Design and synthesis of glucose-templated proline-lysine chimera: polyfunctional amino acid chimera with high prolyl cis amide rotamer population

Article abstract of DOI:10.1016/j.carres.2008.12.024

We describe the synthesis of two glucose-templated proline-lysine chimeras (GlcTProLysCs) that differ in the stereochemistry of the hydroxymethyl substituent at the C-5′ position of the pyrrolidine ring. The key synthetic steps involve C-glycosylation of an exocyclic glucose-based epoxide with allyltributylstannane, which affords functionalized C-ketosides containing an α-hydroxy ester moiety; introduction of an amino group at C-2 through stereoselective reductive amination; and regioselective installation of the azide group at C-6 on the glucose scaffold. Incorporation of these chimeras into the model peptides Ac-GlcTProLysC-NHMe and Ac-GlcTProLysC-OMe demonstrates that the stereochemistry of the hydroxymethyl substituent at the C-5′ position has a profound effect on the equilibrium constant of prolyl amide cis/trans isomerization. The equilibrium constant K_c/t for the peptide mimic Ac-GlcTProLysC-NHMe with C-5′(R) stereochemistry was determined to be 3.03 ± 0.04, while the K_t/c for the C-5′(S) diastereoisomer was 0.56 ± 0.04 in D₂O. Temperature coefficient experiments indicate that the origin of these effects is derived from two critical hydrogen bonds involving the C-5′ hydroxymethyl substituent: one to the N-terminal amide carbonyl group, and the other to the primary amino group in the glucose moiety.

Full text of DOI:10.1016/j.carres.2008.12.024

Carbohydrate Research 344 (2009) 576–585
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Design and synthesis of glucose-templated proline–lysine chimera:
polyfunctional amino acid chimera with high prolyl cis amide rotamer
population
*
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:
We describe the synthesis of two glucose-templated proline–lysine chimeras (GlcTProLysCs) that differ in
the stereochemistry of the hydroxymethyl substituent at the C-50 position of the pyrrolidine ring. The key
synthetic steps involve C-glycosylation of an exocyclic glucose-based epoxide with allyltributylstannane,
Received 10 November 2008
Received in revised form 17 December 2008
Accepted 28 December 2008
Available online 3 January 2009
which affords functionalized C-ketosides containing an a-hydroxy ester moiety; introduction of an amino
group at C-2 through stereoselective reductive amination; and regioselective installation of the azide
group at C-6 on the glucose scaffold. Incorporation of these chimeras into the model peptides Ac-GlcTPro-
LysC-NHMe and Ac-GlcTProLysC-OMe demonstrates that the stereochemistry of the hydroxymethyl sub-
stituent at the C-50 position has a profound effect on the equilibrium constant of prolyl amide cis/trans
isomerization. The equilibrium constant Kc/t for the peptide mimic Ac-GlcTProLysC-NHMe with C-50(R)
stereochemistry was determined to be 3.03 0.04, while the Kt/c for the C-50(S) diastereoisomer was
0.56 0.04 in D2O. Temperature coefficient experiments indicate that the origin of these effects is derived
from two critical hydrogen bonds involving the C-50 hydroxymethyl substituent: one to the N-terminal
amide carbonyl group, and the other to the primary amino group in the glucose moiety.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Glycosyl amino acid
Proline
Lysine
Chimera
1. Introduction
sequences also exist as extended helices8 (polyproline-I and poly-
proline-II) and antimicrobial peptides.9 Furthermore, proline
Conformationally constrained amino acids have found wide
applications as building blocks to study and probe the bioactive
conformation of peptides when binding to receptors.1–3 Among
all naturally occurring amino acids, proline is the only amino acid
with a side chain fused onto the peptide backbone. Its cyclic struc-
ture restricts the rotation about its / dihedral angle, thereby reduc-
ing the energy difference between the prolyl amide cis and trans
isomers. Thus, while most peptide amide bonds exist almost exclu-
sively in the trans form, proline has a much greater propensity to
form cis amide bonds. A variety of factors influence cis/trans isom-
undergoes post-translational modifications to form 4-(R)-hydrox-
yprolines, which are known to contribute to the enhanced stability
of the polyproline-II conformation in both collagenous proteins
and peptides,4 and in plant cell wall glycoproteins.10
Proline analogues displaying the characteristics of other amino
acids are referred to as proline-amino acid chimeras, and have
been used to study the spatial requirements for receptor affinity
and biological activity of both natural amino acids11,12 and pep-
tides.13–15 For example, b-substituted-prolines such as 3-carbo-
xyproline,11a 3-phenylproline,16 and 3-di-methylproline17 combine
amino acid side chain functionality with proline’s conformational
rigidity. In these cases, replacement of the natural amino acids in
peptides by proline-amino acid chimeras provided better under-
standing of the bioactive conformations of peptides binding to
receptors.13–15 While these analogues have proved useful for
inducing specific constraints into amino acids and peptides, their
structures do not permit additional derivatization; a trait that is of-
ten required in drug discovery and lead optimization. Polyfunc-
tional proline-amino acid chimera may overcome these drawbacks.
Our concept for developing such polyfunctional proline-amino
acid chimeras was derived from glycosyl amino acids (GAAs) which
erization of proline; these include electron-withdrawing groups at-
tached to the pyrrolidine ring,4 n?
p
interaction,5 Ar–Pro
*
interactions,6 and steric effects.7 In particular, incorporation of
bulky substituents into the d-position of proline has been shown
to enhance the prolyl amide cis population significantly. cis/trans
Isomerization of proline plays an important role in the formation
of secondary structures in peptides and proteins because proline
induces a reversal in backbone conformation resulting in the for-
mation of reverse turns and disruption of helices and sheets in pro-
teins. Besides the occurrence of proline in b-turns, proline-rich
are defined by an
a-amino acid group [CH(NH2)CO2H] either
* Corresponding author. Fax: +1 204 474 7608.
E-mail address: schweize@ms.umanitoba.ca (F. Schweizer).
directly attached or carbon-linked to the anomeric carbon of a
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.12.024

K. Zhang, F. Schweizer / Carbohydrate Research 344 (2009) 576–585
577
carbohydrate scaffold.18 The relative rigidity of the pyran ring com-
BrCH2CO2Me
OBn
O
OBn
O
LHMDS, THF
bined with the polyfunctional nature of the carbohydrate scaffold
has inspired the design of unusual and conformationally
constrained amino acids and novel peptidomimetics.18 Although
there are many examples of C-glycosyl-glycine, -alanine, -serine,
and -asparagine,18a few proline-based GAAs exist.19
We report here the design and synthesis of spirocyclic glucose-
templated proline–lysine chimeras (GlcTProLysCs), and describe
their properties in peptide mimics. Spirocyclic GlcTProLysCs were
selected on the basis of previous synthetic methodology.20 Bicyclic
GlcTProLysCs combine the molecular features of glucose (pyran-
based polyol) with the unique characteristics of proline or
-78 oC
1'
O
BnO
BnO
BnO
BnO
O
2
82%
CO2Me
BnO
3'
BnO
1
2
OBn
O
β
-face
OSiMe3
CO2Me
BnO
BnO
BnO
A
α-face
OBn
O
OBn
1) allyltributylstannane
TMSOTf, CH2Cl2
3-hydroxyproline and L-lysine (Fig. 1). The characteristics of the ly-
sine side chain including relative length and presence of amino
function are presented on the pyrrolidine ring and are further con-
OH
O
BnO
BnO
BnO
0 o
C
+
BnO
CO2Me
BnO
HO
BnO
(80%)
3
strained by incorporation into the 6-amino-6-deoxy-D-glucose
CO2Me
scaffold. Proline–lysine chimeras were selected due to their fre-
quent occurrence in cationic antimicrobial peptides with polypro-
line conformation.9 To control the prolyl amide cis/trans
isomerization, we were interested in developing GlcTProLysC ana-
logues that contain hydrogen bond forming substituents like a
hydroxymethyl group at the d-position of proline. Previous studies
have shown that bulky substituents at the d-position (including d-
t-butyl proline7 and d,d-dimethyl proline3) enhance the prolyl
amide cis isomer population. However, hydrogen bond forming
groups at the d-position have never been investigated. In addition,
chemical manipulations and derivatization of the polyol scaffold
provide an opportunity to adjust the chemical, physical, and phar-
macodynamic properties of proline-containing peptides.21 This
may provide a novel tool to functionalize extended helical struc-
tures including PP1 and PP2.22 Moreover, incorporation of poly-
hydroxylated amino acids have been shown to induce novel
secondary structures in small peptides. For instance, incorporation
of unprotected sugar amino acids into small peptides such as
gramicidin S23 and opioid peptides24 has prohibited the formation
of the targeted secondary structural motif. Instead, unusual turn
structures were stabilized by intramolecular hydrogen bonds be-
tween sugar hydroxyl groups and the peptidic amide backbone.25
Similar effects may also be observed with GlcTProLysC.
(9%)
4
Scheme 1. Synthesis of the a-allylic intermediate 3.
for optimal yield of target compound 3. The configuration at the
anomeric position in compound 4 was deduced on the basis of ob-
served/unobserved NOE29 contacts (Fig. 2).
Compound 3 served as starting material for the installation of
the amino function at C-2 (Scheme 2). Initially, we attempted to
convert the hydroxyl group at C-2 into an amino function. How-
ever, nucleophilic substitution of C-2-activated sulfonate ester (tri-
flate) with
a variety of nucleophiles including benzylamine,
p-methoxybenzylamine, lithium, and sodium azide at low and ele-
vated temperatures resulted only in trace amounts of the desired
amine. In these cases, unreacted starting material was recovered
(>90%). To avoid these complications, we decided to explore a
reductive amination approach. Alcohol 3 was oxidized to ketone
5 at ꢀ78 °C using a mixture containing trifluoroacetic anhydride,
triethylamine, and dimethylsulfoxide in dichloromethane to pro-
duce ketone 5 in 95% isolated yield.30 In order to confirm the con-
figuration of the product, we performed NOE experiments (Fig. 2).
For instance, subjecting one of the allylic protons to a one-dimen-
sional GOESY experiment showed inter-proton effects to H-3 (7.9%
NOE29) and H-5 (7.1%). This is consistent with the structure 5 bear-
ing an allylic group at the axial position. Subsequently, the ketone
5 was converted into the amino ester 7 in a two-step procedure. At
first, compound 5 was exposed to titanium tetrachloride-promoted
imination using benzylamine in ether to afford the imine 6 in 96%
after chromatographic purification.31 The imine 6 was stereoselec-
tively reduced to amino ester 7 in quantitative yield using sodium
cyanoborohydride in acidified MeOH at 0 °C. The high diastereose-
lectivity could be explained in the Felkin model (Fig. 3),32 in which
the nucleophile approached the imine ion from the less hindered
side (Re face) and resulted in the formation of S-configuration at
C-2 position of compound 7, which was confirmed by the following
NOE experiments.
2. Results and discussion
The synthesis started with the readily available D-glucose-based
lactone 126 (Scheme 1), which reacts with the enolate of methyl
bromoacetate generated from lithium bis-(trimethylsilyl)amide
(LiN(SiMe3)2) in tetrahydrofuran (THF) at ꢀ78 °C to produce the
exocyclic epoxide 2 in 80% yield as a single stereoisomer.27 Tri-
methylsilyl trifluoromethanesulfonate (TMSOTf)-promoted C-gly-
cosylation of epoxide
2
with allyltributylstannane in
dichloromethane followed by hydrolysis of the TMS-ether with tri-
fluoroacetic acid (TFA)-containing wet THF produced a mixture
containing alcohols 3 and 4 (ratio 3:4 = 9:1) in a combined yield
of 89%. Regioselective opening of epoxide 2 proceeded via forma-
tion of oxonium ion (intermediate A) that subsequently underwent
a
-selective C-glycosylation favored by stereoelectronic factors as
observed for similar C-glycosylation reactions.28 It is noteworthy
1.1%
BnO
that slow addition of epoxide 2 to allyltributylstannane is crucial
O
5
O
H
BnO
BnO
2.1%
BnO
BnO
H
CO2Me
3
H
H
H
O
BnO
H
H
2
BnO
7.1%
CO2Me
HO
7.9%
4
5
Figure 2. Assignment of stereochemistry at anomeric carbon in compounds 4 and 5
Figure 1. Glucose-templated proline–lysine chimera (GlcTProLysC).
through 1D NOE experiments (recorded in CDCl3).

578
K. Zhang, F. Schweizer / Carbohydrate Research 344 (2009) 576–585
DMSO, TFAA
TEA, CH2Cl2,
-78 oC
TiCl4,BnNH2
Et2O, 0 oC -rt
OBn
O
OBn
O
NaCNBH3, AcOH
MeOH, 0 o
99%
O
NBn
CO2Me
C
BnO
BnO
BnO
BnO
3
CO2Me
96% (two times)
BnO
BnO
97%
5
6
OBn
O
I2, CH2Cl2/Et2O
0 oC - rt
OBn
O
OBn
O
CO2Me
NBn
CO2Me
NBn
NHBn
CO2Me
BnO
BnO
BnO
BnO
BnO
+
BnO
BnO
BnO
quant.
BnO
7
I
I
(i) AgOAc,
toluene, rt
(ii) K2CO3,
MeOH, rt.
OBn
O
OBn
O
OH
O
CO2Me
Pd(OH)2, H2,
2'
CO2Me
CO2Me
NH
R2
BnO
2'
5'
HCl, MeOH
rt
BnO
BnO
1'
HO
HO
+
BnO
NBn
6'
1'
3'
3'
BnO
NBn
5'
R
HO
R1
BnO
quant.
4'
4'
R1
R2
R = OH (6.5%)
10
R1 = CH2OH; R2 = H (43%)
R1 = H ; R2 = CH2OH (46%)
8
9
11 R1 = CH2OH; R2 = H
12 R1 = H ; R2 = CH2OH
Scheme 2. Preparation of spirocyclic glucose-based proline analogues using a reductive amination route.
hols 8, 9, and 10 in 44%, 45%, and 6% isolated yields, respectively.
H
(S)
(S)
(M)
O
(M)
O
MeO2C
NHBn
Subsequently, exposure of compounds 8 and 10 to catalytic
hydrogenolysis condition using Pearlman’s catalyst provided the
unprotected proline analogues 11 and 12 in quantitative yields,
respectively.
NHBn
H
CO2Me
C2
(L)
C2
(L)
To assign the stereochemistry at C-20, the alcohols 8, 9, and 10
were converted into the acetates 13, 14, and 15 using standard
conditions (acetic anhydride in pyridine, Scheme 3). We selected
the pipecolic acid analogue 15 to assign the stereochemistry at
C-20 (Figure 4). The spirocyclic compound 15 consists of both a
pyranose ring and a piperidine ring. The large coupling constants
for J2,3, J3,4, and J4,5 (>9.0 Hz) in conjunction with inter-proton NOEs
between H-3 and H-5, establish the 4C1 chair conformation of the
sugar ring. The chair conformation of the piperidine ring is de-
duced from the observed vicinal diaxial and long-range coupling
7
6
Figure 3. A rational explanation for stereoselective reductive amination using the
Felkin model.
With amino ester 7 in hand, we installed the pyrrolidine ring by
iodocyclization in dichloromethane to produce an inseparable iso-
meric mixture containing various iodo-compounds. To separate
the compounds from each other, we converted the mixture into
the alcohols 8, 9, and 10 via a two-step process. At first, the mix-
ture was exposed to silver acetate in toluene33 to produce an
inseparable mixture of esters 13, 14, and 15 (Scheme 3) that, by
treatment with potassium carbonate in MeOH, afforded the alco-
constants. For instance, the axial position of protons H-40ax, H-5a0 x
,
and H-60ax can be deduced by their large vicinal diaxial coupling
0
0
0
0
constants (J4 ax,5 ax, J5 ax,6 ax > 10.5 Hz), while the observed long-
0
0
0
0
range coupling constants between J4 eq,6 eq are equal to J2 eq,4 eq
(ꢁ1.0 Hz), confirming the equatorial position of H-20eq, H-4e0 q and
,
H-60eq in the piperidine ring. In 0addition, the observed inter-proton
effects (NOE) between H-5/H-5ax, H-5/H-3, and H-3/H-40ax, together
with the unobserved effect between H-60ax/H-2e0 q using a one-
dimensional GOESY experiment, determined the C-20 (S) configura-
tion (Fig. 4).30,34
OBn
O
Ac2O,
pyridine, rt
quant.
CO2Me
2'
5'
BnO
BnO
1'
8/9
3'
NBn
BnO
4'
R2
R1
2.3%
H
3
Heq
NBn
R
1 = CH2OAc; R2 = H
1 = H ; R2 = CH2OAc
13
14
5
H
O
4'
ax
R
2'
5'
2.7%
2.7%
OBn
O
H
Ac2O,
pyridine, rt
CO2Me
2'
H
Hax
BnO
BnO
1'
10
NBn
6'
quant.
3'
BnO
0.5%
5'
4'
Figure 4. Assignment of stereochemistry at C-20 position in compound 15 through
1D NOE experiment (recorded in C6D6). Some of the substituents in the glucose ring
are omitted for clarity.
OAc
15
Scheme 3. Acylation of compounds 8-10.

K. Zhang, F. Schweizer / Carbohydrate Research 344 (2009) 576–585
579
Once we had established the configuration at C-20 in compound
HO
HO
O
1) H , Pd(OH)
2
HCl, 8 h
,
2
15, we turned our interest to the stereochemistry at C-50 of the spi-
rocyclic proline analogues 13 and 14. Because the iodocyclization
was performed on a single stereoisomer 7, we assume that the ste-
reochemistry at C-20 of the proline analogues 13 and 14 remained
‘S’ based on the previous assignment with piperidine analogue 15.
To discriminate between compounds 13 and 14, we again used
NOE30 experiments (Fig. 5). As an example, the observed inter-pro-
ton effects between H-20/H-50 and H-50/H-5 for compound 13 are
consistent with the C-50 (R) stereochemistry. By comparison, pro-
line analogue 14 did not show any inter-proton effect between
H-20/H-50, which is consistent with a C-50 (S) configuration.
Once we had established the stereochemistry at the C-20- and
C-50-positions in compounds 13 and 14, we then focused on the
conversion of the primary hydroxyl group on the glucose moiety
into an amino function (Scheme 4). Initially, compounds 13 and
14 were exposed to catalytic hydrogenolysis condition using Pearl-
man’s catalyst. The resulting N-debenzylated amine was protected
using di-t-butyl dicarbonate and triethylamine in MeOH to afford
the carbamates 16 and 17 in 90% and 62% yields, respectively,
without acyl migration. The azido group in compounds 16 and
17 was installed by a standard two-step procedure: first, selective
activation of the primary hydroxyl group as sulfonate ester; sec-
ond, nucleophilic substitution with sodium azide in DMF at
80 °C, produced azides 18 and 19 in excellent yields.
O
OMe
NBoc
HO
13, 14
2) TEA, Boc O, 1 h,
HO
R1
16: R = CH OAc, R = H (90%)
2
R2
1
2
2
17: R = H, R = CH OAc (62%)
1
2
2
N
3
O
1) TsCl, pyridine, 12 h
o
O
OMe
HO
HO
2) NaN , DMF, 80 C,
3
NBoc
HO
MeOH,12 h
R
R
2
1
18: R = CH OAc, R = H (92%)
19: R = H, R = CH OAc (94%)
1
2
2
1
2
2
Scheme 4. Installation of the azide function at the C-6 position.
H-20 and methyl group of N-terminus in trans isomer 23b. More-
over, selective inversion of the methyl group of N-terminus in
23b showed inter-proton effects to H-50 (4.54% NOE) and H-60
(3.80% NOE). Similar experiments were performed to assign the
cis/trans isomers in compounds 22, 24, and 25.
In addition, we observed that the 13C NMR chemical shifts of the
a
C
atom of the trans rotamer in compounds 22–25 are high field
Azides 18 and 19 served as starting materials for incorporation
into the peptide mimics 22–25 (Scheme 5), which were used to
study the thermodynamic properties of cis/trans isomerization of
the glucose-templated proline–lysine chimera. In addition, we se-
lected peptide esters 22 and 23 bearing a C-terminal methyl ester
as well as methyl amides 24 and 25 as peptide mimics to study
how the nature of the C-terminal group affects N-terminal prolyl
amide isomerization.
Peptide esters 22 and 23 are prepared from azides 18 and 19
using the synthetic route outlined in Scheme 5. Deprotection of
the N-Boc group in 18 and 19 with trifluoroacetic acid followed
by acetylation using pyridine and acetic anhydride and O-deacety-
lation using sodium methoxide in MeOH produced azides 20 and
21 in 96% and 80% yields, respectively. Attempts to perform selec-
tive N-acylation failed and produced complex reaction mixtures.
Catalytic hydrogenation of azides 20 and 21 using Pearlman’s cat-
alyst produced peptide esters 22 and 23 in quantitative yields. The
N0-methylamides 24 and 25 were prepared from azides 20 and 21
through a two-step procedure: first, methyl esters 20 and 21 were
treated with a concentrated methylamine solution in ethanol to af-
ford N0-methylamide intermediates; second, the azido function
was reduced by catalytic hydrogenation to produce peptide mimics
24 and 25 in 93% and 95% yields, respectively.
shifted (0.75–1.02 ppm) relative to the cis isomer in water. This
is consistent with previous observations made by Lubell and
co-workers7 on other proline-containing peptide mimics and may
serve as a diagnostic tool to assign the trans isomer in cases where
NOE experiments do not allow assignment due to spectral overlap.
The equilibrium constants Kc/t for compounds 22–25 are shown
in Table 1, and were determined by integrating and averaging as
many distinct proton signals as possible for both the major and
minor isomers in the 1H NMR spectra.35 Our results indicate that
compounds 22 and 24 display a higher cis isomer population
relative to their C-50 diastereoisomers 23 and 25, respectively. It
appears that the stereochemistry at C-50 has a profound effect on
N3
HO
O
O
OMe
NAc
1) TFA/CH2Cl2 (1/3), 1 h
HO
18/ 19
HO
R1
2) Ac2O, pyridine, 18 h
3) NaOMe, MeOH, 4 h,
R2
20: R1 = CH2OH, R2 = H (96%)
21: R1 = H, R2 = CH2OH (80%)
The assignments of N-terminal geometry for model peptides
22–25 were made on the basis of NOE experiments in D2O
(Fig. 6). For example, selective inversion of the N-terminal methyl
group in the prolyl amide cis isomer 23a by a selective GOESY34
experiment showed an inter-proton effect to H-20 (5.63% NOE).
By comparison, no inter-proton effect was observed between
.
H2, Pd(OH)2, HCl
MeOH, 20 mins,
HCl
O
NH2
O
OMe
HO
HO
NAc
HO
R1
R2
22: R1 = CH2OH, R 2 = H (quant.)
23: R1 = H, R 2 = CH 2OH (quant.)
CO2Me
.
BnO
BnO
BnO
CO2Me
H
BnO
BnO
BnO
HCl
O
NH2
O
5
O
H
O
2'
0.83%
2'
1) NH2Me, EtOH, 18 h
NHMe
0.73%
CH3
2
NBn
0.17%
HO
HO
BnO
H
NBn
20/ 21
5'
BnO 5'
2) H2, Pd(OH)2, HCl,
MeOH, 20 mins
HO
NAc
H
O
H
O
CH3
O
0.78%
R1
R2
O
24: R1 = CH 2 OH, R2 = H (93%)
25: R1 = H, R2 = CH2OH (95%)
13
14
Figure 5. Assignment of stereochemistry at C-50-position in compounds 13 and 14
through 1D NOE experiment (recorded in C6D6).
Scheme 5. Synthesis of peptide mimics 22–25.

580
K. Zhang, F. Schweizer / Carbohydrate Research 344 (2009) 576–585
O
OMe
H
N
CH3
O
O
2'
OMe
H
5.63%
CH3
O
H
O
O
Cδ
Cβ
N
OCH3
5'
HO
CH3
O
HO
Cγ
H
O
H
H
4.54%
3.80%
6'
*
a
Figure 7. n?
p Interaction (looking down C –N bond).
23a
cis
23b
trans
ues suggesting that these protons are involved in intramolecular
H-bonding in compounds 24 and 25. The low ( d/ T) values ob-
served for OH-2 and OH-3 reflect high solvent exposure of these
hydroxyl groups while the relative high value for OH-4 in 25
may indicate some H-bond interaction with one of the nitrogen
Figure 6. Assignment of cis and trans isomers in compound 23 in D2O using 1D NOE
experiments. The same experiments were used to assign the cis/trans isomers in
compounds 22, 24, and 25.
D
D
the equilibrium of isomerization. Also, the cis prolyl amide popula-
tion in esters 22 and 23 was generally lower than that in N0-meth-
ylamides 24 and 25. Taylor and co-workers have proposed that the
trans conformation of esters is stabilized relative to amides as a re-
sult of increased electron donation from the oxygen lone pair of the
N-terminal amide carbonyl group to the antibonding orbital of the
prolyl C-terminal carbonyl group (Fig. 7).35
lone pairs on NH2-6. The relative high and nearly identical (
Dd/
D
T) values observed for the methyl amide proton in structures
24a, 24b, 25a, and 25b support the notion that this proton is en-
gaged in hydrogen bonding to N-terminal carbonyl group in cis/
trans isomers of both compounds 24 and 25. Moreover, the higher
(Dd/DT) value for the NH2-6 in compound 25 when compared to
that of diastereomer 24 suggests that the amino group in 25 is in-
volved in stabilization of the trans isomer 25b and in the destabi-
lization of the cis isomer 25a relative to 24a and 24b. A similar
trend is observed for the OH-60 position. Isomers 25a and 25b ex-
2.1. Effect of pH
Because compounds 22–25 have an ionizable amino group, we
hibit higher (Dd/DT) values when compared to isomers 24a and
24b suggesting that OH-60 is involved in stabilization of the trans
isomer 25b relative to 24b. Taken together, these results support
the notion that compound 25 is stabilized by an intramolecular
hydrogen bond (60-OH–NH2-6) that is absent in 24. The hydrogen
bond (OH-60–NH2-6) competes with the H-bond (60-OH–
O@C(N)CH3) of the cis isomer 25a resulting in a lower cis popula-
tion of 25a relative to 24a (Fig. 9).
were interested to study the influence of the ionization state on Kt/c
.
Previous studies have indicated that ionizable groups in proximity
to the imide backbone can influence thermodynamics of prolyl
amide cis/trans isomerization.36 We selected three buffer ranges:
pH 2.6, 7.4, and 12.4, to examine the pH effect on the isomerization
of 24 and 25 (Table 2). Our study shows that the prolyl N-terminal
amide cis/trans ratio is not affected by pH and the observed
changes are within the experimental error. Molecular modeling
suggests that the large distance of the ionizable amino function
from the imide function is responsible for the absence of a pH
effect.
3. Conclusions
We have developed a synthetic route to two spirocyclic GlcTPro-
LysCs that differ in the stereochemistry of the hydroxymethyl sub-
stituent at the C-50 position of the pyrrolidine ring.
A key
2.2. Conformational analysis of compounds 24 and 25
intermediate in the synthesis is the glucose-templated C-glycosyl
glycine analogue 7, which bears an additional C-allylic substituent
at the pseudoanomeric position. Compound 7 may find future use
in the synthesis of other carbohydrate-templated amino acids via
derivatization of the allyl group. To study the thermodynamic prop-
erties of prolyl amide cis/trans isomerization, the two GlcTProLysC
analogues were incorporated in peptide mimics Ac-GlcTProLysC-
OMe and Ac-GlcTProLysC-NHMe. Our study indicates that the
stereochemistry at the C-50 position in both peptide mimics has a
The relatively large coupling constants for J2,3, J3,4, and J4,5
(>9.2 Hz) indicate a 4C1 conformation of the pyranose ring in 24
and 25. The conformation of the piperidine ring is expected to be
Cb-exo based on previous studies using 3(S)-hydroxyproline-con-
taining peptide mimics.37 This conformation places the endocyclic
oxygen substituent in an axial position.37b In this conformation, the
pyrrolidine ring will be stabilized by gauche interaction and a sta-
c
b
*
bilizing
r(C -H)?r (C -O) interaction. This conformation is fur-
ther supported by characteristic long-range ‘W’ coupling
constants (J ꢁ 1.0 Hz) between H-20eq and H-4e0 q in both compounds
24 and 25 (Fig. 8).
To explain the different cis/trans ratio in compounds 24 and 25,
we considered intramolecular hydrogen bonding, which can be
Table 2
pH effect on Kc/t for compounds 24 and 25 in D2O
Compounds
pH
7.4
studied by calculating the temperature coefficients (
Dd/DT) of
key exchangeable protons.38 Previous studies have shown that
2.6
12.4
(
D
d/
D
T) > ꢀ3.0 ppb/deg is a diagnostic tool for the detection of
24 ( 0.04)
25 ( 0.04)
3.03
0.61
2.70
0.56
3.03
0.61
intramolecular H-bonding.38 The 1D spectra of compounds 24
and 25 were analyzed between 25 and 45 °C in 5-deg steps in
DMSO-d6 to determine the temperature coefficients (Table 3).
Our results indicate that the protons associated with NH2-6,
OH-60, and NHMe exhibit the highest temperature coefficient val-
Table 1
Cis population (%) and equilibrium constant Kc/t of compounds 22–25 in D2O
Compounds
22
23
24
25
cis ( 3%)
55
19
75
36
Kc/t ( 0.04)
1.22
0.23
3.03
0.56
Figure 8. Pyrrolidine conformation in compounds 24 and 25.

K. Zhang, F. Schweizer / Carbohydrate Research 344 (2009) 576–585
581
4.2. (1R)-2,3,4,6-Tetra-O-benzyl-30(S)-carboxy methyl-
spiro[1,5-anhydro-
-glucitol-1,20-oxirane] (2)
Table 3
Temperature coefficients (Dd/DT, ppb/K) for compounds 24 and 25 in DMSO-d6
D
HO-2
HO-3
HO-4
NH2-6
HO-60
NHMe
Under nitrogen atmosphere, methyl bromoacetate (4.1 mmol)
was dissolved in dry THF (20 mL) and cooled to ꢀ78 °C before lith-
ium bis(trimethylsilyl)-amide (4 mL of a 1 M solution in THF) was
slowly added. The reaction mixture was kept at ꢀ78 °C for an addi-
tional 30 min. Subsequently, a THF solution (5 mL) containing the
lactone 1 (1 mmol) was added over a period of 10 min and kept
at ꢀ78 °C for 1 more hour. The temperature was raised to rt and
stirred for 15 min before a saturated aq NH4Cl soln was added.
The reaction mixture was evaporated under reduced pressure
and the residue was dissolved in dichloromethane and portioned
with water (3 ꢂ 20 mL). The organic layer was dried over anhy-
drous Na2SO4, filtered, concentrated, and purified by flash column
chromatography (hexanes–EtOAc 5:1) to get 2 as a colorless oil
a
a
24
25
cis
trans
ꢀ9.07a
ꢀ2.44b
ꢀ3.80
ꢀ4.28
ꢀ3.29
ꢀ2.76
ꢀ2.44b
ꢀ3.02
ꢀ2.68
ꢀ3.26
a
a
ꢀ8.93a
ꢀ2.44b
cis
trans
ꢀ8.09
ꢀ7.76
ꢀ6.90
ꢀ6.80
ꢀ4.10
ꢀ4.20
ꢀ0.92
ꢀ0.92
a
Not determined.
Overlapped with NHMe.
b
profound effect on the equilibrium constant. For example, incorpo-
ration of GlcTProLysC with ‘R’ configuration at C-50 dramatically in-
creased the cis population (75%) of Ac-GlcTProLysC-NHMe in water,
whereas a smaller augment cis population (34%) was observed in
GlcTProLysC with ‘S’ configuration at C-50. Temperature coefficient
experiments indicate that the hydroxymethyl group at C-50 (S) is in-
volved in H-bonding with the 6-NH2 and vice versa. In contrast, the
same hydrogen bond is absent in the diastereomer with C-50 (R) ste-
reochemistry. Taken together, our results suggest that the cis isomer
ratio in peptide mimic Ac-GlcTProLysC-NHMe having a C-50 (S)
hydroxymethyl substituent is decreased by competing H-bonding
effects between 60-OH–O@C(N)CH3 and 60-OH–6-NH2.
Our work shows for the first time that polar groups capable of
H-bonding in polyhydroxylated spirocyclic proline analogues can
play important roles in controlling the thermodynamics of prolyl
amide cis/trans isomerization. In particular, polar groups such as a
hydroxymethyl group introduced at the d-position of proline are ex-
pected to increase the prolyl N-terminalamide cis isomerin peptides
via H-bonding to the N-terminal amide carbonyl group. Previous
studies have shown that amino acid residues possessing side chains
with hydrogen-bond acceptor and donor moieties are able to stabi-
lize turn conformations when adjacent to proline.39,40 As a result, we
expect that incorporation of GlcTProLysC in bioactive peptides may
induce similar effects. Weare currentlystudying the lysine- and pro-
line- mimetic effects of GlcTProLysC in b-turn-forming peptides.
(500 mg, 82%). ½a D
ꢃ
99.6 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3):
d = 3.53 (m, H-7), 3.63 (dd, H-8a, J = 11.2 Hz, J = 2.1 Hz), 3.68 (dd,
H-8a, J = 11.2 Hz, J = 4.4 Hz), 3.72 (s, 3H), 3.75–3.83 (m, H-5, H-6),
3.90 (s, H-2), 3.95 (d, H-4, J = 9.1 Hz), 4.48 (d, 1H, J = 12.2 Hz),
4.55 (d, 1H, J = 10.7 Hz), 4.58 (d, 1H, J = 12.2 Hz), 4.62 (d, 1H,
J = 11.0 Hz), 4.73 (d, 1H, J = 11.0 Hz), 4.82 (d, 1H, J = 11.2 Hz), 4.84
(d, 1H, J = 10.7 Hz), 4.94 (d, 1H, J = 11.2 Hz), 7.13–7.36 (m, 20H);
13C NMR (75 MHz, CDCl3): d = 52.5, 54.5, 68.3, 73.5, 74.9, 75.2,
75.7, 76.8, 77.0, 77.3, 84.7, 86.29, 127.6–128.5 (aromatic carbons),
137.4, 137.8, 137.9, 138.3, 166.7. Anal. Calcd for C37H38O8: C,
72.77; H, 6.27. Found: C, 72.48; H, 6.54. MS (ES, [M+Na]+) calcd
for C37H38NaO8 633.25, found 633.71.
4.3. Methyl (2S)-hydroxy-2-(1-allyl-2,3,4,6-tetra-O-benzyl-b-D-
glucopyranosyl)-ethanoate (3)
Under a nitrogen atmosphere, to a solution of allyltributylstann-
ane (0.99 mL, 3.15 mmol) in dichloromethane (5 mL) was added
dropwise the solution of trimethylsilyltrifluoromethanesulfonate
(TMSOTf, 0.427 mL, 2.36 mmol) in dichloromethane (5 mL) at 0 °C
followed by the syringe pump-controlled (50 lL/min) addition of
4. Experimental
4.1. General
the solution of epoxide 2 (480 mg, 0.79 mmol) in dichloromethane
(10 mL). And then the mixture was stirred for 1 more hour at rt,
the saturated sodium bicarbonate soln (10 mL) was added to quench
the reaction, followed by the extraction with dichloromethane
(3 ꢂ 15 mL). The organic layer was dried (Na2SO4), filtered, concen-
trated, and treated with trifluoroacetic acid (0.20 mL, 5 equiv) in aq
tetrahydrofuran (THF–H2O 5:1) overnight. The mixture was concen-
trated and purified by flash column chromatography (hexanes–
CH2Cl2–EtOAc from 2:1:0.2 to 2:1:0.4) to get 3 (410 mg, 80%) and 4
All solvents were obtained from a dry solvent system (alumina)
and used without further drying. TLC was performed on E. Merck
Silica Gel 60 F254 with detection by charring with 8% H2SO4 acid.
Silica gel (0.040–0.063 mm) was used for column chromatography.
Melting points are uncorrected.
(46 mg, 9%). Compound 3 ½a D
ꢃ
77.3 (c 1.0, CHCl3); 1H NMR
(300 MHz, CDCl3): d = 2.77 (dd, 1H, J = 16.1 Hz, J = 6.8 Hz), 2.89 (dd,
1H, J = 16.1 Hz, J = 7.2 Hz), 3.48 (br, OH), 3.85–3.65 (m, 7H), 4.04
(dd, 1H, J = 9.8 Hz, J = 8.0 Hz), 4.13 (d, 1H, J = 9.8 Hz), 4.33 (s, 1H),
4.63 (d, 1H, J = 12.5 Hz), 4.68–4.75 (m, 2H), 4.85–4.98 (m, 3H), 5.02
(d, 1H, J = 10.9 Hz), 5.08 (d, 1H, J = 11.4 Hz), 5.28–5.16 (m, 2H),
5.89 (m, 1H), 7.25–7.49 (m, 20H); 13C NMR (75 MHz, CDCl3):
d = 32.0, 52.2, 68.9, 73.5 (2 carbons), 73.6, 75.3, 75.3, 75.6, 78.7,
79.2, 80.6, 84.1, 118.7, 127.7–128.5 (aromatic carbons), 131.7,
138.1, 138.4, 138.4, 138.6, 173.1. Anal. Calcd for C40H44O8: C,
73.60; H, 6.79. Found: C, 73.29; H, 7.04. MS (ES, [M+Na]+) calcd for
C40H44NaO8 675.29, found 675.40.
4.4. Methyl (2S)-hydroxy-2-(1-allyl-2,3,4,6-tetra-O-benzyl-a-D-
glucopyranosyl)-ethanoate (4)
½
a D
84.5 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3): d = 2.62–2.73
(m, 1H), 2.78–2.90 (dd, 1H, J = 15.3 Hz, J = 9.4 Hz), 3.59–3.73 (m,
6H), 3.74–3.81 (dd, 1H, J = 11.0 Hz, J = 3.7 Hz), 3.85 (d, 1H,
J = 9.6 Hz), 3.99 (d, 1H, J = 1.6 Hz), 4.05–4.19 (m, 2H), 4.53 (d, 1H,
ꢃ
Figure 9. Suggested intramolecular H-bonds based on temperature coefficient
experiments for compounds 24 and 25.

582
K. Zhang, F. Schweizer / Carbohydrate Research 344 (2009) 576–585
J = 12.3 Hz), 4.62–4.71 (m, 2H), 4.82–4.95 (m, 4H), 5.00 (d, 1H,
J = 11.0 Hz), 5.10–5.24 (m, 2H), 5.88–6.05 (m, 1H), 7.20–7.40 (m,
20H); 13C NMR (75 MHz, CDCl3): d = 36.6, 52.1, 69.1, 73.0, 73.4,
74.2, 75.1, 75.7, 76.2, 78.4, 78.6, 82.3, 84.0, 119.2, 127.5–128.7
(aromatic carbons), 133.4, 137.3, 138.1, 138.3, 138.5, 170.7. Anal.
Calcd for C40H44O8: C, 73.60; H, 6.79. Found: C, 73.41; H, 6.92.
MS (ES, [M+Na]+); calcd for C40H44NaO8 675.29, found 675.40.
4.7. Methyl (2S)-benzylamino-2-(1-allyl-(2,3,4,6-tetra-O-
benzyl-b- -glucopyranosyl) -ethanoate (7)
D
To an ice-cooled solution of 6 (240 mg, 0.32 mmol) in MEOH
(9 mL) was added NaCNBH3 (128 mg, 1.95 mmol), followed by 98%
AcOH (39 lL, 0.65 mmol). The reaction mixture was stirred for 3 h
at 0 °C and then quenched by the addition of water (5 mL) and ex-
tracted with CH2Cl2 (3 ꢂ 15 mL). The combined organic extracts
were dried (Na2SO4), filtered, concentrated, and purified by flash col-
umn chromatography (hexanes–EtOAc 5:1) to afford 7 (239 mg,
4.5. Methyl 2-oxo-2-(1-allyl-2,3,4,6-tetra-O-benzyl-b-D-
glucopyranosyl)-ethanoate (5)
quant.). ½a D
ꢃ
32.3 (c 1.0, CHCl3); 1H (300 MHz, CDCl3): d = 2.71 (dd,
1H, J = 16.3 Hz, J = 6.0 Hz), 2.84 (dd, 1H, J = 16.3 Hz, J = 7.4 Hz), 3.39
(d, 1H, J = 12.8 Hz), 3.47 (s, 1H), 3.59–3.79 (m, 8H), 3.93 (dd, 1H,
J = 9.5 Hz, J = 8.9 Hz), 4.28 (d, 1H, J = 9.5 Hz), 4.50 (d, 1H,
J = 11.6 Hz), 4.58 (d, 1H, J = 12.0 Hz), 4.65 (d, 1H, J = 10.8 Hz), 4.67
(d, 1H, J = 12.0 Hz), 4.80–4.95 (m, 4H), 5.17–5.04 (m, 2H), 5.77 (m,
1H), 7.45–7.10 (m, 26H); 13C NMR (75 MHz, CDCl3): d = 32.4, 51.5,
51.8, 64.8, 69.1, 73.4, 73.5, 74.9, 75.1, 75.7, 78.8, 79.7, 80.2, 84.7,
118.1, 127.7–128.5 (aromatic carbons), 132.0, 138.2, 138.5, 138.7,
138.8, 139.7, 174.0. Anal. Calcd for C47H51NO7: C, 76.09; H, 6.93; N,
1.89. Found: C, 75.84; H, 7.36; N, 2.37. MS (ES, [M+Na]+) calcd for
C47H51NNaO7 764.37, found 764.32.
Under a nitrogen atmosphere, to a solution of dry dimethyl sulf-
oxide (133 L, 1.88 mmol)) in anhydrous CH2Cl2 (12 mL) cooled
below –65 °C with a dry ice-acetone bath, trifluoroacetic anhydride
(TFAA, 200 L, 1.41 mmol) was slowly added with efficient stirring
l
l
in ca. 10 min. After 10 min below ꢀ65 °C, a solution of compound 3
(307 mg, 0.47 mmol) in CH2Cl2 (8 mL) was added to the mixture in
ca. 15 min. The rate of addition of TFAA or alcohol 3 was controlled
to keep the temperature below ꢀ65 °C. The mixture was stirred be-
low ꢀ65 °C for 40 min, followed by addition of triethylamine
(394 lL, 2.82 mmol) dropwise in ca. 15 min. The reaction was kept
below ꢀ65 °C for 2 more hour. The cooling bath was then removed
and the reaction was allowed to warm up to rt, then quenched by
the addition of H2O (10 mL), and the aqueous layer was back-
washed with CH2Cl2 (2 ꢂ 15 mL). The combined organic solution
was dried with anhydrous Na2SO4, filtered, concentrated, and puri-
fied by flash column chromatography (hexanes–EtOAc 6:1) to get 5
4.8. (1S)-2,3,4,6-Tetra-O-benzyl-10-N-benzyl-50(R)-
hydroxymethylene-spiro[1,5-anhydro-
methyl ester] (8)
D L-proline
-glucitol-1,30-
(296 mg, 97%). ½a D
ꢃ
59.9 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3):
To a solution of 7 (340 mg, 0.46 mmol) in CH2Cl2 and Et2O
(12 mL, 1:1) was added iodine (175 mg, 0.69 mmol) at 0 °C. The
mixture was quenched by the addition of saturated sodium thio-
sulfate soln (5 mL) after overnight. The organic layer was separated
and the aqueous layer was backwashed with CH2Cl2 (2 ꢂ 10 mL),
the combined organic solution was dried with anhydrous Na2SO4
and concentrated followed by dissolution in toluene (15 mL) and
treated with silver acetate (1.146 g, 6.88 mmol) overnight at rt to
get an inseparable mixture of 13, 14, and 15 (323 mg, 93%), which
was hydrolyzed with K2CO3 (73 mg, 1.3 equiv) in MeOH (8 mL) for
1 h at rt, and then quenched by the addition of saturated ammo-
nium chloride (10 mL) and extracted with CH2Cl2 (3 ꢂ 15 mL).
The combined organic solution was dried with anhydrous Na2SO4,
filtered, concentrated, and purified by flash column chromatogra-
phy (hexanes–EtOAc from 4:1 to 2:1) to get 8 (132 mg, 43%), 9
d = 2.66 (dd, 1H, J = 15.6 Hz, J = 8.0 Hz), 3.21 (dd, 1H, J = 15.6 Hz,
J = 5.9 Hz), 3.74 (s, 3H), 3.73–3.66 (m, 2H), 3.91–3.76 (m, 3H),
4.24 (d, 1H, J = 9.6 Hz), 4.51 (d, 1H, J = 11.9 Hz), 4.57 (d, 1H,
J = 10.3 Hz), 4.61 (d, 1H, J = 12.2 Hz), 4.66 (d, 1H, J = 10.7 Hz), 4.73
(d, 1H, J = 10.3 Hz), 4.83–4.89 (m, 3H), 5.09–5.21 (m, 2H), 5.64
(m, 1H), 7.21–7.40 (m, 20H); 13C NMR (75 MHz, CDCl3): d = 31.2,
52.3, 68.7, 73.4, 73.5, 75.3, 75.6, 75.7, 77.8, 80.3, 83.4, 84.5,
119.2, 127.4–128.5 (aromatic carbons), 130.9, 137.7, 138.0, 138.3,
138.5, 164.9, 195.8. Anal. Calcd for C40H42O8: C, 73.83; H, 6.51.
Found: C, 73.43; H, 6.39. MS (ES, [M+Na]+); calcd for C40H42NaO8
673.28, found 673.39.
4.6. Methyl 2-benzyl imino-2-(1-allyl-2,3,4,6-tetra-O-benzyl-b-
D-glucopyranosyl)-ethanoate (6)
(141 mg, 46%), and 10 (20 mg, 6.5%). (8) ½a D
ꢃ
47.7 (c 1.0, CHCl3);
1H NMR (300 MHz, CDCl3): d = 2.13 (dd, 1H, J = 13.9 Hz,
J = 5.4 Hz), 2.60 (dd, 1H, J = 13.9 Hz, J = 11.0 Hz), 3.09 (s, 3H), 3.30
(m, 1H), 3.45–3.85 (m, 11H), 4.02 (d, 1H, J = 13.5 Hz), 4.38 (d, 1H,
J = 12.9 Hz), 4.57–4.71 (m, 4H), 4.83 (d, 1H, J = 11.0 Hz), 4.86 (d,
1H, J = 11.0 Hz), 5.10 (d, 1H, J = 12.7 Hz), 7.10–7.45 (m, 25H); 13C
NMR (75 MHz, CDCl3): d = 27.2, 51.8, 59.0, 60.0, 63.0, 69.5, 72.4,
72.7, 73.5, 74.8, 75.1, 75.5, 76.7, 78.8, 86.1, 88.2, 125.8–128.9 (aro-
matic carbons), 138.0, 138.0, 138.3, 138.8, 138.9, 172.6; HRMS (ES)
calcd for C47H52NO8 [M+H]+ 758.3693, found 758.3687.
Under a nitrogen atmosphere, to an ice-cooled solution of 5
(296 mg, 0.45 mmol)andbenzylamine (148 lL, 1.36 mmol)inanhy-
drous Et2O (15 mL) was added dropwise TiCl4 (0.23 mL of a 1 M solu-
tion in CH2Cl2, 0.23 mmol). After complete addition, the ice bath was
removed and the reaction mixture was stirred for 4 h at rt. After this
period, the resulting suspension was cooled at 0 °C and poured into
1 M sodiumhydroxide solution. Theorganic layer was separated and
the water layer was extracted two times with CH2Cl2 (2 ꢂ 15 mL).
The combined organic layer was dried (Na2SO4), filtered, concen-
trated, and purified by flash column chromatography (hexanes–
EtOAc 6:1) to get the mixture of 5 and 6, which was exposed to the
same procedure again to get 6 (323 mg, 96%). ½aꢃD 30.5 (c 1.0, CHCl3);
1H NMR (300 MHz, CDCl3): d = 2.67 (dd, 1H, J = 15.6 Hz, J = 8.6 Hz),
3.48 (dd, 1H, J = 15.6 Hz, J = 5.3 Hz), 3.86–3.66 (m, 7H), 3.91 (dd,
1H, J = 9.2 Hz, J = 8.9 Hz), 4.16 (d, 1H, J = 9.2 Hz), 4.43–4.56 (m, 3H),
4.59–5.72 (m, 4H), 4.86–4.93 (m, 3H), 5.03–5.16 (m, 2H), 5.77 (m,
1H), 7.15–7.42 (m, 25H); 13C NMR (75 MHz, CDCl3): d = 32.2, 51.6,
58.7, 69.0, 72.9, 73.4, 75.3, 75.5, 75.8, 78.2, 81.5, 82.4, 84.1, 117.9,
127.7–128.5 (aromatic carbons), 132.5, 138.1, 138.2, 138.4, 138.6,
138.7, 163.7, 165.5. Anal. Calcd for C47H49NO7: C, 76.30; H, 6.68; N,
1.89. Found: C, 75.77; H, 7.05; N, 1.86. MS (ES, [M+Na]+) calcd for
C47H49NNaO7 762.34, found 762.38.
4.9. (1S)-2,3,4,6-Tetra-O-benzyl-10-N-benzyl-50(S)-
hydroxymethylene-spiro[1,5-anhydro-
methyl ester] (9)
D L-proline
-glucitol-1,30-
½
a D
ꢃ
ꢀ3.6 (c 1.70, CHCl3); 1H NMR (300 MHz, CDCl3): d = 2.24 (d,
1H, J = 14.6 Hz), 2.83 (dd, 1H, J = 13.9 Hz, J = 10.5 Hz), 3.05–3.23
(br, OH), 3.31 (s, 3H), 3.43 (d, 1H, J = 11.1 Hz), 3.62–3.9 (m, 10H),
4.00 (d, 1H, J = 14.6 Hz), 4.47 (d, 1H, J = 12.5 Hz), 4.54–4.77 (m,
4H), 4.82–4.94 (m, 2H), 5.24 (d, 1H, J = 11.8 Hz), 7.12–7.44 (m,
25H); 13C NMR (75 MHz, CDCl3): d = 27.9, 51.1, 51.7, 61.3, 62.6,
69.1, 72.5, 72.9, 73.0, 73.7, 74.9, 75.7, 77.0, 78.5, 85.7, 86.2,
126.0–128.6 (aromatic carbons), 138.0, 138.2, 138.4, 138.8, 138.9,

K. Zhang, F. Schweizer / Carbohydrate Research 344 (2009) 576–585
583
170.5; HRMS (ES) calcd for C47H52NO8 [M+H]+ 758.3693, found
758.3696.
4.14. (1S)-2,3,4,6-Tetra-O-benzyl-10-N-benzyl-50(S)-
methylenehydroxyl acetate-spiro [1,5-anhydro-
-glucitol-1,30-
-proline methyl ester] (14) (the detailed procedure is the same
as that for 13)
D
L
4.10. (1S)-2,3,4,6-Tetra-O-benzyl-10-N-benzyl-50(S)-hydroxy-
spiro[1,5-anhydro-
D L-pipecolic methyl ester] (10)
-glucitol-1,30-
½
a D
6.2 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3): d = 2.01 (s,
ꢃ
34.7 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3): d = 2.25–2.36
3H), 2.14 (dd, 1H, J = 15.4 Hz, J = 1.1 Hz), 2.82 (dd, 1H, J = 13.9 Hz,
J = 10.0 Hz), 3.26 (s, 3H), 3.58–3.82 (m, 9H), 3.59–3.82 (m, 2H),
4.21 (dd, 1H, J = 10.7 Hz, J = 5.0 Hz), 4.43 (d, 1H, J = 12.6 Hz),
4.57–4.75 (m, 4H), 4.82 (d, 1H, J = 11.2 Hz), 4.86 (d, 1H,
J = 11.2 Hz), 5.15 (d, 1H, J = 12.3 Hz), 7.10–7.40 (m, 25H); 13C
NMR (75 MHz, CDCl3): d = 21.0, 27.7, 51.1, 53.0, 60.0, 67.2, 69.0,
72.9, 73.0, 73.2, 73.7, 75.1, 75.6 (2 carbons), 78.7, 85.9, 86.9,
126.0–128.5 (aromatic carbons), 137.0 (2 carbons), 138.5, 138.9,
139.5, 170.9, 170.9; HRMS (ES) calcd for C49H54NO9 [M+H]+
800.3793, found 800.3793.
½aꢃD
(m, 2H), 2.76 (dd, 1H, J = 11.0 Hz, J = 5.2 Hz), 3.04 (dd, 1H,
J = 10.6 Hz, J = 9.6 Hz), 3.32 (s, 3H), 3.42 (s, 1H), 3.53–3.82 (m,
7H), 3.90–4.07 (m, 2H), 4.58–4.73 (m, 5H), 4.82 (d, 1H,
J = 11.2 Hz), 4.87 (d, 1H, J = 10.9 Hz), 5.10 (d, 1H, J = 11.7 Hz),
7.12–7.39 (m, 25H); 13C NMR (75 MHz, CDCl3) d = 29.7, 30.7,
51.0, 54.5, 59.0, 63.5, 69.5, 69.9, 72.4, 73.3, 73.9, 74.9, 75.4, 78.1,
79.1, 80.4, 84.6, 126.3–128.6 (aromatic carbons), 138.1, 138.2,
138.3, 138.6, 138.7, 170.7; HRMS (ES) calcd for C47H52NO8
[M+H]+ 758.3693, found 758.3686.
4.15. (1S)-2,3,4,6-Tetra-O-benzyl-10-N-benzyl-50(S)-O-acetyl-
spiro[1,5-anhydro-
(the detailed procedure is the same as that for 13)
4.11. (1S)-50(R)-hydroxymethylene-spiro[1,5-anhydro-
glucitol-1,30-
-proline methyl ester] (11)
D-
D L-pipecolic methyl ester] (15)
-glucitol-1,30-
L
Under the nitrogen atmosphere, to the solution of compound 8
(200 mg, 0.25 mmol) in MEOH (10 mL) were added 1 M hydrochlo-
ride acid soln (0.38 mL, 0.38 mmol) and palladium hydroxide
(20 wt % Pd on carbon, 50 mg). The mixture was exposed to hydro-
gen (H2, 10 psi) and stirred for 6 h. The solution was filtrated and
½
a D
44.0 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3): d = 2.05 (s,
ꢃ
3H), 2.32 (dd, 1H, J = 13.6 Hz, J = 11.9 Hz), 2.52 (dd, 1H,
J = 13.7 Hz, J = 4.0 Hz), 2.90 (dd, 1H, J = 10.4 Hz, J = 5.6 Hz), 3.24
(dd, 1H, J = 10.4 Hz, J = 10.8 Hz), 3.29 (s, 3H), 3.46–3.99 (m, 9H),
4.45 (d, 1H, J = 12.9 Hz), 4.59–4.93 (m, 6H), 5.09 (m, 1H), 5.15 (d,
1H, J = 12.4 Hz), 7.10–7.44 (m, 25H); 13C NMR (75 MHz, CDCl3):
d = 21.6, 26.4, 50.2, 50.4, 59.5, 69.7, 72.8, 73.4, 74.4 (2 carbons),
75.4 (2 carbons), 75.8, 78.7, 79.4, 80.9, 85.3, 126.50–128.90 (aro-
matic carbons), 138.8 (3 carbons), 139.2, 139.4, 170.7 (2 carbons);
HRMS (ES) calcd for C49H54NO9 [M+H]+ 800.3793, found 800.3788.
evaporated in vacuum to get the product 11 (75 mg, quant.) ½a D
ꢃ
64.1 (c 1.0, MeOH); 1H NMR (300 MHz, CD3OD): d = 2.13 (dd, 1H,
J = 15.0 Hz, J = 2.8 Hz), 2.41 (dd, 1H, J = 15.0 Hz, J = 10.8 Hz), 3.23–
3.21 (m, 5H), 3.49 (m, 1H), 3.6–3.71 (m, 2H), 3.85–4.00 (m, 4H),
4.22 (s, 1H); 13C NMR (75 MHz, CD3OD): d = 27.3, 54.3, 61.5, 61.9,
63.0, 68.7, 70.8, 71.6, 76.6, 77.0, 88.3, 168.5; HRMS (ES) calcd for
C12H22NO8 [M+H]+ 308.1340, found 308.1343.
4.16. (1S)-10-N-t-butoxycarbonyl-50(R)-methylenehydroxy
acetate–spiro[1,5-anhydro-
ester] (16)
D L-proline methyl
-glucitol-1,30-
4.12. (1S)-50(S)-Hydroxymethylene-spiro[1,5-anhydro-
glucitol-1,30-
-proline methyl ester] (12) (the detailed
procedure is the same as that for 11)
D-
L
To a mixture of 11 (100 mg, 0.13 mmol) and Pd(OH)2 (40 mg,
20 wt % on charcoal) in MEOH (10 mL) was added the solution of
hydrochloride (250 lL of 1M HCl soln, 0.25 mmol) and stirred un-
der H2 (15 psi) for 8 h at rt. The catalyst was removed by filtration
and the solvent was removed under the vacuum. The unprotected
½
a D
75.7 (c 1.10, MeOH); 1H NMR (300 MHz, CD3OD) d = 2.09
ꢃ
(m, 1H), 2.56 (dd, 1H, J = 10.3 Hz, J = 13.6 Hz), 3.24 (m, 1H), 3.30–
3.40 (m, br, 1H, overlapping with solvent peak), 3.47 (m, 1H),
3.57–3.68 (m, 2H), 3.72–3.95 (m, 6H), 4.18–4.33 (m, 2H); 13C
NMR (75 MHz, CD3OD): d = 27.8, 54.2, 62.6, 63.1, 63.2, 69.1, 71.2,
71.7, 76.7, 76.9, 88.2, 167.9; HRMS (ES) calcd for C12H22NO8
[M+H]+ 308.1340, found 308.1348.
product was treated with triethylamine (53 lL, 0.38 mmol) and di-
t-butyl dicarbonate (56 mg, 0.25 mmol) in MeOH (2 mL) for 1 h at
rt. The solvent was removed under vacuum. The crude product was
purified by flash column chromatography (CH2Cl2–MeOH 7:1) to
get 16 (51 mg, 90%).
½
a D =
68.4 (c 1.3, MeOH); 1H NMR
ꢃ
4.13. (1S)-2,3,4,6-Tetra-O-benzyl-10-N-benzyl-50(R)-
methylenehydroxy acetate-spiro [1,5-anhydro-
proline methyl ester] (13)
(300 MHz, CD3OD, two isomers): d = 1.45 (s, 9H), 2.09 (s, 3H),
2.13–2.44 (m, 2H), 3.27–3.46 (m, 3H, partially overlapping with
MeOH peaks), 3.57–3.86 (m, 6H), 4.06 (m, 1H), 4.23 (s, 1H),
4.31–4.45 (m, 1H), 4.53–4.67 (m, 1H); 13C NMR (75 MHz, CD3OD,
two isomers): d = 20.9 (2 carbons), 28.56/28.63 (6 carbons), 29.2/
29.7, 52.9 (2 carbons), 56.2/56.3, 62.8 (2 carbons), 66.0/66.4, 71.2
(2 carbons), 71.4/71.8, 71.5 (2 carbons), 75.9/76.0, 77.3 (2 carbons),
81.8/82.3, 86.2/86.7, 155.6/156.2, 172.0/171.8, 172.7/172.8; HRMS
(ES) calcd for C19H31NNaO11 [M+Na]+ 472.1795, found 472.1783.
D L-
-glucitol-1,30-
To a solution of 8 (60 mg, 0.079 mmol) in pyridine (1 mL) was
added acetic anhydride (37 L, 0.395 mmol) and stirred for 5 h.
The pyridine was removed with high vacuum. The crude product
was purified by flash column chromatography (hexanes–EtOAc
4:1) to get 13 (62 mg, quant.). ½a D
l
ꢃ
50.3 (c 1.0, CHCl3); 1H NMR
(300 MHz, CDCl3): d = 2.05 (s, 3H), 2.18 (dd, 1H, J = 13.0 Hz,
J = 10.8 Hz), 2.33 (dd, 1H, J = 13.6 Hz, J = 5.7 Hz), 3.13 (s, 3H), 3.32
(m, 1H), 3.53 (s, 1H), 3.63–3.78 (m, 6H), 3.83 (d, 1H, J = 14.4 Hz),
4.08 (d, 1H, J = 13.7 Hz), 4.24 (d, 2H, J = 6.0 Hz), 4.42 (d, 1H,
J = 12.3 Hz), 4.58–4.70 (m, 4H), 4.83 (d, 1H, J = 9.9 Hz), 4.86 (d,
1H, J = 9.9 Hz), 5.06 (d, 1H, J = 12.3 Hz), 7.13–7.43 (m, 25H); 13C
NMR (75 MHz, CDCl3): d = 20.9, 30.0, 51.5, 60.4, 60.8, 67.2, 69.4,
72.5, 72.8, 73.5, 75.1, 75.5, 76.0, 76.7, 78.7, 86.1, 87.5, 126.0–
128.8 (aromatic carbons),138.03, 138.04, 138.4, 138.9, 139.3,
171.0, 172.0; HRMS (ES) calcd for C49H54NO9 [M+H]+ 800.3793,
found 800.3794.
4.17. (1S)-10-N-t-butoxycarbonyl-50(S)-methylenehydroxy
acetate-spiro[1,5-anhydro-
ester] (17) (the detailed procedure is the same as that for 16)
D L-proline methyl
-glucitol-1,30-
½
a D
= 15.7 (c 1.35, MeOH); 1H NMR (300 MHz, CD3OD, two iso-
ꢃ
mers): d = 1.43 (s, 9H), 2.09 (s, 3H), 2.14–2.42 (m, 2H), 3.24–3.50
(m, 3H, partially overlapping with MeOH peaks), 3.50–3.87 (m,
6H), 4.04–4.63 (m, 4H); 13C NMR (75 MHz, CD3OD, two isomers):
d = 20.8 (2 carbons), 25.5/ 26.1, 28.5/28.7 (6 carbons), 53.0 (2 car-
bons), 57.2/57.3, 62.6/62.8, 65.8/65.9, 71.2 (2 carbons), 71.35 (2

584
K. Zhang, F. Schweizer / Carbohydrate Research 344 (2009) 576–585
carbons), 71.8/71.4, 75.8/75.9, 77.0/77.1, 81.9/82.3, 88.4/87.4,
155.8/ 156.1, 172.1/171.6, 172.6/172.4; HRMS (ES) calcd for
C19H31NNaO11 [M+Na]+ 472.1795, found 472.1788.
4.21. (1S)-6-Azido-6-deoxy-10-N-acetyl-50(S)-
hydroxymethylene-spiro[1,5-anhydro-D -glucitol-1,30-
L-proline
methyl ester] (21) (the detailed procedure is the same as that
for 20)
4.18. (1S)-6-Azido-6-deoxy-10-N-t-butoxycarbonyl-50(R)-
methylenehydroxy acetate-spiro[1,5-anhydro-
proline methyl ester] (18)
½
a D
= 46.5 (c 0.55, MeOH); 1H NMR (300 MHz, CD3OD, two iso-
ꢃ
D L-
-glucitol-1,30-
mers): d = 1.77 (s, 1.09H), 2.08 (s, 1.91 H), 2.15–2.41 (m, 2H), 3.23–
3.68 (m, 10H, partially overlapping with MeOH peaks), 3.79 (dd,
0.64 H, J = 5.28 Hz, J = 10.95 Hz), 3.96–4.23 (m, 2.36H); 13C NMR
(75 MHz, CD3OD, two isomers): d = 22.04 (22.48), 26.54 (25.35),
52.97 (53.14), 53.13 (53.46), 61.52 (61.38), 64.76 (63.61), 71.14
(71.19), 72.03 (72.44), 72.27 (73.01), 74.68 (74.73), 76.71 (76.62),
87.40 (88.99), 170.78 (171.20), 173.56 (173.50); HRMS (ES) calcd
for C14H22N4NaO8 [M+Na]+ 397.1335, found 397.1348.
To a solution of compound 16 (40 mg, 0.09 mmol) in pyridine
(1 mL) was added p-tolunesulfonyl chloride (42 mg, 0.22 mmol)
and stirred for 12 h at rt. The mixture was concentrated and puri-
fied by flash column chromatography (CH2Cl2–MeOH 10:1) to pro-
vide tosyl ester, which was treated with sodium azide (116 mg,
1.8 mmol) in DMF (1.5 mL) and stirred at 80 °C for 12 h. The mix-
ture was filtered, concentrated, and purified by flash column chro-
4.22. (1S)-6-Amino-6-deoxy-10-N-acetyl-50(R)-
hydroxymethylene-spiro[1,5-anhydro-
methyl ester] HCl salt (22)
matography (CH2Cl2–MeOH 15:1) to get 18 (38 mg, 92%). ½a D
ꢃ
=
D L-proline
-glucitol-1,30-
31.5 (c 1.35, MeOH); 1H NMR (300 MHz, CD3OD, two isomers):
d = 1.44 (s, 9H), 2.10 (s, 3H), 2.14–2.48 (m, 2H), 3.18–3.49 (m, 4H,
partially overlapping with MeOH peaks), 3.58–3.77 (m, 5H),
4.01–4.17 (m, 1H), 4.21 (s, 1H), 4.32–4.48 (m, 1H), 4.52–4.64 (m,
1H); 13C NMR (75 MHz, CD3OD, two isomers): d = 20.9 (2 carbons),
28.6 (6 carbons,), 29.0/29.6, 52.6/52.8, 53.0 (2 carbons), 56.2/56.4,
66.2/66.4, 71.1 (2 carbons), 71.4/71.9, 72.4/72.8, 75.2/75.4, 77.0/
77.1, 81.8/82.3, 86.4/87.0, 155.8/156.3, 171.6/171.7, 172.7/172.8;
HRMS (ES) calcd for C19H30N4 NaO10 [M+Na]+ 497.1860, found
497.1849.
To a solution of 20 (20 mg, 0.053 mmol) and Pd(OH)2 (20 mg,
20 wt % on charcoal) in MeOH (5 mL) was added the solution of
hydrochloride (800 lL of 1 M HCl soln, 0.08 mmol) and stirred un-
der H2 (15 psi) for 20 min at rt. The catalyst was removed by the
regular filtration and the solvent was removed under the vacuum
to afford pure product 22 (20 mg, quant.). ½a D
ꢃ
65.2 (c 0.5, MeOH);
1H NMR (500 MHz, D2O, two isomers): d = 1.86 (s, cis, 1.68H), 1.98
(dd, cis, 0.56H, J = 10.39 Hz, J = 14.57 Hz), 2.04–2.09 (m, trans,
0.44H + 1.32H), 2.36 (dd, cis, 0.56H, J = 6.76 Hz, J = 14.56 Hz), 2.47
(dd, trans, 0.44H, J = 7.42 Hz, J = 14.62 Hz), 2.98 (m, 1H), 3.19–
3.37 (m, 3H), 3.59–3.67 (m, 5H), 3.68–3.74 (m, 1H), 3.81–3.88
(m, 1H), 4.00–4.07 (m, cis, 0.56H), 4.11–4.18 (m, trans, 0.44H).
4.42 (s, cis, 0.56H), 4.44 (s, trans, 0.44H); 13C NMR (75 MHz,
D2O): cis, d = 22.2, 26.7, 40.5, 53.5, 58.5, 62.4, 69.2, 69.7, 70.7,
71.2, 74.4, 86.2, 171.4, 174.4; trans, d = 20.9, 28.8, 40.4, 53.2,
58.4, 63.5, 69.2, 69.7, 69.8, 70.9, 74.4, 85.1, 171.4, 174.5; HRMS
(ES) calcd for C14H25N2O8 [M+H]+ 349.1611, found 349.1623.
4.19. (1S)-6-Azido-6-deoxy-30-N-t-butoxycarbonyl-50(S)-
methylenehydroxy acetate -spiro[1,5-anhydro-D L-
-glucitol-1,30-
proline methyl ester] (19) (the detailed procedure is the same as
that for 18)
½
a D
= 29.1 (c 0.7, MeOH); 1H NMR (300 MHz, CD3OD, two iso-
ꢃ
mers): d = 1.45 (s, 9H), 2.09 (s, 3H), 2.14–2.48 (m, 2H), 3.23–3.44
(m, 3H, partially overlapping with MeOH peaks), 3.50–3.78 (m,
6H), 4.06–4.34 (m, 3H), 4.39–4.63 (m, 1H); 13C NMR (75 MHz,
CD3OD, two isomers): d = 20.8 (2 carbons), 25.6/26.2, 28.5/28.6 (6
carbons,), 53.0 (2 carbons), 53.0/53.1, 57.2 (2 carbons), 65.7/65.9,
71.1 (2 carbons), 71.9/71.5, 72.2/72.3, 74.7/74.8, 76.7/76.8, 82.0/
82.4, 88.8/87.8, 155.8/156.1, 171.9/171.4, 172.6/172.4; HRMS (ES)
calcd for C19H30N4 NaO10 [M+Na]+ 497.1860, found 497.1852.
4.23. (1S)-6-Amino-6-deoxy-10-N-acetyl-50(S)-
hydroxymethylene-spiro[1,5-anhydro-D L-proline
-glucitol-1,30-
methyl ester] HCl salt (23) (the detailed procedure is the same
as that for 22)
½
a D
24.3 (c 0.55, MeOH); 1H NMR (500 MHz, D2O, two isomers):
ꢃ
4.20. (1S)-6-Azido-6-deoxy-10-N-acetyl-50(R)-
hydroxymethylene-spiro[1,5-anhydro-
methyl ester] (20)
d = 1.93 (s, cis, 0.60H), 2.21 (s, trans, 2.40H), 2.29–2.46 (m, 2H),
3.20–3.59 (m, 5H), 3.63–3.82 (m, 5H), 3.85–3.94 (m, 1H), 4.33–
4.41 (m, 1H), 4.43 (s, trans, 0.8H), 4.62 (s, 0.2H); 13C NMR
(75 MHz, D2O): cis, d = 21.8, 25.0, 40.2, 53.6, 59.5, 62.3, 69.3, 69.6,
70.5, 71.2, 74.2, 87.6, 171.4, 174.4; trans, d = 21.3, 25.9, 39.9,
53.2, 59.7, 63.2, 69.3, 69.5, 70.3 (2 carbons), 74.2, 86.3, 170.8,
174.5; HRMS (ES) calcd for C14H25N2O8 [M+H]+ 349.1611, found
349.1618.
D L-proline
-glucitol-1,30-
The compound 18 (30 mg, 0.063 mmol) was dissolved in a mix-
ture of CH2Cl2 and trifluoroacetic acid (1.5 mL/0.5 mL) and stirred
for 1 h at rt. The solution was concentrated at vacuum and then
treated with a mixture of pyridine and acetic acid (1 mL/1 mL)
and stirred for 12 h at rt and then concentrated at vacuum. After
that, it was dissolved in a solution of sodium methoxide in MeOH
(0.1 M, 2 mL) and stirred for 4 h at rt followed by the neutralization
with Amberlite IRC-50S ion-exchange resin (H+). The mixture was
filtered and filtrate was concentrated and purified by the flash col-
umn chromatography (EtOAc–MeOH 6:1) to get compound 20
4.24. (1S)-6-Amino-6-deoxy-10-N-acetyl-50(R)-
hydroxymethylene-spiro[1,5-anhydro-
methyl amide] HCl salt (24)
D L-proline
-glucitol-1,30-
To a solution of methylamine in ethanol (37 wt %, 1 mL) was
added compound 20 (15 mg, 0.04 mmol) and stirred for 18 h at
rt. The mixture was concentrated and purified by flash column
chromatography (CH2Cl2–MeOH 2:1) to quantitatively afford
C-terminal methyl amide intermediate, which was dissolved in a
solution of Pd(OH)2 (15 mg, 20 wt % on charcoal) and 1 M hydro-
chloride acid soln (80 lL, 0.08 mmol). The mixture was stirred un-
der H2 (15 psi) for 20 min at rt. The catalyst was removed by the
regular filtration and the solvent was removed under the vacuum
to afford pure product 24 (14 mg, 93%). ½a D
(22 mg, 96%). ½a D
ꢃ
= 19.2 (c 0.75, MeOH); 1H NMR (300 MHz,
CD3OD, two isomers): d = 1.82 (s, 1.74H), 2.04 (s, 1.26H), 2.05–
2.49 (m, 2H), 3.06–3.34 (m, 4H, partially overlapping with MeOH
peaks), 3.49–3.94 (m, 7H), 4.04–4.16 (m, 1H), 4.20 (s, 0.58H),
4.42 (s, 0.42H); 13C NMR (75 MHz, CD3OD, two isomers):
d = 22.8/21.6, 27.7/29.7, 52.8/52.7, 53.6/53.2, 60.6/60.1, 64.9/65.6,
71.1 (2 carbons), 72.6/71.1, 72.6/72.8, 75.4/75.7, 76.9/77.0, 87.5/
86.4, 171.6/171.9, 173.6 (2 carbons); HRMS (ES) calcd for
C14H22N4NaO8 [M+Na]+ 397.1335, found 397.1351.
ꢃ
54.8 (c 0.35, MeOH);

K. Zhang, F. Schweizer / Carbohydrate Research 344 (2009) 576–585
585
1H NMR (500 MHz, D2O, two isomers): d = 2.02 (s, cis, 2.24H), 2.21
(s, trans, 0.76H), 2.27 (dd, cis, 0.75H, J = 11.31 Hz, J = 14.27 Hz),
2.35–2.44 (m, 1H), 2.53 (dd, trans, 0.25H, J = 6.17 Hz,
J = 14.11 Hz), 2.67–2.83 (m, 3.75H) 2.98–3.05 (m, 0.75H), 3.25
(dd, 0.25H, J = 11.31 Hz, J = 14.27 Hz), 3.30–3.39 (m, 1.25H),
3.41–3.56 (m, 2H), 3.68–3.77 (m, 1.75H). 3.83 (dd, trans, 0.25H,
J = 1.81 Hz, J = 12.43 Hz), 4.09–4.36 (m, 3H); 13C NMR (75 MHz,
D2O): cis, d = 27.2, 30.1, 30.8, 46.5, 62.9 (2 carbons), 64.5, 74.6,
76.0, 77.4, 80.1, 90.3, 176.5, 179.6; trans, 25.9, 30.8, 32.1, 46.4,
62.8, 63.0, 66.0, 74.5, 75.9, 76.5, 78.4, 89.0, 176.6, 179.6; HRMS
(ES) calcd for C14H26N3O7 [M+H]+ 348.1771, found 348.1759.
4. (a) Eberhardt, E. S.; Panasik, N., Jr.; Raines, R. T. J. Am. Chem. Soc 1996, 118,
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4.25. (1S)-6-Amino-6-deoxy-10-N-acetyl-50(S)-
hydroxymethylene–spiro[1,5-anhydro-D L-proline
-glucitol-1,30-
methyl amide] HCl salt (25) (the detailed procedure is the same
as that for 24)
½
a D
19.2 (c 0.40, MeOH); 1H NMR (500 MHz, D2O, two isomers):
ꢃ
d = 1.80 (s, cis, 1.08H), 2.08 (s, trans, 1.92H), 2.16–2.32 (m, 2H), 2.56
(s, trans, 1.92H), 2.62 (s, cis, 1.08H), 3.09–3.54 (m, 6H), 3.62–3.68
(m, 1H), 3.76 (dd, trans, 0.64H, J = 5.26 Hz, J = 11.50 Hz), 3.83 (dd,
cis, 0.36 H, J = 4.60 Hz, J = 11.04 Hz), 4.19 (s, trans, 0.64H),
4.21–4.30 (m, 1.36H); 13C NMR (75 MHz, D2O): cis, d = 26.7, 30.1,
31.2, 45.1, 64.6, 67.3, 74.3, 74.6, 75.5, 77.4, 79.4, 92.6, 175.5,
179.2; trans, d = 26.5, 31.0 (2 carbons), 44.9, 64.7, 68.2, 74.3, 74.5,
75.2, 76.6, 79.4, 91.4, 175.0, 179.1; HRMS (ES) calcd for
C14H26N3O7 [M+H]+ 348.1771, found 348.1766.
4.26. Measurement of equilibrium constant
19. (a) Owens, N.; Braun, C.; Schweizer, F. J. Org. Chem. 2007, 72, 4635; (b) Zhang,
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The calculation was based on the integration of well-resolved
20. Zhang, K.; Mondal, D.; Zhanel, G. G.; Schweizer, F. Carbohydr. Res. 2008, 343,
peaks of the
c-protons, N-terminal methyl group, and a-proton
in 1H NMR.
1644–1652.
21. Haubner, R.; Wester, H.-J.; Burkhart, F.; Senekowitsch-Schmidtke, R.;
Weber, W.; Goodman, S. L.; Kessler, H.; Schwaiger, M. J. Nucl. Med.
2001, 42, 326–336.
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4.27. Temperature coefficient (Dd/DT) experiments
1D 1H NMR spectra of 16 mM solutions of 24 and 25 in 100.0%
Me2SO-d6 were recorded on Bruker AMX500 at 25 °C, and from 25
to 44 °C with increments of 5 °C, using routine techniques. Chem-
ical shift (d) of hydroxyl and amino groups is expressed in ppm and
calibrated with respect to the residual DMSO signal (1H:
2.49 ppm). The chemical shift changes (Dd) at different tempera-
tures were calculated with respect to the chemical shift of hydro-
xyl and amino groups at 25 °C.
25. It has been reported that substitution of D-proline by cis-3-hydroxy-D-proline
(HypC3-OH) in the sequence Boc-Leu-Pro-Gly-Leu-NHMe resulted in novel
pseudo b-turn-like nine-membered ring structure involving an intramolecular
LeuNH?HypC3-OH hydrogen bond: Chakraborty, T. K.; Srinivasu, P.; Vengal
Rao, R.; Kiran Kumar, S.; Kunwar, A. C. J. Org. Chem. 2004, 69, 7399–7402.
26. Gueyrard, D.; Haddoub, R.; Salem, A.; Bacar, N. S.; Goekjian, P. G. Synlett 2005,
520–522.
Acknowledgments
27. (a) Schweizer, F.; Inazu, T. Org. Lett. 2001, 3, 4115–4118; (b) Zhang, K.; Wang, J.;
Sun, Z.; Huang, H.-D.; Schweizer, F. Synlett 2007, 239–242.
28. Dillon, M. P.; Maag, H.; Muszyski, D. M. Tetrahedron Lett. 1995, 36, 5469–
5470.
29. A 40 ms Gaussian pulse with a 560 ms mixing time was used.
30. Huang, S. K.; Omura, K.; Swern, D. J. Org. Chem. 1976, 41, 3329–3331.
31. Boeykens, M.; Kimpe, N. D.; Tehrani, K. A. J. Org. Chem. 1994, 59, 6973–6985.
32. Chérest, M.; Felkin, H.; Prudent, N. Tetrahedron Lett. 1968, 9, 2199–2204.
33. Davies, S. G.; Nicholson, R. L.; Price, P. D.; Roberts, P. M.; Smith, A. D. Synlett
2004, 901–903.
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
studies.
Supplementary data
34. GOESY is a selective 1D NOESY experiment, see: Stonehouse, J.; Adell, P.;
Keeler, J.; Shaka, A. J. J. Am. Chem. Soc. 1994, 116, 6037–6038.
35. Taylor, C. M.; Hardré, R.; Edwards, P. J. B.; Park, J. H. Org. Lett 2003, 5, 4413–
4416. and references therein.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2008.12.024.
36. Dugave, C.; Demange, L. Chem. Rev. 2003, 103, 2475–2532.
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