3-Hydroxyazetidine Carboxylic Acids: Non-Proteinogenic Amino Acids for Medicinal Chemists

Article abstract of DOI:10.1002/cmdc.201200541

The formation from D-glucose of both enantiomers of 2,4-dideoxy-2,4-iminoribonic acid is the first chemical synthesis of unprotected 3-hydroxyazetidine carboxylic acids. The long-term stability of 3-hydroxyazetidine amides is established at acidic and neutral pH and implies their value as non-proteinogenic amino acid components of peptides, providing medicinal chemists with a new class of peptide isosteres. The structure of N,3-O-dibenzyl-2,4-dideoxy-2,4-imino-D-ribonic acid was established by X-ray crystallographic analysis. An N-methylazetidine amide derivative is a specific inhibitor of β-hexosaminidases at the micromolar level, and is only the second example of potent inhibition of any glycosidase by an amide of a sugar amino acid related to an iminosugar.

Full text of DOI:10.1002/cmdc.201200541

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DOI: 10.1002/cmdc.201200541
3-Hydroxyazetidine Carboxylic Acids: Non-Proteinogenic
Amino Acids for Medicinal Chemists
Andreas F. G. Glawar,[a, b] Sarah F. Jenkinson,[a, b] Amber L. Thompson,[c] Shinpei Nakagawa,[d]
Atsushi Kato,*[d] Terry D. Butters,[b] and George W. J. Fleet*[a, b]
The formation from d-glucose of both enantiomers of 2,4-di-
deoxy-2,4-iminoribonic acid is the first chemical synthesis of
unprotected 3-hydroxyazetidine carboxylic acids. The long-
term stability of 3-hydroxyazetidine amides is established at
acidic and neutral pH and implies their value as non-proteino-
genic amino acid components of peptides, providing medicinal
chemists with a new class of peptide isosteres. The structure of
N,3-O-dibenzyl-2,4-dideoxy-2,4-imino-d-ribonic acid was estab-
lished by X-ray crystallographic analysis. An N-methylazetidine
amide derivative is a specific inhibitor of b-hexosaminidases at
the micromolar level, and is only the second example of
potent inhibition of any glycosidase by an amide of a sugar
amino acid related to an iminosugar.
Introduction
Proline 1, containing a pyrrolidine ring, has a smaller loss of
conformational entropy upon folding than other proteinogenic
amino acid constituents and is an important determinant in
the structural features of proteins (Figure 1).[1] Therefore, the
amino acid, first isolated from Convallaria majalis in 1955;[6]
Aze is a constituent of nicotianamine and mugineic acid deriv-
atives, which are iron transporters in plants.[7] Aze occurs in
many other plants, including sugar beet,[8] and in rhizomes of
Polygonatum sibiricum and Polygonatum odoratum used as
supplements in many foods, including tea.[9] Aze is readily mis-
incorporated into proteins as a substitute for proline 1 in
many species, including humans,[10] and causes numerous toxic
effects as well as congenital malformations.[11] There are recent
reports of Aze 4 and its analogues as key components of SAR
studies on azetidine-containing dipeptides as HCMV inhibi-
tors[12] and as modulators of sphingosine phosphate receptor-
1 as a suppressant of autoimmunity,[13] among many other
uses.[14] The rigidity of the four-membered ring makes azeti-
dine carboxylic acids attractive as a family of monomers for fol-
damers[15,16] to complement the use of oxetane amino acids[17]
in the generation of novel secondary structural features in
peptidomimetics. There is therefore current interest in the syn-
thesis of Aze itself[18] and related azetidine amino acids.[19]
The trans-4-hydroxylation of proline 1 by prolyl hydroxylases
to give a trans-4-hydroxyproline 2 (Hyp) in collagen proline
residues changes Gly-Pro-Pro repeats to Gly-Pro-Hyp and is
crucial for the conformational stability of its triple-helical sec-
ondary structure, as studied using collagen-like model pep-
tides.[20] A related family of prolyl hydroxylases (PHDs) regu-
lates the transcriptional complex hypoxia inducible factor (HIF)
by introducing an Hyp moiety into HIF. This Hyp residue in-
creases HIF affinity to the von Hippel Lindau tumor suppressor
~1000-fold, leading to proteasomal HIF degradation. Because
PHD activity is decreased under hypoxic conditions, HIF re-
mains stable and initiates its transcriptional response as central
regulator of mammalian oxygen homeostasis.[21] HIF is activat-
ed in a broad array of ischemic/hypoxic and neoplastic diseas-
es; HIF has provided a focus for efforts to understand the un-
derlying mechanisms of oxygen sensing and signal transduc-
Figure 1. Azetidine-2-carboxylic acid and related amino acids.
use of modified proline isosteres[2] incorporated into peptido-
mimetics is of considerable value to medicinal chemists in the
invention of new pharmacophores.[3] l-Azetidine-2-carboxylic
acid (Aze) 4[4,5] is a naturally occurring non-proteinogenic
[a] A. F. G. Glawar, Dr. S. F. Jenkinson, Prof. G. W. J. Fleet
Chemistry Research Laboratory, Department of Chemistry
University of Oxford, Mansfield Road, Oxford, OX1 3TA (UK)
E-mail: George.fleet@chem.ox.ac.uk
[b] A. F. G. Glawar, Dr. S. F. Jenkinson, Dr. T. D. Butters, Prof. G. W. J. Fleet
Oxford Glycobiology Institute
University of Oxford, South Parks Road, Oxford, OX1 3QU (UK)
[c] Dr. A. L. Thompson
Chemistry Research Laboratory
Department of Chemical Crystallography
University of Oxford, Mansfield Road, Oxford, OX1 3TA (UK)
[d] S. Nakagawa, Prof. A. Kato
Department of Hospital Pharmacy
University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)
E-mail: kato@med.u-toyama.ac.jp
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200541.
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tion.[22] The HIF prolyl hydroxylases are related to the collagen
prolyl hydroxylases; structural studies have been used for the
design of PHD inhibitors aimed at treating anemia and ische-
mic disease.[23] The origin of increased stability of collagen by
4-fluoroproline 3 has been studied.[24] Substrate analogue stud-
ies with 2 and 3 (and their cis-epimers) have shown how PHDs
achieve specificity for hydroxyprolyl/prolyl residues for the C4-
exo/endo prolyl conformations, respectively.[25] Despite the in-
terest in hydroxyprolines,[26] the sole reported preparation of 3-
hydroxy-Aze 5 is by biohydroxylation of Aze with proline hy-
droxylases;[27] other azetidine substrates have been biohy-
droxylated to 3-hydroxyazetidines.[28] There is no previous
report of the chemical synthesis of a 3-hydroxyazetidine-2-car-
boxylic acid with free NH and OH groups in the structure. In
the pursuit of a synthesis of 3-epi-hydroxymugineic acid 7, an
N-alkylated derivative was generated (Figure 2A).[29]
tion of hydroxyazetidine carboxylic acids (and of their fluoro
analogues) would provide further information on the conse-
quences of substitution of proline by Aze and supply com-
pounds of interest to medicinal chemists. It is clear that such
a hydroxylation of an Aze protein would render the protein
potentially conformationally unstable due to a reverse aldol re-
action (Figure 2C).
Additionally, the effect of the hydroxylated azetidines on
a panel of glycosidases was determined; the N-methylamide
11 L is a potent and specific inhibitor of hexosaminidases, and
its enantiomer 11 D is a good inhibitor of a b-glucuronidase.
Although iminosugars are well established as inhibitors of gly-
cosidases and other sugar-metabolizing enzymes, inhibition of
amides of their corresponding amino acids is effectively un-
known. It may also be noted that the azetidine acids 10 are re-
lated to the amino acid bulgecinine 6[33] in the same way the
hydroxyazetidine 5 is related to hydroxyproline 2 (Figure 1).
Results and Discussion
The synthesis of the l-amino acid 10L required cleavage of
the C5ꢀC6 bond in the protected glucose 12 prior to forma-
tion of the azetidine ring (Scheme 1). Selective hydrolysis of
the side chain acetonide in 12 followed by periodate cleavage
and functional group manipulation gave a mixture of anomers
of the protected 3-O-benzylxylose 13 in five steps with an
overall yield of 52%, as previously described.[34]
Reaction of 13 with hydrogen bromide in acetic acid/di-
chloromethane afforded the corresponding bromide, which
upon treatment with methanol in the presence of silver(I) car-
bonate formed the b-pyranoside 14 (61%),[35] from which the
acetyl groups were removed by sodium methoxide in metha-
nol to afford the diol 15 (100%). Esterification of the remaining
hydroxy groups in 15 with trifluoromethanesulfonyl anhydride
in dichloromethane in the presence of pyridine formed the di-
triflate 16b (100%), which with benzylamine in acetonitrile
gave the bicyclic azetidine 17 (85%). Under similar conditions
the corresponding a-pyranoside 16a did not form any bicyclic
product; only b-anomers of such pyranosides cyclized to azeti-
dines in good yield.[36]
Figure 2. A) Published examples; B) synthesis strategy (numbering refers to
the carbon atoms in glucose); C) retro-aldol mechanism.
Herein we report the synthesis of the enantiomers of 2,4-di-
deoxy-2,4-iminoribonic acid 10 and the corresponding N-meth-
ylamides 11 from the protected d-glucose 12, in which the
azetidine ring is formed by double nucleophilic displacements
between C2 and C4 of a glucopyranoside. The l-enantiomer
10L is obtained by cleavage of the C5ꢀC6 bond prior to azeti-
dine ring formation, whereas the d-enantiomer 10D involves
cleavage of the C5ꢀC6 bond after formation of the heterocy-
clic ring (Figure 2B). Protected derivatives of 10 are suitable in-
termediates for the study of highly functionalized azetidine
carboxylic acids as peptidomimetics and as a potential family
of foldamer monomers. It is shown that the amides 11 are
stable at neutral and low pH for periods of six months, but are
vulnerable to aldol cleavage at higher pH (Figure 2C). Similar
mechanisms have rendered methylacetal 8[30] unstable on
standing at room temperature, ketone 9[31] unstable to purifi-
cation on silica gel, and have only allowed for isolation of C3-
hydroxy-protected derivatives.[32] Because Aze 4 is misincorpo-
rated for proline 1 into proteins and because proline hydroxy-
lases oxidize Aze 4 to 5, it may be that the study of incorpora-
Hydrolysis of 17 by aqueous hydrochloric acid in dioxane
gave the lactols 18L. Reduction of 18L by sodium borohy-
dride, followed by purification via an acetylation–deacetylation
sequence to remove the borate complexes, gave the dibenzyl
diol 19 (74%). Removal of the benzyl group by hydrogenolysis
in aqueous dioxane in the presence of palladium on carbon
and hydrochloric acid gave the meso-iminoribitol azetidine 20.
Oxidation of 18L by iodine in methanol in the presence of po-
tassium carbonate[37] produced the methyl ester 21L (84%).
Hydrolysis of 21L by hydrochloric acid in aqueous dioxane
gave the acid 22L (81%) from which the benzyl groups were
removed by hydrogenolysis to afford the unprotected 3-hy-
droxyazetidine carboxylic acid 10L. Treatment of the methyl
ester 21L with methylamine in methanol in the presence of
anhydrous calcium chloride[38] formed the amide 23L (70%)
which, upon hydrogenolysis, formed the unprotected amide
11 L.
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base catalysis; however, the amide 11 is stable indefi-
nitely under neutral or acidic pH.[41]
Among recent studies on the bioactivity of imino-
sugar azetidines,[42] potent inhibition of purine nu-
cleoside phosphorylase[43] and specific inhibition of
non-mammalian glycosidases[44,45] have been report-
ed. Inhibition by the azetidines 10L, 11 L, 10D, 11 D,
and 20 of the following glycosidases were studied
(Table 1):[46] a-glucosidases (EC 3.2.1.20, rice, yeast, As-
pergillus niger), b-glucosidases (EC 3.2.1.21, almond,
bovine liver, Aspergillus niger), a-galactosidase
(EC 3.2.1.22,
coffee
beans),
b-galactosidase
(EC 3.2.1.23, bovine liver), a-mannosidase (EC 3.2.1.24,
Jack bean), b-mannosidase (EC 3.2.1.25, snail), a-l-
rhamnosidase (EC 3.2.1.40, Penicillium decumbens), a-
l-fucosidase (EC 3.2.1.44, bovine kidney), b-glucuroni-
dases (EC 3.2.1.31, Escherichia coli, bovine liver), tre-
halase (EC 3.2.1.28, porcine kidney), amyloglucosidas-
es (EC 3.2.1.3, Aspergillus niger, Rhizopus sp.), b-N-ace-
tylglucosaminidases (EC 3.2.1.52, human placenta,
bovine kidney, Jack beans, HL 60, Aspergillus oryzae),
a-N-acetylgalactosaminidases (EC 3.2.1.49, chicken
liver, Charonia lampas), and b-N-acetylgalactosamini-
dases (EC 3.2.1.53, HL 60, Aspergillus oryzae).
Neither of the enantiomeric acids 10L and 10D
showed any inhibition (IC50 <50% at 1000 mm) of any
glycosidases (Figure 4). The meso-azetidine triol 20
showed good inhibition of yeast a-glucosidase
(IC50 9.5 mm), good inhibition of trehalase (IC50 30 mm)
and rice a-glucosidase (IC50 83 mm), and weak inhibi-
tion of A. niger a-glucosidase (IC50 693 mm) and amy-
loglucosidases [A. niger (IC50 758 mm), Rhizopus sp.
(IC50 274 mm)]. The N-nonyl derivative 26 is a specific
inhibitor of some ceramide-specific glucosyl transfer-
ases and glucosidases.[47]
Scheme 1. a) HBr, 5–108C, AcOH/CH2Cl2 (7:3), 5 h, then Ag2CO3, MeOH, RT, 14 h, 61%;
b) NaOMe, MeOH, 408C, 18 h, 100%; c) Tf2O, py, CH2Cl2, ꢀ20 to ꢀ108C, 1.5 h, 100%;
d) BnNH2, CH3CN, 708C, 2 h, 85%; e) 1,4-dioxane/2m HCl(aq) (1:5), 408C, 17.5 h, 100%;
f) NaBH4, MeOH/1,4-dioxane (3:1), 1.5 h, then Ac2O, py, then NaOMe, MeOH, 74%; g) Pd/
C, H2, H2O/1,4-dioxane (2:1), HCl, 72 h, 100%; h) I2, K2CO3, MeOH, 08C, 1 h, 96%; i) MeNH2,
CaCl2, 458C, 2 h, MeOH, 70%; j) Pd/C, H2, H2O/1,4-dioxane (2:1), HCl, 10 h, 100%; k) HCl,
H2O/1,4-dioxane (2:1), 708C, 3 d, 81%; l) Pd/C, H2, H2O/1,4-dioxane (2:1), HCl, 15 h, 100%.
For the enantiomeric acid 10D and amide 11 D,
the azetidine 25 was formed from the glucose-de-
rived ditriflate 24 as previously described
(Scheme 2).[36] Oxidation of 25 with sodium periodate
in aqueous acetone gave the lactols 18D which,
upon further oxidation by iodine in methanol,
formed the methyl ester 21D (77%). Subsequent
transformations on 21D, identical to those per-
formed on 21L, allowed access to the 3-hydroxy-d-
azetidine carboxylic acid 10D and the corresponding
amide 11 D. The structure of the dibenzyl acid 22D
was firmly established by X-ray crystallographic analy-
sis (Figure 3).[39] NOE studies on the free acid 10L in-
dicated that deprotection of the benzyl groups had
not otherwise changed the stereochemistry of the
Scheme 2. a) NaIO4, H2O/Me2CO (2:1), RT, 1 h; b) I2, K2CO3, MeOH, 08C, 1 h, 77% over two
steps; c) MeNH2, CaCl2, MeOH, 458C, 2 h, 72%; d) Pd/C, H2, H2O/1,4-dioxane (2:1), 12 h,
100%; e) HCl, H2O/1,4-dioxane (2:1), RT!708C, 7 d, 96%; f) Pd/C, H2, H2O/1,4-dioxane
(2:1), 18 h, 100%.
ring.[40] Azetidine carboxylic acid derivatives with an
unprotected hydroxy group at C3, such as the amide
11, are susceptible to retro-aldol reactions under
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hexosaminidases, but is a good inhibitor of E. coli b-glucuroni-
dase (IC50 59 mm).
Conclusions
1. This paper reports the first chemical synthesis of 3-hydroxy-
azetidine carboxylic acids and of protected derivatives suit-
able for incorporation into peptides. The stability of such
moieties is established; the amides, although vulnerable to
ring opening by reverse aldol reactions, are stable at neu-
tral and acidic pH at room temperature for substantial peri-
ods of time. Because of the value of hydroxyprolines as
peptide isosteres, it is almost certain that such hydroxylaze-
tidine carboxylic acids will allow medicinal chemists, for the
first time, to access a new range of novel bioactive peptides
containing highly functionalized azetidines.
Figure 3. X-ray crystal structure of 22D with displacement ellipsoids drawn
at the 50% probability level. Hydrogen atoms are shown as spheres of arbi-
trary radius. The compound crystallizes as the zwitterion with a molecule of
water in the asymmetric unit.
2. The meso-azetidine triol 20 is a very rare example of an imi-
nosugar as a good inhibitor (IC50 9.5 mm) of yeast a-glucosi-
dase. There are many iminosugars that are potent inhibitors
of mammalian glucosidases, but almost all of them show
no significant inhibition (IC50 >1 mm) of yeast a-glucosi-
dase. This observation is consistent with the selective inhib-
ition of non-mammalian glycosidases by azetidine iminosu-
gars.[44,45]
3. Although no glycosidase inhibition was observed for the
acids 10L and 10D, the amide of the azetidine l-ribonic
acid 11 L is a potent and specific inhibitor of hexosamini-
dases, and its enantiomer 11 D is a good inhibitor of a b-
glucuronidase. Although many iminosugars have been
identified as potent glycosidase inhibitors, the inhibition by
the related hydroxyproline and hydroxypipecolic acids has
always been modest. The only examples of any enzyme in-
hibition of amides of such acids reported previously is by
the pipecolic acid amides 29 and 30; the adventitious ob-
servation of the micromolar inhibition of the N-methyla-
mide of the azetidine acid 11 L indicates that such simple
derivatives of polyhydroxylated prolines may be a fertile
field for medicinal chemists to investigate the inhibition of
sugar-metabolizing enzymes.
Figure 4. Glycosidase inhibition studies.
The inhibition of yeast a-glucosidase by the azetidine triol
20 is noteworthy. Iminosugars that are good to potent inhibi-
tors of other a-glucosidases generally show no significant in-
hibition of the yeast enzyme; DAB 27 (IC50 0.15 mm) and DMDP
28 (IC50 0.71 mm) are the only natural iminosugars that are
potent inhibitors of yeast a-glucosidase.[48] Early indica-
tions[44,45] are that azetidines are selective inhibitors of non-
mammalian glycosidases—unlike their pyrrolidine and piperi-
dine counterparts—and may have significance for the develop-
ment of use of such compounds.
4. Further study of such compounds and related azetidines is
likely to reveal interesting bioactivity both as monomers
and as components of peptidomimetics.
The l-ribono-azetidine amide 11 L (IC50 1.4 mm for bovine
kidney b-N-acetylglucosaminidase) is a potent inhibitor of
a number of b-hexosaminidases (Table 1); 11 L is totally specific
and shows no inhibition of any of the other glycosidases. The
only other reported examples of hexosaminidase inhibition by
amides of iminosugar acids is that of the tetrahydroxypipecolic
acid amides 29 and 30 (IC50 11 and 0.09 mm, respectively, for
bovine liver).[49] These observations indicate that the study of
hexosaminidase inhibition by acid amides of iminosugars may
provide a class of specific and potent inhibitors. None of the
amides 11 L, 29, or 30 are inhibitors of a-N-acetylgalactosami-
nidases. The enantiomeric amide 11 D shows no inhibition of
Experimental Section
All commercial reagents were used as supplied. MeOH and pyri-
dine (py) were purchased dry from Aldrich in sure-seal bottles over
molecular sieves. All other solvents were used as supplied (analyti-
cal or HPLC grade) without prior purification. H2O was purified by
a Milli-Q filtration system. Reactions were performed under an at-
mosphere of N2 or Ar unless stated otherwise. Thin-layer chroma-
tography (TLC) was performed on Al sheets coated with 60 F254
silica. Sheets were visualized with a spray of 0.2% w/v Ce(SO4)2 and
5% (NH4)2MoO4 in 2m H2SO4, a spray of 1% w/v KMnO4, 5% w/v
K2CO3, and 0.1% NaOH in H2O. Flash column chromatography was
performed on Sorbsil C60 40/60 silica. Ion-exchange chromatogra-
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208C. The crude bromide was dissolved in MeOH (8 mL)
in a foil-wrapped flask in the presence of Ag2CO3
(637 mg, 2.31 mmol) and stirred in the dark at RT for
14 h. TLC analysis (1:1 cyclohexane/EtOAc) indicated the
formation of a major product (Rf =0.62); the reaction
mixture was filtered through a glass fiber pad and
washed with MeOH. The combined filtrates were evapo-
rated, and the residue (384 mg, 83%) was purified by
flash column chromatography (8:1!7:1 cyclohexane/
EtOAc) to afford the b-pyranoside 14 as a white crystal-
line solid (280 mg, 61%): mp: 93–958C (EtOAc/cyclohex-
ane) [lit. 95.5–968C];[35] [a]D25 =ꢀ56.5 (c=1.3, CHCl3) [lit.
[a]D22 =ꢀ57[35] (c=1.00, CHCl3)]; 1H NMR (400 MHz,
CDCl3): d=2.04 (s, 2ꢁ3H, COCH3), 3.35 (dd, J4,5 =7.1 Hz,
Table 1. Concentration of iminosugars giving 50% inhibition of various glycosidase-
s.[a,b]
Glycosidase / source
b-N-Acetylglucosaminidase
Human placenta
Bovine kidney
Jack bean
HL 60
Aspergillus oryzae
NI (42.8)
NI (46.8)
797
NI (34.7)
NI (21.4)
3.3
1.4
2.8
4.2 [0.892][c]
48
J
J
5,5’ =12.0 Hz, 1H, H5’), 3.45 (s, 3H, OCH3), 3.66 (t, J2,3
3,4 =7.2 Hz, 1H, H3), 4.15 (dd, J4,5 =4.4 Hz, J5,5’ =12.0 Hz,
=
a-N-Acetylgalactosaminidase
Chicken liver
NI (12.5)
NI (13.9)
NI (0)
NI (15.3)
Charonia lampas
1H, H5), 4.40 (d, J1,2 =5.6 Hz, 1H, H1), 4.68 (s, 2H, CH2Ph),
4.92 (td, J4,5 =4.4 Hz, J3,4 =J4,5’ =7.1 Hz, 1H, H4), 4.96 (dd,
b-N-Acetylgalactosaminidase
HL 60
J
1,2 =5.6 Hz, J2,3 =7.3 Hz, 1H, H2), 7.37–7.26 ppm (m, 5H,
ArH); 13C NMR (101 MHz, CDCl3): d=21.1 (C=OCH3), 56.4
(OCH3), 61.2 (C5), 70.3 (C4), 70.9 (C2), 73.4 (CH2Ph), 77.0
(C3), 101.3 (C1), 127.8, 127.9, 128.5 (Ph), 138.0 (ipso-Ph),
169.6 (C=O), 170.0 ppm (C=O); IR (thin film): n˜ =
1745 cmꢀ1 (s, C=O); MS (ESI+): m/z (%): 361 (32) [M+
Na]+, 699 (100) [2M+Na]+; HRMS (ESI+): m/z [M+Na]+
calcd for C17H22NaO7: 361.1258, found: 361.1257.
NI (11.3)
NI (26.0)
18
27
Aspergillus oryzae
b-Glucuronidase
E. coli
59
NI (12.1)
NI (9.0)
NI (0)
Bovine liver
[a] Values of IC50 [mm]. [b] NI: no inhibition (i.e., <50% inhibition at 1000 mm); values
in parentheses: percent inhibition at 1000 mm. [c] Ki value for inhibitor. A complete
table of glycosidase inhibition by the azetidines is provided in the Supporting Infor-
mation.
Methyl 3-O-benzyl-b-d-xylopyranoside (15): A solution
of the protected b-pyranoside 14 (239 mg, 0.70 mmol)
and NaOMe (3.8 mg, 0.07 mmol) in MeOH (4 mL) was
stirred at 408C. After 14 h, TLC analysis (1:1 cyclohexane/
phy was performed using Dowex (50W-X8, H+) eluted with 2m
EtOAc) indicated complete disappearance of the starting material
(Rf =0.68) and formation of a single product (Rf =0.32). The reac-
tion mixture was evaporated to dryness. The residue was taken up
in EtOAc (25 mL), washed with H2O (2ꢁ25 mL) and brine (25 mL),
and the aqueous phase was extracted with EtOAc (2ꢁ50 mL). The
combined organic solutions were dried (MgSO4) and evaporated to
dryness to yield methyl pyranoside 15 as a white crystalline solid
(203 mg, 100%): mp: 102–1048C [lit. 103–1048C];[35] [a]D25 =ꢀ79.8
(c=1.0, CHCl3) [lit. [a]D22 =ꢀ71.5[35] (c=1.00, CHCl3)]; 1H NMR
(400 MHz, CDCl3): d=2.24 (d, J4,OH =3.1 Hz, 1H, 4-OH), 2.47 (d,
2,OH =2.7 Hz, 1H, 2-OH), 3.26 (dd, J4,5’ =9.8 Hz, J5,5’ =11.6 Hz, 1H,
H5’), 3.37 (appt, J2,3 =J3,4 =8.5 Hz, 1H, H3), 3.51 (ddd, J2,OH =2.6 Hz,
1,2 =7.2 Hz, J2,3 =8.8 Hz, 1H, H2), 3.54 (s, 3H, OMe), 3.72 (dddd,
4,OH =3.1 Hz, J4,5 =5.2 Hz, J3,4 =8.4 Hz, J4,5’ =9.7 Hz, 1H, H4), 4.01
(dd, J4,5 =5.2 Hz, J5,5’ =11.6 Hz, 1H, H5), 4.17 (d, J1,2 =7.1 Hz, 1H,
H1), 4.74 (d, Jgem =11.7 Hz, 1H, CH2Ph’), 4.98 (d, Jgem =11.7 Hz, 1H,
CH2Ph), 7.41–7.28 ppm (m, 5H, ArH); 13C NMR (101 MHz, CDCl3):
d=57.3 (OMe), 65.3 (C5), 69.4 (C4), 74.0 (C2), 74.6 (CH2Ph), 83.4
(C3), 104.5 (C1), 128.2, 128.8 (3ꢁPh), 138.6 ppm (ipso-Ph); IR (thin
film): n˜ =3409 cmꢀ1 (m, OH); MS (ESI+): m/z (%): 377 (100) [M+
Na]+, 531 (32) [2M+Na]+; HRMS (ESI+): m/z [M+Na]+ calcd for
C13H18NaO5: 277.1046, found: 277.1045.
NH4OH. Melting points were recorded on a Kofler hot block and
are uncorrected. Optical rotations were recorded on a PerkinElmer
241 polarimeter with a path length of 1 dm. Concentrations are
quoted in g(100 mL)ꢀ1. IR spectra were recorded on a PerkinElmer
1750 IR Fourier transform spectrophotometer using thin films. Only
the characteristic peaks are quoted. Low-resolution MS data (m/z)
were recorded on a Waters LCT Premier spectrometer, and high-
resolution MS data (HRMS m/z) on a Waters ZMD spectrometer;
electrospray ionization (ESI) was the ionization technique used.
NMR spectra were recorded on Bruker AMX 500 (1H: 500 MHz; 13C:
125.7 MHz) and Bruker DPX 400 and DQX 400 spectrometers (1H:
400 MHz; 13C: 100.6 MHz) in the deuterated solvent stated. All
chemical shifts (d) are quoted in ppm, and coupling constants (J)
in Hz. Residual signals from the solvents were used as an internal
reference except in the case of D2O, for which CH3CN was used as
an internal reference. For enantiomeric pairs, the NMR and IR spec-
tra were recorded and found to be identical; hence only the data
for one enantiomer is given.
J
J
J
Synthesis started from 1,2,4-tri-O-acetyl-3-O-benzyl-b-d-xylopyra-
nose 13, which can be prepared from d-glucose according to pub-
lished procedures.[34,35,50]
Methyl 2,4-O-diacetyl-3-O-benzyl-b-d-xylopyranoside (14): HBr
(33% wt in AcOH, 0.63 mL, 3.67 mmol) was added dropwise over
a 20 min period to a stirred solution of triacetate 13 (500 mg,
1.36 mmol) in 7:3 AcOH/CH2Cl2 (5 mL) at 108C, and the reaction
mixture was then cooled to 58C. TLC analysis (1:1 cyclohexane/
EtOAc) after 5 h indicated disappearance of the starting material
(Rf =0.65) and formation of a major product (Rf =0.79). The solu-
tion was diluted with CH2Cl2 (25 mL) and poured into ice water
(25 mL). The organic layer was washed with cold saturated NaCHO3
(2ꢁ25 mL), H2O (25 mL), dried (MgSO4), filtered, and evaporated at
Methyl 3-O-benzyl-2,4-di-O-trifluoromethanesulfonyl-b-d-xylo-
pyranoside (16): Trifluoromethanesulfonic anhydride (0.95 mL,
5.6 mmol) was added slowly to a stirred solution of the pyranoside
15 (357 mg, 1.4 mmol) and py (0.68 mL, 8.4 mmol) in CH2Cl2
(10 mL) at ꢀ208C under N2. The reaction mixture was stirred at
ꢀ108C; after 1.5 h, TLC analysis (1:1 cyclohexane/EtOAc) indicated
complete consumption of the starting material (Rf =0.27) and for-
mation of a major species (Rf =0.78). The reaction mixture was di-
luted with CH2Cl2 (10 mL), washed with HCl(aq) (2m, 3ꢁ20 mL),
dried (MgSO4), filtered, and concentrated to dryness to afford the
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25
crude ditriflate 16 as a clear yellow oil (729 mg, 100%): [a]D
=
washed with H2O (2ꢁ25 mL), saturated NaCHO3(aq) (25 mL) brine
(25 mL), dried (MgSO4), filtered, and concentrated in vacuo to yield
the crude diacetate as colorless oil (65 mg, 0.16 mmol, 96%). The
residue was dissolved in anhydrous MeOH (2 mL); NaOMe (4 mg,
0.07 mmol) was added, and the reaction mixture was stirred at RT
for 20 h. TLC analysis (EtOAc) indicated consumption of starting
material (Rf =0.95) and formation of a major product (Rf =0.54)
along with an additional species (Rf =0.87), which was tentatively
identified as the monoacetate (by MS), and an additional quantity
of NaOMe (4 mg, 0.07 mmol) was added to the reaction mixture.
After a total reaction time of 45 h, TLC analysis (1:1 cyclohexane/
EtOAc) indicated formation of a major species (Rf =0.18) with only
traces of the intermediate (Rf =0.65) remaining, and the reaction
mixture was concentrated in vacuo. The crude was dry loaded
from MeOH/Et3N (99:1) and flash column chromatography (cyclo-
hexane/EtOAc/Et3N 75:29:1!0:99:1) yielded dibenzyl diol 19 as
a clear colorless oil (39 mg, 0.13 mmol, 74%): [a]D25 = +0.23 (c=
ꢀ38.2 (c=1.29, CHCl3); 1H NMR (400 MHz, CDCl3): d=3.53 (s 3H,
OMe), 3.62 (dd, J4,5’ =8.0 Hz, J5,5’ =12.4 Hz, 1H, H5’), 3.91 (appt,
J
2,3 =J3,4 =7.6 Hz, 1H, H3), 4.27 (dd, J4,5 =4.6 Hz, J5,5’ =12.4 Hz, 1H,
H5), 4.53 (d, J1,2 =6.1 Hz, 1H, H1), 4.64 (dd, J1,2 =6.2 Hz, J2,3 =7.6 Hz,
1H, H2), 4.76 (d, Jgem =10.4 Hz, 1H, CH2Ph’), 4.80 (d, Jgem =10.4 Hz,
1H, CH2Ph), 4.85 (apptd, J4,5 =4.7 Hz, J3,4 =J4,5’ =7.7 Hz, 1H, H4),
7.45–7.29 ppm (m, 5H, ArH); 13C NMR (101 MHz, CDCl3): d=57.4
(OMe), 61.4 (C5), 75.5 (CH2Ph), 76.6 (C3), 81.6 (C4), 82.2 (C2), 100.5
(C1), 128.6 128.7, 128.7 (3ꢁPh), 135.7 ppm (ipso-Ph); IR (thin film):
n=fingerprint region only; MS (ESI+): m/z (%): 541 (25) [M+Na]+,
1059 (100) [2M+Na]+; HRMS (ESI+): m/z [M+Na]+ calcd for
C15H16F6NaO9S2: 541.0032, found: 541.0034.
Methyl N,3-O-Dibenzyl-2,4-dideoxy-2,4-imino-b-l-ribopyranoside
(17): Benzylamine (0.77 mL, 7.0 mmol) was added to the crude di-
triflate 16 (729 mg, 1.4 mmol) in CH3CN (7 mL); the reaction mix-
ture was stirred at 65–708C for 2 h when TLC analysis (2:1 cyclo-
hexane/EtOAc) indicated complete disappearance of the starting
material (Rf =0.71) and formation of a major species (Rf =0.56). The
reaction mixture was left to cool, concentrated in vacuo, and the
residue was purified by flash column chromatography (7:1 cyclo-
hexane/EtOAc) to afford the bicyclic azetidine 17 as a light-yellow
oil (388 mg, 85%): [a]D25 =ꢀ30.7 (c=1.21, CHCl3); 1H NMR
(400 MHz, CDCl3): d=3.45 (s, 3H, OMe), 3.55 (dt, J4,5 =J4,5’ =1.4 Hz,
1
1.05, CHCl3); H NMR (400 MHz, CDCl3): d=2.17 (brs, 2H, OH), 3.23
(dt, J1,2 =J1’,2 =J4,5 =J4,5’ =3.2 Hz, J2,3 =J3,4 =6.1 Hz, 2H, H2, H4), 3.29
(dd, J1’,2 =J4,5’ =3.4 Hz, J5,5’ =J1,1’ =11.8 Hz, 2H, H1’, H5’), 3.34 (dd,
J
1,2 =J4,5 =3.0 Hz, J5,5’ =J1,1’ =11.8 Hz, 2H, H1, H5), 3.75 (s, 2H,
NCH2Ph), 4.02 (t, J2,3 =J3,4 =5.4 Hz, 1H, H3), 4.50 (s, 2H, OCH2Ph),
7.40–7.24 ppm (m, 10H, ArH); 13C NMR (101 MHz, CDCl3): d=61.4
(NCH2Ph), 61.9 (C1, C5), 71.6 (C2, C4), 71.7 (C3), 72.1 (OCH2Ph),
127.9, 128.0, 128.1, 128.7, 128.8, 129.1 (10ꢁPh), 137.9, 138.0 ppm
(2ꢁipso-Ph); IR (thin film): n˜ =3405 cmꢀ1 (s, OH); MS (ESI+): m/z
(%): 314 (100) [M+H]+, 336 (98) [M+Na]+, 649 (77) [2M+Na]+;
MS (ESI-): m/z (%): 312 (100) [M-H]ꢀ; HRMS (ESI+): m/z [M+H]+
calcd for C19H24NO3: 314.1751, found: 314.1745.
J
2,4 =4.2 Hz, 1H, H4), 3.65 (dd, J1,2 =0.9 Hz, J2,4 =4.2 Hz, 1H, H2),
3.71 (dd, J4,5’ =1.1 Hz, J5,5’ =10.6 Hz, 1H, H5’), 3.98 (s, 1H, H3), 4.15
(s, 2H, NCH2Ph), 4.34 (dd, J4,5 =1.7 Hz, J5,5’ =10.6 Hz, 1H, H5), 4.64
(d, Jgem =12.1 Hz, 1H, OCH2Ph), 4.64 (d, J1,2 =0.8 Hz, 1H, H1), 4.69
(d, Jgem =12.1 Hz, 1H, OCH2Ph), 7.45–7.18 ppm (m, 10H, ArH,);
13C NMR (101 MHz, CDCl3): d=51.6 (NCH2Ph), 55.9 (OCH3), 62.4 (C5),
63.3 (C4), 66.2 (C2), 71.6 (OCH2Ph), 79.9 (C3), 100.9 (C1), 126.7,
128.0, 128.1, 128.3, 128.4, 128.6 (Ph), 137.8, 139.4 ppm (ipso-Ph); IR
(thin film): n=fingerprint region only; MS (ESI+): m/z (%): 326
(100) [M+H]+, 348 (26) [M+Na]+, 673 (64) [2M+Na]+; HRMS
(ESI+): m/z [M+Na]+ calcd for C20H24NO3: 326.1751, found:
326.1750.
2,4-Dideoxy-2,4-imino-meso-ribitol (20): Concentrated HCl (11.6m,
11 mL, 0.13 mmol) was diluted with H2O (2 mL), premixed with 1,4-
dioxane (1 mL), and added to diol 19 (20 mg, 0.07 mmol). To this
solution was added Pd/C (10% wt, 8 mg), and the reaction vessel
was degassed and charged with H2. After 5 h of vigorous stirring,
TLC analysis (4:1 EtOAc/MeOH) indicated a trace of remaining start-
ing material (Rf =0.81) along with a major species on the baseline
and an intermediate compound (Rf =0.36); similarly, TLC analysis
(14:3:1:1:1 EtOH/py/nBuOH/AcOH/H2O) indicated the presence of
a major product (Rf =0.81) along with a minor component (Rf =
0.91). MS still showed evidence of the starting material along with
a monobenzyl intermediate. After having been re-subjected to the
reaction conditions for an additional 18 h, only a trace of the mon-
obenzyl species remained by TLC analysis; however, MS still indi-
cated its presence until a total of 72 h had passed. The reaction
mixture was then filtered (GF/A glass microfiber), washed with
MeOH (2 mL), concentrated in vacuo, and loaded onto a short
column of Dowex (50W-X8, H+) in which the resin had been pre-
treated by washing with H2O (until eluent was neutral). The crude
was loaded (H2O/1,4-dioxane 2:1) and washed sequentially with
H2O, 1,4-dioxane, EtOH, and H2O again. The product was eluted
with aqueous ammonia (2m) and the ammoniacal fractions con-
centrated in vacuo at RT to yield triol 20 as a viscous gum (11 mg,
100%): [a]D25 = +0.68 (c=0.29, MeOH); 1H NMR (400 MHz, D2O):
N,3-O-Dibenzyl-2,4-dideoxy-2,4-imino-meso-ribitol (19): Bicyclic
azetidine 17 (56 mg, 0.17 mmol) was dissolved in 1,4-dioxane/
HCl(aq) (2m, 1:5, 6 mL) and stirred at 408C for 5 h, after which TLC
analysis (2:1 cyclohexane/EtOAc; sample quenched with Et3N) indi-
cated the consumption of the starting material (Rf =0.49) and the
presence of a major compound (Rf =0.10). The reaction mixture
was diluted with CH2Cl2 (30 mL), washed with saturated NaCHO3(aq)
(25 mL), and the aqueous fraction was extracted with CH2Cl2 (2ꢁ
30 mL). Evaporation of the combined organic fractions to dryness
at 308C yielded the lactol 18L (60 mg, 100%) as a colorless glass,
which was used without further purification. The crude lactol 18L
was dissolved in MeOH/1,4-dioxane (3:1, 4 mL), NaBH4 (8 mg,
0.20 mmol) was added, and the reaction mixture was stirred at RT
for 1.5 h. At this point TLC analysis (EtOAc) indicated disappear-
ance of the lactol (Rf =0.63) and formation of a new major com-
pound (Rf =0.4, streaks to baseline); MS (ESI+) indicated the sole
presence of the desired compound. The reaction mixture was
quenched by addition of a few drops of AcOH and was evaporated
to dryness to give the crude diol 19 (97 mg, 100%). The residue
was dissolved in py/acetic anhydride (1:1 4 mL) and stirred at RT
for 19 h. TLC analysis (3:1 cyclohexane/EtOAc) indicated disappear-
ance of the starting material (Rf =0.0) and appearance of a new
major compound (Rf =0.64), which was identified by MS (ESI+) as
the diacetylated intermediate. The reaction was, on dilution with
toluene (4 mL), evaporated to dryness and co-evaporated with tol-
uene (2ꢁ4 mL). The residue was dissolved in EtOAc (30 mL),
d=3.62 (dd, J=1.4 Hz, J1,2 =J4,5 =4.4 Hz, 4H, H1, H5), 4.05 (dt, J1,2
4,5 =4.4 Hz, J2,3 =J3,4 =7.1 Hz, 2H, H2, H4), 4.27 ppm (t, J2,3 =J3,4
=
=
J
7.1 Hz, 1H, H3); 13C NMR (63 MHz, D2O): d=58.2 (C1, C5), 64.0 (C3),
66.9 ppm (C2, C4); IR (thin film): n˜ =3271 cmꢀ1 (s, OH); MS (ESI-):
m/z (%): 168 (72) [M+Cl]ꢀ, 362 (100); HRMS (ESI+): m/z [M+H]+
calcd for C5H12NO3: 134.0812, found: 134.0807.
Methyl N,3-O-dibenzyl-2,4-dideoxy-2,4-imino-l-ribonate (21L): A
solution of the bicyclic azetidine 17 (136 mg, 0.42 mmol) in 1,4-di-
oxane/HCl(aq) (2m, 1:5, 6 mL) was stirred at 408C for 17.5 h, after
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which TLC analysis (2:1 cyclohexane/EtOAc; sample quenched with
Et3N) indicated the absence of starting material (Rf =0.49) and the
formation of a major product (Rf =0.10). The reaction mixture was
diluted with CH2Cl2 (30 mL) and washed with saturated NaCHO3(aq)
(25 mL). The aqueous fraction was extracted with CH2Cl2 (2ꢁ
30 mL). The combined organic fractions were washed with brine
(40 mL) and dried (MgSO4). Evaporation to dryness yielded bicyclic
lactol 18L (147 mg, 100%) as an unstable colorless glass, which
was used without further purification. A solution of the crude
lactol 18L (102 mg, 0.30 mmol) and K2CO3 (124 mg, 0.90 mmol) in
anhydrous MeOH (4 mL) was stirred at 08C under an atmosphere
of N2. Iodine (99 mg, 0.39 mmol), dissolved by sonication in anhy-
drous MeOH (4 mL), was added dropwise to the stirred reaction
mixture at 08C. MS indicated the formation of the desired product
after 1 h reaction time, and TLC analysis (EtOAc) showed consump-
tion of the starting material (Rf =0.61) and formation of a single
compound (Rf =0.79). The reaction was quenched by the addition
of saturated Na2SO3(aq) (8 mL). The resultant white precipitate was
dissolved in H2O (32 mL), and the aqueous fraction was extracted
with Et2O (4ꢁ50 mL). The combined organic fractions were dried
(MgSO4), filtered, and evaporated in vacuo to yield the title com-
pound 21L (98 mg, 96%). The highest yield was obtained when
the ester 21L was used without further purification in the follow-
ing hydrolysis. However, on one occasion the ester was isolated as
follows: in this case the lactol 18L (147 mg, 0.42 mmol) reacted in
the same way as above to give the crude methyl ester 21L
(136 mg, 95%). Purification by flash column chromatography (cy-
clohexane/EtOAc/Et3N 80:19:1!66:32:1) gave the methyl ester
21L (116 mg, 81%) as a clear colorless oil: [a]D25 =ꢀ58.8 (c=0.80,
cipitate was dissolved in H2O (16 mL), and the aqueous fraction
was extracted with Et2O (4ꢁ30 mL). The organic layer was dried
(MgSO4), filtered, and evaporated to dryness to yield the crude title
compound 21D (84 mg, 100%). Purification by flash column chro-
matography (cyclohexane/EtOAc/Et3N 80:19:1!66:32:1) gave the
methyl ester 21D (60 mg, 77%) as a clear colorless oil. [a]D25 = +
55.5 (c=0.51, CHCl3); HRMS (ESI+): m/z [M+Na]+ calcd for
C20H23NNaO4: 364.1519, found: 364.1514.
N,3-O-Dibenzyl-2,4-dideoxy-2,4-imino-l-ribonic acid (22L): Con-
centrated HCl (11.6m, 11 mL, 0.13 mmol) was diluted with H2O
(4 mL), premixed with 1,4-dioxane (2 mL), added to the methyl
ester 21L (22.5 mg, 0.066 mmol), and stirred at 708C without a con-
denser open to air for three days. The reaction progress was fol-
lowed by MS (starting material found in +ve, title compound in
ꢀve; on completion the title compound was found in both). Fur-
thermore, TLC analysis (EtOAc) indicated the complete disappear-
ance of starting material (Rf =0.79) and appearance of a new com-
pound (Rf =0.0). On completion the reaction mixture was concen-
trated in vacuo. The residue was loaded onto a short column of
Dowex (50W-X8, H+) in which the resin had been pretreated by se-
quential washing with H2O (until eluent was neutral), 1,4-dioxane
and H2O again. The crude was loaded (H2O/1,4-dioxane 2:1) and
washed sequentially with H2O, 1,4-dioxane, and H2O again. The
product was eluted with aqueous ammonia (2m), and the ammo-
niacal fractions were concentrated in vacuo to afford the carboxylic
acid 22L as white crystalline solid (18 mg, 81%): mp: 172–1748C;
1
[a]D25 =ꢀ19.4 (c=0.16, MeOH); H NMR (400 MHz, [D5]py): d=3.61
(appq, J3,4 =J4,5 =4.8 Hz, 1H, H4), 3.76 (d, J4,5 =4.7 Hz, 2H, H5, H5’),
3.97 (d, Jgem =12.9 Hz, 1H, NCH2Ph), 4.10 (d, J2,3 =5.5 Hz, 1H, H2),
4.30 (d, Jgem =12.9 Hz, 1H, NCH2Ph), 4.72 (appt, J2,3 =J3,4 =5.4 Hz,
1H, H3), 4.85 (d, Jgem =11.9 Hz, 1H, OCH2Ph), 4.95 (d, Jgem =11.9 Hz,
1H, OCH2Ph), 7.39–7.24 (m, 6H, ArH), 7.57–7.48 ppm (m, 4H, ArH);
13C NMR (126 MHz, [D5]py): d=62.0 (NCH2Ph), 63.5 (C5), 70.9 (C2),
71.8 (OCH2Ph), 72.1 (C4), 76.1 (C3), 128.1, 128.3, 128.6, 129.0, 129.1,
130.56 (6ꢁPh), 138.7, 139.4 (2ꢁipso-Ph), 174.9 ppm (C=O); IR (thin
film): n˜ =3326 (w, OH), 1634 cmꢀ1 (s, C=O); MS (ESI+): m/z (%): 328
(45) [M+H]+, 350 (100) [M+Na]+; MS (ESI-): m/z (%): 326 (100) [M-
H]ꢀ; HRMS (ESI+): m/z [M+Na]+ calcd for C19H21NNaO4: 350.1363,
found: 350.1356.
1
CHCl3); H NMR (400 MHz, CDCl3): d=2.54 (d, JOH,5 =8.5 Hz, 1H, OH-
5), 3.08 (dd, J4,5’ =2.8 Hz, J5,5’ =11.9 Hz, 1H, H5’), 3.31–3.22 (m, 2H,
H4, H5), 3.66 (s, 3H, OMe), 3.67 (d, J2,3 =5.3 Hz, 1H, H2), 3.70 (d,
J
gem =12.5 Hz, 1H, NCH2Ph), 3.93 (d, Jgem =12.4 Hz, 1H, NCH2Ph),
4.20 (appt, J2,3 =J3,4 =5.3 Hz, 1H, H3), 4.47 (d, Jgem =11.6 Hz, 1H,
OCH2Ph), 4.62 (d, Jgem =11.6 Hz, 1H, OCH2Ph), 7.37–7.24 ppm (m,
10H, ArH); 13C NMR (101 MHz, CDCl3): d=52.1 (OMe), 60.6 (C5),
60.9 (NCH2Ph), 69.7 (C2), 70.6 (C4), 71.9 (OCH2Ph), 72.8 (C3), 127.9,
128.0, 128.1, 128.6, 129.4 (6ꢁPh), 136.7, 137.5 (2ꢁipso-Ph),
171.7 ppm (C=O); IR (thin film): n˜ =3445 (w, OH), 1737 cmꢀ1 (s, C=
O); MS (ESI+): m/z (%): 342 (56) [M+H]+, 364 (100) [M+Na]+, 705
(88) [2M+Na]+; HRMS (ESI+): m/z [M+Na]+ calcd for
C20H23NNaO4: 364.1519, found: 364.1512.
For enantiomer 22D: The protected amino acid 22D was isolated
as a white solid (32 mg, 96%): mp: 177–1788C; [a]D25 = +25.1 (c=
0.66, MeOH); HRMS (ESI+): m/z [M+Na]+ calcd for C19H21NNaO4:
350.1363, found: 350.1357.
For enantiomer 21D: Synthesis started from N,3-O-dibenzyl-2,4-di-
deoxy-2,4-imino-d-talitol 25, prepared according to published pro-
cedures:[36] NaIO4 (117 mg, 0.55 mmol) was added to a solution of
the triol 25 (159 mg, 0.46 mmol) in aqueous acetone (H2O/acetone,
2:1, 6 mL), and the reaction mixture was stirred at RT for 1 h. TLC
analysis (EtOAc) indicated consumption of the starting material
(Rf =0.30) and formation of a single new compound (Rf =0.67).
EtOH (24 mL) was added, the reaction was stirred for 45 min, and
the resultant precipitate was removed by filtration (GF/A glass mi-
crofiber). The filtrate was concentrated in vacuo (only careful heat-
ing) to yield lactol 18D as a clear glass (163 mg, 100%), which was
used directly without further purification. The lactol 18D (70 mg,
0.23 mmol) was dissolved in anhydrous MeOH (3 mL) along with
K2CO3 (95 mg, 0.69 mmol) and stirred at 08C under an atmosphere
of N2. Iodine (76 mg, 0.30 mmol) was dissolved under sonication in
anhydrous MeOH (3 mL) and added dropwise to the stirred reac-
tion mixture at 08C. MS indicated the formation of the desired
product after 45 min of reaction time, and TLC analysis (EtOAc)
showed consumption of the starting material (Rf =0.61) and forma-
tion of a single compound (Rf =0.79). The reaction was quenched
by addition of saturated Na2SO3(aq) (6 mL). The resultant white pre-
2,4-Dideoxy-2,4-imino-l-ribonic acid (10L): Concentrated HCl
(11.6m, 7 mL, 0.08 mmol) was diluted with H2O (2 mL), premixed
with 1,4-dioxane (1 mL), and added to the protected amino acid
22L (15 mg, 0.04 mmol). To this solution was added Pd/C (10% wt,
6 mg), and the reaction vessel was degassed and charged with H2.
After 15 h vigorous stirring, TLC analysis (14:3:1:1:1 EtOH/py/
nBuOH/AcOH/H2O) indicated the presence of a single product (Rf =
0.62). The reaction mixture was filtered (GF/A glass microfiber),
concentrated in vacuo at RT, and loaded onto a short column of
Dowex (50W-X8, H+) in which the resin had been pretreated by se-
quential washing with H2O (until eluent was neutral), 1,4-dioxane,
and H2O again. The crude was loaded (H2O/1,4-dioxane 2:1) and
washed sequentially with H2O, 1,4-dioxane, and H2O again. The
product was eluted with aqueous ammonia (2m), and the ammo-
niacal fractions were concentrated in vacuo at RT to yield the
amino acid 10L as a viscous gum (7 mg, 100%). Data for HCl salt:
[a]D25 =ꢀ9.7 (c=0.11, DMF); 1H NMR (500 MHz, CD3OD): d=3.83
(d, J4,5 =J4,5’ =3.6 Hz, 2H, H5, H5’), 4.28 (dt, J4,5 =J4,5’ =3.5 Hz, J3,4
=
6.7 Hz, 1H, H4), 4.66 (appt, J2,3 =J3,4 =6.6 Hz, 1H, H3), 4.73 ppm (d,
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www.chemmedchem.org
J
2,3 =6.6 Hz, 1H, H2); 13C NMR (126 MHz, CD3OD): d=58.8 (C5), 65.3
CD3OD): d=26.5 (Me), 58.9 (C5), 66.1 (C2), 68.6 (C3), 69.6 (C4),
167.2 ppm (C1); IR (thin film): n˜ =3273 (m, OH), 1673 (s, C=O,
amide I), 1570 cmꢀ1 (m, C=O, amide II); MS (ESI-): m/z (%): 102
(100), 161 (80) [M+H]+, 183 (57) [M+Na]+; HRMS (ESI+): m/z
[M+Na]+ calcd for C6H12N2NaO3: 183.0740, found: 183.0742.
(C2), 68.4 (C3), 69.5 (C4), 168.8 ppm (C=O); IR (thin film): n˜ =3209
(brm, OH, NH), 1733 (s, C=OOH), 1604 (m, CO2ꢀ), 1420 cmꢀ1 (m,
CO2ꢀ).
For enantiomer 10D: The amino acid 10D was isolated as an off-
white viscous gum (15.1 mg, 100%). Data for HCl salt: [a]D25 = +
6.64 (c=0.19, DMF).
For enantiomer 11D: The methylamide 11 D was isolated as
a clear colorless glass (33 mg, 100%). [a]D25 = +7.7 (c=1.29,
MeOH); HRMS (ESI+): m/z [M+Na]+ calcd for C6H12N2NaO3:
183.0740, found: 183.0740.
Methyl
N,3-O-dibenzyl-2,4-dideoxy-2,4-imino-l-ribonamide
(23L): To a 15-mL screw-cap vial was added the methyl ester 21L
(51 mg, 0.15 mmol) along with CaCl2 (17 mg, 0.15 mmol) and anhy-
drous MeOH (5 mL). Methylamine in absolute EtOH (0.37 mL,
3.0 mmol) was added, and the reaction vessel was flushed with
a stream of N2 before being heated at 458C for 2 h. TLC analysis
(1:1 cyclohexane/EtOAc) indicated complete consumption of the
starting material (Rf =0.44) and the formation of a single new prod-
uct (Rf =0.12) along with MS showing only the desired product in
the positive ion mode. The reaction mixture was evaporated to
dryness, and the residue was dissolved in saturated NH4Cl(aq)/H2O
(1:3, 4 mL). The resultant mixture was adjusted to pH 5 with HCl(aq)
(2m) and stirred at RT for 20 min. EtOAc (5 mL) was then added to
the mixture, which was stirred for an additional 10 min. The aque-
ous layer was extracted with EtOAc (3ꢁ10 mL), and the combined
organic fractions were dried (MgSO4), filtered, and evaporated to
give the crude reaction product (51 mg, 100%). The crude was
loaded in CH2Cl2/Et3N (99:1) and purified by flash column chroma-
tography (cyclohexane/EtOAc/Et3N 66:33:1!0:99:1) to yield meth-
ylamide 23L as a white crystalline solid (36 mg, 70%): mp: 97–
Acknowledgements
This work was supported by Cancer Research UK (A.F.G.G.) and
Grant-in-Aid for Scientific Research (No. 23590127) (A.K.) from the
Japanese Society for the Promotion of Science (JSPS). We thank
Mr. N. G. White and Mr. M. J. Langton (Chemistry Research Labo-
ratory, University of Oxford) for assistance with X-ray diffraction
data collection, and Diamond Light Source for an award of
beamtime on I19 (MT7768). We thank Dr. C. Loenarz (Chemistry
Research Laboratory, University of Oxford) for fruitful discussions.
Keywords: amino acids
foldamers · hexosamindase inhibitors
·
azetidines
·
carbohydrates
·
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1
998C; [a]D25 =ꢀ57.3 (c=0.43, CHCl3); H NMR (400 MHz, CDCl3): d=
1.73 (s, 1H, OH), 2.63 (d, JNH,Me =5.0 Hz, 3H, NHMe), 3.44–3.30 (m,
3H, H4, H5, H5’), 3.65 (d, J2,3 =5.1 Hz, 1H, H2), 3.73 (d, Jgem
12.2 Hz, 1H, NCH2Ph), 3.77 (d, Jgem =12.2 Hz, 1H, NCH2Ph), 3.92
(appt, 2,3 =J3,4 =5.1 Hz, 1H, H3), 4.45 (d, gem =11.6 Hz, 1H,
OCH2Ph), 4.83 (d, Jgem =11.7 Hz, 1H, OCH2Ph), 6.54 (brd, JNH,Me
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J
J
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4.1 Hz, 1H, NH), 7.38–7.24 ppm (m, 10H, ArH); 13C NMR (101 MHz,
CDCl3): d=25.7 (NHMe), 61.6 (C5), 61.9 (NCH2Ph), 70.9 (C4), 71.4
(OCH2Ph), 72.6 (C2), 73.9 (C3), 127.9, 128.1, 128.1, 128.5, 128.9,
129.3 (8ꢁPh), 136.9, 137.7 (ipso-Ph), 171.5 ppm (C1); IR (thin film):
n˜ =3343 (m, OH), 1651 (s, C=O, amide I), 1540 cmꢀ1 (m, C=O, ami-
de II); MS (ESI+): m/z (%): 341 (100) [M+H]+, 363 (73) [M+Na]+,
703 (57) [2M+Na]+; MS (ESI-): m/z (%): 375 (100) [M+Cl]ꢀ; HRMS
(ESI+): m/z [M+Na]+ calcd for C20H24N2NaO3: 363.1679, found:
363.1679.
For enantiomer 23D: Starting from N-benzyl-3-O-benzyl-2,4-di-
deoxy-2,4-imino-d-talitol (75 mg, 0.22 mmol), methylamide 23D
was isolated as a white crystalline solid (54 mg, 72%) without inter-
mediate purification. [a]D25 = +58.5 (c=1.04, HCCl3); HRMS (ESI+):
m/z [M+Na]+ calcd for C20H24N2NaO3: 363.1679, found: 363.1680.
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HCl (11.6m, 8 mL, 0.10 mmol) was diluted with H2O (2 mL) premixed
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1
100%): [a]D25 =ꢀ12.2 (c=0.42, MeOH); H NMR (500 MHz, CD3OD):
d=2.82 (s, 3H, Me), 3.83 (d, J4,5 =4.0 Hz, 2H, H5, H5’), 4.28 (dt,
J
4,5 =J4,5’ =3.9 Hz, J3,4 =7.2 Hz, 1H, H4), 4.53 (appt, J2,3 =J3,4 =6.7 Hz,
1H, H3), 4.62 ppm (d, J2,3 =6.6 Hz, 1H, H2); 13C NMR (126 MHz,
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 658 – 666 665

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Received: November 23, 2012
Revised: February 14, 2013
Published online on March 6, 2013
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ChemMedChem 2013, 8, 658 – 666 666