]-Val-NMe₂ 11 and glucose unprotected Ac-Leu-d-Phe-[Glc3′(S)-5′(R)(CH₂OH)HypH]-Val-NMe₂ 13 forms a type VI β-turn conformation. In contrast, the major prolyl amide trans rotamer of tetrapeptide Ac-Leu-d-Phe-[Glc(MOM)₄3′(S)-5′(S)(CH₂OMOM)HypH]-Val-NMe₂ 12 conserves a similar β-turn conformation as the Gramicidin S-based peptide fragment Ac-Leu-d-Phe-Pro-Val-NMe₂ 16.

Full text of DOI:10.1016/j.carres.2010.03.037

Carbohydrate Research 345 (2010) 1114–1122
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Influence of glucose-templated proline mimetics on the b-turn conformation
of the peptide fragment Ac-Leu-D-Phe-Pro-Val-NMe2 found in Gramicidin S
*
Kaidong Zhang, Frank Schweizer
Department of Chemistry, University of Manitoba Winnipeg, Manitoba, Canada R3T 2N2
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of tetrapeptide-based b-turn mimetics containing spirocyclic glucose-templated
3-hydroxyproline hybrids Glc30(S)-50(R)(CH2OH)HypH and Glc30(S)-50(S)(CH2OH)HypH as proline mimet-
Received 23 February 2010
Received in revised form 23 March 2010
Accepted 28 March 2010
Available online 1 April 2010
ics is presented. NMR-based conformational analysis of Ac-Leu-D
-Phe-[Glc30(S)-50(R)(CH2OH)HypH]-Val-
NMe2 and Ac-Leu-D
-Phe-[Glc30(S)-50(S)(CH2OH)HypH]-Val-NMe2 demonstrates the presence of b-turn
conformations. Different turn structures were observed by changing the stereochemistry at 50-position
of Glc30(S)-50(R)(CH2OH)HypH. The major prolyl amide cis isomer of glucose-protected tetrapeptide
Keywords:
Ac-Leu-
-Phe-[Glc30(S)-50(R)(CH2OH)HypH]-Val-NMe2 13 forms a type VI b-turn conformation. In contrast, the
major prolyl amide trans rotamer of tetrapeptide Ac-Leu-
-Phe-[Glc(MOM)430(S)-50(S)(CH2OMOM)-
HypH]-Val-NMe2 12 conserves a similar b-turn conformation as the Gramicidin S-based peptide fragment
Ac-Leu- -Phe-Pro-Val-NMe2 16.
D
-Phe-[Glc(MOM)430(S)-50(R)(CH2OMOM)HypH]-Val-NMe2 11 and glucose unprotected Ac-Leu-
Glycosylamino acid
Proline mimetics
Glycopeptides
Gramicidin S
Carbohydrate-based peptidomimetics
D
D
D
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Over the years, many proline analogues have been synthesized
and used for tuning the peptidyl-prolyl cis/trans isomerization in
peptides and proteins.7 For instance, (2S,5R)-5-tert-butylproline fa-
vors predominantly a cis conformation due to steric hindrance in
short peptide models that adopt a type VI b-turn.8 Another steri-
cally hindered residue, the d,d-dimethylproline has been developed
as a substitute to lock proline in the cis conformation in tripep-
b-Turns play an important role in the folding process of proteins
and peptides,1 and are often involved in molecular recognition pro-
cesses.2 b-Turns consist of four consecutive residues in a non-heli-
cal region, in which the polypeptide chain folds back on itself by
nearly 180°.3 b-Turns are often stabilized by an intramolecular
hydrogen-bond between the carbonyl oxygen of the first residue
(i) and the amide proton of the fourth one (i + 3) (Fig. 1). Many
b-turn mimetics have been developed to study the structural and
biological properties of peptides. In particular, the use of proline
analogues is an attractive strategy in this respect. Due to the con-
formational restrictions imposed by the pyrrolidine ring, the pro-
teinogenic amino acid proline has an exceptional tendency to act
as a turn inducer to generate reverse turn structures like b-turns
and b-hairpins in peptides and proteins.4 In particular, the cis
geometry of the proline N-terminal amide bond induces the type
VI b-turn, in which the proline residue is situated at the i + 2 posi-
tion of the peptide bend. Although the type VI b-turn is a relatively
rare secondary structure, it plays important roles in protein fold-
ing.5 In addition, type VI b-turns have been found in some impor-
tant recognition events of bioactive proteins. For example, a type
VI b-turn conformation has been proposed for thrombin-catalyzed
cleavage of the V3 loop of HIV gp120, a prerequisite to viral
infection.6
tides.9 Azaprolines (AzPro), in which the
a nitrogen, favor the cis conformation of the azaproline-preceding
amide bond by electronic effects.10 Pseudoproline,
Pro, contain-
a-carbon is replaced by
W
ing an oxozolidine or thiazolidine ring, exhibits very high prolyl
amide cis ratios.11
Recently, we have reported the synthesis of spirocyclic
glucosyl-(30-hydroxy-50-hydroxymethyl)proline hybrids (Glc30(S)-
50(CH2OH)HypHs 1 and 2, which differ in the stereochemistry of
the polar hydroxymethyl substitutent at the C-50-position
( Fig. 2).12 These proline analogues exhibit unique features. The
spirocyclic nature of the gluco-derived scaffold constrains the
O
Ri+1
HN
NH
Ri+2
O
O
HN
O
Ri+3
Ri
NH
* Corresponding author. Fax: +1 204 474 7608.
E-mail address: schweize@ms.umanitoba.ca (F. Schweizer).
Figure 1. b-Turn.
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.03.037

K. Zhang, F. Schweizer / Carbohydrate Research 345 (2010) 1114–1122
1115
OH
O
O
OMOM
O
6
1) CbzCl, Na2CO3,
H2O
OMe
NH
COOMe
NCbz
R2
HO
HO
MOMO
MOMO
3'
1 or 2
HO
MOMO
5'
2
2) MOMCl,
CH2Cl2 / DIEA (3/1)
1
R
R
R1
3 R1 = CH2OMOM, R2 = H
4 R1 = H, R2 = CH2OMOM
1
2
83% (2 steps)
72% (2 steps)
1: R = CH OH; R = H
2
1
2
2: R = H; R = CH OH
2
Figure 2. Structure of spirocyclic glucose-3(S)-hydroxyproline hybrids (Glc30(S)-
50(CH2OH)HypHs 1 and 2.
OMOM
LiOH, dioxane
60oC
COOH
NCbz
R1 R2
O
MOMO
MOMO
MOMO
pyrrolidine ring of proline, and introduces artificial post-transla-
tional modifications (hydroxylation + glycosylation). Chemical
manipulations and derivatizations of the glucose-derived polyol
scaffold provide an opportunity to tailor the chemical, physical,
conformational, and pharmacodynamic properties of (Glc30(S)-
50(CH2OH)HypHs-containing peptides as previously shown.13
The model peptide mimics, Ac-(Glc30(S)-50(CH2OH)HypH-OMe
and Ac-(Glc30(S)-50(CH2OH)HypH-NHMe display interesting
conformational properties. For example, when compared to Ac-
Pro-OMe (14% cis) or Ac-(3S)-Hyp-OMe (17% cis), Ac-(Glc30(S)-
50(R)(CH2OH)HypHs-OMe favored the prolyl amide cis rotamer
(53% cis) with an accelerated (20-fold) cis/trans isomerization rate
in water at room temperature. In contrast, the diastereomer
Ac-(Glc30(S)-50(S)(CH2OH)HypHs-OMe disfavors the cis rotamer
(23% cis) with a retarded (10-fold) cis/trans isomerization rate in
water.14 Based on the unique properties of proline mimetics 1
and 2, we developed an interest in exploring the conformational
properties of 1 and 2 in longer model peptides. We selected the tet-
5 R1 = CH2OMOM, R2 = H
6 R1 = H, R2 = CH2OMOM
93%
53%
Scheme 1. Synthesis of MOM-protected [Glc(OMOM)4-30(S)-50(R0)(CH2O MOM)-
HypH] 5 and [Glc(OMOM)4-30(S)-50(S0)(CH2OMOM)HypH] 6.
merization (ꢀ1:1) at the
the two acids could be separated by column chromatography. The
assignment of the -stereochemistry of acid 6 was confirmed by
a-position. Fortunately, the mixture of
a
conversion into methyl ester 4 as described above for compound 5.
2.2. Synthesis of tetrapeptides 11–13
With MOM-protected building block 5 in hand, the synthesis of
the tetrapeptides 11–13 was carried out using solution-phase pep-
tide chemistry (Scheme 2). Dipeptide 7 was prepared via coupling
of NH2-Val-NMe2 to compound 5 (DIEA, TBTU, DMF, 80%). The
N-terminal Cbz group was then removed by catalytic hydrogenol-
ysis in quantitative yield using Pearlman0s catalyst in methanol to
afford the free imine 7 in high yield. Synthesis of tripeptide 9 pro-
rapeptide Ac-Leu-D-Phe-Pro-Val-NMe2, which forms the b-turn
portion of the antibacterial peptide Gramicidin S (GS), as a mod-
el.15 In this peptide, proline occupies the i + 2 position of a b-turn.
Our goal was to study how substitution of the proline residue by
(Glc30(S)-50(CH2OH)HypHs 1 and 2 influences the conformational
and b-turn inducing properties.
ceeded from the dipeptide 7. Coupling with Fmoc-D-Phe-OH (DIEA,
PyBOP, DMF, 82%) provided Fmoc-protected tripeptide, which was
treated with a mixture of piperidine and N,N-dimethylformamide
to produce the tripeptide 9 with an unprotected N-terminus in
quantitative yield. Because of steric hindrance at the N-terminus,
the coupling reagent PyBOP was used to improve the yield.17 The
coupling of tripeptide 9 and Fmoc-Leu-OH was carried out by using
TBTU as the coupling reagent in DMF followed by a deprotection–
acylation procedure to provide MOM-protected tetrapeptide 11 in
92% yield.
The identical synthetic procedure was used for the synthesis of
tetrapeptide 12. Similar yields were obtained in each step except
for the coupling reaction between dipeptide 8 and Fmoc-D-Phe-
OH. In this case, a low yield for tripeptide 10 was obtained and
the dipeptide 8 was recovered in 45% yield. Modifying the reaction
conditions like increasing reaction time, amount of amino acid, and
coupling reagent did not improve the yield.
Finally, exposure of compound 11 to acidic conditions (0.3 N
HCl in MeOH) resulted in complete deblocking of the MOM
protecting groups and afforded unprotected tetrapeptide 13
(Scheme 3) in excellent yield (90%). Unfortunately, applying the
same acidic conditions to tetrapeptide 12 resulted in an insepara-
ble mixture of the dipeptides 14 and 15. We speculate that the
acidic hydrolysis of the prolyl amide bond in 12 is caused by anchi-
meric assistance of the hydroxymethyl substituent at the 50-posi-
tion of proline (Fig. 3). The major cis isomer of prolyl amide A is
stabilized by an intramolecular hydrogen bond between the
50-hydroxymethyl substituent and the carbonyl oxygen of the pro-
lyl amide bond.14 This restricts the 50-hydroxymethyl group into a
geometry in which the carbonyl carbon of the prolyl amide is not
2. Results and discussion
2.1. Synthesis of MOM-protected Glc3(S)HypHs
The use of unprotected mono- and polyhydroxylated amino
acids in peptide chemistry often results in low coupling yields
and difficult purification due to their nucleophilic hydroxyl groups.
To avoid this potential complication, we decided to use methoxy-
methyl (MOM) ethers as temporary hydroxyl protecting groups.16a
The MOM protecting group is relatively small, does not deactivate
adjacent nucleophiles, and can be easily removed under acidic con-
ditions.16 The building block 5 was prepared from 1 via a three-
step procedure (Scheme 1).
Initially, the free imine 1 was treated with benzyl chloroformate
(Cbz-Cl) and sodium carbonate to provide the Cbz-protected carba-
mate followed by the introduction of MOM groups using N,N-diiso-
propylethylamine and chloromethyl methyl ether to provide
MOM-protected intermediate 3 in good yield.16a Subsequently,
lithium hydroxide-based hydrolysis of the methylester in dioxane
produced the desired acid 5 in 88% yield. Surprisingly, only a small
portion of the methyl ester (ꢀ10%) was epimerized at the
a posi-
tion. The stereochemistry at the -position of acid 5 was confirmed
a
by converting it into the corresponding methyl ester 3. Applying
the same procedure to its diastereomer 2 resulted in extended epi-
The acid was treated with 2 equiv iodomethane and 1.2 equiv cesium carbonate
in methanol and stirred for 1 h to provide the corresponding methyl ester.

1116
K. Zhang, F. Schweizer / Carbohydrate Research 345 (2010) 1114–1122
Val NMe2
1) NH2-Val-NMe2 ,
OMOM
O
DIEA, TBTU, DMF, r.t.
MOMO
MOMO
O
5 or 6
2) Pd (OH)2, H2,
MeOH
NH
R2
MOMO
R1
7 R1 = CH2OMOM, R2 = H
80%
85%
8 R1 = H, R2 = CH2OMOM
Val NMe2
OMOM
O
MOMO
MOMO
O
1) Fmoc-D-Phe-OH, DIEA
PyBOP, DMF, r.t.
N
MOMO
D-Phe
H
2) piperidine, DMF
R1
R2
9 R1 = CH2OMOM, R2 = H
80%
10 R1 = H, R2 = CH2OMOM 46%
Val NMe2
OMOM
1) Fmoc-Leu-OH, DIEA
TBTU, DMF
O
MOMO
MOMO
O
N
MOMO
Ac
D-Phe Leu
2) piperidine, Ac2O, DMF
R1
R2
11 R1 = CH2OMOM, R2 = H
12 R1 = H, R2 = CH2OMOM
92%
90%
Scheme 2. Synthesis of protected tetrapeptide Ac-Leu-
50(S)(CH2OMOM)HypH]-Val-NMe2 12.
D D
-Phe-[Glc(OMOM)4-30(S)-50(R)(CH2OMOM)HypH]-Val-NMe2 11 and tetrapeptide Ac-Leu- -Phe-[Glc(OMOM)4-30(S)-
compatible with solution-phase peptide synthesis for Glc30(S)-
50(S)(CH2OH)HypH 1. For reference purposes, the parent peptide
NMe2
O
Val
OH
O
HO
HO
HCl, MeOH
90%
Ac-Leu-D-Phe-Pro-Val-NMe2 16 was also prepared by standard
11
solution-phase peptide chemistry as previously described.15
N
HO
D-Phe Leu Ac
OH
2.3. Conformational analysis of tetrapeptides 11–13 and 16
using NMR spectroscopy
13
Val NMe2
O
Although the reference peptide 16 aggregated in CD2Cl2 at
P10 mM,15 no aggregation was observed for tetrapeptides 11–13
at concentrations 650 mM in CD2Cl2 or water. The low aggregation
tendency of tetrapeptides 11–13 was judged by the fact that the
amide NMR chemical shifts in CD2Cl2 and water of peptides
11–13 were not affected by peptide concentration (650 mM).15
The lower tendency of aggregation of glycosylated peptides has
previously been reported.19
OH
O
HCl, MeOH
HO
HO
12
Ac
83%
Leu D-Phe-H
+
NH
HO
OH
14
15
Scheme 3. Cleavage of MOM protecting groups in compounds 11 and 12.
The NMR spectra of all tetrapeptides were measured at the
same concentration (8 mM). Intramolecular hydrogen bonding
was evaluated by temperature coefficient (Dd/DT) experiments in
subject to nucleophilic attack by the 50-hydroxymethyl group using
a Bürgi–Dunitz trajectory.18 In contrast, the major trans isomer of
the prolyl amide in B makes the prolyl amide carbonyl more acces-
sible for nucleophilic attack (Fig. 3). The synthesis of unprotected
tetrapeptide 13 demonstrates that MOM protecting groups are
DMSO-d6 or H2O–D2O (9:1) because exchangeable protons en-
gaged in intramolecular hydrogen bonds usually show smaller
absolute (Dd/D
T) values.20,23 The prolyl N-terminal geometry and
turn structure were investigated by ROESY experiments.21 The
O
OH
O
Val-NMe2
OH
Val-NMe2
O
HO
HO
O
HO
HO
D-Phe Leu-Ac
O
O
HO
N
HO
N
D-Phe Leu-Ac
O
OH
B
H
A
Figure 3. Rational explanation for the hydrolysis of prolyl amide bond in B. The major cis prolyl amide bond in A is stabilized by an intramolecular hydrogen bond. In contrast
the major trans isomer is prone to acidic hydrolysis via catalytic anchimeric assistance of the hydroxymethyl group.

K. Zhang, F. Schweizer / Carbohydrate Research 345 (2010) 1114–1122
1117
Table 1
Table 2
Chemical shift (d) for amide protons of tetrapeptides 11, 12, 13, 16, and Gramicidin S
in CD2Cl2, DMSO-d6, and H2O–D2O (9:1)
Temperature coefficients (
D
d/D
T, ppb/K) for peptide 11 in DMSO-d6
Leu-NH
Phe-NH
Val-NH
Peptides
Solvent
Leu-NH
Phe-NH
Val-NH
Cis
Trans
ꢁ6.00
ꢁ4.37
ꢁ8.13
ꢁ3.75
ꢁ9.40
d (3J
)
d (3J
)
d (3J
)
H,NH
a
H,NH
a
H,NH
a
ꢁ11.47
11a (cis)
CD2Cl2
DMSO-d6
CD2Cl2
DMSO-d6
CD2Cl2
6.23(8.5)
7.65(9.0)
6.18(8.6)
7.78(8.9)
NDc
6.40(8.3)
8.18(9.7)
6.61(7.7)
8.37(8.6)
NDc
8.20(8.3)
8.12(7.5)
7.03(ND)
7.69(8.2)
NDc
11a (trans)
12 (cis)
(28%) in CD2Cl2. The major prolyl amide cis isomer is confirmed
by a long-range ROESY correlation between H -Phe) and H2 (GlcHypH)
0
D
a
and the absence of ROESY correlation between H
and
DMSO-d6
CD2Cl2
7.65(9.0)
6.88(7.7)
7.79(8.5)
7.98(6.6)
7.75(8.8)
NDc
8.34(8.7)
7.31(7.2)
8.27(8.9)
8.25(7.7)
8.04(8.8)
NDc
7.45(7.0)
6.78(8.2)
7.51(8.1)
9.17(8.1)
8.72(7.3)
NDc
D
a
-Phe)
12 (trans)
13 (cis)
0
H5 (GlcHypH). The presence of a turn conformation in 11 is supported
by long-range ROESY correlations between the Leu-NH or the
N-terminal acetyl group and the C-terminal dimethyl amide
singlets (Fig. 4). In addition, sequential ROESY correlations were
DMSO-d6
H2O–D2O
DMSO-d6
H2O–D2O
DMSO-d6
CD2Cl2
DMSO-d6
CD2Cl2
DMSO-d6
DMSO-d6
13 (trans)
16 (cis)
7.80(9.5)
8.33(8.1)
8.39(5.9)
observed between the neighboring H
and NHPhe as well as
a
(Leu)
NDc
NDc
NDc
0
the H2 (GlcHypH) and NH(Val) indicating their proximity in the major
conformer.
7.74(8.3)
7.76(7.5)
7.79(8.2)
8.32(9.2)
8.43(8.3)
7.12(5.5)
8.37(7.8)
9.05(2.6)
8.12(8.1)
6.85(7.0)
7.78(8.2)
7.21(9.7)
16 (trans)
Gramicidin Sb (trans)
The chemical shifts and coupling constants of the amide
protons in 11 (Table 1) indicate that the cis conformer has a dif-
ferent folding structure when compared to reference peptide 16
in CD2Cl2. The downfield chemical shift (d = 8.20 ppm) and large
temperature coefficient value for Val-NH(Val) (Table 2) support
that the NH(Val) is involved in the formation of an intramolecular
hydrogen bond.
a
b
c
Cis and trans are referred to as prolyl amide rotamers.
Taken from Ref. 15.
ND = not determined.
chemical shifts and coupling constants of the amide protons in tet-
rapeptides 11–13 and 16 are provided in Table 1.
Therefore, a type VI b-turn conformation is suggested for the
prolyl amide cis conformer of 11, in which the type VI b-turn is sta-
bilized by a 10-membered ring hydrogen bond between (C@O)Leu
at i position and NHVal at i + 3 position.5 This hydrogen bond is fur-
ther supported by the observation that the amide proton of Val
2.3.1. Tetrapeptide Ac-Leu-D
-Phe-[Glc(OMOM)430(S)-50(R)
(CH2OH)HypH]-Val-NMe2 (11)
The proline mimetic Glc(OMOM)430(S)-50(R)(CH2OH)HypH in
tetrapeptide 11 coexists as the prolyl amide cis (72%) and trans
undergoes little chemical shift changes (Dd = 0.08 ppm) when dis-
solved in DMSO and CD2Cl2 (Table 1). Due to the low concentration
OMOM
OMOM
OMOM
MOMO
MOMO
MOMO
O
OMOM
MOMO
H
O
H
H
O
H
N
N
OMOM
OMOM
O
H
H
O
O
NH
HN
HN
HN
O
O
H
O
H
NH
O
NH
O
O
N
N
11
12
H
OH
OH
OH
H
HO
H
H
O
N
H
O
N
O
OH
O
HN
O
H
H
HN
O
O
HN
H
O
HN
O
H
NH
NH
O
N
N
O
16
13
Figure 4. Selected 1H–1H ROESY cross-peaks in CD2Cl2 (11, 12, and 16) or H2O (13) as well as potential hydrogen bonds from temperature coefficient experiments in DMSO-d6
for major isomer in peptides 11, 12, and 16 and in H2O–D2O (9:1) for major isomer in 13.

1118
K. Zhang, F. Schweizer / Carbohydrate Research 345 (2010) 1114–1122
Table 5
The equilibrium constant (Kt/c
of trans conformer of 11 in CD2Cl2 (72% cis), a full conformational
analysis of the minor prolyl amide trans isomer was not possible.
However, similar chemical shifts of the amide protons between
the trans isomer of 11 and the trans isomer of 16 support that
these amide protons have a similar chemical environment in
CD2Cl2. Furthermore, the temperature coefficient experiments
(Tables 2 and 3) support that the NHLeu of both trans conformers
a
) of 11, 12, 13 and 16 in various solvents
Compd
CD2Cl2
CD3OD
DMSO-d6
D2O
b
11
12
13
16
0.39
0.33
3.00
0.14
3.17
0.30
1.44
0.67
1.32
—
—
b
13.28
b
—
0.10
b
19.00
—
a
of 11 and 16 were involved in a hydrogen bonding to (C@O)Val
.
Determined by 500 MHz NMR at 25 °C, error is 0.05%.
Not soluble.
b
2.3.2. Tetrapeptide Ac-Leu-
(CH2OH)HypH]-Val-NMe2 (12)
D
-Phe-[Glc(OMOM)430(S)-50(S)
The NMR spectrum showed one major isomer (93%) in CD2Cl2.
0
The ROESY correlation (Fig. 4) between H
and H5 (GlcHypH)
D
(C@O)Leu in its prolyl amide cis conformer. According to amide
temperature coefficient experiments ( d/
T <ꢁ7.45 ppb/K, Table
a
( -Phe)
confirmed its trans geometry. The presence of a turn conformation
in 12 is supported by long-range ROESY correlations between the
Leu-NH or the N-terminal acetyl group and the C-terminal di-
methyl amide singlets ( Fig. 4). Temperature coefficient experi-
ments in DMSO-d6 (Table 3) indicate that NH(Leu) is involved in
hydrogen bonding to the carbonyl group of valine. In addition,
similar chemical shifts observed for the amide protons among
compounds 12, 16, and GS (Table 1) suggest that the b-turn
conformation of 12 resembles the b-turn conformation that occurs
in the parent tetrapeptide. Interestingly, the prolyl amide cis con-
former of 12 may form a type VI b-turn structure on the basis of
chemical shift analysis and hydrogen bond pattern of amide pro-
tons relative to the cis conformer of 11 (Table 1). That is, the ste-
reochemistry at 50-position (d-position) of Glc3(S)HypHs can be
used to control the equilibrium constants of cis/trans isomerization
(72% cis for 11, 93% trans for 12) without disturbing the b-turn
conformation of each isomer (a type VI b-turn for cis conformer;
original b-turn for trans conformer).
D
D
4) it appears that the minor prolyl amide trans isomer of tetrapep-
tide 13 is not involved in hydrogen bonding. Due to the low con-
centration of the prolyl amide trans isomer it was not possible to
observe ROESY correlations between the Leu-NH or the N-terminal
acetyl group and the C-terminal dimethyl amide singlets.
Attempts to investigate the influence of the hydroxyl groups of
the carbohydrate scaffold on the peptide backbone conformation
failed due to poor resolution of the hydroxyl protons in DMSO-
d6.22,23 As indicated above, Glc30(S)-50(R)(CH2OH)HypHs 1 can effi-
ciently increase the cis population of prolyl N-terminus thereby
inducing a type VI b-turn. Derivatization of the remaining hydroxyl
groups as MOM-ethers does not affect prolyl amide cis/trans
isomerization as well as type VI b-turn conformation in tetrapep-
tide 11. In comparison, the MOM-protected diastereoisomer
Glc(OMOM)430(S)-50(S)(CH2OH)HypH stabilizes the prolyl amide
trans conformer resulting in a conserved b-turn structure as ob-
served in reference peptide 16. In conclusion, proline analogues
11–14 can be used to tune the hydrophilic or hydrophobic proper-
ties of peptides without disturbing their major bioactive
conformation.
2.3.3. Tetrapeptide Ac-Leu-
Val-NMe2 (13)
D
-Phe-[Glc30(S)-50(R)(CH2OH)HypH]-
The increased polarity of unprotected tetrapeptide 13 made it
possible to investigate its conformation by NMR in water. ROESY
experiments on compound 13 demonstrate that the major isomer
exhibits a prolyl amide cis conformation (91%) in H2O–D2O (9:1).
This assignment is supported by a ROESY correlation between
0
2.4. Solvent effects
In addition, the influence of different solvents on the geometry
of the prolyl N-terminus was studied (Table 5). The results show
that the equilibrium constant of MOM-protected 11 is not affected
by the solvent polarity and ability of the solvent to donate a
hydrogen bond. The unprotected tetrapeptide 13 also showed the
absence of a strong solvent effect. However, an increased trans
population was observed in aprotic DMSO when compared to
CD3OD and D2O. In contrast, peptide 12 and reference peptide 16
display a solvent effect. In this case the prolyl amide trans rotamer
population is greatly enhanced in nonpolar aprotic solvents and
decreases with an increase in solvent polarity.
H
a
( -Phe) and H2 (GlcHypH (Fig. 4). In comparison with MOM-protected
D
peptide 11, the increased cis population in 13 may be due to the
14
hydrogen bonding between (C@O)(D-Phe) and (50-CH2OH)(GlcHypH)
.
The long-range ROESY correlations between the Leu-NH or the
N-terminal acetyl group and the C-terminal dimethyl amide sing-
lets indicate the presence of a turn conformation in 13 (Fig. 4).
Temperature coefficient experiments (Table 4) indicate that com-
pound 13 exists in a type VI b-turn conformation that is stabilized
by an intramolecular hydrogen bond between the NHVal and
3. Conclusions
Table 3
Temperature coefficients (Dd/DT, ppb/K) for peptides 12 and 16 in DMSO-d6
MOM-protected Glc3(S)HypHs-based proline mimics have been
successfully incorporated into target tetrapeptides using solution-
phase peptide chemistry. In particular, Glc30(S)-50(R)(CH2OH)HypH
demonstrated its potential as a building block in peptide synthesis
while the use of its diastereoisomer Glc30(S)-50(S)(CH2OH)HypH is
limited due to accelerated hydrolysis of its N-terminal amide under
acidic conditions. As predicted by previous work on model peptide
Ac-[Glc30(S)-50(R)(CH2OH)HypH]-NHMe proline mimetic Glc30(S)-
50(R)(CH2OH)HypH dramatically enhances the cis population
Leu-NH
Phe-NH
Val-NH
12
16
Cis
Trans
Cis
ꢁ5.20
ꢁ3.90
ꢁ4.00
ꢁ3.92
ꢁ4.80
ꢁ7.40
ꢁ8.00
ꢁ7.60
ꢁ4.15
ꢁ8.00
ꢁ8.00
ꢁ6.20
Trans
Table 4
Temperature coefficients (
(91%) of the prolyl amide in tetrapeptide Ac-Leu-D
-Phe-[Glc30(S)-
Dd/DT, ppb/K) for peptides 13 in H2O–D2O (9:1)
50(R)(CH2OH)HypH]-Val-NMe2 water. The high cis prolyl amide ra-
tio of Glc30(S)-50(R)(CH2OH)HypH induces a type VI b-turn.
The remarkable high cis prolyl amide induction of Glc30(S)-
50(R)(CH2OH)HypH may find future applications to explore
Leu-NH
Phe-NH
Val-NH
Cis
Trans
ꢁ7.23
ꢁ7.91
ꢁ8.45
ꢁ7.72
ꢁ4.02
ꢁ7.45

K. Zhang, F. Schweizer / Carbohydrate Research 345 (2010) 1114–1122
1119
structure–activity relationships (SARs) of bioactive peptides in
4.3. (1S)-2,3,4,6-Tetra-O-methoxymethyl-10-N-benzyloxycar-
bonyl-50(S)-methyl methoxymethyl ether-spiro[1,5-anhydro-
glucitol-1,30-
-proline methyl ester] (4)
which the cis geometry of prolyl amide or a type VI b-turn is
required for biological activity. Moreover, as demonstrated in
tetrapeptides 11 and 13, chemical modifications on the glucose-
based polyol scaffold can be used to adjust solubility in organic
or polar solvents without affecting peptide folding (type VI b-turn).
This suggests that decoration of the glucose-based polyol scaffold
with hydrophobic/hydrophilic groups in proline mimetic Glc30(S)-
50(R)(CH2OH)HypH may find use to tune the pharmacodynamic
properties of bioactive peptides without affecting their bioactive
conformation. Furthermore, proline mimetics with high cis prolyl
amide population are widely used to solve synthetic problems
caused by peptide aggregation on solid support as shown in the
case of pseudoprolines.11c
D-
L
To a mixture of compound 2 (90 mg, 0.29 mmol) and benzyl
chloroformate (0.21 mL, 1.45 mmol) in water (2 mL) was added
sodium carbonate (93 mg, 0.88 mmol). The reaction was stirred
for 12 h at room temperature and extracted with EtOAc
(5 ꢂ 10 mL). The organic layers were collected, concentrated
and purified by the flash column chromatography (EtOAc–MeOH
4:1) to give a Cbz-protected intermediate (106 mg, 83%). This
compound was treated with 3 mL CH2Cl2 and cooled in an ice
bath under a nitrogen atmosphere, and then diisopropylethyl-
amine (1 mL) was added dropwise, followed by a careful addi-
tion of chloromethyl methyl ether (0.71 mL, 9.36 mmol).
A
significant amount of white smoke formed in the reaction vessel.
The reaction mixture was stirred in the dark for 48 h during
which the solution gradually turned red. After cooling to 0 °C,
saturated aqueous ammonium chloride (5 mL) was added. The
contents were diluted with water and extracted with CH2Cl2
(3 ꢂ 10 mL). The combined organic layers were dried (NaSO4),
filtered, and concentrated. The crude product was purified by
chromatography on silica gel (from EtOAc–hexanes 1:1?EtOAc)
4. Experimental
4.1. General methods
1H and 13C NMR spectra were taken in CD2Cl2, CDCl3, CD3OD,
D2O at 500 MHz and 75 MHz, respectively. ROESY spectra for all
tetrapeptides were recorded at a mixing time of 900 ms. Spectra
of HRMS data were provided by the mass spectrometry laboratory
in the Department of Chemistry at University of Manitoba. The ele-
mental analysis was performed by Guelph Chemical Laboratories
LTD. Analytical thin layer chromatography was performed on
0.20 mm silica gel 60 Å plates. Flash chromatography was per-
to afford the product 4 (125 mg, 72%). Rf = 0.28; [
a]
+12.3 (c
D
1.1, CHCl3); 1H NMR (300 MHz, CDCl3, two isomers): d = 2.23–
2.46 (m, 2H), 3.28–3.82 (m, 25H), 3.90 (dd, 0.38H, J = 9.1 Hz,
J = 4.3 Hz), 4.04 (dd, 0.62H, J = 8.9 Hz, J = 4.4 Hz), 4.16–4.31 (m,
1.62H), 4.36 (s, 0.38H), 4.46–4.84 (m, 10H), 4.91 (d, 0.62H,
J = 12.6 Hz), 5.14 (br s, 0.76H), 5.22 (d, 0.62H, J = 12.6 Hz),
7.23–7.38 (m, 5H); 13C NMR (75 MHz, CDCl3, two isomers):
d = 26.8/27.9, 52.0/52.2, 55.2–56.9 (12 carbons), 66.8/67.0, 67.0/
67.3, 68.1/68.7, 70.2/70.6, 71.9/72.0, 74.8/74.9, 76.7/76.8, 80.5/
80.8, 86.0/87.0, 96.6–98.5 (10 carbons), 127.4–128.4 (10 aro-
matic carbons), 136.2/136.4 (aromatic carbons), 154.6/155.1,
169.7 (2 carbons); HRMS (ES) calcd for C30H48NO15 [M+H]+:
662.3024, found: 662.3034.
formed on 40–63 lm 60 Å silica gel.
4.2. (1S)-2,3,4,6-Tetra-O-methoxymethyl-10-N-benzyloxy
carbonyl-50(R)-methyl methoxymethyl ether-spiro[1,5-anhy-
dro-D L-proline methyl ester] (3)
-glucitol-1,30-
To a mixture of compound 1 (80 mg, 0.26 mmol) and benzyl
chloroformate (0.19 mL, 1.30 mmol) in water (2 mL) was added
sodium carbonate (83 mg, 0.78 mmol). The reaction was stirred
for 12 h at room temperature and extracted with EtOAc
(5 ꢂ 10 mL). The organic layers were collected, concentrated and
purified by the flash column chromatography (EtOAc–MeOH
4:1) to give a Cbz-protected intermediate (103 mg, 90%). This
material was treated with 3 mL CH2Cl2 and cooled in an ice bath
under a nitrogen atmosphere, and then diisopropylethylamine
(1 mL) was added dropwise, followed by a careful addition of
chloromethyl methyl ether (0.71 mL, 9.36 mmol). A significant
amount of white smoke formed in the reaction vessel. The reac-
tion mixture was stirred in the dark for 48 h during which the
solution gradually turned red. After cooling to 0 °C, saturated
aqueous ammonium chloride (5 mL) was added. The contents
were diluted with water and extracted with CH2Cl2 (3 ꢂ 10 mL).
The combined organic layers were dried (NaSO4), filtered, and
concentrated. The crude product was purified by chromatography
on silica gel (from EtOAc–hexanes 1:1?EtOAc) to afford the prod-
4.4. (1S)-2,3,4,6-Tetra-O-methoxymethyl-10-N-benzyloxycar-
bonyl-50(R)-methyl methoxymethyl ether-spiro[1,5-anhydro-
D-
glucitol-1,30-
L-proline] (5)
To a solution of compound 3 (50 mg, 0.076 mmol) in dioxane
(3 mL) was added 2 M lithium hydroxide aqueous solution
(0.38 mL, 0.76 mmol). The reaction mixture was stirred at 60 °C
for 48 h. Afterwards, the solution was cooled to room tempera-
ture and neutralized with Amberlite IRC-50S H+ ion-exchange re-
sin. The mixture was filtered and concentrated to afford the
crude product, which was purified by flash chromatography
(EtOAc–MeOH 20:1) to give pure 5 (Rf = 0.2, 45 mg, 93%). The ste-
reochemistry at the
ing acid 5 into its methyl ester 3. (Reaction conditions: Cs2CO3
(2 equiv), MeI (2.5 equiv), DMF, rt, 3 h.) +53.9 (c 1.0,
a-position was confirmed by simply convert-
[a]
D
uct 3 (128 mg, 83%). Rf = 0.25; [a]
+38.4 (c 1.4, CHCl3); 1H NMR
CH3OH); 1H NMR (300 MHz, CD3OD, two isomers): d = 2.15–
2.27 (m, 1H, J = 13.9 Hz, J = 9.7 Hz), 2.50–2.62 (m, 1H,
J = 13.9 Hz, J = 6.6 Hz), 3.25–3.47 (m, 15H, partially overlapping
with solvent), 3.48–3.92 (m, 7H), 4.00–4.40 (m, 3H), 4.59–4.84
(m, 10H), 5.07–5.18 (br s, 2H), 7.27–7.43 (m, 5H); 13C NMR
(75 MHz, CD3OD, two isomers): d = 32.9/40.0, 55.5–57.2 (12 car-
bons), 67.9/68.4, 68.3 (2 carbons), 70.6/71.4, 72.8/73.3, 73.3 (2
carbons), 76.7/76.9, 77.9 (2 carbons), 81.0/81.4, 86.3/86.8, 97.6–
99.8 (10 carbons), 128.4–129.6 (10 aromatic carbons), 138.2/
137.8 (aromatic carbons), 156.8 (2 carbons), 174.0 (2 carbons);
HRMS (ES) calcd for C29H44NO15 [MꢁH]ꢁ: 646.2711, found:
646.2723.
D
(300 MHz, CDCl3, two isomers): d = 2.08–2.21 (m, 1H), 2.44–2.56
(m, 1H), 3.22–3.44 (m, 15H), 3.48–3.77 (m, 10H), 4.00–4.15 (m,
1.36H), 4.27 (dd, 0.64H, J = 8.9 Hz, J = 3.9 Hz), 4.42 (s, 0.64H),
4.50–4.82 (m, 10.36H), 5.00 (d, 0.64H, J = 13.0 Hz), 5.07–5.18 (m,
1.36H), 7.21–7.36 (m, 5H); 13C NMR (75 MHz, CDCl3, two iso-
mers): d = 31.5/32.9, 52.2/52.3, 55.0–56.5 (12 carbons), 66.7/
66.8, 66.8/67.4, 69.4/70.2, 70.0/70.1, 72.1 (2 carbons), 74.8 (2
carbons), 76.4/76.6, 79.7/80.4, 84.8/85.4, 96.5–98.1 (10 carbons),
127.3–128.5 (10 aromatic carbons), 136.2/136.5 (aromatic car-
bons), 154.4/155.2, 169.9/170.1; HRMS (ES) calcd for
C30H48NO15 [M+H]+: 662.3024, found: 662.3036.

1120
K. Zhang, F. Schweizer / Carbohydrate Research 345 (2010) 1114–1122
4.5. (1S)-2,3,4,6-Tetra-O-methoxymethyl-10-N-benzyloxy-
carbonyl-50(S)-methyl methoxymethyl ether-spiro[1,5-
chromatography (EtOAc–MeOH 30:1) to afford the Cbz-protected
intermediate, which was dissolved in EtOAc (5 mL) and treated
with Pd(OH)2 (20 mg, 20% wt on charcoal) under hydrogenation
condition (H2, 10 psi). The mixture was stirred for 90 min at room
temperature, filtered, and concentrated. The resulting crude prod-
uct was purified by flash chromatography (EtOAc–MeOH 20:1) to
anhydro-D L-proline] (6)
-glucitol-1,30-
To a solution of compound 4 (100 mg, 0.15 mmol) in dioxane
(3 mL) was added 2 M lithium hydroxide aqueous solution
(0.76 mL, 1.5 mmol). The reaction mixture was stirred at 60 °C
for 48 h. Afterwards, the solution was cooled to room temperature
and neutralized with Amberlite IRC-50S H+ ion-exchange resin. The
mixture was filtered and concentrated to afford the crude product,
which was purified by flash chromatography (EtOAc–MeOH 20:1)
to give pure 6 (Rf = 0.22, 51 mg, 53%). The stereochemistry at the
afford the dipeptide 206 (Rf = 0.25, 28 mg, 85%). [a] +54.0 (c 1.0,
D
CH3OH); 1H NMR (500 MHz, CD3OD): d = 0.95–0.97 (m, 6H), 1.78
(dd, 1H, J = 13.2 Hz, J = 6.8 Hz), 2.00–2.07 (m, 1H), 2.48 (dd, 1H,
J = 13.2 Hz, J = 8.2 Hz), 2.97 (s, 3H), 3.20 (s, 3H), 3.30–3.42 (m,
14H, partially overlapping with solvent), 3.46–3.72 (m, 9H), 3.80
(s, 1H), 3.89 (d, 1H, J = 11.4 Hz), 4.57 (d, 1H, J = 4.6 Hz), 4.63–4.84
(m, 10H, partially overlapping with solvent); 13C NMR (75 MHz,
CD3OD): d = 18.4, 19.6, 31.9, 36.1, 36.2, 38.0, 55.4, 55.7, 55.8,
56.8, 57.0, 57.3, 58.0, 68.7, 70.2, 73.0, 76.1, 78.4, 79.3, 83.4, 88.0,
97.9 (2 carbons), 99.7, 99.9, 100.2, 171.4, 173.6; HRMS (ES) calcd
for C28H54N3O13 [M+H]+: 640.3657, found: 640.3649.
a
-position was confirmed by converting acid 6 into its methyl ester
4. (Reaction conditions: Cs2CO3 (2 equiv), MeI (2.5 equiv), DMF, rt,
3 h.) [
D +30.3 (c 1.2, CH3OH); 1H NMR (300 MHz, CD3OD, two iso-
a
]
mers): d = 2.35–2.46 (br s, 2H), 3.28–3.82 (m, 23H, partially over-
lapping with solvent), 4.13–4.31 (m, 2H), 4.56–4.86 (m, 10H),
5.01–5.23 (m, 2H), 7.28–7.43 (m, 5H); 13C NMR (75 MHz, CD3OD,
two isomers): d = 28.1/29.1, 55.5–57.4 (10 carbons), 57.7/58.2,
68.0/68.4, 68.4/68.6, 69.3/69.8, 73.1 (4 carbons), 77.5 (2 carbons),
78.2 (2 carbons), 81.9/82.0, 87.3/88.2, 97.6–100.0 (10 carbons),
128.6–129.6 (10 aromatic carbons), 137.8/138.0 (aromatic car-
bons), 156.8/157.0, 175.3 (2 carbons); HRMS (ES) calcd for
C29H44NO15 [MꢁH]ꢁ: 646.2711, found: 646.2718.
4.8. Tripeptide H-(D)-Phe-[Glc(OMOM)4-30(S)-50(R)(CH2OMOM)
HypH]-Val-NMe2 (9)
To a solution of dipeptide 7 (30 mg, 0.05 mmol) and N,N-diiso-
propylethylamine (43
lL, 0.23 mmol) in DMF (3 mL) were added
the -Fmoc-Phe-H (56 mg, 0.14 mmol) and PyBOP (73 mg,
D
0.14 mmol). The mixture was stirred for 18 h at room temperature
before addition of sodium bicarbonate (16 mg, 0.18 mmol). The
resulting mixture was diluted with water (5 mL) and extracted
with EtOAc (5 ꢂ 10 mL). The combined organic layers were dried
(Na2SO4) and concentrated. The crude product was purified by
flash chromatography (EtOAc–MeOH 20:1) to provide the Fmoc-
protected intermediate, which was dissolved in a mixture of piper-
idine and DMF (0.2 mL + 0.8 mL) and stirred for 1 h at room
temperature. The solution was concentrated and purified by flash
chromatography (EtOAc–MeOH 10:1?6:1; TLC was charred with
4.6. Dipeptide H-[Glc(OMOM)4-30(S)-50(R)(CH2OMOM)HypH]-
Val-NMe2 (7)
The amino acid TFAꢃNH2-Val-NMe2 (44 mg, 0.16 mmol) was dis-
solved in DMF (1 mL) and then added to a solution of acid 5 (35 mg,
0.05 mmol) and N,N-diisopropylethylamine (70 lL, 0.32 mmol) in
DMF (3 mL). The reaction mixture was stirred for 10 min at room
temperature followed by the addition of TBTU (41 mg, 0.11 mmol)
and stirred for another 8 h. Afterwards the reaction was quenched
by the addition of water (8 mL) and the reaction mixture was ex-
tracted with EtOAc (4 ꢂ 10 mL). The combined organic layer was
dried (Na2SO4) and concentrated. The residue was purified by flash
chromatography (EtOAc–MeOH 30:1) to afford the Cbz-protected
intermediate, which was dissolved in EtOAc (5 mL) and treated
with Pd(OH)2 (20 mg, 20% wt on charcoal) under hydrogenation
condition (H2, 10 psi). The mixture was stirred for 90 min at room
temperature, filtered, and concentrated. The resulting crude prod-
uct was purified by flash chromatography (EtOAc–MeOH 20:1) to
iodine) to give tripeptide 9 (Rf = 0.25, 29 mg, 80%). [
a]
+2.0 (c
D
0.4, CH3OH); 1H NMR (500 MHz, CD3OD, two isomers): d = 0.92
(d, 3H, J = 6.6 Hz), 1.04 (d, 3H, J = 7.0 Hz), 1.98–2.06 (m, 1H), 2.19
(dd, 0.30H, J = 14.3 Hz, J = 7.8 Hz), 2.32 (dd, 0.70H, J = 14.0 Hz,
J = 11.7 Hz), 2.40–2.47 (m, 2.80H), 2.51–2.60 (m, 1H), 2.67 (dd,
0.30H, J = 13.7 Hz, J = 7.3 Hz), 2.90–2.94 (m, 1.60H), 3.04 (dd,
0.30H, J = 14.0 Hz, J = 4.9 Hz), 3.13–3.44 (m, 18H, partially overlap-
ping with solvent), 3.49 (m, 0.30H), 3.55–3.98 (m, 6.70H), 4.22–
4.91 (m, 15H), 7.12–7.30 (m, 5H); 13C NMR (75 MHz, CD3OD, two
isomers): d = 19.4 (2 carbons), 19.7/19.8, 31.4/35.7, 32.5/32.6,
35.7/36.0, 38.0/38.2, 41.7/42.3, 55.4–56.9 (14 carbons), 57.1/57.8,
66.7 (2 carbons), 68.5/69.5, 72.3/73.3, 72.8/73.5, 75.9/76.2, 76.8/
77.3, 78.3/78.9, 85.9/86.8, 97.6–99.4 (10 carbons), 127.7–130.8
(10 aromatic carbons), 138.6/139.1 (aromatic carbon), 170.3/
170.6, 173.2/173.3, 178.0/179.0; HRMS (ES) calcd for C37H63
N4O14 [M+H]+: 787.4341, found: 787.4345.
afford the dipeptide 7 (Rf = 0.3, 27 mg, 80%). [
a]
D
+27.4 (c 1.2,
CH3OH); 1H NMR (500 MHz, CD3OD): d = 0.97 (d, 3H, J = 6.9 Hz),
1.01 (d, 3H, J = 6.8 Hz), 1.91 (m, 1H), 2.00–2.13 (m, 2H), 2.94 (s,
3H), 3.20 (s, 3H), 3.30–3.42 (m, 15H, partially overlapping with sol-
vent), 3.46–3.69 (m, 9H), 3.85 (d, 1H, J = 12.0 Hz), 4.55 (d, 1H,
J = 5.3 Hz), 4.62–4.82 (m, 10H); 13C NMR (75 MHz, CD3OD):
d = 18.8, 19.5, 31.7, 31.9, 36.1, 38.1, 55.5, 55.6, 55.7, 56.6, 56.7,
57.0, 57.2, 68.5, 70.7, 71.1, 74.3, 77.5, 78.5, 83.7, 89.2, 97.7, 97.9,
99.6, 99.8, 100.1, 172.8, 174.0; HRMS (ES) calcd for C28H54N3O13
[M+H]+: 640.3657, found: 640.3646.
4.9. Tripeptide H-(D)-Phe-[Glc(OMOM)4-30(S)-50(S)(CH2OMOM)
HypH]-Val-NMe2 (10)
4.7. Dipeptide H-[Glc(OMOM)4-30(S)-50(S)(CH2OMOM)HypH]-Val-
To a solution of dipeptide 8 (50 mg, 0.08 mmol) and N,N-diiso-
NMe2 (8)
propylethylamine (72
lL, 0.38 mmol) in DMF (3 mL) were added
the -Fmoc-Phe-H (93 mg, 0.23 mmol) and PyBOP (122 mg,
D
The amino acid TFAꢃNH2-Val-NMe2 (44 mg, 0.16 mmol) was dis-
solved in DMF (1 mL) and then added to a solution of acid 6 (35 mg,
0.05 mmol) and N,N-diisopropylethylamine (70 lL, 0.32 mmol) in
DMF (3 mL). The reaction mixture was stirred for 10 min at room
temperature followed by the addition of TBTU (41 mg, 0.11 mmol)
and stirred for another 8 h. After this the reaction was quenched by
the addition of water (8 mL) and the reaction mixture was ex-
tracted with EtOAc (4 ꢂ 10 mL). The combined organic layer was
dried (Na2SO4) and concentrated. The residue was purified by flash
0.23 mmol). The mixture was stirred for 18 h at room temperature
before the addition of sodium bicarbonate (27 mg, 0.30 mmol). The
resulting mixture was diluted with water (5 mL) and extracted
with EtOAc (5 ꢂ 10 mL). The combined organic layers were dried
(Na2SO4) and concentrated. The crude product was purified by
flash chromatography (EtOAc–MeOH 20:1) to provide the Fmoc-
protected intermediate, which was dissolved in a mixture of piper-
idine and DMF (0.2 mL + 0.8 mL) and stirred for 1 h at room
temperature. The solution was concentrated and purified by flash

K. Zhang, F. Schweizer / Carbohydrate Research 345 (2010) 1114–1122
1121
chromatography (EtOAc–MeOH 10:1?6:1; TLC was visualized
with iodine) to give tripeptide 10 (Rf = 0.25, 28 mg, 46%). [
product was purified by flash chromatography (CH2Cl2–MeOH
20:1) to provide the Fmoc-protected intermediate, which was
dissolved in a mixture of piperidine and DMF (0.2 mL + 0.8 mL)
and stirred for 1 h at room temperature. The solution was con-
centrated in vacuum. The resulting mixture was dissolved in
a]
D
ꢁ13.2 (c 0.9, CH3OH); 1H NMR (500 MHz, CD3OD, two isomers):
d = 0.87–1.03 (m, 6H), 1.90–2.11 (m, 1.14H), 2.27–2.44 (m,
1.86H), 2.67 (dd, 0.14H, J = 14.9 Hz, J = 7.4 Hz), 2.78 (dd, 0.86H,
J = 12.9 Hz, J = 6.1 Hz), 2.91 (s, 0.42H), 2.93 (s, 2.58H), 2.97–3.11
(m, 2H), 3.28–3.48 (m, 15H, partially overlapping with solvent),
3.49–4.01 (m, 9H), 4.14–4.97 (m, 15H), 7.17–7.27 (m, 5H); 13C
NMR (75 MHz, CD3OD, trans isomer): d = 18.7, 19.7, 32.5, 32.9,
36.0, 38.1, 41.2, 55.4–56.7 (7 carbons), 57.8, 68.9, 71.7, 72.7, 72.9,
76.2, 76.5, 79.3, 86.6, 97.6–99.4 (5 carbons), 127.7–136.6 (5 aro-
matic carbons), 139.1 (aromatic carbon), 170.5, 173.4, 176.8;
HRMS (ES) calcd for C37H63N4O14 [M+H]+: 787.4341, found:
787.4352.
MeOH (2 mL) followed by the addition of pyridine (16
lL,
0.19 mmol) and Ac2O (11 L, 0.12 mmol). The mixture was stir-
l
red for 2 h at room temperature. The solution was concentrated
and purified by flash chromatography (CH2Cl2–MeOH
25:1?15:1; TLC was visualized with iodine) to give tetrapeptide
12 (Rf = 0.21, 29 mg, 90%). [
a]
+29.2 (c 0.8, CH3OH); 1H NMR
D
(500 MHz, CD3OD, two isomers): d = 0.81–1.01 (m, 12H), 1.20–
1.49 (m, 3H), 1.94–2.06 (m, 4H), 2.11 (dd, 0.23H, J = 14.1 Hz,
J = 8.8 Hz), 2.29–2.46 (m, 1.77H), 2.85–3.19 (m, 8H), 3.30–3.54
(m, 16H, partially overlapping with solvent), 3.66–3.85 (m,
6.31H), 3.94 (d, 0.23H, J = 4.5 Hz), 4.0 (d, 0.23H, J = 4.6 Hz), 4.08
(dd, 0.23H, J = 8.2 Hz, J = 4.1 Hz), 4.26–4.88 (m, 14H, partially
overlapping with solvent), 4.97 (d, 0.23H, J = 6.3 Hz), 5.35 (dd,
0.77H, J = 8.5 Hz, J = 6.3 Hz), 7.12–7.26 (m, 5H); 13C NMR
(75 MHz, CD3OD, two isomers): d = 19.3/19.5, 19.6 (2 carbons),
21.8/22.5, 22.6/22.7, 23.1/23.7, 25.8/25.9, 32.1/32.2, 33.0 (2 car-
bons), 36.0/36.1, 37.9/38.2, 38.3/38.6, 42.0/42.5, 52.8–57.5 (18
carbons), 69.0/69.3, 71.6/71.7, 72.8 (2 carbons), 73.7 (2 carbons),
75.7/76.5, 76.0/76.7, 77.2/78.9, 86.6/88.3, 97.0–99.1 (10 carbons),
127.6–130.6 (10 carbons), 138.6 (2 carbons), 170.3/171.3, 172.3/
172.6, 173.2 (2 carbons), 173.7 (2 carbons), 174.2/174.4; HRMS
(ES) calcd for C45H76N5O16 [M+H]+: 942.5287, found: 942.5281.
4.10. Tetrapeptide Ac-Leu-D
-Phe-[Glc(OMOM)4-30(S)-50(R)
(CH2OMOM)HypH]-Val-NMe2 (11)
To a solution of tripeptide 9 (25 mg, 0.03 mmol) and N,N-diiso-
propylethylamine (29 lL, 0.16 mmol) in DMF (3 mL) were added
the Fmoc-Leu-H (34 mg, 0.10 mmol) and TBTU (31 mg, 0.10 mmol).
The mixture was stirred for 18 h at room temperature before the
addition of sodium bicarbonate (11 mg, 0.13 mmol). The resulting
mixture was diluted with water (5 mL) and extracted with EtOAc
(5 ꢂ 10 mL). The combined organic layers were dried (Na2SO4)
and concentrated. The crude product was purified by flash chroma-
tography (CH2Cl2–MeOH 20:1) to provide the Fmoc-protected
intermediate, which was dissolved in a mixture of piperidine and
DMF (0.2 mL + 0.8 mL) and stirred for 1 h at room temperature.
The solution was concentrated in vacuum. The resulting mixture
was dissolved in MeOH (2 mL) followed by the addition of pyridine
4.12. Tetrapeptide Ac-Leu-
Val-NMe2 (13)
D
-Phe-[Glc30(S)-50(R)(CH2OH)HypH]-
(13
l
L, 0.16 mmol) and Ac2O (9
l
L, 0.10 mmol). The mixture was
To a solution of compound 11 (30 mg, 0.03 mmol) in MeOH
(1 mL) was added a 6 M HCl solution (100 L). The mixture was
stirred for 2 h at room temperature. The solution was concentrated
and purified by flash chromatography (CH2Cl2–MeOH 25:1?15:1;
TLC was visualized with iodine) to give tetrapeptide 11 (Rf = 0.27,
l
stirred for 18 h at room temperature and then concentrated. The
crude product was purified by flash chromatography (methylene
chloride–MeOH 5:1) to afford unprotected tetrapeptide 13
(Rf = 0.3, 20 mg, 90%). 1H NMR (500 MHz, D2O, cis isomer):
d = 0.63–1.32 (m, 15H), 1.90 (s, 3H), 1.94–1.99 (m, 1H), 2.30–2.46
(br, 5H), 2.74–2.79 (dd, 1H, J = 12.6 Hz, J = 12.9 Hz), 2.94–3.00
(dd, 1H, J = 17.5 Hz, J = 14.5 Hz), 3.17 (s, 3H), 3.44–3.53 (br, 3H),
3.64–3.87 (m, 5H), 4.04–4.11 (m, 1H), 4.17–4.25 (m, 1H), 4.34–
4.40 (m, 1H), 4.59 (m, 1H), 4.77 (1H, overlapping with solvent),
7.10–7.38 (m, 5H); 13C NMR (75 MHz, CD3OD, cis isomer):
d = 19.4, 19.7, 22.1, 22.3, 23.2, 25.7, 25.9, 31.8, 35.9, 38.2, 38.5,
42.0, 53.0, 54.2, 56.6, 59.9, 60.8, 62.6, 71.0, 71.2, 73.2, 75.3, 77.3,
87.3, 127.7–130.4 (5 carbons), 138.5, 171.7, 173.1, 174.1, 174.5,
174.8; HRMS (ES) calcd for C35H56N5O11 [M+H]+: 722.3976, found:
722.3985.
25 mg, 92%). [
a]
D
ꢁ19.8 (c 1.15, CH3OH); 1H NMR (500 MHz,
CD3OD, two isomers): d = 0.72–1.46 (m, 15H), 1.89–1.92 (m, 3H),
2.03 (m, 1H), 2.26 (dd, 0.29H, J = 14.4 Hz, J = 8.2 Hz), 2.35 (dd,
0.71H, J = 14.2 Hz, J = 11.9 Hz), 2.47 (s, 2.13H), 2.51 (dd, 0.71H,
J = 14.2 Hz, J = 6.9 Hz), 2.68 (m, 1H), 2.83 (dd, 0.29H, J = 14.5 Hz,
J = 8.9 Hz), 2.92 (s, 0.87H), 3.04–3.45 (m, 19H, partially overlapping
with solvent), 3.46–3.76 (m, 4.71H), 3.84–3.89 (m, 1.71H), 3.95–
3.97 (m, 0.29H), 4.05 (d, 0.29H, J = 5.2 Hz), 4.09 (dd, 0.29H,
J = 10.4 Hz, J = 5.2 Hz), 4.19–4.45 (m, 2.71H), 4.54–4.84 (m,
12.71H, partially overlapping with solvent), 5.41–5.45 (m, 0.29H),
7.08–7.25 (m, 5H); 13C NMR (75 MHz, CD3OD, two isomers):
d = 18.5/19.3, 19.7/19.8, 21.9/22.2, 22.5/22.6, 23.2/23.4, 25.7/25.8,
40.0 (2 carbons), 32.3/32.6, 38.0/38.2, 38.8 (2 carbons), 42.2/42.6,
52.6/53.4, 53.1/53.9, 55.4–56.9 (14 carbons), 57.3/57.6, 66.7 (2 car-
bons), 68.5/69.6, 72.1/73.4, 72.7/73.6, 75.6/76.5, 76.7/77.6, 80.1 (2
carbons), 85.9/86.5, 97.5–99.7 (10 carbons), 127.7–130.9 (10 aro-
matic carbons), 137.8/138.4 (aromatic carbon), 170.4/170.6,
172.6/172.9, 173.2/173.3, 173.8/173.9, 174.1/174.9; HRMS (ES)
calcd for C45H76N5O16 [M+H]+: 942.5287, found: 942.5293.
4.13. Attempted acidic hydrolysis of tetrapeptide Ac-Leu-
D-Phe-
[Glc(OMOM)4-30(S)-50(R)(CH2OMOM)HypH]-Val-NMe2 (12)
Exposure of 12 to acidic conditions (2–18 h) as described for
13 required for cleavage of MOM protecting groups resulted in
the formation of dipeptides 14 and 15, which were identified
by MS analysis. (14: ES, calcd for C18H34N3O8 [M+H]+: 420.23,
found: 420.82; 15: ES, C17H25N2O4, [MꢁH]ꢁ: 319.17, found:
319.43.)
4.11. Tetrapeptide Ac-Leu-
(CH2OMOM)HypH]-Val-NMe2 (12)
D
-Phe-[Glc(OMOM)4-30(S)-50(R)
To
a
solution of tripeptide 10 (30 mg, 0.04 mmol) and
L, 0.19 mmol) in DMF (3 mL)
N,N-diisopropylethylamine (35
l
4.14. ROESY experiments
were added the Fmoc-Leu-H (41 mg, 0.12 mmol) and TBTU
(37 mg, 0.12 mmol). The mixture was stirred for 18 h at room
temperature before the addition of sodium bicarbonate (13 mg,
0.16 mmol). The resulting mixture was diluted with water
(5 mL) and extracted with EtOAc (5 ꢂ 10 mL). The combined or-
ganic layers were dried (Na2SO4) and concentrated. The crude
ROESY spectra of tetrapeptides 11–13 and 16 were recorded on
a Bruker AMX500 instrument with 256 points in t1, 2048 points in
t2 and 64 scans per t2 increment. ROESY spectra for all tetrapep-
tides were recorded at a mixing time of 900 ms.

1122
K. Zhang, F. Schweizer / Carbohydrate Research 345 (2010) 1114–1122
5. Reviewed in: (a) Fisher, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 1415–1436; (b)
Liu, J.; Chen, C.-M.; Walsh, C. T. Biochemistry 1991, 30, 2306–2310.
6. Johnson, M. E.; Lin, Z.; Padmanabhan, K.; Tulinsky, A.; Kahn, M. FEBS Lett. 1994,
337, 4–8.
7. Dugave, C.; Demange, L. Chem. Rev. 2003, 103, 2475–2532.
8. Halab, L.; Lubell, W. D. J. Am. Chem. Soc. 2002, 124, 2474–2484. and references
therein.
9. An, S. S. A.; Lester, C. C.; Peng, J.-L.; Li, Y.-J.; Rothwarf, D. M.; Welker, E.;
Thannhauser, T. W.; Zhang, L. S.; Tam, J. P.; Scheraga, H. A. J. Am. Chem. Soc.
1999, 121, 11558–11566.
10. (a) Didierjean, C.; Del Duca, V.; Benedetti, E.; Aubry, A.; Zouikri, M.; Marraud,
M.; Boussard, G. J. Peptide Res. 1997, 50, 451–457; (b) Zouikri, M.; Vicherat, A.;
Aubry, A.; Marraud, M.; Boussard, G. J. Peptide Res. 1998, 52, 19–26.
11. (a) Dumy, P.; Keller, M.; Ryan, D. E.; Rohwedder, B.; Wöhr, T.; Mutter, M. J. Am.
Chem. Soc. 1997, 119, 918–925; (b) Kern, D.; Schutkowski, M.; Drakenberg, T. J.
Am. Chem. Soc. 1997, 119, 8403–8408; (c) Keller, L.; Sager, C.; Dumy, P.;
Schutkowski, M.; Fischer, G. S.; Mutter, M. J. Am. Chem. Soc. 1998, 120, 2710–
2714.
4.15. Temperature coefficient experiments
1D 1H NMR spectroscopy of 8 mM solutions of 11, 12, and 16 in
100.0% Me2SO-d6 and 13 in H2O–D2O (9:1) was recorded on a Bru-
ker AMX500 at 20 °C, and from 20 to 45 °C with increments of 5 °C,
using routine techniques. Chemical shift (d) is expressed in parts
per million (ppm) and calibrated with respect to the residual
DMSO signal (1H: 2.49 ppm) or the TSP (1H: 0.00 ppm) in water.
Acknowledgments
This work was supported by the Natural Sciences and Engineer-
ing Research Council of Canada (NSERC) in the form of a Discovery
Grant. We are grateful to Dr. Kirk Marat for his help with the NMR
experiments.
12. Zhang, K.; Schweizer, F. Synlett 2005, 3111–3115.
13. Owens, N.; Braun, C.; Schweizer, F. J. Org. Chem. 2007, 72, 4635–4643.
14. Zhang, K.; Teklebrhan, R. B.; Schreckenbach, G.; Wetmore, S.; Schweizer, F. J.
Org. Chem. 2009, 74, 3735–3743.
15. Jeannotte, G.; Lubell, W. D. J. Am. Chem. Soc. 2004, 126, 14334–14335.
16. (a) Walker, J. R.; Alshafie, G.; Nieves, N.; Ahrens, J.; Clagett-Dame, M.; Abou-
Issa, H.; Curley, R. W. Bioorg. Med. Chem. 2006, 14, 3038–3048; (b) Sicheral, F.;
Wittmann, V. Angew. Chem., Int. Ed. 2005, 44, 2096–2099.
17. Frerot, E.; Coste, J.; Pantaloni, A.; Dufour, M. N.; Jouin, P. Tetrahedron 1991, 47,
259–270.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2010.03.037.
References
18. Bürgi, H. B.; Dunitz, J. D.; Lehn, J. M.; Wipff, G. Tetrahedron 1974, 30, 1563–
1572.
19. Runkel, L.; Neier, W.; Repinsky, R. B.; Karpusas, M.; Whitty, A.; Kimball, K.;
Brickelmaier, M.; Muldowney, C.; Jones, W.; Goelz, S. E. Pharm. Res. 1998, 15,
641–649.
20. Kopple, K. D.; Ohnishi, M.; Go, A. J. Am. Chem. Soc. 1969, 91, 4264–4272.
21. Misicka, A.; Verheyden, P. M. F.; Van Binst, G. Lett. Pept. Sci. 1998, 5,
375–377.
22. Chakraborty, T. K.; Srinivasu, P.; Vengal Rao, R.; Kiran Kumar, S.; Kunwar, A. C. J.
Org. Chem. 2004, 69, 7399–7402.
23. Leeflang, B. R.; Vliegenthart, J. F. G.; Kroon-Batenburg, L. M. J.; Eijck, B. P.;
Kroon, J. Carbohydr. Res. 1992, 230, 41–61.
1. (a) Richardson, J. S. Adv. Protein Chem. 1981, 34, 167–339; (b) Wilmot, C. M.;
Thornton, J. M. J. Mol. Biol. 1988, 203, 221–232; (c) Rose, G. D.; Gierasch, L. M.;
Smith, J. A. Adv. Protein Chem. 1985, 37, 1–109.
2. Creighton, T. E. Proteins: Structure and Molecular Properties; Freeman: New York,
1983; (b) Rizo, J.; Gierasch, L. Annu. Rev. Biochem. 1992, 61, 387–418.
3. Lewis, P. N.; Momany, F. A.; Scheraga, H. A. Proc. Natl. Acad. Sci. U.S.A. 1971, 68,
2293–2297.
4. Branden, C.; Tooze, J. Introduction to Protein Structure; Garland Publishing: New
York, 1991; (b) Stryer, L. Biochemistry, 4th ed.; W.H. Freeman and Company:
New York, 1999.