Synthesis of lacto-N-tetraose

Article abstract of DOI:10.1016/j.carres.2017.02.001

Human milk oligosaccharides (HMOs) are the third largest macromolecular component of breast milk and offer infants numerous health benefits, most of which stem from the development of a healthy microbiome. Characterization, quantification, and chemical de

Full text of DOI:10.1016/j.carres.2017.02.001

Accepted Manuscript
Synthesis of lacto-N-tetraose
Kelly M. Craft, Steven D. Townsend
PII:
S0008-6215(17)30101-5
10.1016/j.carres.2017.02.001
CAR 7320
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 8 February 2017
Accepted Date: 9 February 2017
Please cite this article as: K.M. Craft, S.D. Townsend, Synthesis of lacto-N-tetraose, Carbohydrate
Research (2017), doi: 10.1016/j.carres.2017.02.001.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of Lacto-N-Tetraose
Kelly M. Craft and Steven D. Townsend*
Department of Chemistry, Institute of Chemical Biology, Vanderbilt University, Nashville, TN, 37235,
United States
Supporting Information Placeholder
ABSTRACT: Human milk oligosaccharides (HMOs) are the third largest macromolecular component
of breast milk and offer infants numerous health benefits, most of which stem from the development of
a healthy microbiome. Characterization, quantification, and chemical derivatization of HMOs remains a
frontier issue in glycobiology due to the challenge of isolating appreciable quantities of homogenous
HMOs from breast milk. Herein, we report the synthesis of the human milk tetrasaccharide lacto-N-
tetraose (LNT). LNT is ubiquitous in human breast milk as it is a core structure common to longer-chain
HMOs and many glycolipids.
1. Introduction
Human milk is among the earliest vehicles for intestinal bacterial colonization.1-3 In addition to labor
and delivery, human milk assists in establishing a microbiota that provides several health benefits for
the breastfed infant. There are an estimated 700 species of bacteria present in human milk at any given
time.1,4-7 The majority of these bacteria are symbiotic or commensal with roles varying from metabolic
functioning to immune enhancing.4 These protective measures are due, in part, to the human milk oligo-
saccharides (HMOs), a family of unconjugated glycans unique to human milk.8-13
HMOs are prebiotics that stimulate the growth of symbiotic bacteria whose colonization in the infant
gut competes with pathogenic colonization.14 This activity is critical to infant health as symbiotic spe-
cies, such as bifidobacteria, aid in digestion, metabolism, immunity, and the production of neurotrans-
mitters that affect behavior and cognition.1,15,16 HMOs also function as anti-adhesive antimicrobial
agents by serving as free flowing decoy receptors. This role allows HMOs to inhibit pathogen adhesion
to host cell ligands thus preventing the first stage of infection. Although the general functionings of
pooled HMOs have been explored, the functional roles of individual HMOs are far less clear due to a
limited access to large quantities of single-entity HMOs.8,11

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HMOs are the third most abundant macromolecule in human milk after lactose and fat. Colostrum,
or the first milk, is produced during the perinatal period and contains ca. 25 g/L of HMOs.17,18 As nurs-
ing progresses and milk matures, the concentration of HMOs decreases. Interestingly, premature milk
features a greater concentration of HMOs than term milk.18 In general, the concentration and molecular
architecture of HMOs vary depending on stage of lactation and Lewis (Le) blood group and secretor sta-
tus of the mother.19
To date, the structures of nearly 200 HMOs have been elucidated.20-22 However, of these, only a
handful of short-chain HMOs can be produced. Moreover, their production is only possible on a multi-
milligram scale. As short-chain HMOs serve as building blocks for long-chain HMOs featuring branch-
ing, fucosylation, and/or sialylation, insufficient access to shorter HMOs has largely limited the access
to long-chain HMOs. Not surprisingly, a number of strategies have been developed to synthesize struc-
turally defined single-entity HMOs.23-30
We recently initiated a program aimed at studying the glycobiology of human milk. As shown in
Figure 1, donor milk from two mothers was fingerprinted using MALDI FT ICR in positive ion mode,
to reveal the structures of HMOs in a mass range of 500-2000 m/z. A mass of 730.241 was observed,
corresponding to the two isomeric tetrasaccharides, lacto-N-tetraose (LNT) and lacto-N-neotetraose
(LNnT). LNT is more common in human milk being present at 8 times the level of LNnT. In contrast,
LNnT is more common in bovine milk and by extension more common in infant formula.28 The relative
intensities of this peak will vary from sample to sample as LNT and/or LNnT undergoes enzymatic
functionalization to larger, fucosylated derivatives. Unlike LNnT, LNT is not currently available at large
scale through chemo-enzymatic synthesis.28 We therefore selected this molecule as a target for chemical
synthesis. Chemical syntheses of several LNT derivatives and a chemical synthesis of LNT have been
previously reported.30-35 Additionally, an elegant chemoenzymatic approach was recently reported which
yielded ca. 20 mg of LNT.36 These preparations, however, are on scales smaller than that featured in the
present work. Herein, we describe the chemical synthesis of homogeneous LNT using an approach that
produces ca. 1 gram of protected tetrasaccharide in a single glycosylation event.
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Figure 1. HMO mass fingerprinting of two human milk samples using MALDI FT ICR. A generic
structural description is given for each molecular ion observed.
As depicted in Figure 1, LNT (1) is a linear tetrasaccharide in which all glycosidic bonds are β-
configured. More specifically, LNT contains lactose (Galβ1-4Glc) at the reducing end, which is elon-
gated by β-1,3 linked lacto-N-biose (Galβ1-3GlcNAc). Our general synthetic strategy envisioned a two-
glycosylation approach wherein the tetrasaccharide could be assembled from galactose-, glucosamine-,
and lactose-derived building blocks 2, 3, and 4 respectively.
Figure 2. Retrosynthetic analysis of 1.
2. Results and discussion
In the opening stages of the program, we synthesized the requisite glucosamine residue 3, which is
related to a previously described building block reported by the Boons laboratory.37 Starting from glu-
cosamine hydrochloride 5, the C-2 amine was protected as its trichloroethyl (Troc) carbamate after
treatment with saturated aqueous sodium bicarbonate solution followed by addition of 2,2,2-
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trichloroethyl chloroformate (Troc-Cl). Following peracetylation, selective removal of the anomeric
acetate was accomplished using 3-(dimethylamino)-1-propylamine (DMAPA).38 Finally, the anomeric
hemiacetal was converted to its triisopropyl silyl ether using standard Corey conditions (imidazole and
DMF)39 to provide β-silyl ether 6 as a single anomer in 54% yield for the 4-step sequence. We initially
attempted to advance substrate 6 to its triol 7 using sodium methoxide in methanol. Unfortunately, in
our hands, we observed compatibility issues with the Troc carbamate. After screening several sets of
conditions, we realized acetate removal could be accomplished cleanly using guanidinium chloride in
the presence of ammonia to arrive at triol 7 in 76% yield. Finally, 7 was converted to the 4,6-
benzylidene acetal upon reaction with benzaldehyde dimethyl acetal and catalytic 10-camphorsulfonic
acid (CSA) to yield glucosamine acceptor 3 in 91% yield.
Scheme 1. Synthesis of Glucosamine Acceptor 8.
We next turned our attention to the synthesis of lacto-N-biose disaccharide 8, which required
uniting a galactose donor with glucosamine acceptor 3. Known galactose-based α-trichloroacetimidate
donor 2240 was prepared in two steps from commercially available galactose pentaacetate and reacted
with acceptor 3. Interestingly, the glycosylation was not as straightforward as anticipated as significant
amounts of orthoester were observed, depending on reaction conditions. As shown in Table 1, our initial
strategy used acetonitrile as the solvent in the hope that it would assist in providing the desired β-
glycoside. Unfortunately, use of acetonitrile and colder reaction temperatures resulted in significant
formation of orthoacetate 9. Ultimately, we discovered formation of the orthoacetate could be alleviated
by using dichloromethane as the solvent alongside catalytic TMSOTf as an activator with warmer reac-
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tion temperatures. Using these conditions, the desired, protected lacto-N-biose disaccharide 8 could be
obtained in 85% yield.
Table 1. Synthesis of Lacto-N-Biose Disaccharide 8 and Unexpected Orthoacetate 9.
Entry
Activator Solvent Temperature (oC) %Yield 9 %Yield 10
1
2
3
4
5
6
7
TMSOTf CH3CN
CH3CN
-40
-40
-10
0
18
0
50
23
40
44
0
BF3 OEt2
TMSOTf CH3CN
TMSOTf CH3CN
TMSOTf CH3CN
TMSOTf CH2Cl2
TMSOTf CH2Cl2
28
36
49
0
r.t.
-78
0
22
0
85
After obtaining gram quantities of 8, the β-silyl ether was removed via treatment with HF in pyr-
idine to yield anomeric hemiacetal 10 in 77% yield. 10 was then converted to α-trichloroacetimidate
donor 11 in 89% yield. With this donor in hand, the campaign to produce protected tetrasaccharide 12
began. Using known lactose acceptor 441, prepared in six steps from lactose octaacetate,42 we attempted
to form the final glycosidic linkage. Unfortunately, we were unable to successfully generate this linkage
despite screening a variety of activators, including TMSOTf, BF3 OEt2, AgOTf, TfOH, and MeOTf, as
well as a number of reaction solvents and reaction temperatures. Ultimately, we concluded our inability
to form the desired bond was due to the rigidity of the benzylidene acetal of the lacto-N-biose based do-
nor and its prohibitive effect on the generation of the requisite oxonium ion. In retrospect, the poor reac-
tivity of glycosyl donors featuring benzylidene acetals is well precedented.43-47
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Scheme 2. Use of Lactose Acceptor 4 in Lacto-N-Tetraose Glycosylation.
To circumvent the poor reactivity of donor 11, we first removed the benzylidene acetal by heating
11 to 80oC and treating with 80% aqueous acetic acid. The resulting diol was then acetylated and the β-
silyl ether removed to yield anomeric hemiacetal 13 in 78% yield over 3 steps. With 13 in hand, we
moved to form α-trichloroacetimidate donor 14. Initial attempts relied on treatment of anomeric hemi-
acetal 13 with 1,8-diazabicylco[5.4.0]undec-7-ene (DBU) and trichloroacetonitirle (Cl3CCN). Surpris-
ingly, these conditions proved unreliable as frequent decomposition was observed. In an effort to lessen
this degradation, potassium carbonate (K2CO3) and prolonged reaction times were used to generate α-
trichloroacetimidate donor 14 in 77% yield.
With the new lacto-N-biose donor 14 in hand, we once again turned our attention to forming the fi-
nal glycosidic linkage. Unfortunately, glycosylation with donor 14 and acceptor 4 did not proceed as
smoothly as expected. While mass spectral data suggested we had formed the desired tetrasaccharide 15,
we were unable to fully characterize the compound as multiple byproducts were observed. One of the
suspected byproducts was the hexasaccharide resulting from glycosylation at both the C-3’ and C-4’ hy-
droxyls of acceptor 4. In an effort to help the glycosylation event proceed more smoothly, we attempted
to better exploit the increased reactivity of the equatorial C-3’ hydroxyl over the C-4’ axial hydroxyl by
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adding the donor dropwise into the reaction vessel containing the acceptor and TfOH. Much to our dis-
appointment, multiple side products were once again observed.
Although we were unable to confirm formation of the hexasaccharide, we hypothesized the glyco-
sylation would be improved by protecting the C-4’ hydroxyl of diol acceptor 4. To do this, we em-
ployed a simple two-step, one-pot procedure to selectively acetylate the axial C-4’ hydroxyl. Treatment
of diol 4 with trimethylorthoacetate followed by treatment with 90% aqueous trifluoroacetic acid fur-
nished known axially acetylated acceptor 16 in a satisfactory 93% yield.48,49 Much to our delight, TfOH
promoted glycosylation of donor 14 with acceptor 16 resulted in formation of the desired fully protected
tetrasaccharide 17 in 94% yield.
With 17 in hand, global deprotection to afford lacto-N-tetraose (1) commenced with Troc removal
accompanied by in situ acetamide formation. To this end, it is important to highlight that the use of acti-
vated Zn dust in a mixed solvent system of acetic acid and acetic anhydride to reductively convert the
N-Troc-protecting group to its corresponding acetamide resulted in partial decomposition and undesira-
bly low yields. To overcome this shortcoming, we moved to less conventional reducing agents, includ-
ing 10% Zn/Pb couple and 10% Cd/Pb couple.50,51 Of these, 10% Zn/Pb couple performed best and re-
ductively cleaved the Troc carbamate without any accompanying decomposition. Following successful
acetamide formation, deacetylation followed by hydrogenolysis furnished lacto-N-tetraose (1) in 64%
yield over the three step deprotection sequence.
Scheme 3. Synthesis of Lacto-N-Tetraose (1).
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In summary, the total synthesis of lacto-N-tetraose has been completed using a linear approach from
three readily synthesized building blocks. Of note is that the key, final glycosylation event was conduct-
ed to produce ca. 1 gram of protected LNT. With this molecule now available at large scale, future direc-
tions in our lab concern studying the conversion of this molecule to other complex HMOs. Progress in
this regard, will be reported in due course.
3. Experimental Section
3.1 General materials and methods
Commercial reagents were used as received. Anhydrous solvents were taken from an MBRAUN solvent
purification system (MB SPS) and stored over 4 Å or 3 Å molecular sieves. All moisture-sensitive reac-
tions were performed in flame- or oven-dried round bottom flasks under an argon atmosphere. All air-
or moisture-sensitive liquids were transferred via oven-dried stainless steel syringes or cannula. Reac-
tion temperatures were monitored and controlled via thermocouple thermometer and corresponding hot
plate stirrer. Flash column chromatography was performed as described by Still et. al. using silica gel
230-400 mesh. Analytical thin-layer chromatography (TLC) was performed on glass-backed Silica gel
60 F254 plates (EMD/Merck KGaA) and visualized using UV, cerium ammonium molybdate stain, and
anisaldehyde stain. 1H NMR spectra were obtained on a Bruker 400 or 600 MHz spectrometer with re-
porting relative to residual solvent signals (CDCl3, 7.26 ppm; CH3OD, 3.31 ppm; D2O, 4.79 ppm). 1H
NMR spectral data are presented as follows: chemical shifts (δ ppm), multiplicity (s=singlet, d=doublet,
dd=doublet of doublets, t=triplet, q=quartet, m=multiplet, br=broad), coupling constants (Hz), integra-
tion, proton assignment. 13C NMR spectra were obtained on a Bruker 100 MHz spectrometer with re-
porting relative to residual solvent signals (CDCl3, 77.16 ppm; CH3OD, 49.0 ppm). 13C NMR spectral
data are presented as follows: chemical shifts (δ ppm), carbon assignment. Proton and carbon assign-
ments were made with the aid of 2D NMR techniques (COSY, HSQC, and HMBC). High resolution
mass spectra were recorded on a high resolution Thermo Electron Corporation MAT 95XP-Trap by use
of electro-spray ionization (ESI) by the Indiana University Mass Spectrometry facility and a SYNAPT
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G2 or SYNAPT G2-S spectrometer (Waters, for TOF-MS) by the McLean lab of Vanderbilt University.
Low resolution mass spectra were recorded on a Thermo Scientific Dionex Ultimate 3000 HPLC system
with MSQ Plus Mass Detector. Optical rotations were obtained using a Perkin Elmer 341 polarimeter.
3.2 Synthetic procedures
3.2.1. (2R,3S,4R,5R,6S)-2-(acetoxymethyl)-5-(((2,2,2-trichloroethoxy)carbonyl)amino)-6-
((triisopropylsilyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (6)
To a suspension of D-(+)-glucosamine hydrochloride 5 (1.0 eq, 3.0 g, 14 mmol) in H2O (30 mL)
was added NaHCO3 (3.0 eq, 3.5 g, 42 mmol) at room temperature and the resulting mixture was stirred
1 H. 2,2,2-trichloroethyl chloroformate (5.0 eq, 9.4 mL, 70 mmol) was then added dropwise over 20
minutes. The reaction was stirred an additional 3 H after which a white solid had formed. The reaction
was filtered, washed with additional H2O (250 mL), and allowed to dry overnight. The crude Troc-
protected glucosamine was coevaporated with benzene (3x) then dissolved in pyridine (130 mL) and
acetic anhydride (5.0 eq, 6.6 mL, 70 mol) was added dropwise over 5 minutes. The resulting solution
was stirred 6 H then was diluted with EtOAc (250 mL), washed with 2 N HCl (6 x 90 mL), brine (1 x 90
mL), dried (MgSO4), filtered, and concentrated in vacuo to yield a pale yellow foam. The crude
tetraacetate was dissolved in THF (70 mL) and 3-(dimethylamino)-1-propylamine (DMAPA) (5.0 eq,
8.8 mL, 70 mmol) was added. The reaction was stirred 1 H and 15 minutes then was diluted with
CH2Cl2 (130 mL), washed with 2 N HCl (2 x 60 mL), dried (MgSO4), filtered, and concentrated in vac-
uo to yield a white foam. The crude anomeric hemiacetal was dissolved in DMF (90 mL) and imidazole
(2.0 eq, 1.9 g, 28 mmol), DMAP (cat), and chlorotriisopropylsilane (1.2 eq, 3.6 mL, 17 mmol) were
added sequentially. The resulting solution was stirred 16 H then additional chlorotriisopropylsilane was
added (1.5 mL). The reaction stirred an additional 22 H then was diluted with EtOAc (180 mL), washed
with H2O (3 x 80 mL), 2N HCl (1 x 80 mL), brine (1 x 80 mL), dried (MgSO4), filtered, and concentrat-
ed in vacuo. The crude residue was purified via flash column chromatography (2:1 hexanes/EtOAc) to
yield anomeric silyl ether 6 (4.87 g, 7.65 mmol, 55% over 4 steps) as a white solid: Rf 0.48 (2:1 hex-
anes/EtOAc); 1H NMR (400 MHz, CDCl3) δ 5.23 (t, J=10.1 Hz , 1H, H-3), 5.06 (d, J=8.6 Hz, 1H, NH),
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5.04 (t, J=9.6 Hz, 1H, H-4), 4.89 (d, J=7.8 Hz, 1H, H-1), 4.75 (d, J=12.0 Hz, 1H, Troc CH), 4.61 (d,
J=12.0 Hz, 1H, Troc CH), 4.15 (d, J=4.2 Hz, 2H, H-6a, H-6b), 3.72-3.61 (m, 2H, H-2, H-5), 2.06 (s,
3H, COCH3), 2.03 (s, 3H, COCH3), 2.02 (s, 3H, COCH3), 1.12-1.01 (m, 21H, TIPS), 1.60 (s, H2O); 13C
(100 MHz, CDCl3) δ 170.9 (COCH3), 170.8 (COCH3), 169.7 (COCH3), 154.1 (Troc CO), 96.1 (C-1),
95.4 (Troc CCl3), 74.8 (Troc CH2), 72.1 (C-3), 71.9 (C-5), 69.1 (C-4), 62.6 (C-6), 58.7 (C-2), 20.8
(COCH3), 17.9 (TIPS), 17.8 (TIPS), 12.3 (TIPS). HRMS (ESI) calcd for C24H40Cl3NO10Si [M+Na]+
658.1385, found 658.1356.
3.2.2. 2,2,2-trichloroethyl ((2S,3R,4R,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl)-2-
((triisopropylsilyl)oxy)tetrahydro-2H-pyran-3-yl)carbamate (7)
To a solution of 6 (1.0 eq, 4.0 g, 6.3 mmol) in CH2Cl2 (60 mL) and MeOH (60 mL), was added
guanidine hydrochloride (5.0 eq, 3.0 g, 31 mmol) and 7 N NH3/MeOH (12.0 eq, 8.4 mL, 75 mmol) se-
quentially. The resulting solution was stirred 21 H then was neutralized with AcOH and concentrated in
vacuo. The crude residue was purified via flash column chromatography (10:1 CHCl3/MeOH) to yield
triol 7 (2.45 g, 4.80 mmol, 76%) as a white solid: Rf 0.41 (10:1 CH2Cl2/MeOH); 1H NMR (400 MHz,
MeOD) δ 4.73 (d, J=7.7 Hz, 1H, H-1), 4.70 (s, 2H, Troc CH, Troc CH), 3.84 (dd, J=2.6, 11.8 Hz, 1H,
H-6a), 3.72 (dd, J=5.0, 11.8 Hz, 1H, H-6b), 3.47-3.33 (m, 3H, H-2, H-3, H-4), 3.23 (ddd, J=2.6, 5.0, 9.0
Hz, 1H, H-5), 1.16-1.07 (m, 21 H, TIPS), 1.98 (s, AcOH); 13C (100 MHz, MeOD) δ 156.9 (Troc CO),
97.7 (C-1), 97.0 (Troc CCl3), 77.7 (C-5), 75.8 (Troc CH2), 75.7 (C-3), 72.3 (C-4), 62.8 (C-6), 61.5 (C-
2), 18.4 (TIPS), 18.4 (TIPS), 13.5 (TIPS). HRMS (ESI) calcd for C18H34Cl3NO7Si [M+Na]+ 532.1068,
found 532.1059.
3.2.3. 2,2,2-trichloroethyl((2R,4aR,6S,7R,8R,8aS)-8-hydroxy-2-phenyl-6-
((triisopropylsilyl)oxy)hexahydropyrano[3,2-d][1,3]dioxin-7-yl)carbamate (3)
To a solution of 7 (1.0 eq, 1.5 g, 2.9 mmol) in CH3CN (30 mL) was added benzaldehyde dime-
thyl acetal (2.0 eq, 0.88 mL, 5.9 mmol). The reaction pH was adjusted between 2-4 using DL-10-
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camphorsulfonic acid and the reaction heated to 60oC. The reaction was stirred 2 H then was neutralized
with Et3N, diluted with EtOAc (70 mL), washed with water (2 x 30 mL), brine (1 x 30 mL), dried
(MgSO4), filtered, and concentrated in vacuo. The crude residue was purified via flash column chroma-
tography (6:1 hexanes/EtOAc) to yield benzylidene acetal 3 (1.6 g, 3.0 mmol, 91%) as a white foam: Rf
1
0.60 (1:1 hexanes/EtOAc); H NMR (400 MHz, CDCl3) δ 7.50-7.48 (m, 2H, aromatic), 7.40-7.36 (m,
3H, aromatic), 5.55 (s, 1H, benzylidene CH), 5.15 (d, J=5.9 Hz, 1H, NH), 4.92 (d, J=7.5 Hz, 1H, H-1),
4.74 (d, J=12.0 Hz, 1H, Troc CH), 4.66 (d, J=11.9 Hz, 1H, Troc CH), 4.29 (dd, J=10.5, 4.9 Hz, 1H, H-
6a), 4.06-4.02 (m, 1H, H-3), 3.78 (t, J=10.2 Hz, 1H, H-6b), 3.58 (t, J=9.2 Hz, 1H, H-4), 3.49-3.36 (m,
13
2H, H-5, H-2), 1.15-1.03 (m, 21H, TIPS); C (100 MHz, CDCl3) δ 154.6 (Troc CO), 137.2 (aromatic),
129.5 (aromatic), 128.5 (aromatic), 126.5 (aromatic), 102.1 (benzylidene CH), 96.2 (C-1), 95.3 (Troc
CCl3), 81.7 (C-4), 75.0 (Troc CH2), 71.0 (C-3), 68.7 (C-6), 66.3 (C-5), 61.3 (C-2), 17.9 (TIPS), 12.3
(TIPS). HRMS (ESI) calcd C25H38Cl3NO7Si [M+Na]+ 620.1381, found 620.1367.
3.2.4. (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(((2R,4aR,6S,7R,8R,8aS)-2-phenyl-7-(((2,2,2-
trichloroethoxy)carbonyl)amino)-6-((triisopropylsilyl)oxy)hexahydropyrano[3,2-d][1,3]dioxin-8-
yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (8)
Donor 2 (1.4 eq, 1.46 g, 3.0 mmol) and acceptor 3 (1.0 eq, 1.27 g, 2.1 mmol) were coevaporated
with benzene (2 x 8 mL) and placed in a vacuum desiccator containing P2O5 overnight. The do-
nor/acceptor mixture was dissolved in CH2Cl2 (14 mL) and the resulting solution was cannulated into a
reaction flask containing 4Å powdered molecular sieves (2.7 g). The mixture was stirred under argon 1
H then cooled to -10oC and TMSOTf (0.1 eq, 0.038 mL in 0.2 mL CH2Cl2) was added. The reaction was
stirred 10 minutes then quenched with Et3N. The reaction was diluted with CH2Cl2, filtered through
celite, dried (MgSO4), filtered, and concentrated in vacuo. The crude residue was purified via flash col-
umn chromatography (2:1 hexanes/EtOAc) to yield diasaccharide 8 (1.59 g, 1.71 mmol, 81%) as a white
foam: Rf 0.35 (1:1 hexanes/EtOAc); 1H NMR (400 MHz, CDCl3) δ 7.49-7.45 (m, 2H, aromatic), 7.40-
7.34 (m, 3H, aromatic), 5.54 (s, 1H, benzylidene CH), 5.29 (d, J=2.8 Hz, 1H, H-4’), 5.19 (dd, J=8.4,
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10.4 Hz, 1H, H-2’), 5.18 (d, J=8.0 Hz, NH), 5.11 (d, J=7.8 Hz, H-1), 4.92 (dd, J=3.4, 10.4 Hz, 1H, H-
3’), 4.69 (s, 2H, Troc CH, Troc CH), 4.68 (d, J=8.6 Hz, H-1’), 4.32 (t, J=9.2 Hz, H-3), 4.27 (dd, J=5.0,
10.6 Hz, 1H, H-6a), 4.11 (q, EtOAc), 4.06 (dd, J=8.4, 10.6 Hz, 1H, H-6’a), 3.83 (dd, J=5.4, 10.7 Hz,
1H, H-6’b), 3.78 (t, J=10.4 Hz, H-6b), 3.72 (t, J=9.2 Hz, 1H, H-4), 3.64 (t, J=6.7 Hz, 1H, H-5’), 3.50-
3.44 (ddd, J=4.9, 9.7, 9.7 Hz, 1H, H-5), 3.28 (q, J=8.0 Hz, 1H, H-2), 2.11 (s, 3H, COCH3), 2.04 (s,
EtOAc), 2.03 (s, 3H, COCH3), 1.95 (s, 3H, COCH3), 1.93 (s, 3H, COCH3), 1.62 (s, H2O), 1.25 (t,
EtOAc), 1.12-1.00 (m, 21H, TIPS), 0.06 (s, silicone grease); 13C (100 MHz, CDCl3) δ 170.4 (COCH3),
170.3 (COCH3), 170.2 (COCH3), 169.6 (COCH3), 153.9 (Troc CO), 137.2 (aromatic), 129.4 (aromatic),
128.5 (aromatic), 126.2 (aromatic), 101.5 (benzylidene CH), 101.0 (C-1’), 95.3 (C-1), 95.2 (Troc CCl3),
80.4 (C-4), 78.2 (C-3), 74.9 (Troc CH2), 71.1 (C-3’), 70.6 (C-5’), 69.4 (C-2’), 68.8 (C-6), 67.0 (C-4’),
66.2 (C-5), 61.0 (C-6’), 60.7 (C-2), 20.9 (COCH3), 20.8 (COCH3), 20.7 (COCH3), 20.7 (COCH3), 17.9
(TIPS), 17.9 (TIPS), 12.3 (TIPS). HRMS (ESI) calcd for C39H56Cl3NO16Si [M+Na]+ 950.2332, found
950.2311.
3.2.5. (3aR,5R,6S,7S,7aR)-5-(acetoxymethyl)-2-methyl-2-(((2R,4aR,6S,7R,8R,8aS)-2-phenyl-7-(((2,2,2-
trichloroethoxy)carbonyl)amino)-6-((triisopropylsilyl)oxy)hexahydropyrano[3,2-d][1,3]dioxin-8-
yl)oxy)tetrahydro-5H-[1,3]dioxolo[4,5-b]pyran-6,7-diyl diacetate (9)
Donor 2 (1.3 eq, 0.16 g, 0.33 mmol) and acceptor 3 (1.0 eq, 0.15 g, 0.25 mmol) were coevapo-
rated with benzene (2 x 4 mL) and placed in a vacuum desicator containing P2O5 overnight. The do-
nor/acceptor mixture was dissolved in CH3CN (1.25 mL) and the resulting solution was cannulated into
a reaction flask containing 4Å powdered molecular sieves (0.4 g). The mixture was stirred under argon
1 H then cooled to -40oC and TMSOTf (1 drop) was added. The reaction was stirred 1 H then quenched
with Et3N. The reaction was diluted with EtOAc (25 mL), filtered through celite, washed with water (3
x 10 mL), brine (1 x 10 mL), dried (MgSO4), filtered, and concentrated in vacuo. The crude residue was
purified via flash column chromotography (2:1 hexanes/EtOAc) to yield orthoacetate 9 (0.115 g, 0.124
1
mmol, 50%) as a white foam: Rf 0.48 (2:1 hexanes/EtOAc); H NMR (400 MHz, CDCl3) δ 7.45-7.43
12

ACCEPTED MANUSCRIPT
(m, 2H, aromatic), 7.35-7.33 (m, 3H, aromatic), 5.82 (d, J=4.8 Hz, 1H, H-1’), 5.51 (s, 1H, benzylidene
CH), 5.33 (dd, J=1.5, 3.2 Hz, 1H, H-4’), 5.26 (d, J=8.7 Hz, 1H, NH), 4.94 (d, J=7.2 Hz, 1H, H-3’), 4.93
(d, J=7.1 Hz, 1H, H-1), 4.83 (d, J=11.9 Hz, 1H, Troc CH), 4.55 (d, J=12.0 Hz, 1H, Troc CH), 4.30-4.23
(m, 3H, H-6a, H-2’, H-5’), 4.11-4.05 (m, 3H, H-6’a, H-3, H-6’b), 3.76 (t, J=10.2 Hz, 1H, H-6b), 3.60 (t,
J=9.3 Hz, 1H, H-4), 3.45-3.36 (m, 2H, H-5, H-2), 2.08 (s, 3H, COCH3), 2.04 (s, 3H, COCH3), 2.01 (s,
3H, COCH3), 1.65 (orthoacetate CH3), 1.25 (m, grease), 0.06 (s, silcone grease), 1.11-1.02 (m, 21H,
13
TIPS); C (100 MHz, CDCl3) δ 170.6 (COCH3), 170.1 (COCH3), 170.0 (COCH3), 154.0 (Troc CO),
137.2 (aromatic), 129.2 (aromatic), 128.3 (aromatic), 126.2 (aromatic), 120.5 (orthoacetate C), 101.7
(benzylidene CH), 98.4 (C-1’), 96.6 (C-1), 95.2 (Troc CCl3), 80.5 (C-4), 75.0 (Troc CH2), 72.5 (C-2’),
71.6 (C-3’), 71.6 (C-3), 69.0 (C-5’), 68.7 (C-6), 66.5 (C-5), 65.8 (C-4’), 61.6 (C-6’), 60.3 (C-2), 25.1
(orthoacetate CH3), 20.9 (COCH3), 20.8 (COCH3), 20.8 (COCH3), 17.9 (TIPS), 17.8 (TIPS), 12.3
(TIPS). HRMS (TOF) calcd for C39H56Cl3NO16Si [M+Na]+ 950.2326, found 950.2365.
3.2.6. (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(((2R,4aR,6R,7R,8R,8aS)-6-hydroxy-2-phenyl-7-(((2,2,2-
trichloroethoxy)carbonyl)amino)hexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)tetrahydro-2H-pyran-
3,4,5-triyl triacetate (10)
To a solution 8 (1.0 eq, 1.0 g, 1.1 mmol) in pyridine (11 mL) cooled to 0oC was added 70% HF
in pyridine (5.4 mL) dropwise over 5 minutes. The solution was stirred 30 minutes then was allowed to
warm to room temperature and stir an additional 4.5 H. The reaction was diluted with water (40 mL)
and extracted with EtOAc (4 x 15 mL). The combined organics were quenched with solid NaHCO3, and
saturated NaHCO3 solution, washed with 2 N HCl (3 x 20 mL), brine (1 x 20 mL), dried (MgSO4), fil-
tered, and concentrated in vacuo. The crude residue was purified via flash column chromatography (2:3
hexanes/EtOAc) to yield anomeric hemiacetal 10 (0.64 g, 0.83 mmol, 77%) (α/β 2.2:1) as a white foam:
1
Rf 0.38 (1:1 hexanes/EtOAc); H NMR (400 MHz, CDCl3) δ (α anomer) 7.48-7.45 (m, 2H, aromatic),
7.38-7.35 (m, 3H, aromatic), 5.55 (s, 1H, benzylidene CH), 5.54 (s, 0.45 H, minor anomer), 5.30-5.25
(m, 2H, H-1, H-4’), 5.19 (dd, J=8.0, 10.4 Hz, minor anomer), 5.17 (dd, J=8.0, 10.4 Hz, H-2’), 4.96 (d,
13

ACCEPTED MANUSCRIPT
J=12.0 Hz, 1H, Troc CH), 4.92 (dd, J=3.4, 10.5 Hz, 1H, H-3’), 4.80 (d, J=11.9 Hz, 0.45 H, minor
anomer), 4.71 (m, 0.45 H, minor anomer), 4.69 (d, J=8.0 Hz, 1H, H-1’), 4.59 (d, J=12.1 Hz, 1H, Troc
CH), 4.43 (m, 0.45 H, minor anomer), 4.33 (dd, J=4.9, 10.5 Hz, 0.45 H, minor anomer), 4.24 (dd,
J=4.8, 10.3 Hz, 1H, H-6a), 4.14-3.98 (m, 4H, H-5, H-2, H-3, H-6’a), 3.83-3.71 (m, 3H, H-6’b, H-6b, H-
4), 3.57 (t, J=6.7 Hz, 1H, H-5’), 3.52 (d, J=3.2 Hz, 1H, OH), 3.53-3.47 (m, 0.9 H, minor anomer), 2.11
(s, 1.35 H, minor anomer), 2.10 (s, 3H, COCH3), 2.06 (s, 3H, COCH3), 1.97 (s, 1.35 H, minor anomer),
1.95 (s, 1.35 H, minor anomer), 1.95 (s, 3H, COCH3), 1.91 (s, 3H, COCH3), 1.75 (br s, 0.45 H, minor
anomer OH), 1.25 (m, grease), 0.06 (s, silicone grease); 13C (100 MHz, CDCl3) δ 170.5 (COCH3), 170.4
(minor anomer), 170.4 (COCH3), 170.3 (COCH3), 169.7 (COCH3), 154.2 (Troc CO), 137.2 (aromatic),
137.0 (minor anomer), 129.5 (minor anomer), 129.4 (aromatic), 128.5 (aromatic), 126.2 (minor
anomer), 126.1 (aromatic), 126.1 (aromatic), 101.6 (minor anomer), 101.6 (benzylidene CH), 101.2 (C-
1’), 95.5 (Troc CCl3), 95.3 (minor anomer), 92.5 (C-1), 80.8 (C-4), 77.3 (C-3), 75.0 (Troc CH2), 71.1
(C-3’), 71.0 (minor anomer), 70.8 (minor anomer), 70.6 (C-5’), 69.6 (C-2’), 69.4 (minor anomer), 69.0
(C-6), 68.7 (minor anomer), 66.9 (C-4’), 66.5 (minor anomer), 62.7 (C-5), 60.9 (C-6’), 54.8 (C-2), 20.8
(COCH3), 20.8 (COCH3), 20.7 (COCH3). HRMS (ESI) calcd for C30H36Cl3NO16 [M+Na]+ 794.0997,
found 794.1010.
3.2.7. (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(((2R,4aR,6R,7R,8R,8aS)-2-phenyl-6-(2,2,2-trichloro-1-
iminoethoxy)-7-(((2,2,2-trichloroethoxy)carbonyl)amino)hexahydropyrano[3,2-d][1,3]dioxin-8-
yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (11)
To a solution of 10 (1.0 eq, 0.35 g, 0.45 mmol) in CH2Cl2 (6.2 mL) cooled to 0oC was added
Cl3CCN (10 eq, 0.45 mL, 4.5 mmol) followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (cat.). The
reaction was stirred 1 H then was allowed to warm to room temperature and stir an additional 1 H. The
reaction was concentrated and the crude residue purified via flash column chromatography (1:1 hex-
anes/ethyl acetate) to yield α-trichloroacetimidate 11 (0.36 g, 0.40 mmol, 89%) as a pale yellow pow-
der: Rf 0.71 (1:1 hexanes/ethyl acetate); 1H NMR (400 MHz, CDCl3) δ 8.78 (s, 1H, imidate NH), 7.52-
14

ACCEPTED MANUSCRIPT
7.45 (m, 2H, aromatic), 7.40-7.35 (m, 3H, aromatic), 6.40 (d, J=3.8 Hz, H-1), 5.60 (s, 1H, benzylidene
CH), 5.32 (d, J=3.0 Hz, 1H, H-4’), 5.21 (dd, J=7.9, 10.4 Hz, H-2’), 5.12 (d, J=8.6 Hz, 1H, NH), 4.97
(dd, J=3.4, 10.5 Hz, H-3’), 4.94 (d, J=11.9 Hz, 1H, Troc CH), 4.80 (d, J=7.9 Hz, 1H, H-1’), 4.56 (d,
J=12.0 Hz, 1H, Troc CH), 4.34 (dd, J=4.6, 10.3 Hz, 1H, H-6a), 4.25 (ddd, J=3.8, 10.2, 10.2 Hz, 1H, H-
2), 4.12-4.04 (m, 2H, H-3, H-6’a), 3.98 (ddd, J=4.7, 9.7, 9.7 Hz, H-5), 3.92-3.84 (m, 2H, H-6’b, H-4),
3.82 (t, J=10.3 Hz, 1H, H-6b), 3.72 (t, J=6.7 Hz, H-5’), 2.12 (s, 3H, COCH3), 2.01 (s, 3H, COCH3),
13
1.96 (s, 3H, COCH3), 1.95 (s, 3H, COCH3), 1.56 (s, H2O), 1.25 (m, grease); C (100 MHz, CDCl3) δ
170.4 (COCH3), 170.3 (COCH3), 169.6 (COCH3), 160.5 (imidate CNH), 154.1 (Troc CO), 136.9 (aro-
matic), 129.5 (aromatic), 128.5 (aromatic), 126.1 (aromatic), 101.6 (benzylidene CH), 100.9 (C-1’),
95.4 (Troc CCl3), 95.3 (C-1), 90.9 (imidate CCl3), 79.8 (C-4), 76.8 (C-3), 74.9 (Troc CH2), 71.0 (C-3’),
70.9 (C-5’), 69.8 (C-2’), 68.6 (C-6), 66.8 (C-4’), 65.2 (C-5), 61.0 (C-6’), 54.4 (C-2), 20.8 (COCH3),
20.7 (COCH3), 20.7 (COCH3). HRMS (ESI) calcd for C32H36Cl6N2O16 [M+Na]+ 937.0094, found
937.0067.
3.2.8. (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(((2R,4aR,6R,7R,8R,8aS)-6-hydroxy-2-phenyl-7-
(((2,2,2trichloroethoxy)carbonyl)amino)hexahydropyrano[3,2-d][1,3]dioxin-8-yl)oxy)tetrahydro-2H-
pyran-3,4,5-triyl triacetate (13)
A solution of 8 (1.0 eq, 1.45 g, 1.56 mmol) in 80% aqueous acetic acid (15 mL) was heated to
80oC and stirred 5 H and 15 minutes. The reaction was diluted with water (40 mL) and extracted with
EtOAc (4 x 15 mL) while solid NaHCO3 was added. The combined organics were washed with saturat-
ed NaHCO3 solution (4 x 20 mL), brine (1 x 20 mL), dried (MgSO4), filtered, and concentrated in vacuo
to yield a white solid. The crude diol was dissolved in pyridine (16 mL) and acetic anhydride (2.5 eq,
0.37 mL, 3.9 mmol) and DMAP (cat.) were added. The reaction was stirred 1 H and 15 minutes then
diluted with EtOAc (50 mL), washed with 2 N HCl (4 x 20 mL), brine (1 x 20 mL), dried (MgSO4), fil-
tered, and concentrated in vacuo to yield a white foam. The crude diacetate was dissolved in pyridine
(15 mL) and the resulting solution cooled to 0oC. 70% HF in pyridine (8 mL) was added dropwise over
15

ACCEPTED MANUSCRIPT
5 minutes. The reaction was stirred 1 H then allowed to warm to room temperature and stir an additional
5 H. The reaction was diluted with water (40 mL) and extracted with EtOAc (4 x 15 mL) while solid
NaHCO3 was added. The combined organics were quenched with saturated NaHCO3 solution, washed
with 2 N HCl (4 x 20 mL), brine (1 x 20 mL), dried (MgSO4), filtered, and concentrated in vacuo. The
crude residue was purified via flash column chromatography (2:3 hexanes/ethyl acetate) to yield ano-
meric hemiacetal 13 (0.94 g, 1.2 mmol, 78% over 3 steps) (α/β 8:1) as a white foam: Rf 0.20 (1:1 hex-
anes/ethyl acetate); 1H NMR (400 MHz, CDCl3) δ (α anomer) 5.36 (m, 1H, NH), 5.35 (d, J=3.2 Hz, 1H,
H-4’), 5.25 (t, J=2.9 Hz, 1H, H-1), 5.05 (dd, J=7.8, 10.4 Hz, 1H, H-2’), 4.99 (t, J=8.8 Hz, H-4), 4.92
(dd, J=3.4, 10.5 Hz, 1H, H-3’), 4.74 (s, 2H, Troc CH, Troc CH), 4.65 (d, J=7.8 Hz, H-1’), 4.24-4.15 (m,
4H, H-6’a, H-5, H-6a, H-6b), 4.07-4.00 (m, 3H, H-3, H-6’b, H-2), 3.90 (dd, J=7.0, 6.7 Hz, 1H, H-5’),
3.35 (d, J=3.0, 1H, OH), 2.14 (s, 3H, COCH3), 2.10 (s, 3H, COCH3), 2.07 (s, 3H, COCH3), 2.06 (s, 3H,
COCH3), 2.06 (s, 3H, COCH3), 1.95 (s, 3H, COCH3); 13C (100 MHz, CDCl3) δ (α anomer) 171.1
(COCH3), 170.6 (COCH3), 170.3 (COCH3), 170.3 (COCH3), 169.8 (COCH3), 169.5 (COCH3), 154.1
(Troc CO), 100.7 (C-1’), 95.3 (Troc CCl3), 92.1 (C-1), 75.4 (C-3), 74.9 (Troc CH2), 70.9 (C-3’), 70.6
(C-5’), 69.1 (C-2’), 68.8 (C-4), 67.9 (C-5), 67.0 (C-4’), 62.5 (C-6), 61.0 (C-6’), 55.2 (C-2), 21.0
(COCH3), 20.9 (COCH3), 20.9 (COCH3), 20.8 (COCH3), 20.8 (COCH3), 20.7 (COCH3). HRMS (ESI)
calcd for C27H36Cl3NO18 [M+Na]+ 790.0896, found 790.0905.
3.2.9. (2R,3R,4S,5S,6R)-2-(((2R,3S,4R,5R,6R)-3-acetoxy-2-(acetoxymethyl)-6-(2,2,2-trichloro-1-
iminoethoxy)-5-(((2,2,2-trichloroethoxy)carbonyl)amino)tetrahydro-2H-pyran-4-yl)oxy)-6-
(acetoxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (14)
To a solution of 13 (1.0 eq, 0.30 g, 0.39 mmol) in CH2Cl2 (5.6 mL) was added potassium car-
bonate (3.0 eq, 1.6 g, 1.2 mmol) and trichloroacetonitirle (10 eq, 0.39 mL, 3.9 mmol) sequentially. The
reaction was stirred 22 H then diluted with CH2Cl2, filtered through celite, and concentrated in vacuo.
The crude residue was purified via flash column chromatography (1:1 hexanes/ethyl acetate) to yield α-
trichloroacetimidate 14 (0.27 g, 0.30 mmol, 77%) as a white foam: Rf 0.38 (1:1 hexanes/ethyl acetate);
16

ACCEPTED MANUSCRIPT
1H (400 MHz, CDCl3) δ 8.83 (s, 1H, imidate NH), 6.31 (d, J=3.7 Hz, 1H, H-1), 5.37 (dd, J=0.8, 3.5 Hz,
1H, H-4’), 5.14-5.05 (m, 3H, H-2’, H-4, NH), 4.96 (dd, J=3.4, 10.4 Hz, 1H, H-3’), 4.78 (d, J=12.0 Hz,
1H, Troc CH), 4.70 (d, J=7.5 Hz, 1H, H-1’), 4.69 (d, 12.2 Hz, 1H, Troc CH), 4.29 (td, J=3.8, 10.1, 10.1
Hz, 1H, H-2), 4.23-4.17 (m, 2H, H-6’a, H-6a), 4.14-4.02 (m, 4H, H-6b, H-5, H-3, H-6’b), 3.96 (t, J=6.7
Hz, 1H, H-5’), 2.15 (s, 3H, COCH3), 2.08 (s, 3H, COCH3), 2.07 (s, 3H, COCH3), 2.06 (s, 3H, COCH3),
2.04 (s, 3H, COCH3), 1.96 (s, 3H, COCH3), 1.66 (s, H2O), 1.25 (m, grease), 0.06 (s, silicon grease); 13C
(100 MHz, CDCl3) δ 170.9 (COCH3), 170.5 (COCH3), 170.2 (COCH3), 170.2 (COCH3), 169.5
(COCH3), 169.5 (COCH3), 160.4 (imidate CNH), 154.0 (Troc CO), 100.6 (C-1’), 95.2 (Troc CCl3), 90.9
(imidate CCl3), 75.8 (C-3), 75.0 (Troc CH2), 70.9 (C-5’), 70.8 (C-3’), 70.3 (C-5), 69.3 (C-2’), 68.0 (C-
4), 67.0 (C-4’), 61.8 (C-6), 61.2 (C-6’), 54.7 (C-2), 20.9 (COCH3), 20.9 (COCH3), 20.8 (COCH3), 20.8
(COCH3), 20.8 (COCH3), 20.7 (COCH3). HRMS (ESI) calcd for C29H36Cl6N2O18 [M+Na]+ 932.9992,
found 932.9962.
3.2.10. (2R,3R,4S,5R,6S)-5-(benzyloxy)-2-((benzyloxy)methyl)-4-hydroxy-6-(((2R,3R,4S,5R,6R)-4,5,6-
tris(benzyloxy)-2-((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)tetrahydro-2H-pyran-3-yl acetate
(16)48,49
To a solution of 4 (1.0 eq, 0.9 g, 1.0 mmol) in CH3CN (10 mL) was added trimethylorthoacetate
(3.0 eq, 0.38 mL, 3.0 mmol) and p-toluene sulfonic acid (0.1 eq, 0.02 g, 0.1 mmol). The reaction was
stirred 25 minutes then 90% trifluroacetic acid (0.36 mL) was added. The resulting solution was stirred
20 minutes then diluted with water (15 mL) and extracted with EtOAc (4 x 5 mL). The combined organ-
ics were washed with a saturated NaHCO3 solution (2 x 7 mL), brine (1 x 7 mL), dried (MgSO4), fil-
tered, and concentrated in vacuo. The crude residue was purified via flash column chromatography (2:1
hexanes/ethyl acetate) to yield axial acetate 16 (0.87 g, 0.94 mmol, 93%) as a white foam: Rf 0.32 (2:1
hexanes/ethyl acetate); 1H (600 MHz, CDCl3) δ 7.34-7.13 (m, 30H, aromatic), 5.30 (d, J=3.2 Hz, 1H, H-
4’), 4.93 (d, J=10.5 Hz, 1H, PhCH), 4.91 (d, J=11.9 Hz, 1H, PhCH), 4.87 (d, J=10.9 Hz, 1H, PhCH),
4.76 (d, J=11.4 Hz, 1H, PhCH), 4.71 (d, J=10.6 Hz, 1H, PhCH), 4.69 (d, J=10.9 Hz, 1H, PhCH), 4.63
17

ACCEPTED MANUSCRIPT
(d, J=11.3 Hz, 1H, PhCH), 4.62 (d, J=12.1 Hz, 1H, PhCH), 4.59 (d, J=12.1 Hz, 1H, PhCH), 4.45 (d,
J=7.8 Hz, 1H, H-1), 4.44 (d, J=7.7 Hz, 1H, H-1’), 4.42 (d, J=11.4 Hz, 1H, PhCH), 4.41 (d, J=12.1 Hz,
1H, PhCH), 4.20 (d, J=12.0 Hz, 1H, PhCH), 3.99 (t, J=9.2 Hz, 1H, H-4), 3.77 (dd, J=4.0, 11.0 Hz, 1H,
H-6a), 3.71 (dd, J=1.2, 10.7 Hz, H-6b), 3.60 (dd, J=3.4, 9.6 Hz, 1H, H-3’), 3.53 (t, J=9.1 Hz, 1H, H-3),
3.48 (t, J=6.6 Hz, 1H, H-5’), 3.45 (dd, J=7.9, 8.9 Hz, 1H, H-2), 3.36 (dd, J=7.9, 9.4 Hz, 1H, H-2’), 3.33
(ddd, J=1.6, 3.8, 9.5 Hz, 1H, H-5), 3.30 (d, J=6.7 Hz, 2H, H-6’a, H-6’b), 2.20 (br s, 1H, OH), 1.99 (s,
13
3H, COCH3), 1.53 (s, H2O); C (100 MHz, CDCl3) δ 171.1 (COCH3), 139.2 (aromatic), 138.7 (aro-
matic), 128.4 (aromatic), 138.3 (aromatic), 138.1 (aromatic), 137.6 (aromatic), 128.6 (aromatic), 128.5
(aromatic), 128.5 (aromatic), 128.4 (aromatic), 128.2 (aromatic), 128.1 (aromatic), 128.0 (aromatic),
128.0 (aromatic), 127.9 (aromatic), 127.8 (aromatic), 127.8 (aromatic), 127.8 (aromatic), 127.7 (aro-
matic), 127.5 (aromatic), 102.7 (C-1), 102.5 (C-1’), 82.9 (C-3), 81.9 (C-2), 80.3 (C-2’), 76.5 (C-4), 75.4
(PhCH2), 75.2 (PhCH2), 75.2 (C-5), 73.6 (PhCH2), 73.4 (PhCH2), 72.6 (C-3’), 72.1 (C-5’), 71.1
(PhCH2), 69.7 (C-4’), 68.3 (C-6), 67.4 (C-6’), 20.9 (COCH3). LRMS calcd for C56H60O12 [M+NH4]+
942.44, found 942.56.
3.2.11. (2R,3R,4S,5S,6R)-2-(((2R,3S,4R,5R,6S)-3-acetoxy-6-(((2R,3S,4S,5R,6S)-3-acetoxy-5-
(benzyloxy)-2-((benzyloxy)methyl)-6-(((2R,3R,4S,5R,6R)-4,5,6-tris(benzyloxy)-2-
((benzyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)tetrahydro-2H-pyran-4-yl)oxy)-2-(acetoxymethyl)-5-
(((2,2,2-trichloroethoxy)carbonyl)amino)tetrahydro-2H-pyran-4-yl)oxy)-6-(acetoxymethyl)tetrahydro-
2H-pyran-3,4,5-triyl triacetate (17)
Donor 14 (1.1 eq, 0.55 g, 0.60 mmol) and acceptor 16 (1.0 eq, 0.50 g, 0.54 mmol) were coevap-
orated with benzene (2 x 6 mL) and placed in a vacuum desiccator containing P2O5 overnight. The do-
nor/acceptor mixture was dissolved in CH2Cl2 (18 mL) and the resulting solution was cannulated into a
reaction flask containing 4Å powdered molecular sieves (1.1 g). The mixture was stirred under argon 1
H then cooled to -10oC and TfOH (cat.) was added. The reaction was stirred 12 minutes then quenched
with Et3N. The reaction was diluted with CH2Cl2, filtered through celite, dried (MgSO4), filtered, and
18

ACCEPTED MANUSCRIPT
concentrated in vacuo. The crude residue was purified via flash column chromatography (1:1 hex-
anes/ethyl acetate) to yield tetrasaccharide 17 (0.85 g, 0.51 mol, 94%) as a white foam: Rf 0.35 (1:1
hexanes/ethyl acetate); 1H (400 MHz, CDCl3) δ 7.48-7.44 (m, 2H, aromatic), 7.38-7.25 (m, 25H, aro-
matic), 7.23-7.16 (m, 3H, aromatic), 5.42 (d, J=3.6 Hz, 1H, H-4’), 5.33 (d, J=3.0 Hz, H-4’’’), 5.01-4.82
(m, 7H, H-3’’’, H-2’’’, Troc CH, PhCH, PhCH, PhCH, PhCH), 4.77-4.69 (m, 5H, PhCH, PhCH, PhCH,
H-1’’, H-4’’), 4.64 (d, J=12.3 Hz, 1H, PhCH), 4.61 (d, J=13.2 Hz, 1H, PhCH), 4.49-4.42 (m, 4H,
PhCH, PhCH, H-1’, H-1), 4.32 (d, J=12.1 Hz, 1H, Troc CH), 4.28 (d, J=11.8 Hz, PhCH), 4.23-4.14 (m,
4H, H-6’’’a, H-1’’’, H-6’’a, H-6’’b), 4.10-4.03 (m, 3H, H-6’’’b, H-4, NH), 3.81-3.77 (m, 2H, H-5’’’, H-
6a), 3.71-3.60 (m, 3H, H-5’’, H-3’, H-6b), 3.58-3.52 (m, 3H, H-5’, H-2’, H-3), 3.49-3.42 (m, 3H, H-3’’,
H-2’’, H-2), 3.36-3.31 (m, 3H, H-6a, H-6b, H-5), 2.13 (s, 3H, COCH3), 2.12 (s, 3H, COCH3), 2.07 (s,
3H, COCH3), 2.02 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 1.96 (s, 3H, COCH3), 1.95 (s, 3H, COCH3),
1.64 (s, H2O), 1.26 (m, grease); 13C (100 MHz, CDCl3) δ 171.0 (COCH3), 170.5 (COCH3), 170.3
(COCH3), 170.3 (COCH3), 169.8 (COCH3), 169.3 (COCH3), 169.3 (COCH3), 153.9 (Troc CO), 139.3
(aromatic), 139.1 (aromatic), 138.7 (aromatic), 138.2 (aromatic), 138.1 (aromatic), 137.6 (aromatic),
128.9 (aromatic), 128.6 (aromatic), 128.5 (aromatic), 128.4 (aromatic), 128.3 (aromatic), 128.2 (aro-
matic), 128.2 (aromatic), 128.1 (aromatic), 128.0 (aromatic), 128.0 (aromatic), 127.9 (aromatic), 127.9
(aromatic), 127.8 (aromatic), 127.7 (aromatic), 127.5 (aromatic), 126.9 (aromatic), 102.6 (C-1), 102.0
(C-1’), 100.9 (C-1’’’), 100.7 (C-1’’), 95.6 (Troc CCl3), 82.8 (C-3), 81.9 (C-2’), 81.7 (C-2), 77.2 (C-3’’),
76.7 (C-3’), 76.0 (C-4), 75.5 (PhCH2), 75.2 (PhCH2), 75.1 (C-5), 74.6 (PhCH2), 74.3 (Troc CH2), 73.7
(PhCH2), 73.7 (PhCH2), 72.8 (C-5’), 71.7 (C-5’’), 71.0 (PhCH2), 70.9 (C-3’’’), 70.5 (C-5’’’), 69.4 (C-
4’), 69.4 (C-4’’), 68.8 (C-2’’’), 68.1 (C-6), 68.0 (C-6’), 66.9 (C-4’’’), 62.5 (C-6’’), 61.1 (C-6’’’), 57.5
(C-2’’), 21.0 (COCH3), 21.0 (COCH3), 20.8 (COCH3), 20.8 (COCH3), 20.8 (COCH3), 20.7 (COCH3),
20.7 (COCH3). HRMS (ESI) calcd for C83H94Cl3NO29 [M+Na]+ 1696.4875, found 1696.4839.
3.2.12. N-((2S,3R,4R,5S,6R)-2-(((2R,3S,4S,5R,6S)-3,5-dihydroxy-2-(hydroxymethyl)-6-
(((2R,3S,4S,5R,6R)-4,5,6-trihydroxy-2-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)oxy)tetrahydro-2H-
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pyran-4-yl)oxy)-5-hydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3-yl)acetamide (1)
To a solution of 17 (1.0 eq, 0.10 g, 0.06 mmol) in THF (1.5 mL) was added acetic acid (1.0 mL)
and acetic anhydride (0.5 mL) followed by 10% Zn/Pb couple solid (0.24 g).50,51 The resulting mixture
was stirred 6 H and 15 min then was diluted with CH2Cl2 (20 mL), filtered through celite, washed with
saturated NaHCO3 solution (3 x 7 mL), brine (1 x 7 mL), dried (MgSO4), filtered, concentrated in vac-
uo, and coevaprotated with toluene to yield a white foam. The crude acetamide was suspended in MeOH
(2 mL) and a concentrated NaOMe solution was added. The reaction was stirred 2 H then neutralized
with Dowex 50Wx8, filtered, and concentrated in vacuo to yield a white solid. The crude heptanol was
suspended in MeOH (2 mL) and H2O (1 mL) and Pd(OH)2 was added (2.0 eq, 0.083 g, 0.119 mmol).
The reaction was stirred under H2 for 3 days then was diluted with H2O and MeOH, filtered through
celite, concentrated in vacuo, coevaporated with toluene, and lyophilized to yield lacto-N-tetraose (1)
1
13
(0.027 g, 0.038 mmol, 64% over 3 steps) as a white solid: [α]20D +13.5 (c 0.26, DMSO); H and C
spectroscopy data were in accordance with literature data.52 HRMS (TOF) calcd for C26H45NO21
[M+Na]+ 730.2382, found 730.3188.
Acknowledgements
The authors would like to acknowledge Vanderbilt University and the Institute of Chemical Bi-
ology for financial support. KMC acknowledges support from the Vanderbilt Chemical Biology Inter-
face (CBI) training program (T32 GM065086) and the Vanderbilt Pre3 (Preventing adverse Pregnancy
outcomes & Prematurity) Initiative for a travel grant. Dr. Michelle Reyzer and Prof. Richard Caprioli
are acknowledged for conducting HRMS analysis of human milk oligosaccharide samples. James C.
Poland and Prof. John McLean are acknowledged for conducting compound HRMS. Jade A. Bing is
acknowledged for her meaningful contributions.
Supplementary Information
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Experimental procedures and characterization data (including spectra for new compounds). This
material is available free of charge via the internet at http://pubs.acs.org
Corresponding Author
* E-mail: steven.d.townsend@vanderbilt.edu
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Highlights
-
-
-
MALDI FT ICR analysis of human milk
Gram Scale synthesis of protected Lacto-N-Tetraose (LNT)
Interesting trapping of acyloxonium ion, resulting from participation of a C2
acetate during a glycosylation event