Glucosamine and glucosamine-6-phosphate derivatives: Catalytic cofactor analogues for the glmS ribozyme

Article abstract of DOI:10.1021/jo400192e

Two analogues of glucosamine-6-phosphate (GlcN6P, 1) and five of glucosamine (GlcN, 2) were prepared for evaluation as catalytic cofactors of the glmS ribozyme, a bacterial gene-regulatory RNA that controls cell wall biosynthesis. Glucosamine and allosamine with 3-azido substitutions were prepared by S_N2 reactions of the respective 1,2,4,6-protected sugars; final acidic hydrolysis afforded the fully deprotected compounds as their TFA salts. A 6-phospho-2-aminoglucolactam (31) was prepared from glucosamine in a 13-step synthesis, which included a late-stage POCl₃-phosphorylation. A simple and widely applicable 2-step procedure with the triethylsilyl (TES) protecting group was developed to selectively expose the 6-OH group in N-protected glucosamine analogues, which provided another route to chemical phosphorylation. Mitsunobu chemistry afforded 6-cyano (35) and 6-azido (36) analogues of GlcN-(Cbz), and the selectivity for the 6-position was confirmed by NMR (COSY, HMBC, HMQC) experiments. Compound 36 was converted to the fully deprotected 6-azido-GlcN (37) and 2,6-diaminoglucose (38) analogues. A 2-hydroxylamino glucose (42) analogue was prepared via an oxaziridine (41). Enzymatic phosphorylation of 42 and chemical phosphorylation of its 6-OH precursor (43) were possible, but 42 and the 6-phospho product (44) were unstable under neutral or basic conditions. Chemical phosphorylation of the previously described 2-guanidinyl-glucose (46) afforded its 6-phospho analogue (49) after final deprotection. This article not subject to U.S. Copyright. Published 2013 by the American Chemical Society.

Full text of DOI:10.1021/jo400192e

Article
pubs.acs.org/joc
Glucosamine and Glucosamine-6-phosphate Derivatives: Catalytic
Cofactor Analogues for the glmS Ribozyme
Jeffrey J. Posakony* and Adrian R. Ferre-D’Amare*
́
́
National Heart, Lung, and Blood Institute, 50 South Drive, MSC 8012 Bethesda, Maryland 20892-8012, United States
S
* Supporting Information
ABSTRACT: Two analogues of glucosamine-6-phosphate
(GlcN6P, 1) and five of glucosamine (GlcN, 2) were prepared
for evaluation as catalytic cofactors of the glmS ribozyme, a
bacterial gene-regulatory RNA that controls cell wall biosyn-
thesis. Glucosamine and allosamine with 3-azido substitutions
were prepared by SN2 reactions of the respective 1,2,4,6-
protected sugars; final acidic hydrolysis afforded the fully
deprotected compounds as their TFA salts. A 6-phospho-2-
aminoglucolactam (31) was prepared from glucosamine in a
13-step synthesis, which included a late-stage POCl3-
phosphorylation. A simple and widely applicable 2-step
procedure with the triethylsilyl (TES) protecting group was
developed to selectively expose the 6-OH group in N-
protected glucosamine analogues, which provided another route to chemical phosphorylation. Mitsunobu chemistry afforded
6-cyano (35) and 6-azido (36) analogues of GlcN-(Cbz), and the selectivity for the 6-position was confirmed by NMR (COSY,
HMBC, HMQC) experiments. Compound 36 was converted to the fully deprotected 6-azido-GlcN (37) and 2,6-diaminoglucose
(38) analogues. A 2-hydroxylamino glucose (42) analogue was prepared via an oxaziridine (41). Enzymatic phosphorylation of
42 and chemical phosphorylation of its 6-OH precursor (43) were possible, but 42 and the 6-phospho product (44) were
unstable under neutral or basic conditions. Chemical phosphorylation of the previously described 2-guanidinyl-glucose (46)
afforded its 6-phospho analogue (49) after final deprotection.
[MurNAcβ1-4GlcNAcβ1-4] repeating disaccharide subunit.10
Disrupting the peptidoglycan polysaccharide assembly via
misregulation of GlmS is a tantalizing prospect, although it is
INTRODUCTION
■
The glmS ribozyme is a catalytic RNA domain that resides in
the 5′ untranslated region (UTR) of the mRNA encoding the
still unclear whether activation or inhibition of the ribozyme by
small molecules would be most effective. The Winkler group
demonstrated in the Gram-positive bacterium Bacillus subtilis
that the glmS ribozyme is indeed essential for peptidoglycan
biosynthesis and that bacteria with a cleavage-defective
ribozyme, analogous to an inhibited ribozyme, were unable to
sporulate or form biofilms.8
The 1.7 Å resolution crystal structure of the glmS ribozyme
in complex with 1 (Figure 1a) shows it binding to a shallow,
solvent-exposed pocket of the RNA in the α-axial anomeric
conformation, with the amine in van der Waals contact with the
5′O of G1, the leaving group of the transesterification
reaction.11 The functional importance of the amine is borne
out by crystal structures of glucose-6-phosphate (18) bound to
the ribozyme.4 Even though this compound is an inhibitor,
rather than an activator, of the ribozyme,12 18 was found to
bind in exactly the same location and also in the α-axial
anomeric conformation.
protein enzyme glucosamine-6-phosphate synthetase (GlmS) in
most Gram-positive bacteria.1 It has attracted considerable
attention because it is the first known example of a natural
ribozyme that uses a small molecule coenzyme (reviewed in refs
2 and 3). The ribozyme employs the amine of GlcN6P (1) as a
general acid−base catalyst to catalyze RNA cleavage through
internal transesterification.4,5 Binding to the ribozyme lowers
the pKa of this functional group by up to 1.6 pH units,
improving the catalytic efficiency of the coenzyme.6,7 In Gram-
positive bacteria, intact mRNAs have a triphosphate moiety at
their 5′ terminus. Ribozyme cleavage of the 5′-UTR of the glmS
mRNA exposes a 5′-OH group, which triggers its degradation
by RNase J1. Because the GlmS protein is unstable, degradation
of its mRNA stops synthesis of 1, thereby allowing negative-
feedback regulation.8
Because it controls the synthesis of 1, an essential metabolic
precursor to the bacterial cell wall, the glmS ribozyme has been
considered a potential drug target.2,9 Established antibiotics
such as the β-lactams and Vancomycin inhibit assembly of the
peptidoglycan component of the bacterial cell wall by
disrupting the peptide cross-linking. For the glycan portion of
the peptidoglycan cell wall biosynthesis, 1 is converted into the
Received: January 28, 2013
Published: April 11, 2013
This article not subject to U.S. Copyright.
Published 2013 by the American Chemical
Society
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requirements for coenzyme function (Figure 1b).1,13,14 The
phosphate of 1 contributes to its binding to the ribozyme as
evidenced by weaker activation by GlcN (2), consistent with
the crystal structure. Carba-sugar analogues of 1 and 2, i.e., 3
and 4, are moderate and weak activators, corroborating the
importance of O5 and the phosphate for binding. The
compound is inactive if the pyranose ring is opened (5).
Substituents that distort the chair conformation result in
inactive analogues (6). Loss of the anomeric hydroxyl or
inversion in configuration at C3 is tolerated (7, 8), but loss of
two hydroxyl groups or introduction of a bulky substituent at
C1 results in loss of activity (9−12). Although methylation of
the amine of GlcN6P (13) or inversion of configuration at C2
(14) is tolerated, activity of the ribozyme requires the amine to
be at position 2 (15). Trimethylation of the amine (16) or its
acetylation (17) yield compounds as inactive as 18.
With insights from previously published chemistry, bio-
chemistry, and X-ray crystallography studies, we sought
improved glmS coenzyme candidates (activators) or inhibitors
through the synthesis of five additional analogues of 2, and two
additional analogues of 1. The 2-NH2 group’s central role in
catalysis led us to analogues with modifications to the 2-
position (hydroxylamino and guanidinyl) or adjacent positions
(3-azido and adjacent amide group in a lactam) that would
modify the acid/base properties at the 2-position. Enzymatic
phosphorylation was investigated but was of limited utility. A
simple, widely applicable procedure for selective exposure of
the 6-OH was developed for chemical phosphorylation and
functional group transformation at the 6-position. Given the
numerous H-bonding opportunities in RNA, changes to the H-
bonding between coenzyme candidate and ribozyme active site
may change the affinity of the coenzyme candidates relative to
1. In principle, all analogues described here alter that pattern,
but azido groups rarely participate in H-bond interactions.15,16
The azides, however, provide opportunities for future
coenzyme candidates through click chemistry, for instance.
RESULTS AND DISCUSSION
■
3-Azido-2-amino Sugars. The presence of a 3-azido group
allows for altered H-bond interactions and 2-NH2 pKa values
compared to GlcN6P. Because allosamine-6-P was the most
active, non-gluco configured coenzyme described to date,13 we
also prepared 3-azidoallosamine (22). GlcN-(Cbz) was
protected by Fisher glycosylation and subsequent introduction
of the 4,6-benzylidene acetal to produce 19 as a mixture of
anomers (Scheme 1). The less soluble β-anomer (20β) was
separated from the mixture by fractional crystallization
(EtOAc) and cyclohexane trituration from a THF solution.
Subsequent recrystallization (EtOAc) and flash chromatog-
raphy of each anomer afforded 20α and 20β in 22.6% and
27.4% yield for 2 steps, respectively. Compound 20β was
converted to its mesylate, which was then displaced by NaN3 to
afford the protected 3-azido-allosamine (21) in 52% yield for 2
steps. Compound 20α was converted to the allo-sugar (23)17
that was then converted to the 23-mesylate (87%). Treatment
with NaN3 in DMF led to a mixture of 24 and an elimination
side-product that were inseparable by flash chromatography.
After deallylation, deallyl-24 (16% for 2 steps) was separated
from the side product. Removal of all protecting groups from
21 and 24 was possible with extended heating in 1:1 TFA/
H2O; however, purer 22 (38% from 21) and 25 (77% from
deallyl-24) were isolated when the anomeric allyl group was
removed first. Azido-substituted aminosugars 22, and 25 can be
Figure 1. (a) Detail of the crystal structure of GlcN6P bound to the
glmS riboyzme.11 Green and red spheres represent a magnesium ion
and water molecules, respectively. Dotted lines indicate hydrogen
bonds. GlcN6P stacks under the nucleobase of RNA residue G1, with
its anomeric oxygen participating in hydrogen bonding interactions
with the scissile phosphate (*), G65, and G66. The catalytically critical
amine of GlcN6P hydrogen bonds with the 5′-O of G1, the base of
U51, and a well-ordered water molecule. The hydroxyls at positions 3
and 4 of GlcN6P make direct and water-mediated hydrogen bonds
with backbone atoms of A50 and U51. The O4 of GlcN6P receives a
hydrogen bond from the nucleobase of C2. Finally, the N1 amide of
G1 hydrogen bonds to the phosphate of the small molecule. (b)
Previously described GlcN6P analogues and their effectiveness as
coenzymes for the glmS ribozyme. (−) = inactive; (+) = weakly active;
(++) = modestly active; (+++) = strongly active.
Analogue studies aiming to discover potent unnatural
coenzymes of the ribozyme have yielded some insight into
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a
Scheme 1. Preparation of the 3-Azido-2-amino Carbohydrates
a
Reagents and conditions: (a) CH2CHCH2OH, H2SO4 (cat.), reflux; (b) PhCH(OMe)2, ( )-camphorsulfonic acid (cat.), DMF; (c) Ms2O, py;
(d) NaN3, DMF, 120 °C; (e) SeO2, AcOH, 1,4-dioxane, 120 °C; (f) 1:1 TFA/H2O, 120 °C; (g) DMSO, Ac2O; (h) NaBH4, MeOH.
a
Scheme 2. 2-Amino-glucono-1,5-lactam-6-phosphate Synthesis
a
Reagents and conditions: (a) DPS-Cl, py; (b) PAB-Cl, BaO, Ba(OH)2·8H2O, DMF, then PAB-I, KOtBu, toluene; (c) SeO2, AcOH, dioxane, reflux;
(d) Dess−Martin reagent, py; (e) 7 N NH3 in MeOH; (f) HCOOH, NaBH3CN, CH3CN, reflux; (g) TBAF, THF, AcOH; (h) POCl3, py, 30 min 0
°C, then H2O; (i) CHCl3, H2O, DDQ, 80 °C; (j) Pd(OH)2/C, NH4·HCOO, H2O/MeOH, reflux. PAB = p-N-(pivaloyl)aminobenzyl; DPS = tert-
butyldiphenylsilyl. A box indicates the major product component from each step; only the major component was carried through to the next step.
added to the two unprotected hexoses with azido and amino
functional groups that have appeared in the literature: 3-azido-
mannosamine18 and 4-azido-galactosamine.19 Aminosugar 25
could be utilized to image glycans that use GlcN6P and GlcN,20
but given that allosamine is a rare aminosugar,21 22 is unlikely
to find such utility.
Chemical and Enzymatic Phosphorylations. The 6-
phosphate of GlcN6P is a key feature that improves coenzyme
binding to the glmS ribozyme, facilitating discrimination among
many possible carbohydrate ligands. Chemical phosphorylation
of several of the remaining coenzyme candidates was
accomplished by POCl3 treatment in pyridine of a selectively
exposed 6-OH group, which was prepared via orthogonal
protection groups (lactam synthesis) or via selective removal of
the 6-O-triethylsilyl (TES) group from a tetrasilylated sugar.
Compounds 22, 25, 32, 42, and 45 were all investigated as
yeast hexokinase (HK) substrates. Only 25 (see Supporting
Information) and 42 (see below) were phosphorylated, despite
the fact that HK accepts a wide variety of glucose-similar
substrates and that changes to the hexose 2- and 3-positions are
well tolerated.22
Phosphoamino Lactam Synthesis. The phosphoamino
lactam (31) analogue of GlcN6P was chosen because of its
relatively inert nature, which could be an advantage for
extended biochemical studies. Lactam 31 lacks an anomeric
OH, is unable to undergo mutarotation, and would not be a
substrate for the next step in peptidoglycan synthesis, namely,
conversion of GlcN6P to its 1-phosphate by phosphoglucos-
amine mutase.23 The 2-amino gluconolactam (32) appeared in
the literature,24,25 but not the phosphorylated analogue (31).
Thus the synthesis of 31 was similar to related molecules24−26
but also included a late-stage phosphorylation of the primary
OH. Protection groups stable to basic (NH3) and acidic
conditions (HCOOH) as well as orthogonality in removing the
anomeric and 6-OH protecting groups were necessary. In
addition, selective, facile removal of the 3,4-O-groups was
desired as to provide an N-Cbz-protected phospho-lactam that
could be HPLC-purified before the final, rapid Cbz removal.
These criteria appeared to be satisfied by the allyl pyranoside
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a
Scheme 3. Selective 6-Deprotection and Derivatization
a
Reagents and conditions: (a) TES-Cl (12 equiv), DMF, DIEA (15 equiv), 60 °C; (b) DCM, MeOH, PPTS (cat.); (c) Ph3P, acetone cyanohydrin,
DIAD, THF; (d) Ph3P, DPPA, DIAD, THF; (e) TFA/H2O (1:1), 110 °C; (f) Pd(OH)2/C, NH4·HCOO, MeOH, H2O, 60 °C.
with 6-O-DPS, and 3,4-bis(p-N-(pivaloyl)aminobenzyl)
(PAB)27 protecting groups.
was not investigated. In conclusion, the chosen protecting
groups were adequate for the carbohydrate to lactam
conversion, but the combined steric demands of the protecting
groups on a carbohydrate scaffold likely led to unsatisfactory
product yields.
Regioselective protection of the 6-OH of 19 using DPS-Cl
proceeded smoothly, producing 26 (93%); however, introduc-
tion of the PAB groups was problematic (Scheme 2). A 7-fold
excess of PAB-Cl in the presence of BaO and Ba(OH)2·8H2O
resulted in an intractable mixture of mono- and bis-PAB
protected sugars (both anomers) and uncharacterized side
products that appeared to originate from the PAB-Cl reagent.
The sugar products were partially purified by chromatography,
and the mixture was treated with a second round of PAB-Cl
and base; unfortunately, the yield of bis-PAB product did not
improve substantially. Numerous modifications to the
benzylation conditions were investigated to improve the yield.
The most successful, PAB-I and KOtBu in toluene, was then
applied to the carbohydrate mixture. After partial purification
by chromatography, the allyl group was removed, followed by
oxidation with Dess−Martin reagent. The result was two
separable lactone isomers (3-step yield: 27a, 2.5%; 27b,
15.3%); 27a had JH2/H3 = 3.2 Hz and very weak COSY
correlation with no scalar coupling between H3 and H4. This
suggests a lactone with skewed conformation, which is perhaps
unsurprising given the steric demands of the protecting groups,
Modifications at the 6-OH of Hexopyranoses. Chem-
ical phosphorylation of the 6-OH in the GlcN analogues
requires N-protection and discrimination between the primary
and secondary hydroxyl groups. Orthogonal protection group
schemes, like that used for the phospholactam synthesis, were
an option for the remaining coenzyme candidates. During the
course of this work, however, a simpler, widely applicable OH-
discrimination scheme was developed using the triethylsilyl
(TES) protecting group. All hydroxyls of GlcN-(Cbz) were
protected using TES-Cl and diisopropylethyamine (DIEA) in
DMF (Scheme 3) to produce 33 (62%). Taking into account
the slightly more polar, incompletely silylated carbohydrates,
90% of the starting material could be accounted for; these could
be resubjected to silylation to improve the yield. Conveniently,
the primary 6-OH was exposed via transilylation (to MeOH)
with catalytic PPTS in MeOH/DCM. Once 33 had been
consumed (TLC), the reaction mixture was loaded directly on
to a silica cartridge and chromatographed; the least polar
product was 34 (58%). In general, the deprotection rate varied
with the amount of PPTS and was different for every per-
silylated substrate. Again, less silylated, more polar carbohy-
drates could be recovered and converted again to 33.
1
or possibly an inversion of configuration at C2. The H NMR
spectra of 27b had too many overlapping peaks to draw any
conclusions on the configuration, but it was the major product
and the synthesis proceeded with it. Lactone 27b was opened
using NH3, producing 28 as 2 separable amides (28a, 80%, and
28b, 6.4%). When mixed with NH3 in MeOH, 27a also
produced two separable amides 28a (19%) and 28c (63%).
Amide 28a was then oxidized with Dess−Martin reagent,
producing two 6-hydroxylactams (29a, 29b); the NMR data
Selectivity for the 6-position was confirmed, and silyl group
migration was ruled out with 35 (64% yield), which was
prepared by a modified Mitsunobu coupling with acetone
cyanohydrin.28 1H, 13C, COSY, and HMBC NMR analyses of
35 showed clear correlation between the C6H (2.9 ppm) and
cyano (118 ppm) 13C peaks. The azido group was introduced
using diphenylphosphorylazide (DPPA)29 and was used to
make coenzyme candidates 37 and 38. Although 36 has not
been previously reported, the 27%, 3-step yield from GlcN-
(Cbz) is similar to the 23%, 3-step conversion of ManN-(Cbz)
1
(1H, 13C, H−COSY) were insufficient to determine config-
uration of the hydroxylactams. However, these two isomers can
be rationalized through epimeric 5-hydroxylactams having the
gluco and idoso configurations;26 in addition, steric demands of
the protecting groups could result in chromatographically
separable hydroxylactams with skewed conformations. Finally,
reductive amidation of 29a followed by removal of the DPS
group with TBAF afforded the 6-OH glucolactam 30a (9%)
and 30b (43%) as the two principal products. NMR spectra of
30b confirmed the gluco configuration, and it was phosphory-
lated with POCl3 in py followed by PAB group removal (DDQ)
to afford the N-Cbz phospholactam (31). Final product
purification by HPLC was followed the removal of the Cbz
group by transfer hydrogenolysis; the solvent was removed by
lyophilization, affording 31 (10%, 3 steps). Whether other,
minor products of 28 and 29 could have been converted to 31
−
to 1,3,4-triacetyl-6-azido-2-N(Cbz)-mannosamine in which N3
displaced a 6-OTs group.30
Treating 36 with 1:1 TFA/H2O afforded 6-azido glucos-
amine 37 (75%). Although the azide of 37 could be reduced via
catalytic hydrogenation over Pd/C or Pd(OH)2/C with
NH4·HCOO or HCOOH, 38 could not be isolated cleanly.
Instead, transfer hydrogenation both reduced the azide and
removed the Cbz group from 36; then the TES groups were
hydrolyzed to afford 38 in 68% yield as the (TFA)2 salt.
Modifications to the 2-Amino Group. The 2-amino
group is critical for the coenzyme function of GlcN6P in
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a
Scheme 4. 2-Hydroxylamino-2-Deoxyglucose Synthesis
a
Reagents and conditions: (a) TES-Cl (12 equiv), DIEA (15 equiv), DMF, 70 °C; (b) p-anisaldehyde, PPTS, toluene, reflux; (c) mCPBA, aq
NaHCO3, DCM; (d) TFA/H2O (4:1); (e) yeast hexokinase, ATP, MgCl2, citric acid·H2O, pH 8.0; (f) cat. CSA, MeOH; (g) POCl3, 2,6-lutidine,
then TFA/H2O (4:1).
a
Scheme 5. Preparation of the 2-Guanidinylglucose-6-phosphate Coenzyme Candidate (49)
a
(a) TES-Cl, DIEA, DMF, 70 °C; (b) cat. PPTS, DCM, MeOH; (c) POCl3/py, then H2O, DMF/AcOH followed by 1 N HCl.
activating the glmS ribozyme. Available evidence point to it as
both a general acid and general base in the cleavage event.6,7
Thus, modification of the 2-amino group expands the
possibilities of finding an activator or inhibitor. To this end,
hydroxylamine- and guanidine-substituted gluco-configured
carbohydrates were prepared and phosphorylated at their 6-
positions.
HPLC31 at pH = 3. Through each purification step, a steady
loss of 44 was apparent (LC−MS) even under the mildly acidic
conditions. After final lyophilization of the product fractions no
44 was present (LC−MS). The decomposition mechanism is
unknown but the presence of the hydroxylamine may facilitate
rearrangement or elimination reactions, which are common in
carbohydrates. These results suggested that 2-hydroxylamino
sugars were stable only under strongly acidic conditions and
were not pursued further because the glmS ribozyme functions
most effectively under slightly basic conditions.
The guanidinylated glucose (45) was prepared using
published procedures.32 Chemical phosphorylation of the di-
BOC precursor (46) was accomplished through the TES
protection, affording 47, and 6-OH deprotection described
above to afford 48 (Scheme 5). Treatment with POCl3/
pyridine followed by acidic TES and BOC removal afforded 49,
which may adopt more conformations than the bicyclic α
anomer according to NMR and MS (see Supporting
Information). This locked-in configuration could be beneficial
for ribozyme binding given that only the α-anomer of GlcN6P
is bound to the ribozyme at the active site.2,6,7
The hydroxylamino analogue was prepared from GlcN
through an oxaziridine intermediate (Scheme 4). The OH
groups of GlcN were protected as their TES-ethers, and 39
(61%) was isolated by chromatography exclusively as the α-
anomer. This was subsequently converted to the imine (40),
which was oxidized (mCPBA) to afford the oxaziridine (41) as
one major (51%, 2 steps) and one minor isomer (5%, 2 steps).
Conversion of 41 to 42 was accomplished with TFA/H2O. The
NMR spectra of isolated 42 indicated α- and β-anomers and
the presence of a putative p-OMe benzylidene adduct in the β-
anomeric configuration in a ratio of 1.6:1.2:1, respectively.
Although 42 in this mixture was phosphorylated by HK, a
steady loss of 44 by LC−MS was apparent under the
phosphorylation conditions. A similar loss of product was
apparent with 42 alone under slightly basic conditions.
Chemical phosphorylation was pursued through the 6-OTES
removal under acidified conditions, MeOH with catalytic
-camphorsulfonic acid, to afford 43 in 47% yield. Treatment
with POCl3/lutidine then TFA/H2O afforded 44. Partial
purification was achieved by passing through a C18 column
(95:5 NH4·HCOO adjusted to pH ∼2.5: MeOH) and 44
eluted at the solvent front, which was subsequently lyophilized.
Compound 44 was then purified by “aqueous normal phase”
In conclusion, analogues of GlcN and GlcN6P were prepared
that provided altered electronic environments around the 2-
amino group, modified amino groups, altered H-bonding
patterns for the ribozyme’s active site, and the scaffold for
additional derivatives via azide derivatization. Further character-
ization and evaluation of the candidates as coenzymes of the
glmS ribozyme will be reported elsewhere.
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101.4, 116.1, 126.3, 127.6, 127.7, 128.0, 128.3, 128.8, 134.4,
137.3, 137.7, 156.0. HRMS (ESI-TOF) m/z: [M + H]+ calcd
for C24H28NO7 442.1860; found 442.1867.
EXPERIMENTAL SECTION
■
2-Propen-1-yl 2-(Benzyloxycarbonyl)amino-2-deoxy-
4,6-O-(phenylmethylene)-D-glucopyranoside (20α, 20β).
Allyl alcohol (200 mL), concd H2SO4 (30 μL), and 2-
(benzyloxycarbonyl)amino-2-deoxyglucose33 (5.28 g, 16.9
mmol) were combined and then refluxed for 2 h. Then,
NaHCO3 (2 g) was added; 1 h later the solution was filtered
and the solvent was removed. The residue was recrystallized
(EtOAc), then the filtrate’s solvent was removed, and the
residue was chromatographed on silica using EtOAc/CH3CN
(3:1). The desired product (two anomers, TLC Rf = 0.45−0.5)
was collected; product recrystallization and chromatography of
the subsequent filtrate was repeated, followed by a final
recrystallization to afford a combined 4.40 g (12.4 mmol,
73.7%) of an off-white powder (19). Under an Ar atmosphere,
19 (4.29 g, 12.1 mmol) was dissolved in anhyd DMF (50 mL).
The reaction flask was cooled in an ice bath, then
-camphorsulfonic acid (100 mg) and benzaldehyde dimethyl
acetal (5.47 mL, 36.3 mmol) were added, and the mixture was
stirred overnight while the solution warmed to rt. The solvent
was removed, and the residue was mixed with DCM (36 mL),
H2O (9 mL), and 5% NaHCO3(aq) (3 mL). After vigorous
stirring (5 min), MeOH (6 mL) and hexanes (25 mL) were
added. The flask was cooled in the freezer for 4 h, and then the
precipitate was filtered off (1st crop); reducing the filtrate
volume afforded two more product crops. The first crop was
recrystallized (EtOAc) to afford 1.62 g of 20β, a portion of
which was chromatographed, which indicated the first crop was
87% pure. The filtrate residue and remaining product crops
were combined and dissolved in a minimum volume of THF.
The β-anomer was triturated with cyclohexane and filtered off.
Both precipitate and filtrate residue were chromatographed,
each on a 50 g silica cartridge (3:1 DCM/EtOAc); 20α eluted
first. Any fractions with 20α/20β mixtures were combined and
subjected to the trituration and chromatography steps. Isolated
yields of purified 20α and 20β, each a white powder, were 1.64
g (3.73 mmol, 22.6% for 2 steps) and 1.99 g (4.52 mmol, 27.4%
for 2 steps), respectively.
2-Propen-1-yl 3-Azido-2-(benzyloxycarbonyl)amino-
2,3-dideoxy-4,6-O-(phenylmethylene)-β-D-allopyrano-
side (21). Compound 20β (1.1 g, 2.5 mmol) was dissolved in
anhyd py, and the reaction flask was cooled in an ice bath.
Then, Ms2O (1.09 g, 6.25 mmol) was added, and the mixture
was stirred for 16 h. The solution was added to H2O (40 mL)
with stirring; 20 min later, the solution was filtered, and the
precipitate was air-dried to afford 1.27 g of an off white powder
(crude 20β-mesylate), which was used without further
purification. NaN3 (45 mg, 0.75 mmol) and 20β-mesylate
(126 mg) were dissolved in anhyd DMF (1 mL). The reaction
vial was heated (hot plate at 110 °C), and mixture was stirred
for 48 h. Then, the solvent was removed, and the residue was
chromatographed on a 12 g silica cartridge (0 to 50% EtOAc in
DCM). The isolated yield of 21 (a white powder, TLC Rf =
0.75 with 1:1 DCM/EtOAc) was 61 mg (0.13 mmol, 52% for 2
steps). The reaction could be scaled up to 1 g of 21 with similar
results. A sample of 21 was recrystallized from DCM/hexanes
for NMR analysis.
1
Data for 20β-mesylate. H NMR (acetone-d6, 600 MHz) δ:
2.97 (s, 3H, SO2-CH3), 3.56 (ddd, J = 10.0, 9.0, 5.0 Hz, 1H,
C5H), 3.74 (dd, J = 9.8, 9.1, 8.3 Hz, 1H, C2H), 3.88 (dd, J =
10.3, 10.3 Hz, 1H, C6H), 3.90 (dd, J = 9.5, 9.5 Hz, 1H, C4H),
4.13 (dddd, J = 13.3, 5.6, 1.5, 1.5 Hz, 1H, allyl-C1H), 4.31 to
4.35 (m, 2H, C6H, allyl-C1H), 4.91 (d, J = 8.3 Hz, 1H, C1H),
4.95 (dd, J = 9.8, 9.8 Hz, 1H, C3H), 5.09 to 5.14 (m, 3H, allyl-
C3H, Bn-CH2), 5.29 (ddd, J = 17.3, 3.5 1.7 Hz, 1H, allyl-C3H),
5.76 (s, 1H, acetal-CH), 6.74 (d, J = 9.1 Hz, 1H, C2-NH), 7.29
to 7.40 (m, 7H, aromatic-H), 7.52 to 7.55 (m, 3H, aromatic-H).
Allyl-C2H multiplet was too broad to observe, but showed up in
COSY. 13C NMR (DMSO-d6, 150 MHz) δ: 38.5, 55.8, 65.2,
65.4, 67.5, 69.5, 78.1, 80.0, 100.08, 100.09, 100.7, 116.5, 125.9,
127.1, 127.5, 127.7, 128.1, 128.3, 128.9, 134.1, 137.0, 137.1,
155.8. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C25H30NO9S 520.1636; found 520.1649.
Data for 21. 1H NMR (acetone-d6, 600 MHz) δ: 3.82 (dd, J
= 10.1, 10.1 Hz, 1H, C6H), 3.87 to 3.93 (m, 2H, C2H, C5H),
4.05 (dd, J = 9.2, 2.4 Hz, 1H, C4H), 4.08 (dddd, J = 13.5, 5.5
Hz, remaining coupling unresolved, 1H, allyl-C1H), 4.28 to
4.33 (m, 2H, C6H, allyl-C1H), 4.46 (dd, J = 3.2, 3.2 Hz, 1H,
C3H), 4.71 (d, J = 8.5 Hz, 1H, C1H), 5.08 to 5.14 (m, 3H, Bn-
CH, allyl-C3H), 5.27 (ddd, J = 17.3, 3.5, 1.6 Hz, 1H, allyl-C3H),
5.74 (s, 0.7H, acetal-CH), 5.86 to 5.9 (m, 0.01H, allyl-C2H),
6.63 (d, J = 8.0 Hz, 0.5H, C2-NH), 7.33 to 7.41 (m, 8H,
aromatic H), 7.52 to 7.54 (m, 2H, aromatic H). 13C NMR
(acetone-d6, 150 MHz) δ: 54.6, 63.4, 65.1, 66.8, 69.4, 70.6, 79.0,
100.2, 102.3, 116.7, 127.1, 128.59, 128.61, 128.8, 129.2, 129.7,
135.3, 138.1, 138.7, 156.6. HRMS (ESI-TOF) m/z: [M + H]+
calcd for C24H27N4O6 467.1925; found 467.1927.
1
Data for 20α. H NMR (CDCl3, 500 MHz) δ: 2.7 (s br,
1H), 3.59 (dd, J = 9.0, 9.0 Hz, 1H), 3.76 (dd, J = 10.0, 10.0 Hz,
1H), 3.85 (ddd, J = 9.6, 9.6, 4.7 Hz, 1H), 3.90 to 4.01 (m, 3H),
4.19 (dddd, J = 12.8, 5.4, 1.4, 1.4 Hz, 1H), 4.27 (dd, J = 10.1,
4.7 Hz, 1H), 4.89 (d, J = 3.2 Hz, 1H), 5.1 to 5.16 (m, 3H), 5.21
(ddd, J = 10.4, 2.3, 1.1 Hz, 1H), 5.28 (ddd, J = 15.7, 2.9, 1.5 Hz,
1H), 5.56 (s, 1H), 5.83 to 5.91 (m, 1H), 7.32 to 7.39 (m, 8H),
7.49 to 7.51 (m, 2H). 13C NMR (CDCl3, 125 MHz) δ: 56.0,
63.0, 67.8, 69.0, 69.4, 70.9, 82.4, 97.7, 102.4, 118.6, 126.7,
128.7, 129.0, 129.7, 133.6, 136.5, 137.5, 157.2. HRMS (ESI-
TOF) m/z: [M + H]+ calcd for C24H28NO7 442.1860; found
442.1867.
1
Data for 20β. H NMR (DMSO-d6, 600 MHz) δ: 2.26 to
3.33 (m, 2H, over H2O peak, C2H, C5H), 3.43 (dd, J = 9.3, 9.3
Hz, 1H, C4H), 3.57 (m, 1H, C3H), 3.72 (dd, J = 10.1, 10.1 Hz,
1H, C6H), 3.99 (dddd, J = 13.7, 5.2 Hz, smaller couplings not
resolved, 1H, allyl-C1H), 4.18 to 4.23 (m, 2H, allyl-C1H, C6H),
4.45 (d, J = 8.4 Hz, 1H, C1H), 4.98 (d, J = 12.7 Hz, 1H, Bn-
CH), [5.06 (d, J = 12.7 Hz, Bn-CH) over 5.07 to 5.15 (m, allyl-
C3H), 2H], 5.23 (ddd, J = 17.2, 3.7, 1.8 Hz, 1H, allyl-C3H),
5.37 (d, J = 5.8 Hz, 1H, C2-NH), 5.59 (s, 1H, acetal-CH), 5.76
to 5.83 (m, 0.2 H, allyl-C2H), 7.27 to 7.38 (m, 8H, aromatic-
H), 7.42 to 7.45 (m, 2H, aromatic-H). 13C NMR (DMSO-d6,
150 MHz) δ: 57.9, 65.1, 66.0, 67.8, 69.1, 70.3, 81.2, 100.6,
General Deallylation Procedure. SeO2 (20 mg, 1.8
mmol) was mixed with AcOH (13.4 μL, 0.24 mmol) and the
allyl pyranoside (0.12 mmol) in anhyd 1,4-dioxane (0.5 mL).
The reaction vial was set on a hot plate (120 °C), and the
mixture was stirred for 2.5 h. Then, the vial was allowed to cool
and the solution was filtered; the solvent was removed from the
filtrate and the residue was chromatographed.
General Acid Hydrolysis Procedure. The hydrolysis
substrate was mixed with TFA/H2O (1 mL/1 mL). The
reaction vial was heated (hot plate at 120 °C), and the mixture
was stirred for 24 h. The heat was removed, then H2O (3 mL)
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The Journal of Organic Chemistry
Article
was added, and the aq phase was extracted 2× with EtOAc (3
mL). The aq phase was frozen and then lyophilized. The
resulting residue was redissolved in H2O (3 mL) and
lyophilized a second time.
Hz, 1H, Bn-CH), 5.17 (unresolved ddd, one J = 1.3 Hz, 1H,
allyl-C3H), 5.24 (unresolved dd, one J = 2.6 Hz, 1H, C3H), 5.38
(ddd, unresolved ddd, one J = 1.7 Hz, 1H, allyl-C3H), 5.77 (s,
1H, acetal-CH), 5.93 to 6.0 (m, 0.3H, allyl-CH), 7.31 to 7.41
(m, 7H, aromatic H), 7.52 to 7.55 (m, 3H, aromatic H). 13C
NMR (acetone-d6, 150 MHz) δ: 39.0, 51.3, 58.9, 67.2, 69.2,
69.4, 76.4, 77.4, 96.9, 102.4, 116.8, 127.1, 128.6, 128.9, 129.1,
129.8, 135.0, 137.8, 138.6, 156.2. HRMS (ESI-TOF) m/z: [M
+ NH4]+ calcd for C25H33N2O9S 537.1901; found 537.1900.
Data for 24. 1H NMR (acetone-d6, 600 MHz) δ: 3.75 (dd, J
= 9.4, 9.4 Hz, 1H, C4H), 3.81 to 3.90 (m, 3H, C2H, C5H, C6H),
4.01 (dd, J = 10.7, 10.1 Hz, 1H, C3H), 4.06 (dddd, J = 13.3, 7.1,
1.4, 1.4 Hz, 1H, allyl-C1H), 4.25 to 4.30 (m, 2H, allyl-C1H,
C6H), 4.94 (d, J = 3.6 Hz, 1H, C1H), 5.12 (s, 2H, Bn-CH2),
5.16 (ddd, J = 10.5, 1.4 Hz, remaining coupling not resolved,
1H, allyl-C3H), 5.37 (ddd, J = 17.9, 3.4, 1.7 Hz, 1H, allyl-C3H),
5.76 (s, 1H, acetal-CH), 5.92 to 5.98 (m, 0.3H, allyl-C2H), 6.71
(d, J = 9.2 Hz, 0.6H, C2-NH), 7.31 to 7.42 (m, 8H, aromatic
H), 7.49 to 7.53 (m, 2H, aromatic H). 13C NMR (acetone-d6,
150 MHz) δ: 55.2, 62.0, 64.0, 66.9, 69.1, 69.3, 81.3, 97.9, 102.1,
117.3, 127.0, 128.6, 128.7, 128.8, 129.2, 129.6, 134.9, 138.1,
138.7, 156.8. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C24H27N4O6 467.1925; found 467.1929.
2-Amino-3-azido-2,3-dideoxyallose (22). The general
conditions for allyl group removal were applied to 21 (55 mg,
0.12 mmol). The residue was chromatographed 2×, each on a
12 g silica cartridge with a 3:1 to 2:3 DCM/EtOAc gradient to
afford 24.5 mg of semipurified product. A portion of this
product (20.5 mg) was subjected to the general acid hydrolysis
conditions, which afforded 12 mg of 22 as the TFA salt (38
μmol, 38% for 2 steps). Injecting a 1:1 MeOH/H2O solution of
22 on a semipreparative C18 column and eluting with 95:5
H2O/MeOH removed the small amount of residual low
polarity material. Compound 22 eluted immediately after the
void volume; the sample was reacidified with HCOOH and
1
then lyophilized, affording an off white powder. H NMR
(D2O, 600 MHz) δ: main product β:α ratio = 6:1, 3.17 (dd, J =
8.4, 3.4 Hz, 6H, βC2H), 3.60 (dd, J = 12.1, 5.6 Hz, 6.5H,
βC6H), 3.66 (dd, J = 12.8, 4.7 Hz, 2H, αC6H), 3.72 to 3.77 (m,
14H, βC5H in here), 3.87 (dd over m, J = 10.6, 3.5 Hz, 8.2H,
βC4H/αC4H/αC5H), 4.23 (dd, J = 3.8, 3.8 Hz, 1.1H, αC3H),
4.28 (dd, J = 3.3, 3.3 Hz, 6H, βC3H), 4.86 (d, J = 8.3 Hz, 6H,
βC1H), 5.16 (d, J = 4.1 Hz, 1H, αC1H). [Signals corresponding
to 2 additional minor carbohydrate products were present: 3.04
(dd, J = 8.7, 3.5 Hz, <0.1H), 3.78 to 3.81 (m, 0.5H), 4.18 (dd, J
= 4.5, 4.2 Hz, 0.3H), 4.38 (dd, J = 7.8, 3.5 Hz, 0.3H), 5.36 (d, J
= 3.5 Hz, 0.1H), 5.49 (d, J = 4.6 Hz, 0.25H)]. 13C NMR (D2O/
DMSO-d6; referenced to DMSO CD3, 150 MHz) δ: 50.2, 54.0,
61.8, 62.1, 62.2, 63.6, 67.8, 68.6, 75.9, 89.6, 92.3, [118.0 (q, J =
291 Hz), 164.3 (q, J = 34.7 Hz), TFA], [172.1 from formic
acid]. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C6H13N4O4
205.0931; found 205.0930.
2-Amino-3-azido-2,3-dideoxyglucose (25). The general
deallylation procedure was applied to the mixture of 24 and the
side product (100 mg) on a 1.9× scale. Then, the crude mixture
was chromatographed on a 12 g silica cartridge with a 10 to
50% EtOAc in DCM gradient to afford 34 mg of semipurified
deallyl product. Final purification from coeluting Se-related
products on a 4 g silica cartridge afforded 27 mg of a white
powder, deallyl-24 (16%, two-step yield from 23-mesylate).
Then deallyl-24 (15 mg, 35 μmol) treated with the general acid
hydrolysis procedure, affording 25 as the TFA salt (a white
1
2-Propen-1-yl 3-Azido-2-(benzyloxycarbonyl)amino-
2,3-dideoxy-4,6-O-(phenylmethylene)-α-D-glucopyrano-
side (24). The conversion of 20α to 23 was carried out as
previously described,34 then 23 (0.62 g, 1.4 mmol) was treated
with Ms2O (0.61 g, 3.5 mmol) in anhyd py (5 mL) as described
for the production of 21. The residue was chromatographed on
a 25 g silica cartridge with 0 to 33% EtOAc in DCM, affording
637 mg of a white powder (23-mesylate, 1.23 mmol, 87.6%
yield). Then, 23-mesylate (0.600 g, 1.16 mmol) was dissolved
in anhyd DMF (5 mL). NaN3 (0.228 g, 3.5 mmol at the start;
75 mg, 1.2 mmol at 58 h) was added, and the reaction was
heated (oil bath, 120 °C) and stirred for 70 h. Then, after
cooling, H2O (20 mL) was added to the mixture. The
precipitate was filtered, washed with H2O (20 mL), and then
air-dried. The precipitate was chromatographed on a 40 g silica
cartridge with a 0 to 30% EtOAc in DCM gradient. An
elimination product consistent with loss of MsOH coeluted
with 24 (TLC Rf = 0.9; 10:1 DCM/EtOAc), affording 0.286 g
of an off-white solid. A portion of 24 was purified for NMR
analysis from the mixture by HPLC on a semipreparative C18
column: A = H2O, B = MeOH, 0 to 1 min (75% B), 1 to 20
min (gradient to 85% B) at 4 mL/min. The product eluting at
tR = 20 min was 24.
solid, 8.6 mg, 27 μmol, 77%). H NMR (D2O, 600 MHz) δ:
[α:β ratio = 2.5:1] 2.84 (dd, J = 10.9, 8.5 Hz, 1H, Hβ2), 3.14
(dd, J = 11.2, 3.3 Hz, 2.5H, Hα2), 3.22−3.24 (m, 1 H), 3.48−
3.52 (m, 1H), 3.54 to 3.70 (overlapping m’s, 9.3H), 3.73
(unresolved dd, J = 12.2 Hz, 2.5H), 3.79 (unresolved dd, J =
12.2 Hz, 1H), 3.81−3.87 (m, 5H), 4.86 (d, J = 8.4 Hz, 1H,
Hβ1), 5.29 (d, J = 3.1 Hz, 2.5H, Hα1). 13C NMR (D2O, 150
MHz) δ: 55.1, 57.3*, 62.8, 63.0*, 64.5, 66.5*, 72.0, 72.4*, 74.2,
79.5*, 91.4, 95.5*, [119.1 (q, J = 290 Hz), 166.0 (q, J = 33 Hz)
TFA]; * = less intense peak, consistent with β-anomer signals.
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C6H13N4O4
205.0931; found 205.0928.
2-Propen-1-yl 2-(Benzyloxycarbonyl)amino-6-O-(tert-
butyldiphenylsilyl)-2-deoxy-D-glucopyranoside (26).
Under an Ar atmosphere, 19 (6.23 g, 17.6 mmol) was
dissolved in anhyd py (75 mL). The reaction flask was cooled
in an ice bath, then DPS-Cl (5.0 mL, 19.4 mmol) was added.
The ice bath was allowed to warm to rt and the reaction
progress was monitored by TLC. More DPS-Cl was added at 6
h (0.685 mL, 2.6 mmol) and at 20 h (0.45 mL, 1.7 mmol).
After 23 h total, the solvent was removed and the residue was
taken up in hexanes (100 mL) and EtOAc (150 mL). The
organic phase was extracted 2× with H2O (100 mL) and then
dried (Na2SO4). The solvent was removed and the residue was
chromatographed on an 80 g silica cartridge with a 1:1 to 0:1
hexanes/EtOAc gradient to afford a mixture of semipure
anomers (TLC Rf = 0.3 and 0.45, 11.02 g, a viscous oil), which
was used without further purification. An 85.5 mg sample of the
semipure mixture was rechromatographed (1:1 to 1:4 hexanes/
EtOAc) to afford pure 26 (73.5 mg, 91% reaction yield) as a
1
Data for 23-mesylate. H NMR (acetone-d6, 600 MHz) δ:
3.03 (s, 3H, SO2CH3), 3.82 (dd, J = 10.3, 10.3 Hz, 1H, C6H),
4.05 to 4.09 (m, 2H, allyl-C1H, C4H), [4.16 (ddd, J = 10.1,
10.0, 5.2 Hz, C5H), 4.20 (ddd, J = 9.1, 4.0, 4.0 Hz, C2H)
overlapping, 2H], 4.29 (dddd, J = 13.4, 4.7, 1.7, 1.7 Hz, 1H,
allyl-C1H), 4.33 (dd, J = 10.3, 5.2 Hz, C6H), 4.97 (d, J = 4.3 Hz,
1H, C1H), 5.10 (d, J = 12.5 Hz, 1H, Bn-CH), 5.12 (d, J = 12.5
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1
viscous oil that crystallized over a period of weeks. H NMR
(CD3OD, 600 MHz) δ: 1.06 (s, 9H, tBu-CH3), 3.45 (m, 2H),
3.65 to 3.74 (m, 2H), 3.89 (ddd, J = 10.9, 10.9, 5.8 Hz, 1H),
3.97 to 4.08 (m, 2H), 4.21 (dd, br, J = 12.8, 4.8 Hz, 0.5H), 4.30
to 4.34 (m, 0.4H), 4.44 (s, v br, 0.3H, βC1H), 4.89 (s br, 0.5 H,
αC1H), 5.07 to 5.16 (m, 3H), 5.23 to 5.31 (m, 1H), 5.88 to
5.95 (m, 0.1H, allyl C2H), 7.25 to 7.45 (m, 11H, aromatic H),
7.65 to 7.75 (m, 4H, aromatic H). 13C NMR (CD3OD, 150
MHz) δ: 20.1, 27.3, 57.2, 59.0, 64.8, 67.4 67.6, 69.2, 70.7, 72.0,
72.2, 73.3, 74.2, 76.1, 78.1, 97.9, 102.1, 117.0, 117.6, 128.6,
128.8, 129.4, 130.7, 134.8, 134.9, 135.4, 135.6, 136.8, 138.2,
138.4, 158.8, 159.0. HRMS (ESI-TOF) m/z: [M + H − allyl
alcohol]+ calcd for C33H42NO7Si 534.2306; found 534.2304.
2-(Benzyloxycarbonyl)amino-6-O-(tert-butyldiphenyl-
silyl)-2-deoxy-3,4-bis-O-(4-(pivaloylamino)benzyl)-D-glu-
cono-1,5-lactone (27a, 27b). Compound 26 (10.8 g, 86%
pure, 15.7 mmol) was dissolved in anhyd DMF (100 mL), then
BaO (13 g), Ba(OH)2·8H2O (5.1 g) and 4-pivaloylaminobenzyl
chloride (PAB-Cl)27 (29 g, 128.5 mmol) were added, and the
mixture was stirred overnight. LC−MS analysis indicated a
mixture of mono- and bis-PAB sugars of both anomers as well
as numerous lower mol wt products that were consistent with
PAB-OH, (PAB)2O, and PAB oligomers. The solid was filtered
off and then washed with DCM. The filtrate volume was
reduced, and some side products were removed by
precipitation in EtOAc. Then filtration of the supernatant
through a plug of silica with EtOAc as eluent afforded the
semipurified product mixture. This was subjected to the
benzylation conditions with PAB-Cl again at 0.5× scale of
that above. LC−MS indicated the reaction had not progressed
much, and the purification scheme above was applied again. An
additional column purification on a 90 g silica cartridge with a
5:1 to 0:1 DCM/EtOAc gradient before proceeding to the
modified benzylation conditions.
again to the general deallylation conditions in dioxane (35 mL)
with SeO2 (0.24 g) and AcOH (0.16 mL). The combined
deallyl product fractions were dissolved in anhyd py (75 mL),
then Dess−Martin periodinane (7.5 g, 17.7 mmol) was added,
and the mixture was stirred for 3 h. Then the solvent was
removed; the residue was taken up in DCM (100 mL) and then
filtered. The filtrate was chromatographed 2×, each on a 220 g
silica cartridge with a 5:1 to 3:1 DCM/EtOAc gradient to afford
semipurified 27a and 27b. Each was again chromatographed 2×
on 120 g silica cartridges with 2:2:1 DCM/hexanes/EtOAc for
27a (TLC Rf = 0.4, 378 mg, 0.41 mmol, 2.6% yield for 3 steps)
and 1:1:1 DCM/hexanes/EtOAc for 27b (TLC Rf = 0.4, 2.33 g,
2.51 mmol, 16.0% yield for 3 steps). Both were stiff white
foams in appearance.
Data for 27a. An HPLC-purified sample (87.5:12.5
CH3CN/H2O isocratic elution on the semipreparative C18
column). 1H NMR (acetone-d6, 600 MHz) δ: 1.06 (s, 9H, tBu-
CH3), 1.29 (s, 9H, tBu-CH3), 1.30 (s, 9H, tBu-CH3), 3.94 (dd,
J = 11.6, 4.3 Hz, 1H, C6H), 3.99 (dd, J = 11.6, 2.3 Hz, 1H,
C6H), 4.12 (d, J = 7.8 Hz, 1H, C4H with very weak COSY
correlation to C3H), 4.24 (d, J = 3.0 Hz, 1H, C3H with very
weak COSY correlation to C4H), 4.44 (d, J = 11.5 Hz, 1H, Bn-
CH), 4.53 (m, 1H, C5H), 4.59 (d, J = 11.9 Hz, 1H, Bn-CH),
4.62 (d, J = 11.6 Hz, 1H, Bn-CH), 4.67 (d, J = 11.9 Hz, 1H, Bn-
CH), 4.89 (dd, J = 7.7, 3.1 Hz, 1H, C2H), 5.12 (d, J = 12.7 Hz,
Bn-CH), 5.14 (d, J = 12.7 Hz, Bn-CH), 6.49 (d, J = 7.7 Hz, 0.05
H, C2-NH), 7.22 (d, J = 8.2 Hz, 2H, aromatic H), 7.27 (d, J =
8.5 Hz, 2H, aromatic H), 7.33 (dd, J = 7.2, 7.2 Hz, 1H, aromatic
H), 7.39 (dd, J = 7.5, 7.5 Hz, 1H, aromatic H), 7.41 to 7.45 (m,
6H, aromatic H), 7.46 to 7.49 (m, 2H, aromatic H), [7.65 (d, J
= 8.5 Hz) overlapping with 7.68 (d, J = 8.5 Hz), 4H, aromatic
H], 7.71 to 7.75 (m, 5H, aromatic H), 8.6 (2 s br, 2H, piv-NH).
13C NMR (acetone-d6, 150 MHz) δ: 19.9, 27.3, 27.8, 40.21,
40.23, 53.8, 63.9, 67.1, 72.0, 72.3, 74.4, 78.2, 80.4, 120.81,
120.84, 128.66, 128.70, 128.72, 128.8, 129.25, 129.28, 129.32,
130.8, 133.3, 133.37, 133.83, 134.05, 140.2, 140.3, 156.8, 169.9,
177.3. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C54H66N3O9Si 928.4563; found 928.4574.
Data for 27b. An HPLC-purified sample (85:15 CH3CN/
H2O isocratic elution on the semipreparative C18 column). 1H
NMR (acetone-d6, 600 MHz) δ: 1.07 (s, 9H, tBu-CH3), 1.29 (s,
9H, tBu-CH3), 1.30 (s, 9H, tBu-CH3), 3.98 to 4.03 (m, 2H),
4.14 to 4.18 (m, 2H), 4.30 to 4.35 (m, 2H), 4.70 (d, J = 10.9
Hz, 1H, Bn-CH), 4.74 (d, J = 11.2 Hz, 1H, Bn-CH), 4.77 (d, J
= 11.2 Hz, 1H, Bn-CH), 4.89 (d, J = 10.9 Hz, 1H, Bn-CH),
5.10 (d, J = 12.5 Hz, 1H, Bn-CH), 5.15 (d, J = 12.5 Hz, 1H, Bn-
CH), 7.19 (d, J = 8.4 Hz, 2H, aromatic H), 7.22 (d, J = 8.3 Hz,
1H, aromatic H), 7.25 (d, J = 8.5 Hz, 2H, aromatic H), 7.32 (m,
1H, aromatic H), 7.34 to 7.50 (m, 9H, aromatic H), 7.65 to
7.68 (m, 4H, aromatic H), 7.71 to 7.76 (m, 4H, aromatic H),
8.63 (s, 1H, piv-NH), 8.65 (s, 1H, piv-NH). 13C NMR
(acetone-d6, 150 MHz) δ: 19.8, 27.2, 27.7, 40.2, 57.4, 63.5, 67.0,
74.8, 76.6, 80.3, 80.5, 120.6, 120.7, 128.7, 128.8, 129.0, 129.2,
130.8, 133.5, 133.8, 133.9, 134.1, 136.4, 136.5, 138.2, 140.0,
140.1, 157.3, 169.2, 177.07, 177.08. HRMS (ESI-TOF) m/z:
[M + H]+ calcd for C54H66N3O9Si 928.4563; found 928.4568.
2-(Benzyloxycarbonyl)amino-6-O-(tert-butyldiphenyl-
silyl)-2-deoxy-3,4-bis-O-(4- (pivaloylamino)benzyl)-D-
gluconamide (28). Lactone 27b (2.19 g, 2.36 mmol) was
dissolved in 7 N NH3 in MeOH (15 mL), and the mixture was
stirred for 30 min. The solvent was removed, and the residue
was chromatographed on a 120 g silica cartridge (3:1 EtOAc/
hexanes) to afford two products, 28a (TLC Rf = 0.4, 1.79g, 1.89
PAB-I. PAB-Cl (14.75 g, 65.4 mmol) and NaI (25 g, 167
mmol) were added to acetone (250 mL), and the mixture was
refluxed for 1 h. Then, the heat was removed, and EtOAc (100
mL) was added. The solution was filtered, and the precipitate
was washed with more EtOAc (100 mL). After solvent removal
the filtrate residue was chromatographed on silica column (5
cm × 25 cm (dia × length) with 1:1 EtOAc/hexanes. Fractions
with PAB-I (TLC Rf = 0.75) were combined and the solvent
was removed to afford 20.1 g (63.3 mmol, 97%) of a light
yellow powder.
Modified Benzylation Conditions. The mixture of mono-
and bis-PAB product mixture was dissolved in dry toluene (200
mL). Then (CH3)3COK (7.3 g, 65 mmol) and PAB-I (20 g, 63
mmol) were added. After stirring overnight, AcOH (2 mL) was
added, and then the solution was filtered. Partial purification
was achieved with a silica column (3.5 cm × 28 cm, dia ×
length) using 3:1 to 0:1 DCM/EtOAc. After solvent removal,
residual PAB-I was removed by adding SiliaMetS Diamine
(Silicycle Inc., 20 g, 1.33 mmol/g) in DCM/MeOH (100
mL:30 mL) and stirring for 24 h.
After filtration and solvent removal, the general deallylation
procedure was applied; in dioxane (150 mL), SeO2 and AcOH
were added in 2 portions, (2.23 and 1.1 g) and (1.6 and 0.78
mL), respectively, at the start and at 18 h. At 24 h, the heat was
removed, and the mixture was chromatographed on a 220 g
silica cartridge, then a 120 g cartridge with 5:1 to 0:1 DCM/
EtOAc gradient. The deallyl product (TLC Rf = 0.2 to 0.25; 5:1
DCM/EtOAc) was kept separate from unreacted starting
material (TLC Rf = 0.6 to Rf = 0.75). The latter was subjected
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Article
mmol, 80.1%) and 28b (TLC Rf = 0.35, 0.143 g, 0.15 mmol,
6.4%); both were white powders.
Data for 28a. An HPLC-purified sample on the semi-
2-(Benzyloxycarbonyl)amino-6-O-(tert-butyldiphenyl-
silyl)-2-deoxy-3,4-bis-O-(4-(pivaloylamino)benzyl)-D-
xylo-hexo-5-ulosonamide (29). Amide 28a (1.25 g, 1.32
mmol) was combined with Dess−Martin periodinane (0.85g,
2.0 mmol) in anhyd py. The mixture was stirred for 7 h, then
diluted with hexanes (40 mL) and EtOAc (40 mL). The
solution was filtered, the solvent was removed from the filtrate
and then the residue was chromatographed on a 220 g silica
cartridge (1:2 to 1:4 hexanes/EtOAc gradient) and then an 80
g silica cartridge (1:1 EtOAc/DCM) to afford 29a (TLC Rf =
0.55 with 1:1 EtOAc/DCM, 0.51 g (0.54 mmol, 41%) and 29b
(TLC Rf = 0.35 with 1:1 EtOAc/DCM, 0.14 g, 0.15 mmol,
11%). Compounds 29a and 29b were white powders.
preparative C18 column (78.5:21.5 MeOH/H2O, 0 to 3.5 min,
1
then gradient to 100% MeOH over 8 min, 3.5 mL/min). H
NMR (CD3OD, 600 MHz) δ: 1.06 (s, 9H, tBu-CH3), 1.27 (s,
9H, tBu-CH3), 1.29 (s, 9H, tBu-CH3), 3.71 (dd, J = 6.5, 6.5 Hz,
1H, C4H), 3.75 (dd, J = 10.3, 4.9 Hz, 1H, C6H), 3.81−3.84 (m,
1H, C5H), 3.87 (dd, J = 10.3, 3.6 Hz, 1H, C6H), 4.39 (dd, J =
5.8, 2.6 Hz, 1H, C3H), 4.46 (d, J = 10.8 Hz, 1H, Bn-CH), 4.51
(d, J = 2.6 Hz, 1H, C2H), [4.55 (d, J = 10.9 Hz, Bn-CH), 4.56
(d, J = 10.9 Hz, Bn-CH), 2H], 4.66 (d, J = 10.8 Hz, 1H, Bn-
CH), 5.06 (s, 2H, Bn-CH2), 7.03 (d, J = 8.4 Hz, 2H, aromatic
H), 7.22 (d, J = 8.5 Hz, 2H, aromatic H), 7.24 to 7.45 (m, 15H,
aromatic H), 7.68 (d, J = 6.8 Hz, 2H, aromatic H), 7.71 (d, J =
6.8 Hz, 2H, aromatic H). 13C NMR (d6-acetone, 125 MHz) δ:
19.8, 27.3, 27.7, 40.1, 57.1, 66.3, 67.1, 73.3, 74.4, 75.1, 80.3,
120.5, 120.6, 128.6, 128.9, 129.16, 129.24, 134.2, 134.3, 134.5,
136.4, 136.5, 137.9, 139.6, 139.9, 157.2, 173.7, 177.0. HRMS
(ESI-TOF) m/z: [M + H]+ calcd for C54H69N4O9Si 945.4828;
found 945.4826.
Data for 29a. An HPLC-purified sample on the semi-
preparative C18 column (80:20 CH3CN:/H2O isocratic
1
elution, 4 mL/min). H NMR (acetone-d6, 600 MHz) δ: 1.12
(s, 9H, tBu-CH3), 1.30 (s, 9H, tBu-CH3), 1.31 (s, 9H, tBu-
CH3), 3.75 (d, J = 10.3 Hz, 1H, C6H), 3.92 (d, J = 10.3 Hz, 1H,
C6H), 4.09 (m, 1H, C4H), 4.25 to 4.30 (m, 2H, C2H and C3H),
[4.57 (d, J = 11.0 Hz, Bn-CH) over 4.58 (s, OH), 2H], 4.74 (s,
2H, Bn-CH2), 4.90 (d, J = 10.9 Hz, 1H, Bn-CH), 5.11 (d, J =
12.6 Hz, 1H, Bn-CH), 5.17 (d, J = 12.6 Hz, 1H Bn-CH), 6.83
(s br, 0.5 H, NH), 6.94 (s, 1H, NH), 7.10 (d, J = 8.5 Hz, 2H,
aromatic H), 7.24 (d, J = 8.5 Hz, 2H, aromatic H), 7.27 to 7.35
(m, 3H, aromatic H), 7.40 to 7.50 (m, 8H, aromatic H), 7.60
(d, J = 8.5 Hz, 2H, aromatic H), 7.63 (d, J = 8.5 Hz, 2H,
aromatic H), 7.70 to 7.74 (m, 4H, aromatic H), [8.54 (s), 8.55
(s), 2H, piv-NH].13C NMR (acetone-d6, 150 MHz) δ: 19.9,
27.4, 27.8, 40.2, 66.8, 67.3, 75.2, 75.6, 79.1, 79.4, 83.9, 120.6,
120.7, 128.56, 128.60, 128.7, 128.8, 129.0, 129.1, 129.2, 130.81,
130.84, 133.7, 133.9, 134.2, 134.6, 136.5, 136.7, 138.4, 139.8,
139.9, 170.2, 177.1, 177.1. HRMS (ESI-TOF) m/z: [M +
NH4]+ calcd for C54H70N5O9Si 960.4937; found 960.4939.
Data for 29b. An HPLC-purified sample on the semi-
preparative C18 column (80:20 CH3CN/H2O isocratic elution,
Data for 28b. 1H NMR (CD3OD, 600 MHz) δ: 1.04 (s, 9H,
tBu-CH3), 1.28 (s, 9H, tBu-CH3), 1.30 (s, 9H, tBu-CH3), 3.45
(dd, J = 10.9, 4.9 Hz, 1H, C6H), 3.80 (dd, J = 10.9, 7.8 Hz, 1H,
C6H), 3.91 (dd, J = 8.3, 1.7 Hz, 1H, C4H), 4.12 (ddd, J = 7.7,
4.9, 1.8 Hz, 1H, C5H), 4.34 (d, J = 1.9 Hz, 1H, C2H), 4.38 (dd,
J = 8.3, 1.8 Hz, 1H, C3H), 4.49 (d, J = 10.6 Hz, 1H, Bn-CH),
4.62 (d, J = 11.0 Hz, 1H, Bn-CH), 4.65 (d, J = 10.6 Hz, 1H, Bn-
CH), 4.81 (d, J = 11.0 Hz, 1H, Bn-CH), 4.89 (d, J = 12.4 Hz,
1H, Bn-CH), 5.03 (d, J = 12.04 Hz, 1H, Bn-CH), 7.16 (d, J =
8.5 Hz, 2H, aromatic H), 7.24 to 7.48 (m, 17H, aromatic H),
7.66 to 7.71 (m, 4H, aromatic H). 13C NMR (CD3OD, 125
MHz) δ: 20.2, 27.5, 27.7, 27.8, 30.7, 40.5, 57.7, 63.3, 68.1, 68.2,
75.5, 75.9, 76.0, 76.2, 80.9, 85.2, 122.2, 122.39, 122.43, 128.7,
128.9, 129.0, 129.06, 129.11, 129.5, 129.7, 130.9, 131.0, 134.7,
135.0, 135.7, 136.4, 136.8, 136.9, 137.1, 137.9, 139.0, 139.2,
158.4, 175.8, 179.8. HRMS (ESI-TOF) m/z: [M + H]+ calcd
for C54H69N4O9Si 945.4828; found 945.4835.
1
4 mL/min). H NMR (acetone-d6, 600 MHz) δ: 1.09 (s, 9H,
tBu-CH3), 1.30 (s, 9H, tBu-CH3), 1.31 (s, 9H, tBu-CH3), 3.82
(d, J = 9.8 Hz, 1H, C6H), 4.03−4.05 (m, 2H, C6H and C4H),
4.11 (dd, J = 8.5, 6.3 Hz, 1H, C3H), 4.61 (dd, J = 9.1, 9.1 Hz,
1H, C2H), 4.66 (d, J = 11.3 Hz, 1H, Bn-CH), 4.71 (s, 2H, Bn-
CH2), 4.74 (d, J = 11.3 Hz, 1H, Bn-CH), 5.10 (d, J = 12.6 Hz,
1H, Bn-CH), 5.12 (d, J = 2.7 Hz, 1H, OH), 5.16 (d, J = 12.6
Hz, 1H, Bn-CH), 7.15 (d br, 1H, NH), 7.19 (d, J = 8.5 Hz,
2H), 7.24 (d, J = 8.5 Hz, 2H, aromatic H), 7.29 to 7.48 (m, 10
H, aromatic H), [7.63 (d, J = 8.5 Hz) overlapping 7.64 (d, J =
8.5 Hz), 4H, aromatic H], 7.75 to 7.78 (m, 4H, aromatic H),
8.58 (s, 1H, piv-NH), 8.60 (s, 1H, piv-NH). 13C NMR
(acetone-d6, 150 MHz) δ: 19.8, 27.3, 27.7, 40.16, 40.18, 66.7,
67.9, 73.9, 74.3, 80.1, 83.3, 85.2, 120.64, 120.65, 128.5, 128.6,
128.66, 128.68, 129.0, 129.1, 129.2, 130.67, 130.70, 133.7,
133.8, 134.0, 134.4, 136.49, 136.50, 138.4, 138.8, 140.0, 169.1,
177.05, 177.09. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C54H67N4O9Si 943.4672; found 943.4665.
5-Amino-2-(benzyloxycarbonyl)amino-2,5-dideoxy-
3,4-bis-O-(4-(pivaloylamino)benzyl)-D-glucono-1,5-lac-
tam (30). Formic acid (0.5 mL), 29a (0.475g, 0.50 mmol), and
NaCNBH3 (55 mg, 0.88 mmol) were mixed in anhyd CH3CN
(10 mL), and the mixture was refluxed for 3.5 h. The solvent
was removed and H2O (50 mL) and hexanes/EtOAc (1:3, 50
mL) were added to the residue. The lactam was then extracted
into the organic phase. Two additional washes of hexanes/
EtOAc were performed, the combined organic phases were
dried (Na2SO4), and then the solvent was removed. Then a 1
Preparation of Gluconamide 28c. Lactone 27a (0.346 g,
0.37 mmol) was dissolved in 7 N NH3 in MeOH (2 mL), and
the mixture was stirred for 30 min. The solvent was removed,
and the residue was chromatographed on a 25 g silica cartridge
(3:1 EtOAc/hexanes) to afford 290 mg 0.3 mmol, 82%) of a
mixture of gluconamide 28a and 28c (TLC Rf = 0.4) in 19%
1
and 63% yields, respectively; both were white solids. 28c: H
NMR (CD3OD, 600 MHz) δ: 1.06 (s, 9H, tBu-CH3), 1.27 (s,
9H, tBu-CH3), 1.29 (s, 9H, tBu-CH3), 3.79 (dd, J = 10.8, 4.3
Hz, 1H, C6H), 3.83 (dd, J = 8.0, 1.7 Hz, 1H, C4H), 3.89−3.93
(m, 2H, C5H, C6H), 4.21 (dd, J = 8.3, 1.7 Hz, 1H, C3H), 4.38
(d, J = 10.5 Hz, 1H, Bn-CH2), 4.44 (d, J = 10.5 Hz, 1H, Bn-
CH2), 4.55 (s, 2H, Bn-CH2), 4.60 (d, J = 8.3 Hz, 1H, C2H),
5.03 (d, J = 12.4 Hz, 1H, Bn-CH2), 5.14 (d, J = 12.5 Hz, 1H,
Bn-CH2), 6.99 (d, J = 8.4 Hz, 2H, aromatic H), 7.22−7.28 (m,
5H, aromatic H), 7.31−7.38 (m, 8H, aromatic H), 7.40−7.45
(m, 4H, aromatic H), 7.66−7.69 (m, 4H, aromatic H), 7.97 (s,
0.1H, Piv-NH), 8.54 (s, 0.04H, Piv-NH). 13C NMR (CD3OD,
150 MHz) δ: 20.1, 27.5, 27.77, 27.80 40.49, 40.52, 56.3, 66.6,
67.8, 72.4, 74.7, 74.8, 79.0, 79.4, 122.2, 122.3, 128.8, 128.87,
128.91, 129.0, 129.5, 129.7, 129.8, 130.9, 131.0, 134.4, 134.6,
135.0, 135.4, 136.87, 136.93, 138.1, 139.3, 158.2, 170.3, 175.8,
179.78, 179.81. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C54H69N4O9Si 945.4828; found 945.4833.
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M solution of TBAF in THF (625 μL) was treated with AcOH
(250 μL); the lactam product residue was redissolved in
CH3CN (6 mL), and this was added to the TBAF solution.
After 36 h of stirring, the reaction was incomplete (LC−MS).
More 1 M TBAF (625 μL) was mixed with AcOH (250 μL),
and the solution was added to the reaction mixture, which was
then heated to 80 °C for 3 h. The solvent was removed, and
then the residue was chromatographed on a 80 g silica cartridge
(10:0 to 10:1 EtOAc/MeOH), and then fractions with
coeluting products were rechromatographed on a 25 g silica
cartridge (20:1 to 10:1 EtOAc/MeOH) to afford 30a (TLC Rf
= 0.7 with 10:1 EtOAc/MeOH, 30 mg, 44 μmol, 8.8%, 2 steps)
and 30b (TLC Rf = 0.45 with 10:1 EtOAc/MeOH, recrystal-
lized from EtOAc, 147 mg, 0.213 mmol, 43%, 2 steps) and two
other minor lactam components that were not characterized.
Compounds 30a and 30b were white powders.
20:1 to 0:1 (50 mM NH4·COO(aq)/MeOH); product fractions
with 31-N-(CBz) (determined by MS) were pooled and then
lyophilized. The residue was dissolved in H2O (4 mL) and this
was passed through a DEAE-Sepharose Fast Flow column (1.5
cm dia × 8 cm length). The flow-through was collected to
afford 32-N-(CBz) (1.5 mg, 4.8 μmol, 12% after lyophilization),
then eluted with 140 mM NH4·HCO3; 31-N-(CBz) eluted in
the first column volume of NH4·HCO3 and the DDQ-related
compounds were retained on the column. Final purification on
the analytical anion exchange column using an identical elution
profile afforded 31-N-(CBz)·NH4 (4.0 mg, 10 μmol, 26% for 2
steps) after lyophilization. Then, 31-N-(CBz)·NH4 (4 mg, 10
μmol), ammonium formate (20 mg), and Pd(OH)2/C (5 mg)
were combined in H2O (1 mL). The mixture was heated for 20
min at 80 °C, then the solution was filtered, and the filtrate was
lyophilized 2× to afford 31 (2.6 mg, 10 μmol, 99%), which was
a white powder. 1H NMR (D2O, 600 MHz): 3.58 (ddd, J = 8.6,
5.3, 2.7 Hz, 1H, C5H), 3.94 (ddd, J = 8.4, 8.4, 1.4 Hz, 1H,
C4H), 3.96−4.03 (m, 3H, C2H, C3H, C6H), 4.09 (ddd, J = 11.1,
5.5, 2.8 Hz, 1H, C6H). 13C NMR DEPTQ (D2O, 150 MHz) δ:
56.9 (+), 59.2 (+, d, J = 7.2 Hz), 66.4 (−, d, J = 4.1 Hz), 71.1
(+), 73.0 (+), 170.7 (−). HRMS (ESI-TOF) m/z: [M + H]+
calcd for C6H14N2O7P 257.0533; found 257.0528.
1
Data for 30a. H NMR (CD3OD, 600 MHz) δ: [1.28 (s),
1.29 (s), 18H, tBu-CH3], 3.74 (dd, J = 10.9, 3.9 Hz, 1H, C6H),
3.85 to 3.95 (m, 1H, C5H), 3.98 to 4.03 (m, 1H, C6H), 4.17 to
4.22 (m, 1H, C4H), 4.30 (dd, J = 7.2, 4.2 Hz, 1H, C3H), [4.50
(d br, J = 10.9 Hz, C2-NH), 4.55 (d br, J = 9.8 Hz, Bn-CH)
over (s br, Bn-CH), 3H], 4.63 (s br, 2H, C2H, ring NH), [5.11
(d, J = 12.7 Hz, 1H), 5.13 (d, J = 12.7 Hz, 1H), Bn-CH2), 7.21
(d br, J = 7.7 Hz, 2H, aromatic H), 7.26 to 7.36 (m, 7H,
aromatic H), [7.48 (d, J = 2.8 Hz), 7.49 (d, J = 2.8 Hz) 4H,
aromatic H]. 13C NMR (DMSO-d6, 150 MHz) δ: 27.2, 58.5,
59.5, 62.4, 63.0, 63.6, 66.1, 66.3, 70.7, 70.8, 71.6, 79.9, 80.7,
80.8, 81.5, 119.9, 120.0, 126.9, 127.3, 127.6, 127.8, 128.1, 128.3,
128.4, 132.5, 132.7, 136.7, 138.7, 138.8, 154.2, 154.7, 170.3,
170.6, 176.4. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C38H49N4O8 689.3545; found 689.3548.
General Silylation Conditions. Under an Ar atmosphere,
the silylation substrate (3 mmol) and diisopropylethylamine
(7.8 mL, 45 mmol, 15 equiv) were mixed in dry DMF (20 mL).
Then, chlorotriethylsilane (6 mL, 36 mmol, 12 equiv) was
added, and the reaction flask was heated in an oil bath at 60 °C
for 4 h. The solvent was removed, and the residue was
chromatographed.
Triethylsilyl 2-Deoxy-2-((phenylmethoxy)carbonyl)-
amino-3,4,6-tris-O-(triethylsilyl)-D-glucopyranoside (33).
The general silylation conditions were used with GlcN-(CBz)
(0.107 g, 0.34 mmol), DMF (2.5 mL), diisopropylethylamine
(1.25 mL), and TES-Cl (0.8 mL). Chromatography on a 40 g
silica cartridge (1:1 to 0:1 hexanes/DCM then 1:1 to 1:1
DCM/EtOAc) afforded 33 (TLC Rf = 0.5; 10:1 hexanes,E-
tOAc, 0.16 g, 0.21 mmol, 62%). A tris-TES product
(incompletely silylated) product (TLC Rf = 0.25) was also
collected (45 mg), and this was set aside for recovery by
resilylation; additional bis-TES products can also be isolated
and recovered, accounting for an additional 20−30% of the
starting material. The reaction could be scaled to 3 mmol with
1
Data for 30b. H NMR (CD3OD, 600 MHz) δ: [1.28 (s),
1.29 (s) 18H, tBu-CH3], 3.45 (m, 1H, C5H), 3.58 (dd, J = 11.2,
5.2 Hz, 1H, C6H), 3.73 (dd, J = 11.2, 2.7 Hz, 1H, C6H), 3.78
(dd, J = 8.8, 8.8 Hz, 1H, C4H), 3.93 (dd, J = 9.3, 9.3 Hz, 1H,
C3H), 4.06 (d, J = 9.9 Hz, 1H, C2H), [4.62 (d, J = 11.1 Hz),
4.64 (d, J = 11.0 Hz), 2H, Bn-CH2], 4.68 (d, J = 11.1 Hz, 1H,
Bn-CH), 4.82 (d, below H2O peak, Bn-CH), 5.04 (d, J = 12.3
Hz, 1H, Bn-CH), 5.10 (d, J = 12.3 Hz, 1H, Bn-CH), 7.20 (d, J
= 8.5 Hz, 2H, aromatic H), 7.23−7.36 (m, 7H, aromatic H),
7.46 (d, J = 8.5 Hz, 2H, aromatic H), 7.49 (d, J = 8.5 Hz, 2H,
aromatic H). 13C NMR (d6-DMSO, 150 MHz) δ: 27.2, 55.5,
56.0, 60.8, 65.3, 73.1, 73.4, 76.8, 80.1, 119.8, 119.9, 120.0,
127.6, 127.7, 127.8, 128.1, 128.3, 133.0, 133.1, 137.12, 138.7,
138.8, 156.4, 168.7, 176.36, 176.38. HRMS (ESI-TOF) m/z:
[M + H]+ calcd for C38H49N4O8 689.3545; found 689.3559.
2,5-Diamino-2,5-dideoxy-D-glucono-1,5-lactam-6-
phosphate (31). Lactam 30b (66 mg, 96 μmol) was dissolved
in anhyd py (1 mL), and the reaction vial was cooled in an ice
bath. Then, POCl3 (13.4 μL, 0.14 mmol) was added; 45 min
later H2O (0.25 mL) was added. After 30 min, the solvent was
removed, and the residue was chromatographed on a 40 g
1
similar yields. H NMR (acetone-d6, 600 MHz) δ: [anomeric
H-signals indicate a 4:1 ratio α:β anomers], 0.62 to 0.72 (m,
18H, Si-CH2), 0.73 to 0.79 (m, 6H, Si-CH2), 0.94 to 1.03 (m,
36H, CH3), 3.25 to 3.30 (m, 0.2H, βC5H), 3.31 to 3.37 (m,
0.2H, βC2H), 3.63 (ddd, J = 9.7, 9.7, 3.2 Hz, 0.8 H, αC2H),
3.67 to 3.75 (m, 1.8H), 3.81 to 3.85 (m, 1.2H), 3.88 (dd, J =
9.3, 7.7 Hz, 0.8H, αC3H), 3.92 (dd, J = 11.2, 3.3 Hz, 1H, C6H),
4.84 (d, J = 7.4 Hz, 0.2 H, βC1H), [5.06 (d, J = 12.4 Hz), 5.07
(s), 5.11 (d, J = 12.4 Hz), 2H, Bn-CH2], 5.16 (d, J = 3.2 Hz,
0.8H, αC1H), 5.77 (d, J = 10.1 Hz, 0.01H, αC2-NH), 6.31 (d, J
= 9.5 Hz, 0.03H, βC2-NH), 7.31 to 7.41 (m, 5H, aromatic H).
13C NMR (acetone-d6, 150 MHz) δ: 5.15, 5.19, 5.4, 5.9, 6.01,
7.0, 7.10, 7.2, 7.4, 58.8, 57.8, 60.8, 62.6, 63.3, 66.7, 66.9, 72.8,
72.9, 74.4, 74.9, 76.9, 78.0, 92.9, 96.7, 128.55, 128.63, 128.90,
128.94, 129.03, 129.07, 138.0, 138.1, 155.8, 156.8. HRMS (ESI-
TOF) m/z: [M + H − TES−OH]+ calcd for C32H60NO6Si3
638.3723; found 638.3731.
SiliaBond-C18 with a 20:1 to 0:1 (50 mM NH4·COO(aq)
/
MeOH) gradient. Fractions containing monophospho-30b
(determined by LC−MS) were pooled, and the mixture was
lyophilized; some unreacted 30b and a bis-phospho adduct of
30b coeluted with the desired product. Then 40% of the
lyophilized residue was mixed with DDQ (41 mg, 0.18 mmol)
were combined with CHCl3 (3 mL), H2O (1 mL) and 5% aq
NaHCO3 (300 μL). The mixture was heated for 6 h at 70 °C
and then stored at −20 °C for 1h. The solution was filtered,
and then the aq phase was separated and lyophilized. The
residue was chromatographed on a 40 g SiliaBond-C18 with
Triethylsilyl 2-Deoxy-2-((phenylmethoxy)carbonyl)-
amino-3,4-bis-O-(triethylsilyl)-α-D-glucopyranoside (34).
Compound 33 (0.5 g, 0.65 mmol) was dissolved in a mixture of
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DCM (7.5 mL) and MeOH (2.5 mL). Then, PPTS (2 mg) was
added, and the reaction was monitored by TLC (10:1 DCM/
EtOAc). After 1 h, no 33 (TLC Rf = 0.5) remained and a new
spot had appeared (TLC Rf = 0.2, 34). The mixture was loaded
directly on a 40 g silica cartridge and chromatographed (1:1 to
0:1 hexanes/DCM, then 1:0 to 1:1 DCM/EtOAc gradient).
Compound 34 was a colorless oil (0.25 g, 0.38 mmol, 58%);
the α-anomer was the sole, isolated product. More polar mono-
and bis-silylated products from this reaction can be subjected
again to the silylation conditions to reclaim another 30% of the
product as 33. 1H NMR (acetone-d6, 600 MHz) δ: 0.65 (q, J =
8.0 Hz, 6H, Si-CH2), 0.69 (q, J = 8.0 Hz, 6H, Si-CH2), 0.76 (q,
J = 7.9 Hz, 6H, Si-CH2), 0.96 (overlapping t’s, J = 8.0 Hz, 18H,
CH3), 1.01 (t, J = 8.1 Hz, 9H, CH3), 3.56 (dd, J = 7.7, 4.7 Hz,
1H, OH), 3.62 (ddd, J = 9.8, 9.8, 3.2 Hz, 1H, C2H), 3.69 (ddd,
J = 8.8, 2.9, 3.1 Hz, 1H, C5H), 3.70 to 3.79 (m, 3H, C4H, C6H,
C6H), 3.88 (dd, J = 9.5, 7.8 Hz, 1H, C3H), 5.07 (J = 12.3 Hz,
1H, Bn-CH), 5.11 (d, J = 12.3 Hz, 1H, Bn-CH), 5.14 (d, J = 3.2
Hz, 1H, αC1H), 5.76 (d, J = 10.3 Hz, 0.04 H, C2-NH), 7.30 to
7.39 (m, 5H, aromatic H). 13C NMR (acetone-d6, 150 MHz) δ:
5.1, 5.9, 6.0, 6.9, 7.4, 57.9, 61.6, 67.0, 72.9, 74.4, 74.8, 93.0,
128.7, 128.9, 129.2, 138.0, 156.9). HRMS (ESI-TOF) m/z: [M
+ H − TES − OH]+ calcd for C26H46NO6Si2 524.2858; found
524.2866.
residue was chromatographed on a 40 g silica cartridge using a
20:1 to 3:1 hexanes/EtOAc gradient. The first product to elute
from the column was 36 affording an isolated yield of 0.196 g
1
(a colorless oil, 0.29 mmol, 76%). H NMR (acetone-d6, 600
MHz) δ: 0.66 to 0.73 (m, 12H, Si-CH2), 0.75 (q, J = 7.9 Hz,
6H, Si-CH2), 0.95 (t, J = 7.9 Hz, 9H, CH3), 0.98 (t, J = 8.0 Hz,
9H, CH3), 1.01 (t, J = 8.0 Hz, 9H, CH3), 3.47 (dd, J = 13.1, 5.5
Hz, 1H, C6H), 3.58 (dd, J = 13.1, 2.7 Hz, 1H, C6H), 3.63 (dd, J
= 8.6, 8.3 Hz, 1H, C4H), 3.68 (ddd, J = 9.9, 9.9, 3.2 Hz, 1H,
C2H), 3.87 to 3.90 (m, 2H, C2H, C5H), 5.08 (d, J = 12.4 Hz,
1H, Bn-CH), 5.12 (d, J = 12.4 Hz, 1H, Bn-CH), 5.20 (d, J = 3.2
Hz, 1H, αC1H), 7.33 to 7.39 (m, 5H, aromatic H). 13C NMR
(acetone-d6, 150 MHz) δ: 5.1, 5.97, 6.02, 6.9, 7.3, 7.4, 52.4,
57.9, 67.2, 73.2, 74.4, 74.6, 92.9, 128.8, 129.0, 129.2, 137.9,
156.9. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C32H60NO6Si3 638.3723; found 638.3727.
2-Amino-6-azido-2,6-dideoxy-D-glucose (37). Com-
pound 36 (30 mg, 44 μmol) was treated with the standard
acid hydrolysis conditions on 1/4-scale and heating and stirring
were applied for only 3 h; the yield of 37, a white powder, was
10.5 mg (33 μmol, 75%) as the TFA salt. 1H NMR (D2O, 600
MHz) δ: [anomeric H-signals indicate a 2.5:1 ratio α:β
anomers and less prominent peaks for other forms of this sugar,
e.g., furanoses], 2.91 (dd, J = 10.6, 8.5 Hz, 1H, βC2H), 3.21
(dd, J = 10.6, 3.6 Hz, 2.6H, αC2H), 3.39 (dd, J = 9.5, 9.5 Hz,
3.6 H, αC2H), [overlapping dd’s: 3.45 (J = 13.4, 5.8 Hz), 3.47
(J = 13.6, 5.5 Hz) 3.6H], 3.50 to 3.61 (m, 6H), 3.77 (dd, J =
10.6, 9.2 Hz, 2.4 H, αC3H), 3.97 to 4.08 (m, 0.75 H), 4.21 (d, J
= 2.6 Hz, 0.24 H), 4.29 (d, J = 5.4 Hz, 0.24 H), 4.84 (d, J = 8.5
Hz, 1H, βC1H), 5.33 (d, J = 3.6 Hz, 2.1 H, αC1H), [6.16 (d, J =
5.4 Hz), 6.18 (d, J = 5.5 Hz) 0.2H]. 13C NMR (D2O/DMSO-
d6, 150 MHz; referenced to DMSO) δ: 52.2, 55.6, 55.7, 58.2,
65.6, 68.4, 70.9, 71.85, 71.91, 72.0, 73.3, 74.7, 76.2, 82.1, 90.7,
94.3, 105.3, [117.9 (q, J = 292.4 Hz), 164.0 (q, J = 35.5 Hz),
TFA]. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C6H13N4O4
205.0931; found 205.0932.
Triethylsilyl 6-Cyano-2,6-dideoxy-2-((phenylmeth-
oxy)carbonyl)amino-3,4-bis-O-(triethylsilyl)-α-D-gluco-
pyranoside (35). Under an Ar atmosphere, 34 (44 mg, 67
μmol) was dissolved in anhyd THF (2 mL). Then, five
additions of Ph3P (39 mg, 0.15 mmol), diisopropyl
azodicarboxylate (29.5 μL, 0.15 mmol), and acetone cyanohy-
drin (9.2 μL, 0.1 mmol) each were made. The first addition was
at the reaction start, and the remaining additions were made
over the next 36 h. The reaction progress was monitored by
LC−MS. At 38 h, the solvent was removed, and the residue was
chromatographed on a 25 g silica cartridge with a 0 to 25%
EtOAc in DCM gradient. The fractions with unreacted 34 and
those with 35 were identified by MS. Solvent removal from
these fractions afforded 34 (8 mg, 18%) and 35 (23.5 mg, 35
2,6-Diamino-2,6-dideoxy-D-glucose (38). Compound 36
(50 mg, 73 μmol) was mixed with ammonium formate (26 mg)
and Pd(OH)2/C (10 mg) in MeOH (1 mL). The mixture was
heated (60 °C) and stirred for 1 h, and then the Pd(OH)2 was
filtered off and washed with MeOH (0.5 mL). Then, H2O (5
mL) and TFA (1 mL) were added to the filtrate. The mixture
was stirred for 16 h and then extracted with EtOAc (2 × 2 mL).
The aqueous phase was lyophilized 2×, affording a viscous
resin, 38·(TFA)2 with (NH4·O2C2F3)6·TFA0.5·H2O0.5 (MW =
1258.56 g/mol; 50 μmol, 68%) based on NMR and elemental
1
μmol; 64% yield on 82% conversion). H NMR (acetone-d6,
600 MHz) δ: 0.67 to 0.75 (overlapping q’s, J = 7.9, 7.8 Hz,
12H, Si-CH2), 0.78 (q, J = 7.8 Hz, 6H, Si-CH2), 0.95 to 0.99
(overlapping t’s, J = 7.9, 7.8 Hz, 18 H, CH3), 1.03 (t, J = 7.9 Hz,
9H, CH3), 2.84 (dd, J = 17.0, 6.7 Hz, 1H, C6H), 2.90 (J = 17.0,
4.1 Hz, 1H, C6H), 3.58 (dd, J = 8.0, 8.0 Hz, 1H, C4H), 3.70
(ddd, J = 10.0, 10.0, 3.2 Hz, 1H, C2H), 3.92 (dd, J = 8.9, 7.7 Hz,
1H, C3H), 3.98 (ddd, J = 8.0, 6.7, 4.1 Hz, 1H, C5H) 5.09 (d, J =
12.3 Hz, 1H, Bn-CH), 5.12 (d, J = 12.3 Hz, 1H, Bn-CH), 5.21
(d, J = 3.1 Hz, 1H, αC1H), 5.85 (d, J = 10.1 Hz, 0.75 H, C2-
NH), 7.32 to 7.42 (m, 5H, aromatic H). 13C NMR (acetone-d6,
150 MHz) δ: 5.1, 5.99, 6.02, 6.9, 7.27, 7.32, 21.6, 57.7, 67.1,
70.0, 74.4, 76.1, 92.6, 118.7, 128.8, 129.0, 129.2, 137.9, 156.9.
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C33H60N2O6Si3
665.3832; found 665.3826. See Supporting Information for
HMBC and HMQC data.
Triethylsilyl 6-Azido-2,6-dideoxy-2-((phenylmeth-
oxy)carbonyl)amino-3,4-bis-O-(triethylsilyl)-α-D-gluco-
pyranoside (36). Under an Ar atmosphere, 34 (0.25 g, 0.38
mmol) was dissolved in anhyd THF (11 mL). Then, Ph3P
(0.39 g, 1.5 mmol), diisopropyl azodicarboxylate (0.275 mL,
1.4 mmol), and diphenylphosphorylazide (0.3 mL, 1.4 mmol)
were added in two equal portions, the first to initiate the
reaction, and the second was added 60 min later. Stirring was
continued for 6 h. Then the solvent was removed and the
1
analyses. H NMR (D2O, 600 MHz) δ: [2:1 ratio of α:β
anomers] 3.16 (dd, J = 10.6, 8.5 Hz, 0.5H, Hβ2), 3.22 to 3.30
(m, 1.5H), 3.45 (dd, J = 10.6, 3.6 Hz, 1H, Hα2), 3.49 to 3.62
(m, 3H), 3.78 to 3.83 (m, 1H), 4.01 (dd, J = 10.4, 9.2 Hz, 1H,
Hα3), 4.18 (ddd, J = 9.5, 9.5, 2.7 Hz, 1H, Hα5), 5.08 (d, J = 8.5
Hz, 0.5 H, Hβ1), 5.57 (d, J = 3.5 Hz, 1H, Hα1). 13C NMR
(D2O, 150 MHz) δ: 43.2, 57.0, 59.4, 70.3, 72.2, 74.3, 74.5, 74.8,
92.0, 95.6, [119.2 (q, J = 290.0 Hz), 165.7 (q, J = 35.0 Hz),
from TFA]. Anal. Calcd for C23H41.5F25.5N8O21.5: C, 21.95; H,
3.32; N, 8.90; F, 38.49. Found: C, 21.76; H, 3.17; N, 8.89; F,
38.55. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C6H15N2O4
179.1026; found 179.1028.
Triethylsilyl 2-Amino-2-deoxy-3,4,6-tris-O-(triethylsil-
yl)-α-D-glucopyranoside (39). The general silylation con-
ditions were applied to glucosamine·HCl (2× scale, 1.29 g, 6
mmol); a second addition of N,N-diisopropylethylamine (5.2
mL, 30 mmol) and chlorotriethylsilane (4 mL, 24 mmol) was
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made at 90 min. After another 60 min at 60 °C, solvent removal
followed by chromatography on an 80 g cartridge (10:1 to 0:1
of hexanes/EtOAc gradient) afforded 39 (TLC Rf = 0.45 with
5:1 hexanes/EtOAc, a colorless oil, 2.33 g, 3.66 mmol, 61%),
Triethylsilyl 2-Deoxy-2-((4-methoxyphenyl)-oxaziri-
din-2-yl)-3,4-bis-O-(triethylsilyl)-α-D-glucopyranoside
(43). Oxaziridine 41 (100 mg, 0.13 mmol) was dissolved in
MeOH (2.5 mL). Then DL-10-camphorsulfonic acid (100 μL of
a 1 mg/mL solution in MeOH) was added, and the mixture was
stirred for 10 min. The solvent volume was reduced to 0.5 mL,
and then the mixture was loaded directly on to a 25 g silica
cartridge and chromatographed (DCM/EtOAc gradient: 100:0
to 9:1 in 2.5 column volumes then to 0:1 in 4 column
volumes). The first compound to elute was unreacted 41 (4.5
mg, 4.5%; TLC Rf = 0.95 with 20:1 DCM/EtOAc), which was
followed by 43 (a viscous oil, TLC Rf = 0.85, 38 mg, 58 μmol,
45%). Another band (TLC Rf = 0.45, 20 mg) was consistent
(MS) with bis-O-(triethylsilyl) sugar(s). Subsequent elution
with 4:1 EtOAc/MeOH eluted the final product(s) from the
column that were consistent (MS) with mono-O-(triethylsilyl)
sugar(s) (10 mg, 23 μmol, 18%). 43: 1H NMR (acetone-d6, 600
MHz) δ: 0.37 to 0.52 (m, 6H, Si-CH2), 0.76 to 0.83 (m, 15H,
SiCH2CH3 and Si-CH2), 0.87 to 0.92 (m, 6H, Si-CH2), 1.03 (t,
J = 7.9 Hz, 9H, SiCH2CH3), 1.10 (t, J = 7.9 Hz, 9H,
SiCH2CH3), 2.16 (dd, J = 9.4, 3.0 Hz, 1H, C2H), 3.41 (dd, J =
6.7, 5.2 Hz, 1H, C6H), 3.66 (dd, J = 8.6, 8.6 Hz, 1H, C4H), 3.69
to 3.74 (m, 3H, OH, C6H, C5H), 3.83 (s, 3H, O-CH3), 4.43
(dd, J = 9.2, 8.3 Hz, 1H, C3H), 4.84 (s, 1H, oxaziridine CH),
5.38 (d, J = 2.9 Hz, 1H, αC1H), 6.97 (d, J = 8.7 Hz, 2H,
aromatic H), 7.46 (d, J = 8.7 Hz, 2H, aromatic H). 13C NMR
(acetone-d6, 125 MHz) δ: 5.1, 5.9, 6.1, 6.8, 7.4, 7.6, 29.6, 55.6,
61.6, 73.0, 74.86, 74.91, 76.7, 76.8, 93.2, 114.4, 128.3, 130.4,
162.1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C32H62NO7Si3 656.3829; found 656.3830.
1
exclusively as it is α-anomer. H NMR (C6D6, 500 MHz) δ:
0.60 (q, J = 7.8 Hz, 6H), 0.715 (overlapping q’s, J = 7.9 Hz,
6H), 0.83 to 0.94 (m, 12H), 0.98 (t, J = 8.0 Hz, 9H), 1.07 to
1.05 (overlapping t’s, J = 8.0 Hz, 27H), 2.55 (dd, J = 8.6, 3.2
Hz, 1H), 3.86 to 3.92 (m, 4H), 4.08 (dd, J = 11.1, 2.8 Hz, 1H),
4.99 (d, J = 3.3 Hz, 1H). 13C NMR (CD3CN, 125 MHz) δ: 5.0,
5.1, 5.8, 6.4, 6.9, 7.1, 7.4, 7.5, 58.1, 62.5, 72.5, 74.0, 78.0, 95.4.
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C30H70NO5Si4
636.4326; found 636.4318.
Triethylsilyl 2-Deoxy-2-(4-methoxyphenylmethyli-
dene)-amino-3,4,6-tris-O-(triethylsilyl)-α-D-glucopyrano-
side (40). Compound 39 (2.33 g, 3.66 mmol) was mixed with
p-anisaldehyde (0.67 mL, 5.5 mmol) and PPTS (15 mg) in
toluene (40 mL). The mixture was refluxed 2 h; water was
removed using a Dean−Stark trap. Then, the solvent was
removed, and the residue was chromatographed on a 50 g silica
cartridge (10:1 to 5:1 hexanes/EtOAc gradient). The first
compound to elute (TLC Rf = 0.4; 10:1 hexanes/EtOAc, some
hydrolysis of the imine occurred on the TLC plate) was 40 (a
1
colorless oil, 2.52 g, 3.34 mmol, 91.3%). H NMR (acetone-d6,
500 MHz) δ: 0.58 to 0.76 (m, 18H), 0.81 to 0.86 (m, 6H), 0.93
(t, J = 7.9 Hz, 9H), 1.01 to 1.22 (m, 27H), 3.16 (dd, J = 9.4, 3.1
Hz, 1H), 3.75 (dd, J = 9.5, 8.5 Hz, 1H), [3.94 (s) overlapping
3.85 to 3.96 (m), 6H], 4.44 (dd, J = 9.4, 8.0 Hz, 1H), [α-
anomeric H: 5.02 (d, J = 3.2 Hz); β-anomeric H: 4.82 (d, J =
7.3 Hz); α:β ratio =17:1, 1H], 7.08 (d, J = 8.8 Hz, 2H), 7.83 (d,
J = 8.8 Hz, 2H), 8.34 (s, 1H). 13C NMR (CD3CN, 125 MHz)
δ: 5.17, 5.21, 5.99, 6.00, 6.88, 6.95, 7.0, 7.2, 7.4, 56.0, 63.1, 73.2,
74.1, 75.9, 77.9, 96.0, 114.7, 130.4, 130.9, 162.6, 163.2. HRMS
(ESI-TOF) m/z: [M + H]+ calcd for C38H76NO6Si4 754.4744;
found 754.4739.
2-Deoxy-2-(N-hydroxyl-amino)-D-glucose (42). Oxazir-
idine 41 (125 mg, 0.162 mmol) was dissolved in TFA/H2O (2
mL/0.5 mL). The mixture was stirred for 5 h, then another 2.5
mL of H2O was added, and stirring was continued overnight.
Then DCM (5 mL) was added, and the mixture was stirred for
2 h. The organic phase was discarded, and the aq phase was
lyophilized (2×) to afford 30 mg of a mixture of 42 and a
benzylidene acetal of 42; the mixture was an off-white powder.
NMR analysis showed one α-anomeric and two β-anomeric
Triethylsilyl 2-Deoxy-2-((4-methoxyphenyl)-oxaziri-
din-2-yl)-3,4,6-tris-O-(triethylsilyl)-α-D-glucopyranoside
(41). Imine 40 (2.52 g, 3.34 mmol) was dissolved in DCM (20
mL), and then saturated aq NaHCO3 (3.3 mL) and m-CPBA
(70%, 1.14 g) were added. The solution was stirred vigorously
for 2 h; the organic and aqueous layers were separated, and the
latter was extracted with DCM (20 mL). The combined organic
layers were dried (Na2SO4); after solvent removal the residue
was chromatographed on a 50 g silica cartridge (1:1 to 1:0
DCM/hexanes gradient). A minor product of the reaction with
the same MS as 41 eluted first (Rf = 0.65, typically 5% yield)
and was discarded. The yield of the main product of 41 was
1.44 g (a viscous oil, TLC Rf = 0.5 with 2:1 DCM/hexanes,
1
sugars. H NMR (D2O, 600 MHz) δ: the C1H, C2H, and C3H
signals, respectively, inside [ ]: α-anomer [5.69 (d, J = 3.5 Hz,
0.8H), 3.61 (dd, J = 10.8, 3.5 Hz, 0.8H)], β-anomer no. 1 [5.33
(d, J = 7.9 Hz, 0.5 H), 3.85 (in m), 4.29 (dd, J = 10.0, 8.8 Hz,
0.5H)], β-anomer no. 2 [5.22 (d, J = 8.2 Hz, 0.6H), 3.25 (dd, J
= 10.7, 8.2 Hz, 0.6H), 4.1 (dd, J = 10.7, 8.7 Hz, 0.6H)], 7.92 (s,
0.5 H). Aromatic H’s: 7.19 (d, J = 9.1 Hz, 1H), 8.33 (d, J = 9.1
Hz, 1H). Based on integration values, β-anomer no. 1
corresponds with the putative p-OMe benzylidene acetal of
42, which is in a 1:3 ratio with 42. The remaining signals
[3.56−3.65 (m, 4.4H), 3.81−3.88 (m, 3H), 3.91−4.06 (m,
6.5H)] overlapped and no further interpretation was possible.
13C NMR (D2O, 150 MHz) δ: 58.2, 63.0, 63.2, 63.3, 66.4, 67.6
70.3, 70.5, 72.4, 72.6, 72.8, 73.3, 74.1, 78.66, 78.73, 82.0, 90.2,
93.2, 95.3, 117.1, 118.1, 120.0, 124.3, 135.3, 145.5. HRMS
(ESI-TOF) m/z: [M + H]+ calcd for C6H14NO6 196.0816;
found 196.0813. Putative p-OMe benzylidene acetal: [M + H]+
calcd for C14H20NO7 314.1234; found 314.1232.
1
1.86 mmol, 56%). H NMR (acetone-d6, 500 MHz) δ: 0.38 to
0.54 (m, 6H, Si-CH2), 0.65 (q, J = 7.9 Hz, 6H, Si-CH2), 0.77 to
0.84 (m, 15H, Si-CH2, SiCH2CH3), 0.91 (t, J = 7.7 Hz, 6H, Si-
CH2), 1.00 (t, J = 7.9 Hz, 9H, SiCH2CH3), 1.04 (t, J = 7.9 Hz,
9H, SiCH2CH3), 1.10 (t, J = 7.8 Hz, 9H, SiCH2CH3), 2.17 (dd,
J = 9.4, 3.1 Hz, 1H, C2H), 3.65 (dd, J = 9.2, 8.3 Hz, 1H, C4H),
3.72 (ddd, J = 9.3, 3.7, 1.9 Hz, 1H, C5H), 3.80 (dd, J = 11.4, 1.9
Hz, 1H, C6H), 3.84 (s, 3H, OCH3), 3.88 (dd, J = 11.4, 3.8 Hz,
1H, C6H), 4.42 (dd, J = 9.2, 8.4 Hz, 1H, C3H), 4.89 (s, 1H,
oxaziridine CH), 5.42 (d, J = 3.1 Hz, 1H, αC1H), 6.97 (d, J =
8.7 Hz, 2H, aromatic H), 7.48 (d, J = 8.7 Hz, 2H, aromatic H);
13C NMR (acetone-d6, 125 MHz) δ: 5.1, 5.2, 5.9, 6.1, 6.8, 7.1,
7.4, 7.6, 56.6, 62.7, 73.0, 74.9, 75.0, 76.7, 76.8, 91.1, 114.4,
128.3, 130.4, 162.1. HRMS (ESI-TOF) m/z: [M + H]+ calcd
for C38H76NO7Si4 770.4693; found 770.4691.
Triethylsilyl 2-[[Bis[[(1,1-dimethylethoxy)carbonyl]-
amino]methylene]amino]-2-deoxy-3,4,6-tris-O-(triethyl-
silyl)-α-D-glucopyranoside (47). The general silylation
conditions were applied to 4532 (0.505g, 1.2 mmol, 0.6×
scale) with the addition of DMAP (50 mg). After solvent
removal, the mixture was chromatographed 3× on 90 g silica
4741
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cartridges (100:7.5 to 10:1 hexanes/EtOAc) to separate the
main product of 47 (TLC Rf = 0.5 100:7.5 hexanes/EtOAc)
exclusively as the α-anomer from two minor products of 47
(TLC Rf = 0.55, 27 mg; Rf = 0.45, 45 mg). The isolated yield of
the main 47 isomer was 675 mg (a viscous oil, 0.77 mmol,
mL injections per run and a gradient of 0−200 mM
NH4·HCO3. The fractions with 49-BOC were lyophilized 2×
to afford 0.251 g of an off-white powder. Then 49-BOC (125
mg) was dissolved in 0.25 M HCl (5 mL), the reaction vial was
heated to 95 °C for 1 h, and then the mixture was allowed to
cool. The solution was neutralized with saturated NaHCO3 ;
TLC analysis (5:4:1 nPrOH/concd NH4OH/H2O) indicated
the conversion from 49-BOC (Rf = 0.75) to 49 (Rf = 0.25).
The sample was lyophilized and then purified by “Aqueous
Normal Phase” HPLC31 on a semipreparative Cogent Diamond
Hydridecolumn (Microsolv, 1 × 25 cm, 4 μm; A = 50 mM aq
NH4·HCO3, B = CH3CN; 0 to 3 min (95% B), 3 to 13 min
(gradient to 30% B)). The CH3CN was removed from product
containing fractions on a rotary evaporator with a water bath at
50 °C, and then the remaining solution was lyophilized to
afford purified 49 (a white powder, 13.1 mg, 46 μmol, 30%
1
64%); the minor products were discarded. H NMR (acetone-
d6, 500 MHz) δ: 0.62 to 0.71 (m, 18H, Si-CH2), 0.74 to 0.79
(m, 6H, Si-CH2), 0.94 to 1.02 (m, 36H, SiCH2CH3), 1.42 (s,
9H, tBu-CH3), 1.55 (s, 9H, tBu-CH3), 3.70 to 3.75 (m, 2H),
3.84 (dd, J = 11.4, 2.0 Hz, 1H), 3.91 to 3.96 (m, 2H), 4.22
(ddd, J = 9.4, 9.4, 3.2 Hz, 1H, C2H), 5.19 (d, J = 3.2 Hz, 1H,
αC1H), 8.56 overlapping d’s, J = 9.4 Hz, 1H, C2-NH), 11.80 (s,
0.2 H, guanidine NH(BOC)). 13C NMR (acetone-d6, 125
MHz) δ: 5.1, 5.8, 5.9, 6.9, 7.1, 7.3, 7.4, 28.0, 28.4, 56.5, 62.5,
72.5, 74.8, 74.9, 79.0, 84.0, 92.2, 153.8, 157.1, 164.6. HRMS
(ESI-TOF) m/z: [M + H]+ calcd for C41H88N3O9Si4 878.5592;
found 878.5591.
1
from 48). H NMR (D2O, 600 MHz) δ: 3.70 (m, 0.2H), 3.91
(dd, J = 8.4, 2.5 Hz, 1H), 3.93 to 4.05 (m, 1.6 H), 4.06 (dd, J =
8.0, 4.3 Hz, 0.2 H), 4.08 to 4.20 (m, 2.8H), 4.25 to 4.28 (m,
0.2H), 5.02 (d, J = 4.3 Hz, 0.2 H), 5.06 (d, J = 2.9 Hz, 0.8 H),
5.07 (d, J = 2.3 Hz, 0.2H), 6.87 (s, 0.8H), 6.90 (s, 0.2H). 13C
NMR DEPTQ (D2O, 150 MHz) δ: (+) 66.8, (+) 66.9, (+)
68.3, (−) 69.0 (d, J = 5.0 Hz), (−) 69.1 (d, J = 5.3 Hz), (+)
72.6 (d, J = 7.5 Hz), (+) 73.7 (d, J = 7.6 Hz), (+) 74.3 (d, J =
6.3 Hz), (+) 74.9, (+) 75.2, (+) 77.8 (d, J = 5.7 Hz), (−) 113.1,
(−) 130.1, (−) 150.7. HRMS (ESI-TOF) m/z: [M + H]+ calcd
for C7H14N3O7P 284.0642; found 284.0637
Triethylsilyl 2-[[Bis[[(1,1-dimethylethoxy)carbonyl]-
amino]methylene]amino]-2-deoxy-3,4-bis-O-(triethylsil-
yl)-α-D-glucopyranoside (48). Compound 47 (0.65 g, 0.74
mmol) was dissolved in a mixture of CH2Cl2 (10 mL) and
MeOH (20 mL). Then PPTS was added (20 mg), and the
mixture was stirred for 12 h. The solvent was removed, and the
residue was chromatographed on a 90 g silica cartridge (10:1 to
5:1 hexanes/EtOAc). Unreacted 47 (TLC 0.182 g, 28%, Rf =
0.85 with 10:1 hexanes/EtOAc) and 48 (a viscous oil, 0.25 g,
0.33 mmol, Rf = 0.25, 45%, or 61% on 72% conversion) were
isolated. 1H NMR (C6D6, 500 MHz) δ: [NMR data suggest the
presence of two species, both α-anomers, consistent with 2
conformers and in a 1:0.16 ratio] 0.55 (q, J = 7.9 Hz, 4H, Si-
CH2), 0.63 to 0.75 (m, 2H, Si-CH2), 0.79 to 0.88 (m, 12H, Si-
CH2), 0.94 (t, J = 8.0 Hz, 6H, SiCH2CH3), [1.02 to 1.08 (m)
overlapping with 1.11 (t, J = 8.0 Hz), 21H, SiCH2CH3], [1.20
(s) and 1.24 (s), 9H, tBu-CH3], [1.44 and 1.45 (s), 9H, tBu-
CH3], [3.74 to 3.80 (m) and 3.83 to 3.88 (m) and 3.91 to 3.95
(m), 4H], [4.04 (dd, J = 10.3, 5.6 Hz), 4.11 (dd, J = 10.2, 5.2
Hz), 4.16 (dd, J = 9.9, 8.1 Hz) and 4.26 (m), 2H], [4.59 (ddd, J
= 8.5, 6.5, 2.7 Hz, and 4.71 (ddd, J = 9.7, 9.8, and 3.4 Hz), 1H,
C2H], [5.30 (d, J = 3.4 Hz) over 5.31 (d, J = 2.7 Hz), 1H,
αC1H], [8.75 (d, J = 9.7 Hz) and 9.06 (d, J = 8.5 Hz) 1H, C2-
NH], [12.27 (s) and 12.51 (s), 1H]. 13C NMR (C6D6, 125
MHz) δ: 4.8, 4.9*, 5.3*, 5.4*, 5.7, 6.7, 7.1*, 7.2*, 7.3, 7.4, 27.6,
27.7*, 28.3, 31.9, 55.4*, 56.4, 61.8, 62.5*, 70.9*, 72.5, 73.3,
73.8*, 74.6, 77.3*, 78.7*, 78.8, 82.4*, 82.6, 90.7*, 92.3, 153.0*,
153.6, 156.8, 157.3*, 164.2*, 164.7. * = smaller peaks adjacent
to larger peaks, supporting the presence of a conformer. HRMS
(ESI-TOF) m/z: [M + H]+ calcd for C35H74N3O9Si3 764.4727;
found 764.4729.
2-[(Aminoiminomethyl)amino]-2-deoxy-D-glucose-6-
phosphate (49). Compound 48 (0.23 g, 0.30 mmol) was
dissolved in anhyd py (3 mL). Then POCl3 (35 μL, 0.375
mmol) was added, and the mixture was stirred for 40 min, at
which point H2O (0.5 mL) was added. After 5 min, the solvent
was removed; then the residue was mixed with DMF/H2O/
AcOH (3.2 mL/1.6 mL/0.8 mL), and the mixture was stirred
for 12 h. The solvent was removed, and the mixture was taken
up in H2O (5 mL) and EtOAc (5 mL); the pH of the aq phase
was adjusted to 7 with 1 N NaOH. The biphasic mixture was
shaken, and after separation, the organic phase was discarded;
the aq phase was washed 2× more with EtOAc (5 mL ea).
Then 49, as the mono-BOC adduct (49-BOC), was isolated by
anion exchange chromatography on a DEAE-Sepharose Fast
Flow column (1.5 cm dia × 8 cm length) with 6 injections of 2
ASSOCIATED CONTENT
* Supporting Information
■
S
General experimental information, copies of 1D and 2D NMR
spectra, MS/HRMS and HPLC analyses. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: jeff.posakony@nih.gov; adrian.ferre@nih.gov.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank N. Tjandra and his group (National
Heart, Lung and Blood Institute, NHLBI) for assistance with
NMR experiments. D.-Y. Lee of the NHLBI Biochemistry Core
Facility, R. Levine (NHLBI), P. Gafken (Proteomics Core at
Fred Hutchinson Cancer Research Center, FHCRC) and D.
Shen’s group (University of Washington) assisted with MS and
LC−MS experiments. We also thank M. Roth and J. Simon
(both FHCRC) for the generous loan of equipment and
reagents. This research was supported in part by the Howard
Hughes Medical Institute, the Bill and Melinda Gates
foundation and the Intramural Research Program of the NIH,
National Heart, Lung and Blood Institute.
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