Synthesis of Tumor-Associated LeaLex Hexasaccharides: Instability of a Thiol-Containing Oligosaccharide in Mass Spectrometry and Hypermetalation Detected by ESI FAIMS

Article abstract of DOI:10.1021/acs.joc.5b01190

We report the efficient synthesis of three analogues of the tumor-associated carbohydrate antigen Le^aLe^x. This hexasaccharide was prepared as a soluble inhibitor hexyl glycoside, as a 6-aminohexyl glycoside for conjugation to protein

Full text of DOI:10.1021/acs.joc.5b01190

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Synthesis of TumorꢀAssociated LeaLex hexasaccharides: Instability of a
Thiolꢀcontaining Oligosaccharide in Mass Spectrometry and Hyperꢀ
metalation detected by ESI FAIMS.
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Mickaël Guillemineau,† Jadwiga Lyczko, Wojciech Gabryelski, FranceꢀIsabelle Auzanneau*
Department of Chemistry, University of Guelph, Guelph, Ontario, N1G 2W1 Canada.
Abstract
We report the efficient synthesis of three analogues of the tumor associated carbohydrate
antigen LeaLex. This hexasaccharide was prepared as a soluble inhibitor hexyl glycoside, a 6ꢀ
aminohexyl glycoside for conjugation to proteins, and as 6ꢀthiohexyl glycoside for
immobilization to a gold surface. These three analogues were obtained from a common
hexasaccharide intermediate and isolated pure following efficient deprotection reactions that
involved metal dissolving conditions. While all other intermediates and analogues gave the
expected molecular ions in ESI HRMS, the 6ꢀthiohexyl glycoside final compound gave a
complex spectrum in which no signal matched the molecular ion. Using ESI FAIMS HRMS, we
were able to prevent ion dissociation reactions and obtained high quality spectral data. The ions
detected could be characterized unambiguously from their accurate masses and gave insight into
the behavior of the thiohexyl analogue in the gas phase. These results indicate that the 6ꢀ
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thiohexyl glycoside lost water and led to the formation of “hyperꢀmetalated” species which we
propose are cyclic.
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Introduction
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Although not yet successful, the use of Tumor Associated Carbohydrate Antigens
(TACAs) to develop antiꢀcancer vaccines has led to intense research efforts in the past two
decades.1 Of particular interest to our group is the TACA LeaLex which has been shown to be
overꢀexpressed on the surface of squamous lung carcinoma (SLC) cells glycoproteins as well as
secreted soluble mucins from SLC cells.2 While the nonreducing end Lea trisaccharide is also
displayed by normal cells,3 monoclonal antibody (mAb) 43ꢀ9F raised in mice against human
SLC cells was shown to recognize preferentially LeaLex over Lea and to recognize an epitope
primarily displayed on SLC cells and associated with the tumorigenicity of these cells.2, 3b, 4
Thus, we are exploring the possibility of using the epitope recognized by mAb 43ꢀ9F to develop
an antiꢀcancer vaccine. To identify the smallest LeaLex fragments that still displays this epitope,
we have already chemically prepared numerous fragments of the natural hexasaccharide using
stepwise approaches and attempting to limit the deprotection steps leading to the desired final
compounds.5 We report here the efficient and convergent chemical synthesis of analogues 2–4 of
the LeaLex TACA. Hexylglycoside 2 will be used as soluble inhibitor in competitive ELISA
studies while aminohexyl glycoside 3 will be conjugated to tetanus toxoid for immunizations and
BSA for immobilization on ELISA plates. Thiohexylglycoside 4 will be immobilized to a gold
chip for the detection of antibodies by surface plasmon resonance (SPR).
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HO
OH
OH
C'
O
OH
O
OH HO
O
6
7
8
9
OH
O
HO
O
O
B
A'
HO
HO
O
O
B'
A
O(CH2)6R
O
O
HO
AcHN
AcHN
OH
OH
O
C
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OH
HO
1 LeaLex
2 R = H
3 R = NH3+AcO-
4 R = SH
To the best of our knowledge, while fragments of the TACA were also prepared in Ling’s
laboratory,6 only one enzymatic synthesis of the LeaLex glycosphingolipid has been reported in
the literature by Hakomori.4d Interestingly, while we confirmed the structure of analogues 2 and
3 easily by ESI HRMS, data for the 6ꢀthiohexyl glycoside 4 did not show the expected molecular
ion. Indeed, the results presented here suggest that the hexylꢀ6ꢀthiol aglycone in hexasaccharide
4 gave a molecular ion which was too labile to be detected. Interpretation of the data obtained
from electrospray high resolution mass spectrometry (ESI HRMS) was complicated by gas phase
reactions involving the primary thiol and which led to the formation of dissociation species and
species accommodating multiple sodium and ammonium ions. Using electrospray highꢀfield
asymmetric waveform ion mobility spectrometry (ESI FAIMS),7 we were able to suppress the
ion dissociation reactions and obtain high quality spectral data that was easily interpreted and
suggested that the 6ꢀthiohexyl glycoside 4 lost water and led to the formation of “hyperꢀ
metalated” cyclic species. Interestingly, ESI FAIMS HRMS on analogues 2 and 3 did not lead to
either loss of water nor hypermetalation.
Results and Discussion
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The rapid assembly of the LeaLex hexasaccharide employed known monosaccharide
3
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6
building blocks 58, 69, 710, 811 and 912 and is shown on Scheme 1.
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OPiv
O
OBn
O
AcO
ClAcO
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HO
ClAcO
O(CH2)6Cl
AcO
AcHN
OC(NH)CCl3
O
6
5
Ph
O
O
SEt
OBn
O
ClAcO
OBn
BnO
TCAHN
OC(NH)CCl3
8
7
OAc
O
AcO
AcO
AcO
OC(NH)CCl3
9
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OPiv
O
AcO
O
Ph
O
OPiv
O
OBn
O
AcO
RO
7
O
5
6
O
BF3.OEt2
40 °C
OBn
O
B
A'
R2O
O
TMSOTf
A
R1O
O(CH2)6Cl
O
AcO
TCAHN
0 °C
ClAcO
O(CH2)6Cl
AcO
AcHN
1
2
AcHN
10 R = ClAc (79%)
11
12
13
14
R = R = ClAc (79%)
1
R = ClAc, R2 = H (36%)
(H2N)2CS
(H2N)2CS
1
2
R = H (65%)
R = R = H (7-77%)
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OPiv
O
AcO
Ph
O
OPiv
AcO
OBn
O
R2O
O
O
OBn
O
1
O
O
A'
B
AcO
R O
RO
O
B
O
A'
A
O
O
O
O
AcO
O(CH2)6Cl
A
O
1. ClAcCl
B'
TCAHN
AcO
O(CH2)6Cl
9
, rt
AcO
14
TCAHN
O
AcHN
O
AcHN
2. (i) 8, MeOTf,
(ii) AcOH/Ac2O
C
AcO
OAc
O
BF3.OEt2
C
OBn
OBn
OBn
BnO
OBn
BnO
1
R , R2 = >CHPh (52%)
15 R = ClAc (84%)
16 R = H (88%)
17
18
(H2N)2CS
HCl/NaCNBH3
R
1 = H, R2 = Bn (62%)
BnO
OBn
OBn
C'
O
OPiv
O
OBn AcO
1. Na/NH3(l)
2
OBn
O
19
(61%)
8
1. , MeOTf
2. AcOH/Ac2O AcO
O
AcO
O
O
2. Ac2O in MeOH
18
B
A'
O
O
B'
A
O(CH2)6R1
O
R2HN
Na/NH3(l)
2
(89%)
3 (61%)
O
AcO
20
21
AcO
AcHN
OAc
O
C
OBn
OBn
BnO
1. MeONa/MeOH
2. Na/NH3(l)
4
(90%)
22
19 R1 = Cl, R2 = TCA (76%)
20 R1 = Cl, R2 = Ac (95%)
21 R1 = N3, R2 = Ac (quant)
22 R1 = SAc, R2 = Ac (93%)
Zn/AcOH
KSAc
NaN3
SCHEME 1. Synthesis of hexasaccharides 2–4 from building blocks 5–9.
Glycosylation of acceptor 6 with donor 5 (4 equiv) activated with BF3·OEt2 (2 equiv) at
40 °C gave disaccharide 10 in very good yield (79%). Selective deꢀchloroacetylation at Oꢀ3’of
disaccharide 10 was achieved in 65% yield using the conditions that we have established
previously on a similar analogue5c (1.2 equiv of thiourea at 55 °C). The position of the free
hydroxyl group in acceptor 11 was confirmed by the upfield shift of Hꢀ3’, from 4.79 ppm in 10
to 3.54 ppm in the 1H NMR spectrum. Acceptor 11 was then glycosylated with 3 equiv of
trichloroacetimidate donor 7 activated at 0 °C with TMSOTf (2 equiv) and the desired
trisaccharide 12 was obtained in 79%. While studying the reactivity of Oꢀ3A and Oꢀ3A’ in diol
14, we have reported that galactosylation at Oꢀ3A’ was mismatched while that at Oꢀ3A was
matched.13 Further studies are onꢀgoing in our laboratory to explain these results but we expected
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that the introduction of the galactosyl B’ would require harsh reaction conditions which may lead
to the loss of a fucosyl unit if it was introduced first at Oꢀ3A. Thus, we attempted the selective
deꢀchloroacetylation at the nonꢀreducing end Oꢀ3A’ of trisaccharide 12 using the conditions
applied for the preparation of disaccharide 11. While after 7 h analytical RPꢀHPLC showed 80%
conversion to acceptor 13 with only 12% of diol 14 and 8% of starting material, chromatography
of the crude mixture gave the desired trisaccharide 13 in only 36% yield, while diol 14 was
obtained in 7% yield. Faced with this poor yield, the synthetic strategy was revised and
fucosylation at the reducing end was scheduled to occur before the galactosylation at Oꢀ3A’.
Both chloroacetates were removed (excess thiourea) to give the previously characterized13 diol
14 (77%). The diol was then first selectively chloroacetylated13 at the reducing end Oꢀ3A’, and
the resulting acceptor was fucosylated at Oꢀ3A with donor 8 (3 equiv) activated by MeOTf (5
equiv). The crude mixture was treated with 25% AcOH in Ac2O to convert any methyl imidate14
into Nꢀacetyl group and the desired tetrasaccharide 15 was isolated in 84% yield.
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Dechloroacetylation of tetrasaccharide 15 with an excess thiourea gave the previously
characterized13 tetrasaccharide acceptor 16 in 88% yield, which was submitted to galactosylation
with donor 9. As anticipated harsh reaction conditions were required to obtain 17: excess
BF3·OEt2 (2 equiv) at room temperature and 5 equiv of donor 9. Due to coꢀmigration of the
products and degraded donor, the reaction was best monitored by RPꢀHPLC after quenching of
an aliquot with NEt3. One hour of reaction was found to be ideal to obtain the best yield of
desired tetrasaccharide 17 (52%) while avoiding excessive degradation and loss of the fucosyl
unit, however unreacted acceptor 16 was also recovered in 20% yield. Regioselective opening of
the benzylidene acetal in pentasaccharide 17 then gave pentasaccharide acceptor 18 in 62%
yield. Fucosylation of 18 with 5 equiv of donor 9 activated with MeOTf was followed by
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treatment of the crude mixture with 25% AcOH in Ac2O and gave the desired protected LeaLex
hexasaccharide 19 in excellent 76% yield. Reduction of the trichloroacetamide into an acetamide
was achieved using with zinc in acetic acid under sonication at 50 °C for 7 h and gave
hexasaccharide 20 in excellent yield. Portions of the 6ꢀchlorohexyl glycoside 20 were then
converted to the 6ꢀazidohexyl and 6ꢀacetylthiohexyl glycosides 21 and 22 through the
nucleophilic displacement of the chlorine atom using excess sodium azide or potassium
thioacetate, respectively, in DMF at 80 °C. The 6ꢀazidohexyl glycoside 21 was obtained
quantitatively while the 6ꢀacetylthiohexyl glycoside 22 was isolated in 93% yield. Hexyl
hexasaccharide 2 was then prepared from either the trichloroacetamido intermediate 19 or the Nꢀ
acetamido hexasaccharide 20. Full deprotection of hexasaccharide 20 using dissolving metal
conditions led to reduction of the chlorohexyl to an hexyl chain and removal of all protecting
groups including the trichloracetamido at Cꢀ2A’ giving the free amine.5b, c, 9, 15 The free amine
was acetylated through work up of the reaction mixture with acetic anhydride and
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hexasaccharide 2 was isolated pure in 61% yield after chromatography on a Biogel® P2 column
eluted with milliꢀQ water. Alternatively dissolving metal conditions applied to chlorohexyl
glycoside 20 gave the desired hexasaccharide 2 in 89% yield. Deprotection of the 6ꢀazidohexyl
glycoside 21 in the same conditions gave the 6ꢀaminohexyl glycoside hexasaccharide 3 as the
ammonium acetate salt in 61% yield after purification on a Biogel® P2 column eluted with 0.05
M ammonium acetate and repeated freezeꢀdrying. We have shown9 that under dissolving metal
condition an Sꢀacetyl hexyl chain underwent desulfurization to give the corresponding hexyl
glycoside. Thus hexasaccharide 22 first underwent Zemplèn deacetylation and the crude reaction
product was then submitted to dissolving metal conditions to give the 6ꢀthiohexyl hexasaccharide
4 in 90% yield after purification on a Biogel® P2 column eluted with milliꢀQ water.
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High resolution mass spectrometry along with H NMR spectroscopy that showed a
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triplet at 0.85 ppm corresponding to the hexyl methyl group, confirmed the structure of the hexyl
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glycoside 2. Similarly, the structure of aminohexyl glycoside 3 was confirmed by HRMS and by
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+
the presence of a triplet at 2.97 ppm corresponding to the terminal CH2NH3 in the 1H NMR. In
contrast, while by analogy to hexasaccharides 2 and 3 NMR spectroscopy unequivocally
supported the structure and purity of thiol 4, no molecular ion could be seen in HRMS when the
sample was analyzed at three different facilities. Interestingly, the chemical shift measured for
the SCH2 appeared 0.19 ppm lower field than that (2.70 pm) of the analogue hexyl disulfide
glycoside Lex dimer (see 23 Figure 2) that we have reported.9 We observed that this triplet at
2.89 ppm matched that of a minor compound formed when preparing the Lex analogue and that
we assumed to be the corresponding hexyl thiol glycoside. We thus concluded that
hexasaccharide 4 was likely a thiol rather than a disulfide dimer.
Figure 1 shows the results from the high resolution mass spectrometry (HRMS) analysis
of 6ꢀthiohexyl glycoside 4 using two different techniques. As can be seen on the inserted full
spectra, electrosprayꢀfield asymmetric waveform ion mobility spectrometry (ESI FAIMS) high
resolution mass spectrometry7d gave a simple spectrum (Figure 1A, insert) with excellent signalꢀ
toꢀnoise ratio. The expanded spectrum (Figure 1A) shows the detection of several major doubly
charged ions formed from 6ꢀthiohexyl glycoside 4 after loss of water (M–H2O). Elemental ion
compositions (determined from high resolution accurate mass measurements, see supporting
information) showed that while two sodium ions provided the +2 net ion charge, up to four
additional sodium ions were also added.
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FIGURE 1. High resolution mass spectra recorded for 6ꢀthiohexyl glycoside 4. A)
Electrospray highꢀfield asymmetric waveform ion mobility spectrometry high resolution
mass spectrometry (ESI FAIMS HRMS); B) electrospray high resolution mass
spectrometry (ESI HRMS).
In addition to the sodium adducts, Figure 1A shows the formation of hyperꢀmetalated ammonium
adducts giving signals to the left of the major sodium adduct ions. The conventional ESI HRMS
analysis of 6ꢀthiohexyl glycoside 4 (Figure 1B, insert) gave a much more complex spectrum than
the ESI FAIMS HRMS with over 100 major ions detected. The many ions present can be
attributed to dissociation products of labile doubly and singly charged sodium adducts of the
glycoside and the spectral interpretation is complicated by the relatively poor signalꢀtoꢀnoise
ratio and overlapping ions of similar m/z. However, we were able to find singly charged ions of
hyperꢀmetalated species that had also lost water and were in agreement with the results given by
the ESI FAIMS HRMS for doubly charged species (Figure 1B).
To investigate the impact of the thiol group in hexasaccharide 4 on the formation of the
ions observed by ESI FAIMS HRMS, we also studied the hexyl and aminohexyl hexasaccharides
2 and 3 as well as the previsouly reported9 hexyl disulfide glycoside Lex dimer (23). The full ESI
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FAIMS spectra are given in the supporting information while expanded regions showing the
most abundant ions that were detected in these experiments are given in Figure 2. Only doubly
charged ions were detected that were identified from elemental ion composition (see supporting
information) as proton, ammonium or sodium adduct, as well as mixed proton/sodium or
ammonium adducts (Figure 2). Interestingly, none of the compounds underwent loss of water
and no hypemetalated species were seen for either the hexyl or amino glycosides 2 and 3, or the
disulfide dimer 23.
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FIGURE 2. ESI FAIMS HRMS for A) hexyl
glycoside 2; B) aminohexylglycoside 3; and
hexyl disulfide glycoside Lex dimer 23.
“Hyperꢀmetalation” of biomolecules in ESI MS was first named and described by Pope et al.
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for the cyclic peptide Gramicidin S which was shown to add four or more sodium in the +2
charge state.16 The authors attributed the formation of these adducts to the displacement of
protons from the Gramicidin S amide nitrogen atoms leading to salts and leaving the net charge
of the ion unchanged. While, there have been several other reports on the hyperꢀmetalation of
peptides,17 we could only find one such report involving carbohydrates. Indeed, Madhusuanan
reported multiple lithium exchanges while studying cyclodextrins by ESI MS under lithium
cationization.18 In our study, the fact that no molecular ion was detected for hexyl thiol 4 while
we encountered no such issue when analyzing hexasaccharides 2 and 3 or the reported9 analogue
hexyl disulfide glycoside Lex dimer 23, suggests that the hexyl thiol chain played a major role in
the loss of water and ions being formed. Since cyclic species such as cylcodextrins or Gramicidin
S seem to be more prone to hyperꢀmetalation through the trapping and intramolecular
coordination of metal ions, we propose that the molecular ion generated from hexyl thiol 4
underwent cyclization involving the thiol group and leading to the concomitant loss of a water
molecule. This may have been following protonation (Figure 3, path a) or adduction of a sodium
ion on a hydroxyl group (Figure 3, path b) forming the molecular ion and subsequent
nucleophilic attack by the thiol displayed at the end of a long and flexible hexyl chain (Figure 3).
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(CH2)6
SH
Path a
H+
Path b
Na+
OH
4
(CH2)6
(CH2)6
S
S
H
H
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Na+
OH
OH2
[M+Na]+
- Na+
[M+H]+
- H3O+
- H2O
(CH2)6
S
FIGURE 3. Proposed pathways leading to the
loss of water in hexyl thiol glycoside 4
This cyclic species may trap sodium ions which are present in the sample solution at trace
levels from the last synthetic step [NH3(l)/Na] and undergo hyperꢀmetalation through proton
displacement from the hydroxyl groups and intramolecular coordination of sodium ions.
Conclusion
We are reporting the first fully chemical preparation of three analogues of the TACA
LeaLex. Building on our experience, the synthetic scheme proved straightforward efficient and
convergent and the final intermediates were easily deprotected using dissolving metal conditions.
Most interestingly, we have observed that the hexasaccharide bearing a long hexyl 6ꢀthiol
aglycone at the reducing end did not give the expected molecular ion in ESI HRMS and
underwent considerable ion dissociation. Using the more recent ESI FAIMS HRMS technique
proved to be invaluable. FAIMS is a gasꢀphase atmospheric pressure separation technique,7a
which was developed for use with MS.7b It operates between the ESI source and the MS
detection behaving as an ion filter that separates ions according to their differential ion mobility in
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alternating low and high electric fields generated in the gap between two closely spaced electrodes.7c
Coupled with “soft” QꢀTOF mass spectrometry, it is capable of detecting extremely labile ions with
minimal fragmentation. Mass calibration was carried out using formaldehyde, as described
recently for use with “soft” QꢀTOF mass spectrometry19 and it appeared that all ions detected for
compounds 2–4 and 23 were doubly charged. Hexyl glycoside 2¸ aminohexyl glycoside 3, and
hexyl disulfide analogue 23 gave stable doubly charged ions through protonation, or adduction of
ammonium or sodium. In contrast, and based on the ions that could be unequivocally assigned
through their accurate masses, we observed that the molecular ion formed from the 6ꢀthiohexyl
glycoside 4 was highly unstable. We propose that loss of a water equivalent occurred in the gas
phase with the concomitant formation of cyclic species involving the nucleophilic thiol present at
the end of the long hexyl chain. The resulting cyclic structure then became hyperꢀmetalated
through sodium exchange at the hydroxyl groups. While there are a few reports describing
hyperꢀmetalation of peptides during ESI HRMS,16ꢀ17 it is rarely observed during the analysis of
oligosaccharides. Indeed, while multiple lithium exchange has been observed during ESI MS of
cyclodextrins under lithium cationization, openꢀended maltohexoses treated in the same
conditions gave barely detectable signals for hyperꢀlithiated species.18 We thus conclude that the
formation of the cyclic ion may have favored hyperꢀmetalation through the efficient coordination
of the sodium ions by the hydroxyl groups brought in closer proximity.
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Experimental Section
Electrospray highꢀfield asymmetric waveform ion mobility spectrometry high
resolution mass spectrometry (ESI FAIMS HRMS) on hexasaccharides 2–4 and disulfide
23. A ~10 µM solution of the compound to be analyzed was prepared using methanol/water (9/1
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v/v) containing 0.1 mM ammonium acetate. The solution was infused into a nanospray source at
a flow rate of 400 nL/min using the autoꢀsampler and pump from a nanoꢀflow capillary UPLC
system. Ionization was carried out in positive mode with a spray voltage of 5000 V. The sample
was analyzed using an FAIMS analyzer with 20% CO2 in N2 as a carrier and desolvation gas and
a dispersion voltage of 4000 V. Detection was carried out using a QꢀTOF mass spectrometer. Ion
transmission conditions at low ion kinetic energy were optimized in order to minimize ion
dissociation during the analysis. The sample cone voltage, extraction cone voltage, and collision
energy voltage were set at 20 V, 1.0 V and 4 V, respectively. After acquisition, data were
summed, smoothed two times using the SavitzkyꢀGolay method and then centered. The mass
calibration was carried out using formaldehyde, as described recently for use with “soft” QꢀTOF
mass spectrometry.19 Mass accuracies were <5 ppm, and were sufficient to determine the
elemental composition of the product ions presented. The product ions are listed in the
Supporting Information.
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6ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ(2,4ꢀdiꢀOꢀacetylꢀ3ꢀOꢀchloroacetylꢀ6ꢀOꢀpivaloylꢀβꢀ
Dꢀ
galactopyranosyl)ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀchloroacetylꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranoside (10). A stirred
D
solution of known9 alcohol 6 (905 mg, 1.787 mmol) and known8 galactosyl trichloroacetimidate
5 (4.07 g, 7.150 mmol, 4 equiv) in dry CH2Cl2 (45 mL) was heated to 40 °C under N2. Freshly
distilled BF3·OEt2 (449 µL, 3.573 mmol, 2 equiv) was added to the mixture, which was stirred
for 1 h at 40 °C, then quenched with NEt3 (598 µL, 4.290 mmol, 2.4 equiv). The reaction was
diluted with CH2Cl2 (55 mL) and washed with saturated aq NaHCO3 (1 × 100 mL). The aq layer
was reꢀextracted with CH2Cl2 (2 × 30 mL) and the combined organic layers were dried and
concentrated. The product was purified by column chromatography (EtOAc/hexanes, 1:1) to give
the disaccharide 10 (1.29 g, 79%) pure as a slightly yellowish amorphous foam. [α]D –4.5 (c 1.0,
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CH2Cl2). 1H NMR (400 MHz, CDCl3, 297 K): δH 7.40–7.30 (m, 5 H, Ar), 5.65 (d, J = 9.1 Hz, 1
H, NHA), 5.19 (d, J = 3.3 Hz, 1 H, Hꢀ4B), 5.14 (dd, J = 9.0, 10.2 Hz, 1 H, Hꢀ3A), 4.94 (dd, J =
8.0, 10.3 Hz, 1 H, Hꢀ2B), 4.79 (dd, J = 3.5, 10.3 Hz, 1 H, Hꢀ3B), 4.75 (d, J = 12.0 Hz, 1 H,
CHHPh), 4.49 (d, J = 8.0 Hz, 1 H, Hꢀ1A), 4.39 (d, J = 12.0 Hz, 1 H, CHHPh), 4.33 (d, J = 8.0
Hz, 1 H, Hꢀ1B), 4.09–3.98 (m, 4 H, Hꢀ6Bab, COCH2Cl), 3.92–3.86 (m, 5 H, Hꢀ2A, Hꢀ4A,
OCHHCH2, COCH2Cl), 3.70 (m, 2 H, Hꢀ6Aab), 3.54 (t, J = 6.7 Hz, 1 H, Hꢀ5B), 3.50 (t, J = 6.6
Hz, 2 H, CH2Cl), 3.46 (m, 1 H, Hꢀ5A), 3.42 (m, 1 H, OCHHCH2), 2.11, 1.95 (2 s, 6 H, 2
OCOCH3), 1.92 (s, 3 H, NHCOCH3), 1.73 (m, 2 H, O(CH2)4CH2CH2Cl), 1.55 (m, 2 H,
OCH2CH2(CH2)3CH2Cl), 1.41 (m, 2 H, O(CH2)3CH2(CH2)2Cl), 1.32 (m, 2 H,
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O(CH2)2CH2(CH2)3Cl), 1.18 (s, 9 H, C(CH3)3). 13C NMR (100 MHz, CDCl3, 297 K): δC 177.8,
170.4, 170.2, 169.1, 167.5, 166.6 (C=O), 137.7 (quat Ar), 128.7, 128.2 (Ar), 100.9 (Cꢀ1A), 99.9
(Cꢀ1B), 74.4 (Cꢀ3A), 74.3 (Cꢀ4A), 74.2 (Cꢀ5A), 73.7 (CH2Ph), 72.5 (Cꢀ3B), 70.5 (Cꢀ5B), 69.5
(OCH2CH2), 68.9 (Cꢀ2B), 67.3 (Cꢀ6A), 66.5 (Cꢀ4B), 60.6 (Cꢀ6B), 53.7 (Cꢀ2A), 45.0 (CH2Cl),
40.9, 40.4 (COCH2Cl), 38.8 (C(CH3)3), 32.5 (O(CH2)4CH2CH2Cl), 29.2
(OCH2CH2(CH2)3CH2Cl), 27.1 (C(CH3)3), 26.5 (O(CH2)3CH2(CH2)2Cl), 25.2
(O(CH2)2CH2(CH2)3Cl), 23.3 (NHCOCH3), 20.7, 20.6 (OCOCH3). HRESIMS (m/z): [M+Na]+
calcd for C40H56Cl3NO16Na 934.2562, found 934.2585.
6ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ(2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀpivaloylꢀβꢀ
Dꢀ
galactopyranosyl)ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀchloroacetylꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranoside (11).
D
Disaccharide 10 (6.75 g, 7.392 mmol) was dissolved in a mixture of pyridine and EtOH (2:1, 180
mL), thiourea (675.2 mg, 8.870 mmol, 1.2 equiv) was added and the solution was heated to 55
°C for 4 h, then allowed to come to rt. The reaction mixture was diluted with CHCl3 (500 mL)
and washed with HCl 2 N (1 × 600 mL). The aq layer was reꢀextracted with CHCl3 (5 × 100 mL)
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and the combined organic layers were dried and concentrated. The product was purified by
column chromatography (CHCl3/MeOH, 20:1) to give alcohol 11 (4.04 g, 65%) pure as a yellow
amorphous foam. [α]D –3.1 (c 1.0, CH2Cl2). 1H NMR (400 MHz, CDCl3, 296 K): δH 7.38–7.27
(m, 5 H, Ar), 5.57 (d, J = 9.0 Hz, 1 H, NHA), 5.18–5.12 (m, 2 H, Hꢀ3A, Hꢀ4B), 4.76–4.68 (m, 2
H, Hꢀ2B, CHHPh), 4.50 (d, J = 8.0 Hz, 1 H, Hꢀ1A), 4.43 (d, J = 12.0 Hz, 1 H, CHHPh), 4.34 (d,
J = 8.0 Hz, 1 H, Hꢀ1B), 4.13–3.98 (m, 4 H, Hꢀ6Bab, COCH2Cl), 3.92–3.80 (m, 3 H, Hꢀ2A, Hꢀ
4A, OCHHCH2), 3.75 (m, 2 H, Hꢀ6Aab), 3.58–3.49 (m, 6 H, Hꢀ5A, Hꢀ3B, Hꢀ5B, OCHHCH2,
CH2Cl), 2.48 (d, J = 6.0 Hz, 1 H, OHꢀ3B), 2.14, 2.04 (2 s, 6 H, 2 OCOCH3), 1.93 (s, 3 H,
NHCOCH3), 1.73 (m, 2 H, O(CH2)4CH2CH2Cl), 1.56 (m, 2 H, OCH2CH2(CH2)3CH2Cl), 1.41 (m,
2 H, O(CH2)3CH2(CH2)2Cl), 1.33 (m, 2 H, O(CH2)2CH2(CH2)3Cl), 1.19 (s, 9 H, C(CH3)3). 13C
NMR (100 MHz, CDCl3, 296 K): δC 177.9, 170.9, 170.8, 170.1, 167.4 (C=O), 137.9 (quat Ar),
128.6, 128.0, 127.9 (Ar), 101.0 (Cꢀ1A), 99.9 (Cꢀ1B), 74.5 (Cꢀ3A), 74.5 (Cꢀ4A), 74.4 (Cꢀ5A),
73.6 (CH2Ph), 73.1 (Cꢀ2B), 71.5 (Cꢀ3B), 70.9 (Cꢀ5B), 69.5 (OCH2CH2), 69.4 (Cꢀ4B), 67.5 (Cꢀ
6A), 61.4 (Cꢀ6B), 53.9 (Cꢀ2A), 45.0 (CH2Cl), 40.9 (COCH2Cl), 38.8 (C(CH3)3), 32.5
(O(CH2)4CH2CH2Cl), 29.3 (OCH2CH2(CH2)3CH2Cl), 27.1 (C(CH3)3), 26.5
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(O(CH2)3CH2(CH2)2Cl), 25.2 (O(CH2)2CH2(CH2)3Cl), 23.4 (NHCOCH3), 21.0, 20.7 (OCOCH3).
HRESIMS (m/z): [M+H]+ calcd for C38H56Cl2NO15 836.3027, found 836.3003.
6ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ[2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀpivaloylꢀ3ꢀOꢀ(4,6ꢀOꢀ
benzylideneꢀ3ꢀOꢀchloroacetylꢀ2ꢀdeoxyꢀ2ꢀtrichloroacetamidoꢀβꢀ
Dꢀglucopyranosyl)ꢀβꢀDꢀ
galactopyranosyl]ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀchloroacetylꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranoside (12).
D
Disaccharide glycosyl acceptor 11 (40.0 mg, 0.04781 mmol) and known10 glycosyl donor 7 (90.8
mg, 0.1434 mmol, 3 equiv) were dissolved in dry CH2Cl2 (2.5 mL) under N2. After cooling the
mixture to 0 °C, freshly distilled TMSOTf (17.3 µL, 0.09562 mmol, 2 equiv) was added to the
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reaction, which was stirred at 0 °C for 40 min, then quenched with NEt3 (16.0 µL, 0.1147 mmol,
2.4 equiv). The reaction was diluted with CH2Cl2 (15 mL) and washed with saturated aq
NaHCO3 (1 × 15 mL). The aq layer was reꢀextracted with CH2Cl2 (3 × 10 mL) and the combined
organic layers were dried and concentrated. The product was purified by column
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chromatography (EtOAc/hexanes, 1:1) to give trisaccharide 12 (49.2 mg, 79%) pure as a yellow
amorphous foam. [α]D –8.0 (c 1.0, CH2Cl2). 1H NMR (600 MHz, CDCl3, 295 K): δH 7.40–7.27
(m, 10 H, Ar), 7.19 (d, J = 8.8 Hz, 1 H, NHA’), 5.95 (d, J = 9.2 Hz, 1 H, NHA), 5.52 (t, J = 9.9
Hz, 1 H, Hꢀ3A’), 5.47 (s, 1 H, >CHPh), 5.24 (d, J = 3.4 Hz, 1 H, Hꢀ4B), 5.07 (t, J = 9.1 Hz, 1 H,
Hꢀ3A), 4.91 (dd, J = 8.3, 9.7 Hz, 1 H, Hꢀ2B), 4.73 (d, J = 8.0 Hz, 1 H, Hꢀ1A’), 4.69 (d, J = 12.1
Hz, 1 H, CHHPh), 4.42 (d, J = 12.0 Hz, 1 H, CHHPh), 4.36 (d, J = 7.7 Hz, 1 H, Hꢀ1A), 4.31 (dd,
J = 4.9, 10.4 Hz, 1 H, Hꢀ6Aa’), 4.27 (d, J = 8.0 Hz, 1 H, Hꢀ1B), 4.11–3.92 (m, 7 H, Hꢀ2A, Hꢀ
6Bab, 2 COCH2Cl), 3.87–3.80 (m, 2 H, Hꢀ4A, Hꢀ2A’), 3.78 (m, 1 H, OCHHCH2), 3.72–3.66 (m,
2 H, Hꢀ4A’, Hꢀ6Ab’), 3.64 (m, 2 H, Hꢀ6ab), 3.59–3.54 (m, 2 H, Hꢀ3B, Hꢀ5B), 3.52–3.47 (m, 3
H, Hꢀ5A’, CH2Cl), 3.40 (m, 1 H, Hꢀ5A), 3.35 (m, 1 H, OCHHCH2), 2.04, 1.99 (2 s, 6 H, 2
OCOCH3), 1.92 (s, 3 H, NHCOCH3), 1.72 (m, 2 H, O(CH2)4CH2CH2Cl), 1.53 (m, 2 H,
OCH2CH2(CH2)3CH2Cl), 1.39 (m, 2 H, O(CH2)3CH2(CH2)2Cl), 1.31 (m, 2 H,
O(CH2)2CH2(CH2)3Cl), 1.21 (s, 9 H, C(CH3)3). 13C NMR (125 MHz, CDCl3, 295 K): δC 178.0,
170.1, 170.0, 169.0, 167.6, 167.5, 162.1 (C=O), 137.8, 136.5 (quat Ar), 129.2, 128.5, 128.3,
128.0, 125.9 (Ar), 101.2 (>CHPh) 101.0 (Cꢀ1A), 100.2 (Cꢀ1A’), 100.1 (Cꢀ1B), 92.1 (CCl3), 78.2
(Cꢀ4A’), 76.2 (Cꢀ3B), 74.4 (Cꢀ5A), 74.3 (Cꢀ3A), 74.1 (Cꢀ4A), 73.6 (CH2Ph), 72.3 (Cꢀ3A’), 71.0
(Cꢀ5B), 70.6 (Cꢀ2B), 69.5 (OCH2CH2), 68.6 (Cꢀ4B), 68.2 (Cꢀ6A’), 67.3 (Cꢀ6A), 66.0 (Cꢀ5A’),
61.6 (Cꢀ6B), 56.6 (Cꢀ2A’), 52.8 (Cꢀ2A), 45.0 (CH2Cl), 40.8, 40.5 (COCH2Cl), 38.8 (C(CH3)3),
32.4 (O(CH2)4CH2CH2Cl), 29.2 (OCH2CH2(CH2)3CH2Cl), 27.0 (C(CH3)3), 26.5
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(O(CH2)3CH2(CH2)2Cl), 25.2 (O(CH2)2CH2(CH2)3Cl), 23.2 (NHCOCH3), 21.1, 20.7 (OCOCH3).
HRESIMS (m/z): [M+H]+ calcd for C55H71Cl6N2O21 1305.2680, found 1305.2687.
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6ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ[2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀpivaloylꢀ3ꢀOꢀ(4,6ꢀOꢀ
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benzylideneꢀ2ꢀdeoxyꢀ2ꢀtrichloroacetamidoꢀβꢀ
benzylꢀ3ꢀOꢀchloroacetylꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranoside (13) and 6ꢀchlorohexyl 2ꢀacetamidoꢀ
4ꢀOꢀ[2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀpivaloylꢀ3ꢀOꢀ(4,6ꢀOꢀbenzylideneꢀ2ꢀdeoxyꢀ2ꢀtrichloroacetamidoꢀβꢀ
ꢀglucopyranosyl)ꢀβꢀ ꢀgalactopyranosyl]ꢀ6ꢀOꢀbenzylꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranoside (14).
Dꢀglucopyranosyl)ꢀβꢀDꢀgalactopyranosyl]ꢀ6ꢀOꢀ
D
D
D
D
Method A: Trisaccharide 12 (514.9 mg, 0.394 mmol) was dissolved in a mixture of pyridine and
EtOH (2:1, 21 mL), thiourea (36 mg, 0.473 mmol, 1.2 equiv) was added and the solution was
heated to 55 °C for 7 h, then allowed to come to rt. The reaction mixture was diluted with CHCl3
(100 mL) and washed with HCl 2 N (1 × 100 mL). The aq layer was reꢀextracted with CHCl3 (5
× 25 mL) and the combined organic layers were dried and concentrated. Reversedꢀphase HPLC
of the residue (CH3CN/H2O, 40:60 to 95:5, 30 minutes) gave trisaccharide 13 (178.8 mg, 36%)
pure as a white amorphous foam and trisaccharide 14 (33 mg, 7%) pure as a slightly yellowish
amorphous foam. Analytical data for diol 14 were reported previously.13
Method B: Trisaccharide 12 (500 mg, 0.382 mmol) was dissolved in a mixture of pyridine and
EtOH (1:1, 40 mL), thiourea (291 mg, 3.823 mmol, 10 equiv) was added and the solution was
heated to 70 °C for 19 h, then allowed to come to rt. The reaction mixture was diluted with
CHCl3 (150 mL) and washed with HCl 2 N (1 × 150 mL). The aq layer was reꢀextracted with
CHCl3 (5 × 20 mL) and the combined organic layers were dried and concentrated. The product
was purified by column chromatography (CH2Cl2/MeOH, 20:1) to give diol 14 (338.2 mg, 77%)
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pure as a slightly yellowish amorphous foam. Analytical data for diol 14 were reported
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previously.13
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Analytical data for 13: [α]D –7.2 (c 1.0, CH2Cl2). 1H NMR (CDCl3, 600 MHz, 296 K): δH 7.46–
7.42 (m, 2 H, Ar), 7.39–7.28 (m, 8 H, Ar), 7.20 (d, J = 7.2 Hz, 1 H, NHA’), 5.74 (d, J = 9.0 Hz,
1 H, NHA), 5.51 (s, 1 H, >CHPh), 5.26 (d, J = 3.4 Hz, 1 H, Hꢀ4B), 5.11 (t, J = 9.1 Hz, 1 H, Hꢀ
3A), 5.06 (d, J = 8.0 Hz, 1 H, Hꢀ1A’), 4.92 (dd, J = 8.2, 9.8 Hz, 1 H, Hꢀ2B), 4.70 (d, J = 12.1
Hz, 1 H, CHHPh), 4.44 (d, J = 7.7 Hz, 1 H, Hꢀ1A), 4.39 (d, J = 12.1 Hz, 1 H, CHHPh), 4.34–
4.28 (m, 2 H, Hꢀ3A’, H6Aa’), 4.20 (d, J = 8.0 Hz, 1 H, Hꢀ1B), 4.12–4.01 (m, 3 H, Hꢀ6Ba,
COCH2Cl), 3.92 (dd, J = 8.1, 11.5 Hz, 1 H, Hꢀ6Bb), 3.87–3.80 (m, 2 H, Hꢀ2A, Hꢀ4A), 3.77 (m, 1
H, OCHHCH2), 3.70 (t, J = 9.9 Hz, 1 H, Hꢀ6Ab’), 3.66 (m, 2 H, Hꢀ6Aab), 3.61 (dd, J = 3.4,
10.0 Hz, 1 H, Hꢀ3B), 3.54–3.40 (m, 6 H, Hꢀ5A, Hꢀ5B, Hꢀ4A’, Hꢀ5A’, CH2Cl), 3.37–3.28 (m, 2
H, Hꢀ2A’, OCHHCH2), 3.12 (br s, 1 H, OHꢀ3A’), 2.03, 2.02 (2 s, 6 H, 2 OCOCH3), 1.91 (s, 3 H,
NHCOCH3), 1.72 (m, 2 H, O(CH2)4CH2CH2Cl), 1.52 (m, 2 H, OCH2CH2(CH2)3CH2Cl), 1.40 (m,
2 H, O(CH2)3CH2(CH2)2Cl), 1.30 (m, 2 H, O(CH2)2CH2(CH2)3Cl), 1.20 (s, 9 H, C(CH3)3). 13C
NMR (125 MHz, CDCl3, 296 K): δC 178.0, 170.2, 170.0, 169.2, 167.4, 162.2 (C=O), 137.8,
136.9 (quat Ar), 129.3, 128.6, 128.3, 128.1, 126.2 (Ar), 101.8 (>CHPh), 100.8 (Cꢀ1A), 100.1 (Cꢀ
1B), 99.5 (Cꢀ1A’), 92.4 (CCl3), 81.3 (Cꢀ4A’), 76.4 (Cꢀ3B), 74.3 (Cꢀ5A), 74.1 (Cꢀ3A), 73.9 (Cꢀ
4A), 73.6 (CH2Ph), 71.1 (Cꢀ5B), 70.6 (Cꢀ2B), 69.4 (OCH2CH2), 68.9 (Cꢀ4B), 68.6 (Cꢀ3A’),
68.4 (Cꢀ6A’), 67.3 (Cꢀ6A), 66.0 (Cꢀ5A’), 61.6 (Cꢀ6B), 59.8 (Cꢀ2A’), 53.4 (Cꢀ2A), 45.0 (CH2Cl),
40.8 (COCH2Cl ), 38.8 (C(CH3)3), 32.4 (O(CH2)4CH2CH2Cl), 29.2 (OCH2CH2(CH2)3CH2Cl),
27.1 (C(CH3)3), 26.5 (O(CH2)3CH2(CH2)2Cl), 25.1 (O(CH2)2CH2(CH2)3Cl), 23.3 (NHCOCH3),
21.1, 20.7 (OCOCH3). HRESIMS(m/z): [M + H]+ calcd for C53H70Cl5N2O20 1229.2965, found
1229.2960.
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6ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ[2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀpivaloylꢀ3ꢀOꢀ(4,6ꢀOꢀ
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benzylideneꢀ3ꢀOꢀchloroacetylꢀ2ꢀdeoxyꢀ2ꢀtrichloroacetamidoꢀβꢀ
D
ꢀglucopyranosyl)ꢀβꢀ
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ꢀ
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galactopyranosyl]ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀ ꢀfucopyranosyl)ꢀ2ꢀdeoxyꢀβꢀ
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ꢀ
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glucopyranoside (15). Chloroacetyl chloride (111 µL, 1.394 mmol, 1 equiv) was slowly added
at 0 °C to a stirred solution of the trisaccharide 14 (1.61 g, 1.394 mmol) in anhyd CH2Cl2 (280
mL) containing dry pyridine (225 µL, 2.79 mmol, 2 equiv) under N2. The reaction was left at 0
°C for 2 h, then washed sequentially with HCl 2 N (1 × 100 mL) and saturated aq NaHCO3 (1 ×
100 mL). The aq layers were reꢀextracted with CH2Cl2 (2 × 50 mL) and the combined organic
layers were dried and concentrated to give the previously described13 selectively
chloroacetylated trisaccharide (1.72 g, quant.) as a slightly yellowish amorphous foam. The
trisaccharide (1.72 g, 1.397 mmol) and known11 thioglycoside donor 8 (2.00 g, 4.191 mmol, 3
equiv) in dry CH2Cl2 (170 mL) containing freshly activated molecular sieves 4 Å (17 g) were
stirred under N2 for 3 h at rt. MeOTf (790 µL, 6.984 mmol, 5 equiv) was added to the reaction
mixture, which was stirred at rt for 30 min, then quenched with NEt3 (1.17 mL, 8.381 mmol, 6
equiv) and filtered over Celite®. The solids were washed with CH2Cl2 (4 × 50 mL) and the
combined filtrate and washings were washed with saturated aq NaHCO3 (1 × 300 mL). The aq
layer was reꢀextracted with CH2Cl2 (2 × 50 mL) and the combined organic layers were dried and
concentrated. The resulting residue was dissolved in a mixture of Ac2O and AcOH (3:1, 100 mL)
and the solution was stirred at rt for 75 min, then coꢀconcentrated with toluene (3 × 80 mL). The
product was purified by column chromatography (EtOAc/hexanes, 4:6 then 1:1) to give
tetrasaccharide 15 (1.94 g, 84%) pure as white amorphous glass. [α]D –55.2 (c 1.0, CH2Cl2). 1H
NMR (CDCl3, 600 MHz, 295 K): δH 7.43–7.39 (m, 2 H, Ar), 7.37–7.20 (m, 23 H, Ar), 6.85 (d, J
= 8.8 Hz, 1 H, NHA’), 5.92 (d, J = 7.6 Hz, 1 H, NHA), 5.50 (s, 1 H, >CHPh), 5.46 (t, J = 10.3
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Hz, 1 H, Hꢀ3A’), 5.24 (d, J = 3.1 Hz, 1 H, Hꢀ4B), 5.07 (d, J = 3.1 Hz, 1 H, Hꢀ1C), 4.98–4.91 (m,
2 H, Hꢀ2B, CHHPh), 4.85–4.77 (m, 3 H, Hꢀ1A, 2 CHHPh), 4.74–4.68 (m, 3 H, Hꢀ1A’, 2
CHHPH), 4.65 (d, J = 11.8 Hz, 1 H, CHHPh), 4.62 (d, J = 12.0 Hz, 1 H, CHHPh), 4.42–4.38 (m,
2 H, Hꢀ1B, CHHPh), 4.33 (dd, J = 4.9, 10.4 Hz, 1 H, Hꢀ6Aa’), 4.23 (m, 1 H, Hꢀ5C), 4.11–3.98
(m, 5 H, Hꢀ3A, Hꢀ6Ba, Hꢀ2C, COCH2Cl), 3.94 (dd, J = 6.8, 11.0 Hz, 1 H, Hꢀ6Bb), 3.89ꢀ3.72
(m, 7 H, Hꢀ4A, Hꢀ6Aab, Hꢀ3C, Hꢀ2A’, Hꢀ4A’, Hꢀ6Ab’), 3.68 (m, 1 H, OCHHCH2), 3.64 (dd, J
= 3.5, 10.0 Hz, 1 H, Hꢀ3B), 3.61–3.56 (m, 2 H, Hꢀ5B, Hꢀ4C), 3.55–3.48 (m, 3 H, Hꢀ2A, Hꢀ5A,
Hꢀ5A’), 3.45 (t, J = 6.7 Hz, 2 H, CH2Cl), 3.35 (m, 1 H, OCHHCH2), 2.02, 1.97 (2 s, 6 H, 2
OCOCH3), 1.76 (s, 3 H, NHCOCH3), 1.67 (m, 2 H, O(CH2)4CH2CH2Cl), 1.45 (m, 2 H,
OCH2CH2(CH2)3CH2Cl), 1.33 (m, 2 H, O(CH2)3CH2(CH2)2Cl), 1.23 (m, 2 H,
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O(CH2)2CH2(CH2)3Cl), 1.18 (s, 9 H, C(CH3)3), 1.11 (d, J = 6.4 Hz, 3 H, Hꢀ6C). 13C NMR (125
MHz, CDCl3, 295 K): δC 177.7, 170.1, 169.7, 169.1, 167.4, 162.1 (C=O), 138.9, 138.5, 138.1,
136.5 (quat Ar), 129.3, 128.4, 128.3, 128.2, 127.8, 127.6, 127.5, 127.3, 127.0, 126.0 (Ar), 101.4
(>CHPh), 100.1 (Cꢀ1A’), 99.5 (Cꢀ1A), 99.3 (Cꢀ1B), 96.8 (Cꢀ1C), 92.0 (CCl3), 79.8 (Cꢀ3C), 78.1
(Cꢀ4A’), 77.2 (Cꢀ4C), 76.6 (Cꢀ2C), 75.1 (Cꢀ3B), 74.4 (CH2Ph), 74.2 (Cꢀ5A), 73.7 (Cꢀ4A), 73.5
(CH2Ph) 73.2 (CH2Ph), 72.9 (Cꢀ3A), 72.6 (CH2Ph), 71.9 (Cꢀ3A’), 70.9 (Cꢀ2B), 70.7 (Cꢀ5B),
69.3 (OCH2CH2), 68.7 (Cꢀ6A), 68.6 (Cꢀ4B), 68.2 (Cꢀ6A’), 66.5 (Cꢀ5C), 66.3 (Cꢀ5A’), 61.0 (Cꢀ
6B), 56.6 (Cꢀ2A’), 54.6 (Cꢀ2A), 45.0 (CH2Cl), 40.4 (COCH2Cl), 38.7 (C(CH3)3), 32.5
(O(CH2)4CH2CH2Cl), 29.2 (OCH2CH2(CH2)3CH2Cl), 27.1 (C(CH3)3), 26.5
(O(CH2)3CH2(CH2)2Cl), 25.2 (O(CH2)2CH2(CH2)3Cl), 23.1 (NHCOCH3), 21.1, 20.7 (OCOCH3),
16.6 (Cꢀ6C). HRESIMS (m/z): [M + H]+ calcd for C80H98Cl5N2O24 1645.495, found 1645.490.
6ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ[2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀpivaloylꢀ3ꢀOꢀ(4,6ꢀOꢀ
benzylideneꢀ2ꢀdeoxyꢀ2ꢀtrichloroacetamidoꢀβꢀ
D
ꢀglucopyranosyl)ꢀβꢀ
D
ꢀgalactopyranosyl]ꢀ6ꢀOꢀ
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benzylꢀ3ꢀOꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀLꢀfucopyranosyl)ꢀ2ꢀdeoxyꢀβꢀDꢀglucopyranoside (16).
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Tetrasaccharide 15 (1.94 g, 1.177 mmol) was dissolved in a mixture of pyridine and EtOH (1:1,
100 mL), thiourea (896 mg, 11.77 mmol, 10 equiv) was added and the solution was heated to 70
°C for 7 h, then allowed to come to rt. The reaction mixture was diluted with CHCl3 (200 mL)
and washed with HCl 2 N (1 × 300 mL). The aq layer was reꢀextracted with CHCl3 (5 × 60 mL)
and the combined organic layers were dried and concentrated. The product was purified by
column chromatography (CH2Cl2/MeOH, 20:1) to give the previously described13 alcohol 16
(1.62 g, 88%) pure as a white amorphous foam.
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6ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ{2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀpivaloylꢀ3ꢀOꢀ[4,6ꢀOꢀ
benzylideneꢀ3ꢀOꢀ(2,3,4,6ꢀtetraꢀOꢀacetylꢀβꢀ
trichloroacetamidoꢀβꢀ ꢀglucopyranosyl]ꢀβꢀ
Oꢀbenzylꢀαꢀ ꢀfucopyranosyl)ꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranoside (17). Freshly distilled BF3·OEt2
D
ꢀgalactopyranosyl)ꢀ2ꢀdeoxyꢀ2ꢀ
D
Dꢀgalactopyranosyl}ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀ(2,3,4ꢀtriꢀ
L
D
(16 µL, 0.1273 mmol, 2 equiv) was added to a stirred solution of alcohol 16 (100 mg, 0.06365
mmol) and known12 galactosyl trichloroacetimidate 9 (156.8 mg, 0.3182 mmol, 5 equiv) in dry
CH2Cl2 (5 mL) at rt under N2. The reaction was left to proceed for 1 h at rt, then quenched with
NEt3 (21.2 µL, 0.1528 mmol, 2.4 equiv). The solvent was evaporated and a column
chromatography (EtOAc/hexanes, 1:1 then 7:3) followed by a reverseꢀphase HPLC
(CH3CN/H2O, 75:25 to 85:15, 30 min) of the residue gave recovered glycosyl acceptor 16 (20.1
mg, 20%) and pentasaccharide 17 (62.5 mg, 52%) pure as an amorphous colorless glass. [α]D –
29.0 (c 1.0, CH2Cl2). 1H NMR (CDCl3, 600 MHz, 295 K): δH 7.47–7.43 (m, 2 H, Ar), 7.38–7.22
(m, 23 H, Ar), 6.98 (d, J = 7.0 Hz, 1 H, NHA’), 5.90 (d, J = 7.6 Hz, 1 H, NHA), 5.53 (s, 1 H,
>CHPh), 5.29 (d, J = 2.8 Hz, 1 H, Hꢀ4B’), 5.26 (d, J = 3.4 Hz, 1 H, Hꢀ4B), 5.19 (d, J = 7.8 Hz, 1
H, Hꢀ1A’), 5.12 (dd, J = 7.9, 10.3 Hz, 1 H, Hꢀ2B’), 5.06 (d, J = 3.7 Hz, 1 H, Hꢀ1C), 4.96–4.92
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(m, 2 H, Hꢀ2B, CHHPh), 4.90 (dd, J = 3.4, 10.4 Hz, 1 H, Hꢀ3B’), 4.83 (d, J = 11.9 Hz, 1 H,
CHHPh), 4.82–4.78 (m, 2 H, Hꢀ1A, CHHPh), 4.75–4.68 (m, 3 H, Hꢀ1B’, 2 CHHPH), 4.66 (d, J
= 11.9 Hz, 1 H, CHHPh), 4.60 (d, J = 12.1 Hz, 1 H, CHHPh), 4.52 (t, J = 9.7 Hz, 1 H, Hꢀ3A’),
4.40–4.36 (m, 2 H, Hꢀ1B, CHHPh), 4.33 (dd, J = 4.9, 10.6 Hz, 1 H, Hꢀ6Aa’), 4.23 (m, 1 H, Hꢀ
5C), 4.11ꢀ4.05 (m, 3 H, Hꢀ3A, Hꢀ2C, Hꢀ6Ba’), 4.03 (dd, J = 7.0, 11.3 Hz, 1 H, Hꢀ6Ba), 3.97
(dd, J = 6.9, 11.3 Hz, 1 H, Hꢀ6Bb’), 3.91 (dd, J = 6.8, 11.1 Hz, 1 H, Hꢀ6Bb), 3.86 (dd, J = 2.5,
10.1 Hz, 1 H, Hꢀ3C), 3.84–3.80 (m, 2 H, Hꢀ4A, Hꢀ6Aa), 3.79–3.64 (m, 6 H, Hꢀ3B, Hꢀ6Ab, Hꢀ
4A’, Hꢀ6Ab’, Hꢀ5B’, OCHHCH2), 3.57 (s, 1 H, Hꢀ4C), 3.55–3.44 (m, 6 H, Hꢀ2A, Hꢀ5A, Hꢀ5B,
Hꢀ5A’, CH2Cl), 3.35 (m, 1 H, OCHHCH2), 3.24 (m, 1 H, Hꢀ2A’), 2.09, 2.02, 1.98, 1.93, 1.92,
1.76 (6 s, 18 H, 6 OCOCH3), 1.74 (s, 3 H, NHCOCH3), 1.67 (m, 2 H, O(CH2)4CH2CH2Cl), 1.45
(m, 2 H, OCH2CH2(CH2)3CH2Cl), 1.33 (m, 2 H, O(CH2)3CH2(CH2)2Cl), 1.23 (m, 2 H,
O(CH2)2CH2(CH2)3Cl), 1.18 (s, 9 H, C(CH3)3), 1.11 (d, J = 6.5 Hz, 3 H, Hꢀ6C). 13C NMR (125
MHz, CDCl3, 295 K): δC 177.6, 170.3, 170.1, 169.4, 161.9 (C=O), 139.0, 138.9, 138.6, 138.1,
136.8 (quat Ar), 129.4, 128.5, 128.4, 128.3, 128.2, 127.8, 127.6, 127.5, 127.4, 127.1, 126.2 (Ar),
101.6 (>CHPh), 99.5 (Cꢀ1A, Cꢀ1B), 99.0 (Cꢀ1A’), 98.5 (Cꢀ1B’), 96.9 (Cꢀ1C), 92.2 (CCl3), 79.8
(Cꢀ3C), 78.1 (Cꢀ4A’), 77.1 (Cꢀ4C), 76.6 (Cꢀ2C), 75.9 (Cꢀ3B), 75.0 (Cꢀ3A’), 74.4 (CH2Ph), 74.1
(Cꢀ5A), 73.7 (Cꢀ4A), 73.4 (CH2Ph) 73.3 (CH2Ph), 72.9 (Cꢀ3A), 72.6 (CH2Ph), 70.9 (Cꢀ5B, Cꢀ
3B’), 70.7 (Cꢀ5B’), 70.4 (Cꢀ2B), 69.3 (OCH2CH2), 69.1 (Cꢀ4B), 68.8 (Cꢀ2B’), 68.7 (Cꢀ6A), 68.4
(Cꢀ6A’), 66.8 (Cꢀ4B’), 66.5 (Cꢀ5C), 66.2 (Cꢀ5A’), 61.4 (Cꢀ6B’), 60.9 (Cꢀ6B), 59.1 (Cꢀ2A’), 54.7
(Cꢀ2A), 45.0 (CH2Cl), 38.7 (C(CH3)3), 32.5 (O(CH2)4CH2CH2Cl), 29.2
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(OCH2CH2(CH2)3CH2Cl), 27.1 (C(CH3)3), 26.6 (O(CH2)3CH2(CH2)2Cl), 25.2
(O(CH2)2CH2(CH2)3Cl), 23.1 (NHCOCH3), 21.2, 20.8, 20.7, 20.6, 20.5 (OCOCH3), 16.7 (Cꢀ6C).
HRESIMS (m/z): [M + NH4]+ calcd for C92H118Cl4N3O32 1916.6453, found 1916.6445.
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6ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ{2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀpivaloylꢀ3ꢀOꢀ[6ꢀOꢀbenzylꢀ3ꢀOꢀ
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(2,3,4,6ꢀtetraꢀOꢀacetylꢀβꢀ
glucopyranosyl]ꢀβꢀ ꢀgalactopyranosyl}ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀ
fucopyranosyl)ꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranoside (18). A solution of the benzylidene acetal 17
Dꢀgalactopyranosyl)ꢀ2ꢀdeoxyꢀ2ꢀtrichloroacetamidoꢀβꢀDꢀ
D
Lꢀ
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D
(117 mg, 0.06150 mmol) in dry THF (6 mL) containing freshly activated molecular sieves 3 Å
(900 mg), NaCNBH3 (58.0 mg, 0.9225 mmol, 15 equiv) and methyl orange indicator (2 mg) was
stirred under N2 for 1 h at rt and cooled to 0 °C. A 2 M solution of HCl in Et2O (461 µL, 0.9225
mmol, 15 equiv) was added drop wise to the reaction mixture at 0 °C until the methyl orange
indicator turned pink, remained as such for 10 min, and H2(g) was no longer generated. The
reaction was stirred at rt for 2.5 h, more NaCNBH3 (58.0 mg, 0.9225 mmol, 15 equiv) and 2 M
solution of HCl in Et2O (461 µL, 0.9225 mmol, 15 equiv) were added and the reaction was left to
proceed at rt for an additional hour, then was filtered over Celite®. The solids were washed with
THF (3 × 15 mL) and the combined filtrate and washings were concentrated. The residue was
dissolved in CH2Cl2 (30 mL) and washed with saturated aq NaHCO3 (1 × 30 mL). The aq layer
was reꢀextracted with CH2Cl2 (3 × 10 mL) and the combined organic layers were dried and
concentrated. Column chromatography (CH2Cl2/MeOH, 30:1) followed by RP HPLC
(CH3CN/H2O, 75:25 to 85:15, 30 min) of the residue gave alcohol 18 (73.0 mg, 62%) pure as an
amorphous white foam. [α]D –24.2 (c 1.0, CH2Cl2). 1H NMR (CDCl3, 600 MHz, 295 K): δH
7.36–7.20 (m, 25 H, Ar), 6.86 (d, J = 7.2 Hz, 1 H, NHA’), 5.89 (d, J = 7.4 Hz, 1 H, NHA), 5.35
(d, J = 3.2 Hz, 1 H, Hꢀ4B’), 5.33 (d, J = 3.5 Hz, 1 H, Hꢀ4B), 5.20 (dd, J = 8.0, 10.5 Hz, 1 H, Hꢀ
2B’), 5.04 (d, J = 3.7 Hz, 1 H, Hꢀ1C), 4.97–4.83 (m, 6 H, Hꢀ1A, Hꢀ2B, Hꢀ1A’, Hꢀ3B’, 2
CHHPh), 4.79 (d, J = 11.8 Hz, 1 H, CHHPh), 4.71 (d, J = 11.8 Hz, 1 H, CHHPh), 4.67 (d, J =
11.9 Hz, 1 H, CHHPh), 4.64 (d, J = 12.5 Hz, 1 H, CHHPh),4.62–4.57 (m, 3 H, CH2Ph, CHHPh),
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4.51 (d, J = 8.0 Hz, 1 H, Hꢀ1B’), 4 34 (d, J = 12.1 Hz, 1 H, CHHPh), 4.30–4.24 (m, 2 H, Hꢀ1B,
Hꢀ5C), 4.18 (dd, J = 7.6, 10.0 Hz, 1 H, Hꢀ3A’), 4.12–4.03 (m, 4 H, Hꢀ3A, Hꢀ2C, Hꢀ6Bab’), 4.01
(dd, J = 5.9, 11.3 Hz, 1 H, Hꢀ6Ba), 3.95 (t, J = 6.5 Hz, 1 H, Hꢀ5B’), 3.90–3.79 (m, 3 H, Hꢀ6Bb,
Hꢀ3C, Hꢀ6Aa’), 3.81–3.76 (m, 2 H, Hꢀ4A, Hꢀ6Aa), 3.75–3.63 (m, 5 H, Hꢀ3B, Hꢀ6Ab, OHꢀ4A’,
Hꢀ6Ab’, OCHHCH2), 3.58 (s, 1 H, Hꢀ4C), 3.53–3.43 (m, 6 H, Hꢀ5A, Hꢀ5B, Hꢀ4A’, Hꢀ5A’,
CH2Cl), 3.41–3.32 (m, 2 H, Hꢀ2A, OCHHCH2), 3.21 (m, 1 H, Hꢀ2A’), 2.14, 2.06, 2.02, 2.00,
1.95, 1.89 (6 s, 18 H, 6 OCOCH3), 1.72 (s, 3 H, NHCOCH3), 1.67 (m, 2 H, O(CH2)4CH2CH2Cl),
1.46 (m, 2 H, OCH2CH2(CH2)3CH2Cl), 1.33 (m, 2 H, O(CH2)3CH2(CH2)2Cl), 1.23 (m, 2 H,
O(CH2)2CH2(CH2)3Cl), 1.17 (s, 9 H, C(CH3)3), 1.11 (d, J = 6.4 Hz, 3 H, Hꢀ6C). 13C NMR (125
MHz, CDCl3, 295 K): δC 177.6, 170.4, 170.1, 170.0, 169.5, 169.4, 161.8 (C=O), 139.0, 138.9,
138.6, 138.2, 137.8 (quat Ar), 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.6, 127.5, 127.4,
127.3 (Ar), 100.7 (Cꢀ1B’), 99.5 (Cꢀ1A), 99.3 (Cꢀ1B), 98.3 (Cꢀ1A’), 97.0 (Cꢀ1C), 92.1 (CCl3),
81.4 (Cꢀ3A’), 79.9 (Cꢀ3C), 77.1 (Cꢀ4C), 76.6 (Cꢀ2C), 75.4 (Cꢀ5A’), 75.1 (Cꢀ3B), 74.4 (CH2Ph),
74.1 (Cꢀ5A), 73.6 (Cꢀ4A), 73.5 (CH2Ph) 73.4 (CH2Ph), 73.3 (CH2Ph), 73.0 (Cꢀ3A), 72.5
(CH2Ph), 71.1 (Cꢀ5B, Cꢀ5B’), 70.9 (Cꢀ2B), 70.7 (Cꢀ3B’), 69.7 (Cꢀ6A’), 69.4 (Cꢀ4B), 69.3
(OCH2CH2), 69.2 (Cꢀ4A’), 68.5 (Cꢀ6A, Cꢀ2B’), 66.7 (Cꢀ4B’), 66.4 (Cꢀ5C), 61.6 (Cꢀ6B), 61.2
(Cꢀ6B’), 58.5 (Cꢀ2A’), 55.1 (Cꢀ2A), 45.0 (CH2Cl), 38.7 (C(CH3)3), 32.5 (O(CH2)4CH2CH2Cl),
29.2 (OCH2CH2(CH2)3CH2Cl), 27.1 (C(CH3)3), 26.6 (O(CH2)3CH2(CH2)2Cl), 25.2
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(O(CH2)2CH2(CH2)3Cl), 23.1 (NHCOCH3), 21.3, 20.9, 20.8, 20.6, 20.5 (OCOCH3), 16.7 (Cꢀ6C).
HRESIMS (m/z): [M + H]+ calcd for C92H117Cl4N2O32 1901.634, found 1901.628.
6ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ{2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀpivaloylꢀ3ꢀOꢀ[6ꢀOꢀbenzylꢀ3ꢀOꢀ
(2,3,4,6ꢀtetraꢀOꢀacetylꢀβꢀ
D
ꢀgalactopyranosyl)ꢀ4ꢀOꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀ
Lꢀfucopyranosyl)ꢀ2ꢀ
deoxyꢀ2ꢀtrichloroacetamidoꢀβꢀ
D
ꢀglucopyranosyl]ꢀβꢀ ꢀgalactopyranosyl}ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀ
D
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(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀLꢀfucopyranosyl)ꢀ2ꢀdeoxyꢀβꢀDꢀglucopyranoside (19). A solution of the
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glycosyl acceptor 18 (32.6 mg, 0.01712 mmol) and known11 thioglycoside donor 8 (40.9 mg,
0.08559 mmol, 5 equiv) in dry CH2Cl2 (2 mL) containing freshly activated molecular sieves 4 Å
(100 mg) was stirred under N2 for 1 h 30 min at rt. MeOTf (9.7 µL, 0.08559 mmol, 5 equiv) was
added to the reaction mixture, which was stirred at rt for 40 min, then quenched with NEt3 (14.3
µL, 0.1027 mmol, 6 equiv) and filtered over Celite®. The solids were washed with CH2Cl2 (3 ×
10 mL) and the combined filtrate and washings were washed with saturated aq NaHCO3 (1 × 15
mL). The aq layer was reꢀextracted with CH2Cl2 (2 × 10 mL) and the combined organic layers
were dried and concentrated. The resulting residue was dissolved in a mixture of Ac2O and
AcOH (3:1, 2 mL) and the solution was stirred at rt for 16 h, then coꢀconcentrated with toluene
(3 × 3 mL). Column chromatography (EtOAc/hexanes, 4:6 then 1:1) followed by RP HPLC
(CH3CN/H2O, 90:10 to 95:5, 30 min) of the residue gave hexasaccharide 19 (30.1 mg, 76%) pure
as an amorphous white foam. [α]D –46.0 (c 1.0, CH2Cl2). 1H NMR (CDCl3, 600 MHz, 295 K): δH
7.41–7.39 (m, 2 H, Ar), 7.36–7.20 (m, 38 H, Ar), 6.72 (d, J = 8.2 Hz, 1 H, NHA’), 5.92 (d, J =
7.4 Hz, 1 H, NHA), 5.32 (d, J = 3.2 Hz, 1 H, Hꢀ4B’), 5.20 (d, J = 3.6 Hz, 1 H, Hꢀ4B), 5.08 (dd, J
= 8.2, 10.4 Hz, 1 H, Hꢀ2B’), 5.06 (d, J = 3.7 Hz, 1 H, Hꢀ1C), 5.03 (d, J = 3.7 Hz, 1 H, Hꢀ1C’),
4.97–4.90 (m, 3 H, Hꢀ2B, 2 CHHPh), 4.87–4.77 (m, 6 H, Hꢀ1A, Hꢀ3B’, 4 CHHPh), 4.76–4.65
(m, 6 H, Hꢀ1A’, Hꢀ1B’, 4 CHHPh), 4.66–4.62 (m, 2 H, 2 CHHPh), 4.59 (d, J = 12.1 Hz, 1 H,
CHHPh), 4.46 (m, 1 H, Hꢀ5C’), 4.41 (m, 2 H, CH2Ph), 4 35 (d, J = 12.1 Hz, 1 H, CHHPh), 4.29
(d, J = 8.0 Hz, 1 H, Hꢀ1B), 4.22 (m, 1 H, Hꢀ5C), 4.14–3.98 (m, 7 H, Hꢀ3A, Hꢀ6Ba, Hꢀ2C, Hꢀ
3A’, Hꢀ6Bab’, Hꢀ2C’), 3.96 (t, J = 7.9 Hz, 1 H, Hꢀ4A’), 3.90–3.85 (m, 3 H, Hꢀ6Bb, Hꢀ3C, Hꢀ
3C’), 3.83–3.78 (m, 3 H, Hꢀ4A, Hꢀ6Aa, Hꢀ5B’), 3.77 (dd, J = 4.0, 10.7 Hz, 1 H, Hꢀ6Aa’), 3.74–
3.62 (m, 5 H, Hꢀ3B, Hꢀ6Ab, Hꢀ6Ab’, Hꢀ4C’, OCHHCH2), 3.60–3.52 (m, 3 H, Hꢀ4C, Hꢀ2A’, Hꢀ
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5A’), 3.50 (m, 1 H, Hꢀ5A), 3.48–3.43 (m, 4 H, Hꢀ2A, Hꢀ5B, CH2Cl), 3.35 (m, 1 H, OCHHCH2),
2.04, 1.99, 1.92, 1.87, 1.85 (6 s, 18 H, 6 OCOCH3), 1.74 (s, 3 H, NHCOCH3), 1.67 (m, 2 H,
O(CH2)4CH2CH2Cl), 1.45 (m, 2 H, OCH2CH2(CH2)3CH2Cl), 1.33 (m, 2 H,
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O(CH2)3CH2(CH2)2Cl), 1.26–1.21 (m, 5 H, Hꢀ6C’, O(CH2)2CH2(CH2)3Cl), 1.16 (s, 9 H,
C(CH3)3), 1.09 (d, J = 6.4 Hz, 3 H, Hꢀ6C). 13C NMR (125 MHz, CDCl3, 295 K): δC 177.7, 170.1,
169.9, 169.8, 169.7, 169.6, 169.4, 161.3 (C=O), 139.0, 138.9, 138.5, 138.2, 138.1, 137.9 (quat
Ar), 128.5, 128.4, 128.3, 128.2, 127.9, 127.7, 127.6, 127.5, 127.4, 127.3, 127.0 (Ar), 100.0 (Cꢀ
1B’), 99.5 (Cꢀ1A), 99.2 (Cꢀ1B), 98.6 (Cꢀ1A’), 97.3 (Cꢀ1C’), 96.8 (Cꢀ1C), 92.3 (CCl3), 80.5 (Cꢀ
3C’), 79.8 (Cꢀ3C), 77.2 (Cꢀ4C), 76.6 (Cꢀ2C), 76.4 (Cꢀ3A’, Cꢀ4C’), 76.6 (Cꢀ5A’, Cꢀ2C’), 74.7
(CH2Ph), 74.5 (Cꢀ3B), 74.5 (CH2Ph), 74.4 (CH2Ph), 74.0 (Cꢀ5A), 73.6 (Cꢀ4A), 73.5 (CH2Ph),
73.2 (CH2Ph), 72.9 (CH2Ph), 72.8 (Cꢀ3A), 72.6 (CH2Ph), 72.2 (CH2Ph), 71.8 (Cꢀ4A’), 71.5 (Cꢀ
2B), 71.2 (Cꢀ5B), 70.8 (Cꢀ3B’), 70.5 (Cꢀ5B’), 69.3 (OCH2CH2), 68.7 (Cꢀ6A), 68.6 (Cꢀ4B), 68.1
(Cꢀ2B’), 67.8 (Cꢀ6A’), 66.7 (Cꢀ4B’), 66.5 (Cꢀ5C, Cꢀ5C’), 61.6 (Cꢀ6B), 60.1 (Cꢀ6B’), 58.2 (Cꢀ
2A’), 54.5 (Cꢀ2A), 45.0 (CH2Cl), 38.7 (C(CH3)3), 32.5 (O(CH2)4CH2CH2Cl), 29.2
(OCH2CH2(CH2)3CH2Cl), 27.1 (C(CH3)3), 26.6 (O(CH2)3CH2(CH2)2Cl), 25.2
(O(CH2)2CH2(CH2)3Cl), 23.1 (NHCOCH3), 21.2, 20.7, 20.6, 20.5 (OCOCH3), 16.8 (Cꢀ6C’), 16.7
(Cꢀ6C). HRESIMS (m/z): [M + H]+ calcd for C119H145Cl4N2O36 2317.833, found 2317.827.
6ꢀChlorohexyl 2ꢀacetamidoꢀ4ꢀOꢀ{2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀpivaloylꢀ3ꢀOꢀ[6ꢀOꢀbenzylꢀ3ꢀOꢀ
(2,3,4,6ꢀtetraꢀOꢀacetylꢀβꢀ
acetamidoꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranosyl]ꢀβꢀ
Oꢀbenzylꢀαꢀ ꢀfucopyranosyl)ꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranoside (20). To a solution of
D
ꢀgalactopyranosyl)ꢀ4ꢀOꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀ
Lꢀfucopyranosyl)ꢀ2ꢀ
D
D
ꢀgalactopyranosyl}ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀ(2,3,4ꢀtriꢀ
L
D
hexasaccharide 19 (62.6 mg, 0.02698 mmol) dissolved in AcOH (3.0 mL) was added freshly
activated Zn (176 mg, 2.698 mmol, 100 equiv). The reaction mixture was heated to 50 °C under
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sonication for 4 h, more Zn (176 mg, 2.698 mmol, 100 equiv) was added and the reaction was
left to proceed at 50 °C under sonication for an additional 3 h, then filtered over Celite®. The
solids were washed with CH2Cl2 (2 × 50 mL) and the combined filtrate and washings were
washed with saturated aq NaHCO3 (1 × 60 mL). The aq layer was reꢀextracted with CH2Cl2 (3 ×
30 mL) and the combined organic layers were dried and concentrated to give Nꢀacetamide 20
(56.8 mg, 95%) pure as a white amorphous foam. [α]D –41.8 (c 1.0, CH2Cl2). 1H NMR (CDCl3,
600 MHz, 295 K): δH 7.44–7.41 (m, 2 H, Ar), 7.38–7.21 (m, 38 H, Ar), 5.79 (d, J = 7.1 Hz, 1 H,
NHA), 5.57 (d, J = 7.3 Hz, 1 H, NHA’), 5.33 (d, J = 3.2 Hz, 1 H, Hꢀ4B’), 5.25 (d, J = 3.6 Hz, 1
H, Hꢀ4B), 5.09–5.02 (m, 3 H, Hꢀ1C, Hꢀ2B’, Hꢀ1C’), 4.98–4.91 (m, 4 H, Hꢀ1A, 3 CHHPh),
4.90–4.84 (m, 3 H, Hꢀ2B, Hꢀ3B’, 1 CHHPh), 4.83–4.76 (m, 4 H, Hꢀ1A’, 3 CHHPh), 4.75–4.71
(m, 2 H, 2 CHHPh), 4.70–4.60 (m, 6 H, Hꢀ1B’, Hꢀ5C’, 4 CHHPh), 4.45 (d, J = 12.1 Hz, 1 H,
CHHPh), 4.41–4.33 (m, 4 H, Hꢀ5C, Hꢀ3A’, 2 CHHPh), 4.31 (d, J = 8.2 Hz, 1 H, Hꢀ1B), 4.17–
4.07 (m, 4 H, Hꢀ3A, Hꢀ2C, Hꢀ6Ba’, Hꢀ2C’), 4.05–3.98 (m, 2 H, Hꢀ6Ba, Hꢀ6Bb’), 3.94–3.90 (m,
2 H, Hꢀ3C, Hꢀ3C’), 3.89–3.79 (m, 5 H, Hꢀ4A, Hꢀ6Bb, Hꢀ4A’, Hꢀ6Aa’, Hꢀ5B’), 3.78–3.71 (m, 4
H, Hꢀ6Aab, Hꢀ4C’, OCHHCH2), 3.67 (m, 1 H, Hꢀ6Ab’), 3.61 (s, 1 H, Hꢀ4C), 3.57 (m, 1 H, Hꢀ
5A’), 3.50–3.42 (m, 5 H, Hꢀ5A, Hꢀ3B, Hꢀ5B, CH2Cl), 3.37 (m, 1 H, OCHHCH2), 3.25 (br s, 1 H,
Hꢀ2A), 3.06 (br s, 1 H, Hꢀ2A’), 2.06, 2.00, 1.94 (4 s, 12 H, 4 OCOCH3), 1.93 (s, 3 H,
NHCOCH3), 1.88, 1.80 (2 s, 6 H, 2 OCOCH3), 1.72–1.66 (m, 5 H, NHCOCH3,
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O(CH2)4CH2CH2Cl), 1.48 (m, 2 H, OCH2CH2(CH2)3CH2Cl), 1.36 (m, 2 H,
O(CH2)3CH2(CH2)2Cl), 1.29–1.22 (m, 5 H, Hꢀ6C’, O(CH2)2CH2(CH2)3Cl), 1.17 (s, 9 H,
C(CH3)3), 1.15 (d, J = 6.5 Hz, 3 H, Hꢀ6C). 13C NMR (125 MHz, CDCl3, 295 K): δC 177.7, 170.5,
170.1, 170.0, 169.8, 169.6, 169.4, 169.0 (C=O), 139.0, 138.8, 138.6, 138.5, 138.3, 137.8 (quat
Ar), 128.5, 128.4, 128.3, 128.2, 128.0, 127.8, 127.6, 127.5, 127.4, 127.3, 127.0 (Ar), 100.3 (Cꢀ
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1B’), 99.4 (Cꢀ1A’), 99.3 (Cꢀ1A), 99.2 (Cꢀ1B), 97.4 (Cꢀ1C’), 97.3 (Cꢀ1C), 80.6 (Cꢀ3C’), 80.0 (Cꢀ
3C), 77.1 (Cꢀ4C), 76.7 (Cꢀ2C), 76.3 (Cꢀ4C’), 75.9 (Cꢀ3B, Cꢀ3A’), 75.6 (Cꢀ2C’), 75.3 (Cꢀ5A’),
74.7 (CH2Ph), 74.3 (CH2Ph), 74.2 (Cꢀ5A), 74.0 (CH2Ph), 73.9 (Cꢀ4A), 73.6 (CH2Ph), 73.5
(CH2Ph), 73.3 (Cꢀ3A), 73.2 (CH2Ph), 72.4 (Cꢀ4A’, CH2Ph), 72.2 (CH2Ph), 71.2 (Cꢀ5B), 71.0 (Cꢀ
2B, Cꢀ3B’), 70.3 (Cꢀ5B’), 69.4 (Cꢀ4B, OCH2CH2), 68.5 (Cꢀ2B’), 68.1 (Cꢀ6A, Cꢀ6A’), 66.6 (Cꢀ
4B’), 66.2 (Cꢀ5C, Cꢀ5C’), 61.8 (Cꢀ6B), 60.0 (Cꢀ6B’), 57.8 (Cꢀ2A’), 56.4 (Cꢀ2A), 45.0 (CH2Cl),
38.7 (C(CH3)3), 32.5 (O(CH2)4CH2CH2Cl), 29.2 (OCH2CH2(CH2)3CH2Cl), 27.1 (C(CH3)3), 26.5
(O(CH2)3CH2(CH2)2Cl), 25.1 (O(CH2)2CH2(CH2)3Cl), 23.6, 23.1 (NHCOCH3), 21.0, 20.8, 20.6,
20.5 (OCOCH3), 16.8 (Cꢀ6C’), 16.7 (Cꢀ6C). HRESIMS (m/z): [M + NH4]+ calcd for
C119H151ClN3O36 2232.977, found 2232.968.
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6ꢀAzidohexyl 2ꢀacetamidoꢀ4ꢀOꢀ{2,4ꢀdiꢀOꢀacetylꢀ6ꢀOꢀpivaloylꢀ3ꢀOꢀ[6ꢀOꢀbenzylꢀ3ꢀOꢀ
(2,3,4,6ꢀtetraꢀOꢀacetylꢀβꢀ
acetamidoꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranosyl]ꢀβꢀ
Oꢀbenzylꢀαꢀ ꢀfucopyranosyl)ꢀ2ꢀdeoxyꢀβꢀ ꢀglucopyranoside (21). NaN3 (17.6 mg, 0.2706
D
ꢀgalactopyranosyl)ꢀ4ꢀOꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀ
Lꢀfucopyranosyl)ꢀ2ꢀ
D
D
ꢀgalactopyranosyl}ꢀ6ꢀOꢀbenzylꢀ3ꢀOꢀ(2,3,4ꢀtriꢀ
L
D
mmol, 10 equiv) was added to a solution of the hexasaccharide 20 (60 mg, 0.02706 mmol) in
DMF (6 mL) and the reaction mixture was heated to 80 °C for 20 h. The solvent was evaporated,
the residue was dissolved in CH2Cl2 (30 mL) and washed with water (2 × 30 mL). The aq layer
was reꢀextracted with CH2Cl2 (3 × 10 mL) and the combined organic layers were dried and
concentrated to give the azido 21 (60.2 mg, quant) pure as a white foam. [α]D –46.5 (c 1.0,
CH2Cl2). 1H NMR (CDCl3, 600 MHz, 295 K): δH 7.44–7.41 (m, 2 H, Ar), 7.38–7.21 (m, 38 H,
Ar), 5.79 (d, J = 7.2 Hz, 1 H, NHA), 5.56 (d, J = 7.3 Hz, 1 H, NHA’), 5.33 (d, J = 3.2 Hz, 1 H,
Hꢀ4B’), 5.25 (d, J = 3.6 Hz, 1 H, Hꢀ4B), 5.09–5.02 (m, 3 H, Hꢀ1C, Hꢀ2B’, Hꢀ1C’), 4.98–4.91
(m, 4 H, Hꢀ1A, 3 CHHPh), 4.90–4.84 (m, 3 H, Hꢀ2B, Hꢀ3B’, 1 CHHPh), 4.83–4.76 (m, 4 H, Hꢀ
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1A’, 3 CHHPh), 4.75–4.71 (m, 2 H, 2 CHHPh), 4.70–4.60 (m, 6 H, Hꢀ1B’, Hꢀ5C’, 4 CHHPh),
4.45 (d, J = 12.1 Hz, 1 H, CHHPh), 4.41–4.33 (m, 4 H, Hꢀ5C, Hꢀ3A’, 2 CHHPh), 4.31 (d, J = 8.2
Hz, 1 H, Hꢀ1B), 4.17–4.07 (m, 4 H, Hꢀ3A, Hꢀ2C, Hꢀ6Ba’, Hꢀ2C’), 4.05–3.98 (m, 2 H, Hꢀ6Ba, Hꢀ
6Bb’), 3.94–3.90 (m, 2 H, Hꢀ3C, Hꢀ3C’), 3.89–3.79 (m, 5 H, Hꢀ4A, Hꢀ6Bb, Hꢀ4A’, Hꢀ6Aa’, Hꢀ
5B’), 3.78–3.71 (m, 4 H, Hꢀ6Aab, Hꢀ4C’, OCHHCH2), 3.67 (m, 1 H, Hꢀ6Ab’), 3.61 (s, 1 H, Hꢀ
4C), 3.57 (m, 1 H, Hꢀ5A’), 3.50–3.42 (m, 3 H, Hꢀ5A, Hꢀ3B, Hꢀ5B), 3.37 (m, 1 H, OCHHCH2),
3.26 (br s, 1 H, Hꢀ2A), 3.20 (t, J = 6.9 Hz, 2 H, CH2N3), 3.06 (br s, 1 H, Hꢀ2A’), 2.06, 2.00, 1.94
(4 s, 12 H, 4 OCOCH3), 1.93 (s, 3 H, NHCOCH3), 1.88, 1.80 (2 s, 6 H, 2 OCOCH3), 1.68 (s, 3 H,
NHCOCH3), 1.55–1.44 (m, 4 H, O(CH2)4CH2CH2N3, OCH2CH2(CH2)3CH2N3), 1.31–1.21 (m, 7
H, Hꢀ6C’, O(CH2)3CH2(CH2)2N3, O(CH2)2CH2(CH2)3N3), 1.17 (s, 9 H, C(CH3)3), 1.15 (d, J =
6.4 Hz, 3 H, Hꢀ6C). 13C NMR (125 MHz, CDCl3, 295 K): δC 177.7, 170.5, 170.1, 170.0, 169.8,
169.6, 169.4, 169.0 (C=O), 139.0, 138.8, 138.6, 138.5, 138.3, 137.8 (quat Ar), 128.5, 128.4,
128.3, 128.2, 128.0, 127.7, 127.6, 127.5, 127.4, 127.3, 127.0 (Ar), 100.3 (Cꢀ1B’), 99.4 (Cꢀ1A’),
99.3 (Cꢀ1A), 99.2 (Cꢀ1B), 97.4 (Cꢀ1C’), 97.3 (Cꢀ1C), 80.6 (Cꢀ3C’), 80.0 (Cꢀ3C), 77.1 (Cꢀ4C),
76.7 (Cꢀ2C), 76.3 (Cꢀ4C’), 75.9 (Cꢀ3B, Cꢀ3A’), 75.6 (Cꢀ2C’), 75.3 (Cꢀ5A’), 74.7 (CH2Ph), 74.3
(CH2Ph), 74.2 (Cꢀ5A), 74.0 (CH2Ph), 73.9 (Cꢀ4A), 73.6 (CH2Ph), 73.5 (CH2Ph), 73.3 (Cꢀ3A),
73.2 (CH2Ph), 72.4 (Cꢀ4A’, CH2Ph), 72.2 (CH2Ph), 71.2 (Cꢀ5B), 71.0 (Cꢀ2B, Cꢀ3B’), 70.3 (Cꢀ
5B’), 69.4 (Cꢀ4B, OCH2CH2), 68.5 (Cꢀ2B’), 68.1 (Cꢀ6A, Cꢀ6A’), 66.6 (Cꢀ4B’), 66.2 (Cꢀ5C, Cꢀ
5C’), 61.8 (Cꢀ6B), 60.0 (Cꢀ6B’), 57.8 (Cꢀ2A’), 56.4 (Cꢀ2A), 51.3 (CH2N3), 38.7 (C(CH3)3), 29.2
(OCH2CH2(CH2)3CH2N3), 28.7 (O(CH2)4CH2CH2N3), 27.1 (C(CH3)3), 26.4
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(O(CH2)3CH2(CH2)2N3), 25.4 (O(CH2)2CH2(CH2)3N3), 23.5, 23.1 (NHCOCH3), 21.0, 20.8, 20.6,
20.5 (OCOCH3), 16.8 (Cꢀ6C’), 16.7 (Cꢀ6C). HRESIMS (m/z): [M + H]+ calcd for C119H148N5O36
2222.990, found 2222.987.
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