# A concise synthesis of putative carrageenan precursor substrates to examine extracellular matrix biosynthesis in red algae

Fayliesha Spyker, Elizabeth Ficko-Blean, Keith Stubbs

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Fayliesha Spyker, Elizabeth Ficko-Blean, Keith Stubbs. A concise synthesis of putative carrageenan precursor substrates to examine extracellular matrix biosynthesis in red algae. Carbohydrate Research, 2025, 558, pp.109687. 10.1016/j.carres.2025.109687. hal-05338982

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Submitted on 30 Oct 2025

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 A concise synthesis of putative carrageenan precursor substrates to examine
2 extracellular matrix biosynthesis in red algae

3 Fayliesha Spyker,\*a Elizabeth Ficko-Blean,\b Keith A. Stubbs\*a,c

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aSchool of Molecular Sciences, The University of Western Australia, 35 Stirling Hwy, Crawley, 6009, Australia

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bCNRS, Sorbonne Université, UMR8227, Laboratory of Integrative Biology of Marine Models, Station Biologique de Roscoff, Roscoff, F-29680, France

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Email: fayliesha.spyker@research.uwa.edu.au, keith stubbs@uwa.edu.au
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**Abstract**

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**Keywords**

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Glycosylation, carrageenan, trichloroacetimidate, biosynthesis, extracellular matrix, galactan.

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**Highlights**

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* Synthetic route to provide putative substrates for examination of carrageenan biosynthesis

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* Expedient method to form alternating α- and β-glycosidic linkages with D-galactose

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* Amenable procedure for preparing substrates with varying saccharide lengths

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Graphical Abstract

<figure><img src="image_2.png" /></figure>

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## 1. Introduction

The Rhodophyta are an ancient eukaryotic photosynthetic phylum near the base of the Archaeplastida lineage. Red macroalgae, also called red seaweed, are multicellular organisms within the Rhodophyta. Red macroalgae have extracellular matrix (ECM) compositions that differ importantly from their Archaeplastida plant and green algal relatives [1]. Some red macroalgae have sulfated galactans (e.g. carrageenans and agars) [1] in their ECMs and a structural model for the red macroalgal ECM has been proposed [2].

Carrageenans are linear D-galactans with alternating α-1,3- and β-1,4-glycosidic linkages. The backbone may also be decorated with sulfonato, methyl, or pyruvyl groups. A unique modification, found only in red algae, is the formation of the 3,6-anhydro-bridge, resulting in the bicyclic sugar 3,6-anhydro-D-galactose. Carrageenans are hybrid polymers that contain various carrageenan motifs, including differing regiospecific sulfations and the presence or absence of the 3,6-anhydro-bridge (Figure 1A) [3, 4]. The 3,6-anhydro-bridge is the major driver of the gelling properties of carrageenans [5]. These properties are thought to be important to the algae during harsh environmental conditions, such as waves and tides, providing flexibility and protecting against osmotic shock and desiccation [6]. Carrageenan complexity also protects against microbial degradation, though some bacteria have developed highly specific enzymatic systems to degrade the polymer [7-11]. Research into carrageenan degradation has resulted in defined molecular tools for the characterisation of enzyme activity [12], specificity [13], and mechanism [8].

Due to their natural gelling properties, carrageenans have become industrially relevant hydrocolloids, with applications as gelling, thickening, and stability agents in the pharmaceutical, cosmetic, and food industries [5]. Carrageenans and their derivatives have also demonstrated bioactivities, as shown by *in vitro* anti-tumour and antiviral studies [14-16], and antihyperlipidemic [17] and antiviral [18] studies *in vitro*.
68 *vivo*. Some carrageenan derivatives have shown direct binding to human antibodies and galectins [19]
69 and may also act as biostimulants in agriculture [20].

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71 Despite the carrageenan structures from red algae being relatively well-characterised, little is known
72 about the enzymes involved in their biosynthesis or remodelling. A hypothesised enzymatic pathway
73 describing carrageenan biosynthesis was published in 1979 [21] and later updated [3, 19]. To date, the
74 only biosynthetic activity that has been biochemically elucidated is the formation of the 3,6-anhydro-
75 bridgeto give the 3,6-anhydro-D-galactose residue [22, 23]. The publication of the genome of the
76 carrageenophyte red alga *Chondrus crispus* [24] and transcriptomic analyses [25] have allowed
77 identification of further enzyme targets that may be involved in carrageenan biosynthesis and
78 modification.
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80 Initial investigations into carrageenan biosynthesis suggest three main enzyme types: D-
81 galactosyltransferases, D-sulfotransferases and D-galactose-6-sulfurylases [3, 19, 21, 22, 24, 25]. The
82 preferred hypothetical pathway suggests sequential α-1,3- and β-1,4-D-galactosyltransferase activities
83 using UDP-D-Gal as the activated substrate to form the carrageenan backbone, followed by 6-O-
84 sulfurylation performed by a highly processive sulfotransferase. This 6-O-sulfated polysaccharide may
85 then undergo further sulfurylation depending on the carrageenan motif. The D-galactose-6-sulfurylases
86 catalyse the removal of the 6-O-sulfate to form the 3,6-anhydro bridge in the last step of carrageenan
87 biosynthesis; the only step in carrageenan biosynthesis to be biochemically characterised [22, 23]. Since
88 the galactosyltransferases and sulfotransferases require precursor substrates that are not available
89 commercially, the development of novel chemical tools to further interrogate this pathway would prove
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92 Carrageenan oligosaccharides have commonly been obtained from natural starting materials via partial
93 hydrolysis procedures [26-28]. These methods, however, are tedious and low-yielding, often with
difficult separation and isolation of products. We identified two studies into the chemical synthesis of carrageenan oligosaccharides that have been undertaken [29, 30]. Of note, is an effort focused on the incorporation of sulfonato moieties on a tetrasaccharide structure [30]. Further, carrageenan-like disaccharide motifs have been previously synthesised [31-33]. A chemical synthesis approach was used to generate undecorated putative carrageenan precursor substrates of varying lengths and with alternating glycosidic linkages for carrageenan biosynthesis investigations. These carrageenan-based oligosaccharides are α- and β-methyl glycosides containing two (1 and 2, respectively), three (3 and 4, respectively), and four (5 and 6, respectively), D-galactose subunits (Figure 1B) with alternating linkages varying with the D-galactose in the α- or β-configuration on the non-reducing end.

<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_4.png" /></figure>

**Figure 1.** a) Examples of some common carrageenans: iota (ι-), kappa (κ-), and lambda (λ-) carrageenan; b) Putative precursor substrates for the investigation of the carrageenan biosynthetic pathways.
## 2. Results and discussion

A key design consideration in the synthesis of the target compounds **1-6** was the formation of the α-1,3- and β-1,4-glycosidic linkages between the D-galactose monomers. In the first instance, the disaccharides **1** and **2** were targeted to establish the efficiency of the glycosylation methods. Previously, **1** has been prepared as a mixture [34] and fully reported [33], and **2** prepared via an enzymatic method [35]. Employing the trichloroacetimidate **7** [36] with a participating group at the 2-O-position for the formation of the β-linkages, and the known donor **8** (Kiso's donor) [37] for the formation of the α-linkages, with the appropriate acceptors **9** [38] and **10** [39], respectively, gave the disaccharides **11** and **12** [13] in excellent yield (Scheme 1), both utilising trifluoromethanesulfonate-catalysed processes. To prepare **1**, deacetylation of **11** gave tetrol **13**, and a final deprotection step gave the desired disaccharide **1**. For **2**, the tribenzoate **12** was treated with hydrogen fluoride in pyridine to allow for removal of the silyl group to give the known diol **14** [13]. The synthesis of **12** and **14** are described in detail in [13]. Subsequent removal of the benzylidene moiety afforded the novel tetrol **15** in excellent yield. A final deprotection step, using sodium methoxide in methanol, gave the desired disaccharide **2**.

<figure><img src="image_5.png" /><figcaption>**Scheme 1.** a) TMSOTf, 4 Å MS, CH₂Cl₂, -20 °C to 0 °C; b) NaOMe, MeOH, rt; c) H₂, Pd/C, MeOH, 35 °C; d) TMSOTf, 4 Å MS, CH₂Cl₂, 0 °C; e) 70% w/w HF-pyridine, THF, rt; f) AcOH:H₂O (4:1), 80 °C; g) NaOMe, MeOH, 25 °C.</figcaption></figure>
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With the two putative disaccharide substrates in hand, attention could be turned to the synthesis of **3**, with the formation of the α-1,3-linkage addressed first. To that end, glycosylation of donor **8** [37] with the acceptor **16** [40] gave the disaccharide **17** (Scheme 2). Treatment of **17** with hydrogen fluoride in pyridine gave the diol **18**, and removal of the benzylidene moiety, akin to the preparation of **15**, afforded the novel tetrol **19**. A subsequent deprotection step, using sodium methoxide in methanol, gave **20**, which was then acetylated to give the heptaacetate **21**. Subsequent removal of the benzyl glycoside afforded the hemiacetals **22**, which previously have been prepared via a different method [41]. With **22** in hand, the trichloroacetimidate **23** could be synthesised using trichloroacetonitrile under basic conditions.

<figure><img src="image_6.png" /></figure>

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**Scheme 2.** a) TMSOTf, 4 Å MS, CH₂Cl₂, 0 °C; b) 70% w/w HF-pyridine, THF, rt; c) AcOH:H₂O (4:1), 80 °C; d) NaOMe, MeOH, 28 °C; e) Ac₂O, pyridine, rt; f) H₂, Pd/C, MeOH, rt; g) Cl₃CCN, DBU, CH₂Cl₂, 0 °C.

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Glycosylation of donor **23** with acceptor **9** [38] gave the trisaccharide **24**, with the desired β-1,4-linkage successfully formed (Scheme 3). Deprotection steps then ensued: first deacetylation to give **25**, and subsequent debenzylation to afford **3**. Interestingly, the reaction to remove the benzyl protecting groups seemed to stall, despite further additions of catalyst. It was decided to purify the reaction mixture, which gave a modest yield of **3** (50%). In an effort to increase the yield, the residues from the chromatography
148 were again put under hydrogenolysis conditions and pleasingly, after purification, more of 3 was
149 isolated, increasing the yield to 86% overall.

<figure><img src="image_7.png" /></figure>

150 **Scheme 3.** a) TMSOTf, 4 Å MS, CH₂Cl₂, -20 °C to 0 °C; b) NaOMe, MeOH, 35 °C; c) H₂, Pd/C, 151 MeOH, 40 °C.

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With the donors **7** and **23** in hand, attention could be directed toward the synthesis of the desired molecules **4** and **6**. To this end, disaccharide **12** could conveniently be transformed into the benzylated glycosyl acceptor **26** over seven steps (Scheme 4). Initially, removal of the benzylidene moiety gave the diol **27**, which was then acetylated to give **28**. Treatment of **28** with hydrogen fluoride in pyridine allowed for removal of the silyl group to give the diol **29**. The diol **29** was then protected with a benzylidene group, affording **30**, in preparation for a future ring-opening step. In order to increase the reactivity of the 4'-*O*- position for the upcoming glycosylation, **30** was deprotected using sodium methoxide in methanol to give **31** and subsequently benzylated, affording **32**. A final acid-mediated reductive ring-opening of the benzylidene gave the desired glycosyl acceptor **26**.
<figure><img src="image_8.png" /></figure>

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<figure><img src="image_9.png" /><figcaption>a 82%</figcaption></figure>

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<figure><img src="image_10.png" /><figcaption>b 94%</figcaption></figure>

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<figure><img src="image_11.png" /><figcaption>c 99%</figcaption></figure>

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<figure><img src="image_12.png" /></figure>

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<figure><img src="image_13.png" /><figcaption>e 94%</figcaption></figure>

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<figure><img src="image_14.png" /><figcaption>f 94%</figcaption></figure>

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<figure><img src="image_15.png" /><figcaption>g 82%</figcaption></figure>

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**Scheme 4.** a) AcOH:H₂O (4:1), 80 °C; b) Ac₂O, pyridine, rt; c) 70% w/w HF-pyridine, THF, rt; d) PhCH(OMe)₂, CSA, MeCN, rt; e) NaOMe, MeOH, 40 °C; f) BnBr, NaH, DMF, rt; g) NaCNBH₃, HCl/Et₂O, 4 Å MS, THF, rt.

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Having successfully synthesised acceptor **26**, glycosylation with donor **7** [36] was attempted and this successfully formed trisaccharide **33** (Scheme 5). Deprotection steps then ensued: first deacetylation to give **34**, and subsequent debenzylation to afford **4**. Similar to what was observed in the synthesis of **3**, multiple reactions were needed to maximise the yield of **4**, which resulted in an overall yield of 84%. For **6**, donor **23** and acceptor **26** could be utilised and, upon glycosylation, they gave the desired tetrasaccharide **35** (Scheme 5). All that remained then were the final deprotection steps: deacetylation of **35** gave **36** in good yield and, finally, debenzylation of **35** afforded **6**.

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<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

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<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

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<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

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<figure><img src="image_22.png" /></figure>

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<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

35

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

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<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

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**Scheme 5.** a) 7, TMSOTf, 4 Å MS, CH₂Cl₂, -20 °C to 0 °C; b) 23, TMSOTf, 4 Å MS, CH₂Cl₂, -20 °C to 0 °C; c) NaOMe, MeOH, 30 °C; d) H₂, Pd/C, MeOH, 40 °C.

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With putative substrates **1-4** and **6** in hand, attention was now turned to the synthesis of tetrasaccharide **5**. For the synthesis of **5**, a stepwise approach was taken, starting from the reducing end of the oligosaccharide (Scheme 6). Glycosylation of the known donor **37** [42] and acceptor **9** [38] gave the disaccharide **38** with the desired β-1,4-linkage. The chloroacetyl moiety at the 3'-*O*-position of **38** allowed for selective deprotection, affording **39**, in preparation for the next glycosylation reaction.
<figure><img src="image_29.png" /></figure>

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<figure><img src="image_30.png" /></figure>

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<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

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<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

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<figure><img src="image_35.png" /></figure>

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<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

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<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

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<figure><img src="image_40.png" /></figure>

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<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

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<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

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**Scheme 6.** a) TMSOTf, 4 Å MS, CH₂Cl₂, -20 °C to 0 °C; b) (NH₂)₂CS, NaHCO₃, Bu₄NI, THF, 55 °C; c) **8**, TMSOTf, 4 Å MS, CH₂Cl₂, -20 °C to 0 °C; d) 70% w/w HF-pyridine, THF, rt; e) PhCH(OMe)₂, CSA, MeCN, rt; f) NaOMe, MeOH, 40 °C; g) BnBr, NaH, DMF, rt; h) NaCNBH₃, HCl/Et₂O, 4 Å MS, THF, rt.

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The next glycosidic linkage was formed using Kiso's donor **8** [37] together with **39** (Scheme 6), which pleasingly gave **40** in good yield. Removal of the silyl group afforded the diol **41** and subsequent protection with a benzylidene moiety gave **42**. Again, as for **30**, the benzoyl and acetyl groups were removed from **42** to give **43**, which was subsequently benzylated, affording **44**. A final acid-catalysed reductive ring-opening of the benzylidene gave the desired trisaccharide glycosyl acceptor **45**.

The final glycosylation required for the synthesis of **5** involved donor **7** [36] and acceptor **45**, with this reaction yielding tetrasaccharide **46** (Scheme 7). Following on from this, deacetylation gave **47** and a final debenzylation process afforded **5** in an overall yield of 77%.
<figure><img src="image_45.png" /></figure>

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<figure><img src="image_46.png" /></figure>

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<figure><img src="image_47.png" /></figure>

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<figure><img src="image_48.png" /></figure>

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<caption>**Scheme 7.** a) 7, TMSOTf, 4 Å MS, CH<sub>2</sub>Cl<sub>2</sub>, -20 °C to 0 °C; b) NaOMe, MeOH, rt; c) H<sub>2</sub>, Pd/C, MeOH, 40 °C.</caption>

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**3. Conclusions**

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The undecorated carrageenan motifs **1-6** have been successfully synthesised using an expedient and mild route to provide a rational panel of D-galactose-based substrates with alternating α-1,3 and β-1,4-linkages and either an α-1,3 or β-1,4 linked D-galactosyl unit on the non-reducing end. These small molecule substrates, that mimic putative natural carrageenan backbone precursors [3, 19, 21]. They have the potential to act as artificial substrates to provide insight into the biosynthesis of carrageenans by assessing the enzymatic action of red algal sulfotransferases and galactosyltransferases. They may also be of interest for studies on enzymes involved in their degradation. Overall, this work provides a stepwise chemical assembly of carrageenan backbone precursors with alternating glycosidic linkages. The procedures are modular and streamlined and are amenable for preparing a wide range of substrates with varying saccharide lengths and aglycons.
217 **4. Experimental**

218 **4.1. General methods**

219 ¹H and ¹³C (¹H decoupled) nuclear magnetic resonance (NMR) spectra were obtained at 298 K on
220 Bruker AV400 (400 MHz for ¹H and 100.6 MHz for ¹³C), Bruker AV500 (500 MHz for ¹H and
221 125.8 MHz for ¹³C) or Bruker AV600 (600 MHz for ¹H and 150.9 MHz for ¹³C) spectrometers. Solvents
222 used for NMR were: deuteriochloroform (CDCl₃) with CHCl₃ (¹H, δ 7.26) or CDCl₃ (¹³C, δ 77.16) used
223 as internal standards, tetradeuteriomethanol (CD₃OD) with CD₂HOD (¹H, δ 3.31) or CD₃OD (¹³C,
224 δ 49.00) used as internal standards, and deuterium oxide (D₂O) with DHO (¹H, δ 4.79) or CH₃OH (¹³C,
225 δ 49.50) used as internal standards. High resolution mass spectra were recorded with a Thermo Orbitrap
226 Exploris 120 LCMS spectrometer, using ESI method in positive mode, with samples run by staff at the
227 mass spectrometry facility. Infrared spectra were recorded with a Perkin-Elmer Spectrum One or Two
228 FT-IR spectrometer fitted with a PerkinElmer Universal ATR sampling accessory. Flash
229 chromatography was performed on Davisil® silica gel with the specified solvents. Thin layer
230 chromatography was performed on Merck silica gel 60 F254 aluminium-backed plates that were stained
231 by heating (>200 °C) with 5% v/v H₂SO₄ in EtOH. All solvents except toluene, CH₂Cl₂, THF, DMF and
232 MeCN were distilled prior to use. Dimethylformamide and MeCN were dried over 4 Å molecular sieves
233 before use. Toluene, CH₂Cl₂, and THF and were obtained from a PureSolv™ MD 5 Solvent Purification
234 System.
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236 **4.2 Synthesis**

237 **4.2.1 Methyl 4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-benzyl-α-D-galactopyranoside 11**

239 To a solution of the trichloroacetimidate 7 (386 mg, 0.783 mmol) and the alcohol 9 (303 mg, 0.652 mmol) in CH₂Cl₂ (7 mL) was added molecular sieves 4 Å (0.9 g) and the mixture was stirred at room temperature for 30 min. To the mixture was then added TMSOTf (17 µL, 0.094 mmol) at -20 °C
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and the mixture was stirred and allowed to return to 0 °C over 1 hr. The reaction was then quenched with NEt₃, and the mixture filtered through Celite. The filtrate was washed with water (20 mL), brine (20 mL), dried (MgSO₄), filtered, and concentrated. The resultant residue was purified by flash chromatography (EtOAc:hexanes, 3:7) to give the disaccharide **11** as a white foam (448 mg, 86%). R<sub>f</sub> 0.37 (EtOAc:hexanes, 2:3); <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>): δ 7.39-7.23 (m, 15H), 5.35 (dd, *J* = 3.5, 1.0 Hz, 1H), 5.17 (dd, *J* = 10.5, 7.8 Hz, 1H), 4.99 (dd, *J* = 10.5, 3.5 Hz, 1H), 4.81 (d, *J* = 11.7 Hz, 1H), 4.77 (d, *J* = 11.9 Hz, 1H), 4.69 (d, *J* = 7.8 Hz, 1H), 4.66 (d, *J* = 11.7 Hz, 1H), 4.62 (d, *J* = 3.5 Hz, 1H), 4.59 (d, *J* = 11.9 Hz, 1H), 4.57 (d, *J* = 12.0 Hz, 1H), 4.51 (d, *J* = 12.0 Hz, 1H), 4.08 (dd, *J* = 11.1, 7.4 Hz, 1H), 4.01-3.95 (m, 2H), 3.92-3.83 (m, 2H), 3.81-3.74 (m, 2H), 3.69 (dd, *J* = 10.4, 5.0 Hz, 1H), 3.61 (dd, *J* = 10.4, 6.8 Hz, 1H), 3.36 (s, 3H), 2.13 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H), 1.80 (s, 3H); <sup>13</sup>C NMR (100.6 MHz, CDCl<sub>3</sub>): δ 170.5, 170.4, 170.3, 169.9, 138.7, 138.6, 138.5, 128.60, 128.57, 128.5, 128.1, 127.9, 127.8, 127.7, 127.6, 102.3, 98.6, 78.3, 76.9, 73.9, 73.8, 73.5, 71.0, 70.4, 70.1, 69.3, 69.2, 67.0, 61.3, 55.5, 20.9, 20.80, 20.78, 20.76; FT-IR (ATR): ν 1745 (s) cm<sup>-1</sup>; HR-MS (ESI) *m/z* [M+Na]<sup>+</sup> calculated for C<sub>42</sub>H<sub>50</sub>NaO<sub>15</sub><sup>+</sup>: 817.3042, found: 817.3033.

256

4.2.2 Methyl 2,3,6-tri-O-benzyl-4-O-(β-D-galactopyranosyl)-α-D-galactopyranoside **13**

To a solution of the disaccharide **11** (434 mg, 0.546 mmol) in MeOH (10 mL) was added 1 M NaOMe in MeOH (0.2 mL, 0.2 mmol) at 0 °C and the mixture was stirred at room temperature for 2 h. The reaction mixture was then neutralised with Amberlite resin (IR-120, H⁺ form) and the mixture was filtered and concentrated. The resultant residue was purified by flash chromatography (MeOH:CHCl₃, 1:19) to give the tetrol **13** as a white foam (327 mg, 96%). R<sub>f</sub> 0.47 (MeOH:CHCl₃, 1:9); <sup>1</sup>H NMR (400 MHz, CD₃OD): δ 7.44-7.23 (m, 15H), 4.79-4.74 (m, 3H), 4.72 (d, *J* = 3.7 Hz, 1H), 4.67 (d, *J* = 11.7 Hz, 1H), 4.59-4.55 (m, 2H), 4.53 (d, *J* = 11.9 Hz, 1H), 4.30 (d, *J* = 3.0 Hz, 1H), 4.06 (dd, *J* = 10.2, 3.7 Hz, 1H), 3.93-3.87 (m, 2H), 3.82-3.76 (m, 2H), 3.72-3.68 (m, 2H), 3.62 (dd, *J* = 10.6, 7.0 Hz, 1H), 3.52 (dd, *J* = 9.7, 7.6 Hz, 1H), 3.43-3.38 (m, 2H), 3.36 (s, 3H); <sup>13</sup>C NMR (100.6 MHz, CD₃OD): δ 139.9, 139.8, 139.6, 129.5, 129.40, 129.37, 129.2, 129.1, 128.83, 128.82, 128.78, 128.6, 105.4, 99.9,
268 79.6, 77.7, 76.8, 76.0, 75.0, 74.5, 74.2, 74.1, 73.4, 71.2, 70.6, 70.1, 62.4, 55.6; FT-IR (ATR): v 3414

269 (br) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₃₄H₄₂NaO₁₁⁺: 649.2619, found: 649.2616.

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271 **4.2.3 Methyl 4-O-(β-D-galactopyranosyl)-α-D-galactopyranoside 1** [33]

272 To a solution of the tetrol **13** (315 mg, 0.503 mmol) in MeOH (10 mL) was added 10% w/w Pd/C

273 (22 mg) and the mixture was stirred at 35 °C under an atmosphere of H₂. Further additions of 10% w/w

274 Pd/C were made: 30 mg at 24 h, 55 mg at 5 d, and 18 mg at 6 d. At 7 d the reaction mixture was filtered

275 through Celite and concentrated. The resultant residue was purified by flash chromatography

276 (CHCl₃:MeOH:H₂O, 5:4:1) to give the title compound **1** as a white foam (106 mg, 59%). The ¹H and

277 ¹³C NMR data were consistent with those found in the literature [33].

278

279 **4.2.4 Methyl 2-O-benzoyl-3-O-(2,3-di-O-benzoyl-α-D-galactopyranosyl)-β-D-galactopyranoside 15**

280 To the diol **14** (526 mg, 0.695 mmol) was added AcOH:H₂O (4:1, 25 mL) and the resultant solution was

281 stirred at 80 °C for 1.5 h. The reaction mixture was then concentrated, co-evaporating with toluene

282 (3 x 15 mL). The resultant residue was purified by flash chromatography (MeOH:CHCl₃, 2:23) to give

283 the tetrol **15** as a white foam (448 mg, 96%). R<sub>f</sub> 0.39 (MeOH:CHCl₃, 1:9); ¹H NMR (400 MHz, CDCl₃):

284 δ 8.14–8.10 (m, 2H), 7.99–7.93 (m, 4H), 7.64–7.58 (m, 1H), 7.55–7.46 (m, 4H), 7.41–7.31 (m, 4H),

285 5.70 (dd, *J* = 10.7, 3.7 Hz, 1H), 5.57 (dd, *J* = 10.7, 3.0 Hz, 1H), 5.52 (dd, *J* = 9.5, 8.0 Hz, 1H), 5.48 (d, *J*

286 = 3.7 Hz, 1H), 4.49 (d, *J* = 8.0 Hz, 1H), 4.25 (d, *J* = 3.0 Hz, 1H), 3.98 (dd, *J* = 9.5, 3.3 Hz, 1H), 3.95–

287 3.92 (m, 1H), 3.90–3.83 (m, 2H), 3.69 (dd, *J* = 11.8, 4.9 Hz, 1H), 3.57–3.52 (m, 1H), 3.47 (s, 3H), 3.46–

288 3.42 (m, 2H); ¹³C NMR (100.6 MHz, CDCl₃): δ 166.4, 165.8, 165.6, 133.9, 133.7, 133.5, 130.0, 129.91,

289 129.85, 129.8, 129.4, 128.9, 128.79, 128.78, 128.6, 102.2, 96.1, 79.0, 74.2, 70.9, 70.7, 70.1, 69.8, 69.0,

290 67.0, 62.9, 62.2, 56.9; FT-IR (ATR): v 3446 (br), 1714 (s) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated

291 for C₃₄H₃₆NaO₁₄⁺: 691.1997, found: 691.1990.

292

293 **4.2.5 Methyl 3-O-(α-D-galactopyranosyl)-β-D-galactopyranoside 2** [35]
To a solution of the tetrol **15** (434 mg, 0.649 mmol) in MeOH (20 mL) was added 1 M NaOMe in MeOH (0.5 mL, 0.5 mmol) at 0 °C and the mixture was kept at 25 °C for 27 h. The reaction mixture was then neutralised with Amberlite resin (IR-120, H⁺ form) and the mixture was filtered and concentrated.
The resultant residue was purified by flash chromatography (CHCl₃:MeOH:H₂O, 5:4:1) to give the title compound **2** as a white foam (192 mg, 83%). The ¹H and ¹³C NMR data were consistent with those found in the literature [35].

4.2.6 Benzyl 2-O-benzoyl-3-O-{2,3-di-O-benzoyl-4,6-O-[bis(tert-butyl)silylene]-α-D-galactopyranosyl}-4,6-O-benzylidene-β-D-galactopyranoside **17**

To a solution of the trichloroacetimidate **8** (4.21 g, 6.26 mmol) and the alcohol **16** (2.47 g, 5.34 mmol) in CH₂Cl₂ (125 mL) was added molecular sieves 4 Å (7.2 g) and the mixture was stirred at room temperature for 30 min. To the mixture was then added TMSOTf (100 µL, 550 µmol) at 0 °C and the mixture was stirred at that temperature for 1 h. The reaction was then quenched with NEt₃ and the mixture filtered through Celite. The filtrate was washed with water (100 mL), brine (100 mL), dried (MgSO₄), filtered, and concentrated. The resultant residue was purified by flash chromatography (EtOAc:hexanes, 3:7) to give the disaccharide **17** as a white foam (4.85 g, 93%). R_f 0.55 (EtOAc:hexanes, 2:3); ¹H NMR (400 MHz, CDCl₃): δ 8.04–7.99 (m, 2H), 7.97–7.92 (m, 2H), 7.82–7.77 (m, 2H), 7.65–7.59 (m, 1H), 7.53–7.45 (m, 3H), 7.37–7.12 (m, 13H), 7.02–6.96 (m, 2H), 5.76 (dd, *J* = 10.2, 7.9 Hz, 1H), 5.61 (d, *J* = 3.8 Hz, 1H), 5.51 (dd, *J* = 10.6, 3.8 Hz, 1H), 5.42 (dd, *J* = 10.6, 3.0 Hz, 1H), 5.09 (s, 1H), 4.86 (d, *J* = 12.7 Hz, 1H), 4.65 (d, *J* = 12.7 Hz, 1H), 4.61 (d, *J* = 7.9 Hz, 1H), 4.56 (d, *J* = 3.0 Hz, 1H), 4.30 (dd, *J* = 12.2, 1.5 Hz, 1H), 4.14 (d, *J* = 3.3 Hz, 1H), 3.98 (dd, *J* = 10.2, 3.3 Hz, 1H), 3.97 (dd, *J* = 12.2, 1.4 Hz, 1H), 3.76 (dd, *J* = 12.7, 1.4 Hz, 1H), 3.68–3.66 (m, 1H), 3.53 (dd, *J* = 12.7, 2.0 Hz, 1H), 3.40–3.38 (m, 1H), 1.02 (s, 9H), 0.83 (s, 9H); ¹³C NMR (100.6 MHz, CDCl₃): δ 166.8, 165.9, 165.2, 137.4, 137.2, 133.5, 133.3, 133.1, 130.1, 130.0, 129.9, 129.8, 129.7, 128.9, 128.7, 128.6, 128.42, 128.40, 128.38, 128.1, 128.0, 127.8, 126.2, 100.5, 99.4, 95.5, 76.9, 72.8, 70.9, 70.8, 70.1,
319 69.8, 69.2, 68.9, 67.3, 66.7, 27.5, 27.3, 23.3, 20.8; FT-IR (ATR): ν 1722 (s) cm⁻¹; HR-MS (ESI) m/z
320 [M+Na]⁺ calculated for C₅₅H₆₀O₁₄NaSi⁺: 995.3650, found: 995.3641.

321

322 4.2.7 Benzyl 2-O-benzoyl-3-O-(2,3-di-O-benzoyl-α-D-galactopyranosyl)-4,6-O-benzylidene-β-D-
323 galactopyranoside 18

324 To a solution of the disaccharide **17** (4.84 g, 4.97 mmol) in THF (110 mL) was added 70% w/w
325 hydrogen fluoride in pyridine (2.4 mL, 92 mmol) at 0 °C and the mixture was stirred at room
326 temperature for 1.5 h. The reaction was then quenched with sat. aq. NaHCO₃ at 0 °C and the resultant
327 mixture was extracted with CHCl₃ (300 mL). The organic extract was washed with brine (50 mL), dried
328 (MgSO₄), filtered, and concentrated. The resultant residue was purified by flash chromatography
329 (EtOAc:hexanes, 4:1) to give the diol **18** as a white foam (3.62 g, 87%). Rf 0.20 (EtOAc:hexanes, 7:3);
330 ¹H NMR (400 MHz, CDCl₃): δ 8.09–8.05 (m, 2H), 7.95–7.91 (m, 2H), 7.80–7.76 (m, 2H), 7.66–7.60
331 (m, 1H), 7.53–7.45 (m, 3H), 7.36–7.10 (m, 13H), 7.05–6.99 (m, 2H), 5.77 (dd, *J* = 10.1, 7.9 Hz, 1H),
332 5.63 (d, *J* = 3.4 Hz, 1H), 5.53 (dd, *J* = 10.7, 4.2 Hz, 1H), 5.49 (dd, *J* = 10.7, 3.4 Hz, 1H), 5.17 (s, 1H),
333 4.87 (d, *J* = 12.7 Hz, 1H), 4.66 (d, *J* = 12.7 Hz, 1H), 4.63 (d, *J* = 7.9 Hz, 1H), 4.31 (dd, *J* = 12.2, 1.4 Hz,
334 1H), 4.20–4.17 (m, 1H), 4.17 (d, *J* = 3.4 Hz, 1H), 3.99 (dd, *J* = 10.1, 3.4 Hz, 1H), 3.97 (dd, *J* = 12.2, 1.5
335 Hz, 1H), 3.84–3.79 (m, 1H), 3.56–3.38 (m, 3H), 2.92–2.87 (m, 1H), 1.99–1.92 (m, 1H); ¹³C NMR
336 (100.6 MHz, CDCl₃): δ 166.7, 165.5, 165.3, 137.3, 137.2, 133.5, 133.39, 133.37, 130.1, 130.0, 129.8,
337 129.7, 129.6, 128.9, 128.7, 128.6, 128.5, 128.4, 128.1, 128.0, 127.8, 126.1, 100.6, 99.4, 95.3, 76.7, 72.7,
338 70.8, 70.4, 69.8, 69.2, 69.1, 68.9, 66.6, 63.2; FT-IR (ATR): ν 3485 (br), 1713 (s) cm⁻¹; HR-MS (ESI)
339 m/z [M+Na]⁺ calculated for C₄₇H₄₄O₁₄Na⁺: 855.2629, found: 855.2654.

340

341 4.2.8 Benzyl 2-O-benzoyl-3-O-(2,3-di-O-benzoyl-α-D-galactopyranosyl)-β-D-galactopyranoside **19**

342 To the diol **18** (2.71 g, 3.25 mmol) was added AcOH:H₂O (4:1, 130 mL) and the resultant solution was
343 stirred at 80 °C for 1.5 h. The reaction mixture was then concentrated, co-evaporating with toluene
344 (3 x 20 mL). The resultant residue was purified by flash chromatography (EtOAc:hexanes, 9:1) to give
the tetrol **19** as a white foam (2.09 g, 86%). R<sub>f</sub> 0.31 (EtOAc:hexanes, 9:1); ¹H NMR (400 MHz, CDCl<sub>3</sub>): δ 8.08–8.03 (m, 2H), 7.99–7.93 (m, 4H), 7.66–7.59 (m, 1H), 7.55–7.46 (m, 4H), 7.41–7.32 (m, 4H), 7.23–7.12 (m, 5H), 5.68 (dd, *J* = 10.7, 3.8 Hz, 1H), 5.63–5.55 (m, 2H), 5.44 (d, *J* = 3.8 Hz, 1H), 4.83 (d, *J* = 12.7 Hz, 1H), 4.65 (d, *J* = 12.7 Hz, 1H), 4.56 (d, *J* = 8.0 Hz, 1H), 4.26 (d, *J* = 3.0 Hz, 1H), 3.94–3.81 (m, 4H), 3.68 (dd, *J* = 11.8, 4.8 Hz, 1H), 3.51–3.46 (m, 1H), 3.44–3.40 (m, 2H); <sup>13</sup>C NMR (100.6 MHz, CDCl<sub>3</sub>): δ 166.3, 165.8, 165.5, 137.1, 133.9, 133.7, 133.5, 130.0, 129.9, 129.84, 129.79, 129.4, 128.9, 128.8, 128.7, 128.6, 128.5, 128.0, 127.9, 99.7, 96.2, 79.1, 74.3, 70.9, 70.7, 70.5, 69.9, 69.8, 69.0, 67.0, 63.0, 62.3; FT-IR (ATR): ν 3442 (br), 1715 (s) cm<sup>-1</sup>; HR-MS (ESI) *m/z* [M+Na]<sup>+</sup> calculated for C<sub>40</sub>H<sub>40</sub>NaO<sub>14</sub><sup>+</sup>: 767.2310, found: 767.2308.

354

### 4.2.9 Benzyl 3-O-(α-D-galactopyranosyl)-β-D-galactopyranoside 20

To a solution of the tetrol **19** (2.07 g, 2.78 mmol) in MeOH (50 mL) was added 1 M NaOMe in MeOH (0.8 mL, 0.8 mmol) at 0 °C and the mixture was kept at 28 °C for 3 d. The reaction mixture was then neutralised with Amberlite resin (IR-120, H<sup>+</sup> form) at room temperature, and the mixture was filtered and concentrated. The resultant residue was purified by flash chromatography (CHCl<sub>3</sub>:MeOH:H<sub>2</sub>O, 12:7:1) to give the polyol **20** as a white foam (1.12 g, 93%). R<sub>f</sub> 0.60 (CHCl<sub>3</sub>:MeOH:H<sub>2</sub>O, 5:4:1); ¹H NMR (400 MHz, CD<sub>3</sub>OD): δ 7.45–7.40 (m, 2H), 7.36–7.23 (m, 3H), 5.05–5.01 (m, 1H), 4.94 (d, *J* = 11.8 Hz, 1H), 4.68 (d, *J* = 11.8 Hz, 1H), 4.37 (d, *J* = 7.4 Hz, 1H), 4.25–4.19 (m, 1H), 4.06 (d, *J* = 3.1 Hz, 1H), 3.93–3.89 (m, 1H), 3.86–3.62 (m, 8H), 3.54–3.48 (m, 1H); <sup>13</sup>C NMR (100.6 MHz, CD<sub>3</sub>OD): δ 139.1, 129.3, 129.2, 128.7, 103.8, 97.6, 80.0, 76.4, 72.2, 71.7, 71.4, 71.2, 71.0, 70.2, 66.7, 62.8, 62.5; FT-IR (ATR): ν 3289 (br) cm<sup>-1</sup>; HR-MS (ESI) *m/z* [M+Na]<sup>+</sup> calculated for C<sub>19</sub>H<sub>28</sub>NaO<sub>11</sub><sup>+</sup>: 455.1524, found: 455.1526.

367

### 4.2.10 Benzyl 2,4,6-tri-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl)-β-D-galactopyranoside 21

369
To a solution of the polyol **20** (1.10 g, 2.54 mmol) in pyridine (15 mL) was added Ac₂O (8.5 mL, 370 90 mmol) at 0 °C and the mixture was kept at room temperature for 24 h. The reaction was then 371 quenched with MeOH, and the mixture was concentrated, co-evaporating with toluene (3 x 20 mL). The 372 resultant residue was purified by flash chromatography (EtOAc:hexanes, 1:1) to give the heptaacetate **21** 373 as a white foam (1.77 g, 96%). Rf 0.30 (EtOAc:hexanes, 1:1); ¹H NMR (400 MHz, CDCl₃): δ 7.38–7.27 374 (m, 5H), 5.44 (d, *J* = 3.1 Hz, 1H), 5.36 (d, *J* = 3.2 Hz, 1H), 5.31 (dd, *J* = 10.3, 7.9 Hz, 1H), 5.29–5.22 375 (m, 2H), 5.12 (dd, *J* = 10.8, 3.2 Hz, 1H), 4.90 (d, *J* = 12.4 Hz, 1H), 4.63 (d, *J* = 12.4 Hz, 1H), 4.40 (d, *J* 376 = 7.9 Hz, 1H), 4.25–4.12 (m, 4H), 4.01–3.92 (m, 1H), 3.85 (dd, *J* = 10.3, 3.1 Hz, 1H), 3.82–3.77 (m, 377 1H), 2.16 (s, 3H), 2.12 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 1.94 (s, 3H), 1.91 (s, 3H); ¹³C 378 NMR (100.6 MHz, CDCl₃): δ 170.6, 170.32, 170.31, 170.28, 170.03, 169.98, 169.2, 136.8, 128.6, 379 128.2, 128.0, 99.9, 93.4, 73.0, 70.9, 70.6, 69.6, 68.1, 67.4, 67.1, 66.6, 65.1, 61.8, 61.7, 20.91, 20.88, 380 20.74, 20.70; FT-IR (ATR): ν 1740 (s) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₃₃H₄₂NaO₁₈⁺: 381 749.2263, found: 749.2257.

4.2.11 2,4,6-Tri-*O*-acetyl-3-*O*-((2,3,4,6-tetra-*O*-acetyl-*α*-D-galactopyranosyl)-D-galactopyranose **22** [41]

To a solution of the heptaacetate **21** (1.76 g, 2.42 mmol) in MeOH (30 mL) was added 10% w/w Pd/C (109 mg) and the mixture was stirred at room temperature for 5 h under an atmosphere of H₂. The reaction mixture was then filtered through Celite and concentrated. The resultant residue was purified by flash chromatography (EtOAc:hexanes, 3:2) to give the hemiacetals **22** as a white foam (950 mg, 62%, *α*:*β* anomeric ratio 9:1). Rf 0.41, 0.33 (EtOAc:hexanes, 7:3); ¹H NMR (400 MHz, CDCl₃) *α* anomer: δ 5.57 (dd, *J* = 3.5, 3.5 Hz, 1H), 5.47 (dd, *J* = 3.2, 1.4 Hz, 1H), 5.43 (d, *J* = 3.2 Hz, 1H), 5.33–5.27 (m, 385 2H), 5.21 (dd, *J* = 10.8, 3.2 Hz, 1H), 5.08 (dd, *J* = 10.8, 3.5 Hz, 1H), 4.47 (ddd, *J* = 7.3, 5.2, 1.2 Hz, 386 1H), 4.40–4.33 (m, 2H), 4.27 (dd, *J* = 11.4, 5.2 Hz, 1H), 4.11 (dd, *J* = 11.3, 6.5 Hz, 1H), 4.06 (dd, *J* = 387 11.3, 6.7 Hz, 1H), 4.00 (dd, *J* = 11.4, 7.3 Hz, 1H), 2.96 (dd, *J* = 3.5, 1.1 Hz, 1H), 2.154 (s, 3H), 2.148 (s, 388 3H), 2.14 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 1.96 (s, 3H); ¹³C NMR (100.6 MHz, CDCl₃) *α* 389 anomer: δ 170.8, 170.7, 170.5, 170.4, 170.24, 170.20, 170.0, 92.7, 90.7, 69.7, 68.4, 67.8, 67.5, 67.1, 390 391 392 393 394 395
396 66.7, 66.2, 62.2, 62.1, 20.92, 20.90, 20.86, 20.83, 20.77, 20.75; FT-IR (ATR): ν 3461 (br), 1740 (s) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₂₆H₃₆NaO₁₈⁺: 659.1794, found: 659.1792.

397

398

399 4.2.12 2,4,6-Tri-*O*-acetyl-3-*O*-[(2,3,4,6-tetra-*O*-acetyl-α-D-galactopyranosyl)-α-D-galactopyranosyl]trichloroacetimidate 23

401 To a solution of the hemiacetals **22** (600 mg, 0.943 mmol) in CH₂Cl₂ (10 mL) were added Cl₃CCN (1.4 mL, 14 mmol) and DBU (0.17 mL, 1.1 mmol) at 0 °C and the mixture was kept at that temperature for 2 h. The reaction mixture was then concentrated. The resultant residue was purified by flash chromatography (EtOAc:hexanes, 9:11) to give the trichloroacetimidate **23** as a white foam (664 mg, 90%). R_f 0.37 (EtOAc:hexanes, 1:1); ¹H NMR (400 MHz, CDCl₃): δ 8.66 (s, 1H), 6.65 (d, J = 3.5 Hz, 1H), 5.51 (d, J = 3.2 Hz, 1H), 5.44 (dd, J = 3.1, 1.5 Hz, 1H), 5.34–5.20 (m, 4H), 4.44 (ddd, J = 8.6, 5.5, 1.3 Hz, 1H), 4.41 (dd, J = 10.8, 3.1 Hz, 1H), 4.37–4.33 (m, 1H), 4.20–4.13 (m, 2H), 4.05 (dd, J = 11.3, 6.6 Hz, 1H), 3.98 (dd, J = 11.0, 5.5 Hz, 1H), 2.16 (s, 3H), 2.14 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.96 (s, 3H); ¹³C NMR (100.6 MHz, CDCl₃): δ 170.5, 170.4, 170.3, 170.21, 170.19, 169.93, 169.86, 160.9, 93.7, 93.3, 91.0, 69.6, 68.8, 68.5, 67.6, 67.3, 66.8, 66.4, 65.7, 61.5, 60.7, 20.9, 20.8, 20.77, 20.76, 20.5; FT-IR (ATR): ν 3304 (w), 1745 (s), 1686 (m) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₂₈H₃₆³⁵Cl₃NNaO₁₈⁺: 802.0890, found: 802.0890.

413

414 4.2.13 Methyl 4-*O*-[(2,4,6-tri-*O*-acetyl-3-*O*-[(2,3,4,6-tetra-*O*-acetyl-α-D-galactopyranosyl)-β-D-galactopyranosyl]-2,3,6-tri-*O*-benzyl-α-D-galactopyranoside 24

416 To a solution of the trichloroacetimidate **23** (642 mg, 0.822 mmol) and the alcohol **9** (322 mg, 0.693 mmol) in CH₂Cl₂ (8 mL) was added molecular sieves 4 Å (1.2 g) and the mixture was stirred at room temperature for 30 min. To the mixture was then added TMSOTf (17 µL, 0.094 mmol) at -20 °C and the mixture was stirred and allowed to return to 0 °C over 1 hr. The reaction was then quenched with NEt₃, and the mixture filtered through Celite. The filtrate was washed with water (40 mL), brine (40 mL), dried (MgSO₄), filtered, and concentrated. The resultant residue was purified by flash
chromatography (EtOAc:hexanes, 1:1) to give the trisaccharide **24** as a white foam (673 mg, 90%). R<sub>f</sub> = 0.33 (EtOAc:hexanes, 1:1); ¹H NMR (400 MHz, CDCl<sub>3</sub>): δ 7.42-7.23 (m, 15H), 5.43 (d, J = 3.1 Hz, 1H), 5.32 (d, J = 3.1 Hz, 1H), 5.28-5.11 (m, 4H), 4.80 (d, J = 11.7 Hz, 1H), 4.79 (d, J = 12.0 Hz, 1H), 4.69-4.58 (m, 4H), 4.57 (d, J = 11.9 Hz, 1H), 4.51 (d, J = 11.9 Hz, 1H), 4.30-4.19 (m, 2H), 4.08 (dd, J = 11.2, 7.3 Hz, 1H), 4.02 (d, J = 2.5 Hz, 1H), 4.00-3.78 (m, 6H), 3.73-3.65 (m, 2H), 3.61 (dd, J = 10.3, 6.8 Hz, 1H), 3.36 (s, 3H), 2.13 (s, 6H), 2.06 (s, 3H), 2.01 (s, 3H), 1.944 (s, 3H), 1.942 (s, 3H), 1.87 (s, 3H); ¹³C NMR (100.6 MHz, CDCl<sub>3</sub>): δ 170.5, 170.4, 170.3, 170.2, 170.03, 169.97, 169.4, 138.7, 138.6, 128.6, 128.51, 128.50, 128.1, 128.00, 127.98, 127.8, 127.7, 127.6, 102.3, 98.6, 93.8, 78.4, 76.9, 76.5, 73.9, 73.7, 73.6, 73.4, 70.5, 70.3, 70.1, 69.3, 67.8, 67.4, 66.8, 66.7, 65.0, 61.5, 61.2, 55.5, 20.91, 20.87, 20.84, 20.82, 20.75, 20.7; FT-IR (ATR): ν 1743 (s) cm<sup>-1</sup>; HR-MS (ESI) *m/z* [M+Na]<sup>+</sup> calculated for C<sub>54</sub>H<sub>66</sub>NaO<sub>23</sub><sup>+</sup>: 1105.3887, found: 1105.3887.

433

4.2.14 Methyl 2,3,6-tri-*O*-benzyl-4-*O*-[3-*O*-(α-D-galactopyranosyl)-β-D-galactopyranosyl]-α-D-galactopyranoside **25**

To a solution of the trisaccharide **24** (655 mg, 0.605 mmol) in MeOH (10 mL) was added 1 M NaOMe in MeOH (0.2 mL, 0.2 mmol) at 0 °C and the mixture was stirred at room temperature for 3 h and then at 35 °C for 5 d. The reaction mixture was then neutralised with Amberlite resin (IR-120, H⁺ form) at room temperature, and the mixture was filtered and concentrated. The resultant residue was purified by flash chromatography (MeOH:CHCl₃, 3:17) to give the polyol **25** as a white foam (410 mg, 86%). R<sub>f</sub> = 0.51 (MeOH:CHCl₃, 1:4); ¹H NMR (400 MHz, CD₃OD): δ 7.45-7.40 (m, 2H), 7.39-7.23 (m, 13H), 5.03-5.00 (m, 1H), 4.78-4.73 (m, 3H), 4.72 (d, J = 3.7 Hz, 1H), 4.66 (d, J = 11.8 Hz, 1H), 4.62 (dd, J = 4.3, 3.4 Hz, 1H), 4.57 (d, J = 11.9 Hz, 1H), 4.53 (d, J = 11.9 Hz, 1H), 4.31-4.24 (m, 2H), 4.08-4.01 (m, 2H), 3.95-3.86 (m, 3H), 3.84-3.81 (m, 2H), 3.79 (dd, J = 10.6, 4.9 Hz, 1H), 3.76-3.66 (m, 4H), 3.65-3.59 (m, 3H), 3.44-3.39 (m, 1H), 3.35 (s, 3H); ¹³C NMR (100.6 MHz, CD₃OD): δ 139.9, 139.8, 139.6, 129.5, 129.41, 129.36, 129.3, 129.2, 128.9, 128.83, 128.79, 128.6, 105.3, 99.9, 97.6, 79.7, 79.6, 77.8,
447 76.4, 76.0, 74.6, 74.2, 74.1, 72.0, 71.8, 71.4, 71.3, 71.2, 70.6, 70.2, 66.5, 62.9, 62.3, 55.6; FT-IR (ATR):
448 ν 3388 (br) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₄₀H₅₂NaO₁₆⁺: 811.3148, found: 811.3142.

449

450 **4.2.15** Methyl 4-*O*-[3-*O*-(*α*-D-galactopyranosyl)-*β*-D-galactopyranosyl]-*α*-D-galactopyranoside **3**

451 To a solution of the polyol **25** (398 mg, 0.505 mmol) in MeOH (10 mL) was added 10% w/w Pd/C
452 (60 mg) and the mixture was stirred at 40 °C under an atmosphere of H₂. Further additions of 10% w/w
453 Pd/C were made: 72 mg at 2 d, 66 mg at 3 d, and 40 mg at 6 d. At 8 d the reaction mixture was filtered
454 through Celite and concentrated. The resultant residue was purified by flash chromatography
455 (CHCl₃:MeOH:H₂O, 5:5:1) to give the title compound **3** as a white foam (130 mg, 50%). The remaining
456 residue of the reaction was treated under the same conditions to give additional mass (94 mg, 36%). Rf
457 0.21 (CHCl₃:MeOH:H₂O, 5:4:1); ¹H NMR (400 MHz, D₂O): δ 5.16 (d, *J* = 3.9 Hz, 1H), 4.88–4.85 (m,
458 1H), 4.66 (d, *J* = 7.8 Hz, 1H), 4.26–4.24 (m, 1H), 4.24–4.19 (m, 1H), 4.18 (d, *J* = 3.0 Hz, 1H), 4.03 (dd,
459 *J* = 3.3, 0.9 Hz, 1H), 3.99–3.94 (m, 2H), 3.93–3.90 (m, 2H), 3.89–3.67 (m, 10H), 3.43 (s, 3H); ¹³C NMR
460 (100.6 MHz, D₂O): δ 105.0, 100.1, 96.0, 79.3, 78.0, 75.6, 71.6, 70.72, 70.67, 70.65, 69.9, 69.8, 69.4,
461 68.9, 65.5, 61.64, 61.58, 61.57, 55.8; FT-IR (ATR): ν 3344 (br) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺
462 calculated for C₁₉H₃₄NaO₁₆⁺: 541.1739, found: 541.1741.

463

464 **4.2.16** Methyl 2-*O*-benzoyl-3-*O*-{2,3-di-*O*-benzoyl-4,6-*O*-[bis(*tert*-butyl)silylene]-*α*-D-
galactopyranosyl}-*β*-D-galactopyranoside **27**

465
466 To the disaccharide **12** (3.31 g, 3.69 mmol) was added AcOH:H₂O (4:1, 250 mL) and the resultant
467 solution was stirred at 80 °C for 3 h. The reaction mixture was then concentrated, co-evaporating with
468 toluene (3 x 20 mL). The resultant residue was purified by flash chromatography (EtOAc:hexanes, 3:2)
469 to give the diol **27** as a white foam (2.46 g, 82%). R<sub>f</sub> 0.33 (EtOAc:hexanes, 3:2); ¹H NMR (400 MHz,
470 CDCl<sub>3</sub>): δ 8.10–8.06 (m, 2H), 8.02–7.95 (m, 4H), 7.64–7.59 (m, 1H), 7.58–7.53 (m, 1H), 7.53–7.46 (m,
471 3H), 7.44–7.34 (m, 4H), 5.73 (dd, *J* = 10.6, 3.8 Hz, 1H), 5.51–5.44 (m, 2H), 5.40 (d, *J* = 3.8 Hz, 1H),
472 4.61 (d, *J* = 3.0 Hz, 1H), 4.48 (d, *J* = 8.0 Hz, 1H), 3.97 (dd, *J* = 9.7, 3.4 Hz, 1H), 3.89–3.83 (m, 2H),
473 3.70–3.50 (m, 5H), 3.48 (s, 3H), 1.03 (s, 9H), 0.86 (s, 9H); ¹³C NMR (100.6 MHz, CDCl₃): δ 166.3, 166.0, 165.4, 133.9, 133.6, 133.3, 129.87, 129.85, 129.82, 129.76, 129.0, 128.83, 128.78, 128.5, 102.1, 96.2, 79.1, 74.1, 71.0, 70.7, 70.5, 68.9, 67.9, 67.0, 66.4, 62.4, 56.8, 27.5, 27.3, 23.3, 20.8; FT-IR (ATR): ν 3546 (br), 1719 (s) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₄₂H₅₂NaO₁₄Si⁺: 831.3019, found: 831.3018.

478

479 4.2.17 Methyl 4,6-di-*O*-acetyl-2-*O*-benzoyl-3-*O*-{2,3-di-*O*-benzoyl-4,6-*O*-[bis(*tert*-butyl)silylene]-*α*-D-galactopyranosyl}-*β*-D-galactopyranoside **28**

481 To a solution of the diol **27** (2.44 g, 3.02 mmol) in pyridine (20 mL) was added Ac₂O (2.3 mL, 24 mmol) at 0 °C and the mixture was kept at room temperature for 3 d. The reaction was then quenched with MeOH, and the mixture was concentrated, co-evaporating with toluene (3 x 20 mL). The resultant residue was purified by flash chromatography (EtOAc:hexanes, 3:7) to give the diacetate **28** as a white foam (2.54 g, 94%). R_f 0.27 (EtOAc:hexanes, 3:7); ¹H NMR (400 MHz, CDCl₃): δ 8.10–8.05 (m, 2H), 8.03–7.98 (m, 2H), 7.94–7.89 (m, 2H), 7.62–7.57 (m, 1H), 7.54–7.44 (m, 4H), 7.41–7.36 (m, 2H), 7.35–7.29 (m, 2H), 5.71 (dd, *J* = 10.7, 3.6 Hz, 1H), 5.53–5.45 (m, 2H), 5.43 (d, *J* = 3.3 Hz, 1H), 5.25 (dd, *J* = 10.7, 3.2 Hz, 1H), 4.54 (d, *J* = 8.0 Hz, 1H), 4.23 (d, *J* = 3.2 Hz, 1H), 4.16 (dd, *J* = 11.3, 6.3 Hz, 1H), 4.11 (dd, *J* = 10.1, 3.3 Hz, 1H), 4.07–4.00 (m, 2H), 3.87–3.82 (m, 1H), 3.72–3.64 (m, 2H), 3.52 (s, 3H), 2.03 (s, 3H), 1.40 (s, 3H), 1.03 (s, 9H), 0.81 (s, 9H); ¹³C NMR (100.6 MHz, CDCl₃): δ 170.6, 170.0, 166.4, 165.8, 165.1, 133.5, 133.2, 133.1, 130.1, 130.01, 129.99, 129.92, 129.87, 129.7, 128.8, 128.5, 128.4, 102.4, 93.8, 73.3, 71.0, 70.7, 70.6, 70.4, 67.7, 67.1, 66.5, 64.9, 61.5, 57.1, 27.5, 27.3, 23.3, 20.9, 20.7, 19.8; FT-IR (ATR): ν 1724 (s) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₄₆H₅₆NaO₁₆Si⁺: 915.3230, found: 915.3224.

495

496 4.2.18 Methyl 4,6-di-*O*-acetyl-2-*O*-benzoyl-3-*O*-*(2,3-di-*O*-benzoyl-*α*-D-galactopyranosyl)-*β*-D-galactopyranoside **29**

497
To a solution of the diacetate **28** (2.53 g, 2.83 mmol) in THF (55 mL) was added 70% w/w hydrogen fluoride in pyridine (1.1 mL, 42 mmol) at 0 °C and the mixture was stirred at room temperature for 1 h.

The reaction was then quenched with sat. aq. NaHCO₃ at 0 °C and the resultant mixture was extracted with EtOAc (100 mL). The organic extract was washed with water (50 mL), brine (50 mL), dried (MgSO₄), filtered, and concentrated, co-evaporating with toluene (3 x 30 mL). The resultant residue was purified by flash chromatography (EtOAc:hexanes, 4:1) to give the diol **29** as a white foam (2.11 g, 99%). Rf 0.38 (EtOAc:hexanes, 9:1); ¹H NMR (400 MHz, CDCl₃): δ 8.18–8.14 (m, 2H), 8.01–7.96 (m, 2H), 7.91–7.87 (m, 2H), 7.64–7.58 (m, 1H), 7.54–7.43 (m, 4H), 7.40–7.34 (m, 2H), 7.34–7.28 (m, 2H), 5.68 (dd, *J* = 10.8, 3.6 Hz, 1H), 5.55–5.49 (m, 2H), 5.44 (d, *J* = 3.3 Hz, 1H), 5.36 (dd, *J* = 10.8, 3.1 Hz, 1H), 4.57 (d, *J* = 7.9 Hz, 1H), 4.20–4.14 (m, 2H), 4.06 (dd, *J* = 11.3, 6.7 Hz, 1H), 3.91–3.86 (m, 2H), 3.84 (d, *J* = 3.1 Hz, 1H), 3.70 (dd, *J* = 12.1, 5.1 Hz, 1H), 3.64 (dd, *J* = 12.1, 4.2 Hz, 1H), 3.52 (s, 3H), 2.02 (s, 3H), 1.42 (s, 3H); ¹³C NMR (100.6 MHz, CDCl₃): δ 170.6, 170.1, 166.2, 165.3, 165.2, 133.7, 133.4, 133.3, 130.1, 130.0, 129.79, 129.76, 129.4, 128.8, 128.54, 128.50, 102.3, 93.6, 73.2, 71.0, 70.6, 70.4, 69.6, 69.4, 67.8, 65.0, 63.2, 61.8, 57.1, 20.8, 19.8; FT-IR (ATR): ν 3489 (br), 1723 (s) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₃₈H₄₀NaO₁₆⁺: 775.2209, found: 775.2205.

4.2.19

Methyl 4,6-di-*O*-acetyl-2-*O*-benzoyl-3-*O*-(*2*,*3*-di-*O*-benzoyl-4,6-*O*-benzylidene-*α*-D-galactopyranosyl)-*β*-D-galactopyranoside **30**

To a solution of the diol **29** (1.66 g, 2.21 mmol) in MeCN (20 mL) were added PhCH(OMe)₂ (0.5 mL, 3.3 mmol) and CSA (40 mg, 0.17 mmol) and the mixture was stirred at room temperature for 1.5 h. The reaction was then quenched with NEt₃ at 0 °C and the reaction mixture was concentrated. The resultant residue was purified by flash chromatography (EtOAc:hexanes, 1:1) to give the disaccharide **30** as a white foam (1.64 g, 88%). Rf 0.24 (EtOAc:hexanes, 2:3); ¹H NMR (400 MHz, CDCl₃): δ 8.17–8.11 (m, 2H), 8.02–7.97 (m, 2H), 7.94–7.89 (m, 2H), 7.69–7.62 (m, 1H), 7.58–7.34 (m, 8H), 7.33–7.27 (m, 5H), 5.73 (dd, *J* = 10.9, 3.5 Hz, 1H), 5.57–5.50 (m, 2H), 5.48 (dd, *J* = 10.9, 3.5 Hz, 1H), 5.42 (d, *J* = 3.4 Hz, 1H), 5.27 (s, 1H), 4.57 (d, *J* = 8.0 Hz, 1H), 4.20–4.02 (m, 5H), 3.89–3.83 (m, 1H), 3.72–3.68 (m, 1H),
3.57–3.50 (m, 4H), 2.02 (s, 3H), 1.48 (s, 3H); ¹³C NMR (100.6 MHz, CDCl₃): δ 170.6, 169.9, 166.1, 165.8, 165.0, 137.5, 133.7, 133.3, 130.2, 130.0, 129.89, 129.87, 129.8, 129.6, 129.1, 128.9, 128.4, 128.3, 126.2, 102.4, 100.6, 94.6, 74.0, 73.8, 71.0, 70.7, 68.8, 68.7, 68.0, 65.2, 62.6, 61.6, 57.2, 20.8, 19.8; FT-IR (ATR): ν 1738 (s) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₄₅H₄₄NaO₁₆⁺: 863.2522, found: 863.2523.

529

530 **4.2.20** Methyl 3-*O*-(4,6-*O*-benzylidene-*α*-D-galactopyranosyl)-*β*-D-galactopyranoside **31**

531 To a solution of the disaccharide **30** (1.61 g, 1.91 mmol) in MeOH (40 mL) was added 1 M NaOMe in MeOH (0.8 mL, 0.8 mmol) at 0 °C and the mixture was stirred at 40 °C for 44 h. The reaction mixture was then neutralised with AcOH at room temperature, and the mixture was concentrated. The resultant residue was purified by flash chromatography (MeOH:CHCl₃, 3:17) to give the polyol **31** as a white foam (799 mg, 94%). R<sub>f</sub> 0.40 (MeOH:CHCl₃, 1:4); ¹H NMR (400 MHz, CD₃OD): δ 7.56–7.49 (m, 2H), 7.38–7.31 (m, 3H), 5.60 (s, 1H), 5.11 (d, *J* = 3.6 Hz, 1H), 4.27 (dd, *J* = 3.5, 1.0 Hz, 1H), 4.24–4.21 (m, 1H), 4.19 (d, *J* = 7.3 Hz, 1H), 4.16 (dd, *J* = 12.5, 1.5 Hz, 1H), 4.10 (dd, *J* = 12.5, 1.7 Hz, 1H), 4.07 (dd, *J* = 2.9, 0.9 Hz, 1H), 4.01 (dd, *J* = 10.2, 3.5 Hz, 1H), 3.93 (dd, *J* = 10.2, 3.6 Hz, 1H), 3.79 (dd, *J* = 11.3, 6.7 Hz, 1H), 3.75 (dd, *J* = 11.3, 5.5 Hz, 1H), 3.66 (dd, *J* = 9.7, 2.9 Hz, 1H), 3.61 (dd, *J* = 9.7, 7.3 Hz, 1H), 3.56–3.50 (m, 4H); ¹³C NMR (100.6 MHz, CD₃OD): δ 139.8, 129.8, 129.0, 127.5, 106.0, 102.3, 98.3, 80.1, 78.2, 76.3, 70.8, 70.4, 70.1, 66.7, 64.6, 62.4, 57.3; FT-IR (ATR): ν 3482 (br) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₂₀H₂₈NaO₁₁⁺: 467.1524, found: 467.1522.

543

544 **4.2.21** Methyl 2,4,6-tri-*O*-benzyl-3-*O*-(2,3-di-*O*-benzyl-4,6-*O*-benzylidene-*α*-D-galactopyranosyl)-*β*-D-galactopyranoside **32**

545 To a solution of the polyol **31** (788 mg, 1.77 mmol) in DMF (12 mL) were added NaH 60% w/w dispersion in oil (535 mg, 13.4 mmol) and BnBr (1.2 mL, 10 mmol) at room temperature and the mixture was stirred for 2.5 h, with a further addition of BnBr (0.11 mL, 0.93 mmol) at 1.5 h. The reaction was then quenched with MeOH at 0 °C and the mixture was concentrated. The residue was
taken up in EtOAc (60 mL) and was washed with water (30 mL), brine (30 mL), dried (MgSO₄), filtered, and concentrated. The resultant residue was purified by flash chromatography (EtOAc:hexanes, 1:3) to give the disaccharide **32** as a white foam (1.49 g, 94%). R<sub>f</sub> 0.45 (EtOAc:hexanes, 3:7); ¹H NMR (400 MHz, CDCl<sub>3</sub>): δ 7.50-7.46 (m, 2H), 7.45-7.40 (m, 2H), 7.37-7.14 (m, 24H), 7.13-7.08 (m, 2H), 5.28 (s, 1H), 5.19 (d, *J* = 3.3 Hz, 1H), 5.07 (d, *J* = 11.6 Hz, 1H), 4.93 (d, *J* = 10.7 Hz, 1H), 4.89 (d, *J* = 11.4 Hz, 1H), 4.80-4.66 (m, 3H), 4.46 (d, *J* = 11.9 Hz, 1H), 4.43-4.36 (m, 3H), 4.30-4.26 (m, 1H), 4.15 (dd, *J* = 10.2, 3.3 Hz, 1H), 4.03 (dd, *J* = 10.2, 3.3 Hz, 1H), 3.90-3.84 (m, 2H), 3.83-3.78 (m, 2H), 3.77-3.71 (m, 2H), 3.61-3.50 (m, 6H), 3.29 (dd, *J* = 12.6, 1.6 Hz, 1H); <sup>13</sup>C NMR (100.6 MHz, CDCl<sub>3</sub>): δ 139.1, 138.9, 138.8, 138.4, 138.2, 138.1, 129.0, 128.6, 128.5, 128.4, 128.2, 128.11, 128.05, 127.92, 127.87, 127.8, 127.7, 127.2, 126.5, 105.4, 101.0, 96.1, 78.6, 77.7, 76.2, 75.7, 75.4, 74.8, 74.7, 74.6, 73.6, 73.5, 72.7, 71.8, 69.3, 69.0, 62.5, 57.1; FT-IR (ATR): ν 3031 (w), 2860 (w) cm<sup>-1</sup>; HR-MS (ESI) *m/z* [M+Na]<sup>+</sup> calculated for C<sub>55</sub>H<sub>58</sub>NaO<sub>11</sub><sup>+</sup>: 917.3871, found: 917.3865.

4.2.22 Methyl 2,4,6-tri-*O*-benzyl-3-*O*-((2,3,6-tri-*O*-benzyl-*α*-D-galactopyranosyl)-*β*-D-galactopyranoside

To a solution of the disaccharide **32** (1.47 g, 1.64 mmol) in THF (25 mL) were added molecular sieves 4 Å (1.5 g) and 95% w/w NaCNBH₃ (1.1 g, 17 mmol) and the mixture was stirred at room temperature. To the mixture was then added dropwise 2 M HCl in Et₂O (8 mL, 16 mmol) and the mixture was stirred for 30 min. The reaction mixture was then diluted with CH₂Cl₂ (20 mL), neutralised with NEt₃, and filtered through Celite. The filtrate was washed with water (2 x 100 mL), sat. aq. NaHCO₃ (100 mL), brine (100 mL), dried (MgSO₄), filtered, and concentrated. The resultant residue was purified by flash chromatography (EtOAc:hexanes, 3:7) to give the alcohol **26** as a colourless oil (1.21 g, 82%). R<sub>f</sub> 0.59 (EtOAc:hexanes, 2:3); ¹H NMR (400 MHz, CDCl₃): δ 7.40-7.10 (m, 30H), 5.19 (d, *J* = 3.4 Hz, 1H), 5.03 (d, *J* = 11.6 Hz, 1H), 4.89 (d, *J* = 10.8 Hz, 1H), 4.83 (d, *J* = 11.6 Hz, 1H), 4.72-4.63 (m, 3H), 4.55 (d, *J* = 10.8 Hz, 1H), 4.47-4.32 (m, 5H), 4.26-4.22 (m, 1H), 4.21-4.18 (m, 1H), 4.00 (dd, *J* = 9.8, 3.4 Hz, 1H), 3.91-3.84 (m, 2H), 3.82-3.74 (m, 3H), 3.59-3.48 (m, 6H), 3.48-3.38 (m, 2H), 2.85 (s, 1H);
576 $^{13}$C NMR (100.6 MHz, CDCl$_3$): δ 139.03, 138.96, 138.4, 138.3, 138.24, 138.19, 128.6, 128.54, 128.45,
577 128.43, 128.35, 128.3, 128.2, 128.14, 128.10, 128.0, 127.93, 127.90, 127.85, 127.8, 127.7, 127.6, 127.3,
578 105.4, 95.5, 78.5, 77.9, 77.4, 75.8, 75.2, 74.64, 74.59, 73.6, 73.4, 73.2, 72.7, 72.1, 70.2, 69.0, 68.6, 68.0,
579 57.2; FT-IR (ATR): ν 3469 (br) cm$^{-1}$; HR-MS (ESI) m/z [M+Na]$^+$ calculated for C$_{55}$H$_{60}$NaO$_{11}$$^+$:
580 919.4028, found: 919.4026.

581
582 4.2.23 Methyl 3-O-[4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-benzyl-α-D-
583 galactopyranosyl]-2,4,6-tri-O-benzyl-β-D-galactopyranoside 33

584 To a solution of the trichloroacetimidate 7 (321 mg, 0.652 mmol) and the alcohol 26 (487 mg,
585 0.543 mmol) in CH2Cl2 (7 mL) was added molecular sieves 4 Å (1.0 g) and the mixture was stirred at
586 room temperature for 30 min. To the mixture was then added TMSOTf (11 µL, 0.061 mmol) at -20 °C
587 and the mixture was stirred and allowed to return to 0 °C over 1 hr. The reaction was then quenched
588 with NEt3, and the mixture filtered through Celite. The filtrate was washed with water (40 mL), brine
589 (40 mL), dried (MgSO4), filtered, and concentrated. The resultant residue was purified by flash
590 chromatography (EtOAc:hexanes, 7:13) to give the trisaccharide 33 as a white foam (555 mg, 83%). Rf
591 0.38 (EtOAc:hexanes, 2:3); 1H NMR (500 MHz, CDCl3): δ 7.38-7.08 (m, 30H), 5.34 (d, J = 3.5 Hz,
592 1H), 5.19 (d, J = 2.6 Hz, 1H), 5.16 (dd, J = 10.5, 7.8 Hz, 1H), 4.97 (dd, J = 10.5, 3.5 Hz, 1H), 4.95 (d, J
593 = 11.3 Hz, 1H), 4.88 (d, J = 11.2 Hz, 1H), 4.75 (d, J = 11.4 Hz, 1H), 4.67 (d, J = 11.7 Hz, 1H), 4.65 (d,
594 J = 11.1 Hz, 1H), 4.62-4.58 (m, 2H), 4.56 (d, J = 11.7 Hz, 1H), 4.46-4.31 (m, 6H), 4.12 (d, J = 6.8 Hz,
595 1H), 4.07 (dd, J = 11.2, 7.5 Hz, 1H), 3.98 (dd, J = 11.2, 6.1 Hz, 1H), 3.91-3.84 (m, 3H), 3.81-3.70 (m,
596 4H), 3.61 (dd, J = 10.8, 7.5 Hz, 1H), 3.56-3.41 (m, 7H), 2.14 (s, 3H), 2.01 (s, 3H), 1.98 (s, 3H), 1.80 (s,
597 3H); 13C NMR (125.8 MHz, CDCl3): δ 170.5, 170.4, 170.3, 169.8, 139.3, 139.0, 138.9, 138.5, 138.3,
598 138.2, 128.6, 128.52, 128.48, 128.34, 128.26, 128.2, 128.1, 128.00, 127.97, 127.9, 127.80, 127.76,
599 127.4, 127.3, 127.2, 105.3, 102.2, 95.1, 78.5, 78.4, 77.2, 76.9, 76.6, 75.0, 74.6, 74.5, 73.5, 73.4, 73.2,
600 72.7, 72.6, 71.0, 70.4, 70.1, 69.3, 69.0, 68.8, 66.9, 61.3, 57.1, 20.9, 20.82, 20.80, 20.76; FT-IR (ATR): ν
601 1747 (s) cm-1; HR-MS (ESI) m/z [M+Na]⁺ calculated for C69H78NaO20⁺: 1249.4979, found: 1249.4979.
602

603

4.2.24 Methyl 2,4,6-tri-O-benzyl-3-O-[2,3,6-tri-O-benzyl-4-O-(β-D-galactopyranosyl)-α-D-galactopyranosyl]-β-D-galactopyranoside **34**

604

605

606

607

608

609

610

611

612

613

614

615

616

617

618

619

To a solution of the trisaccharide **33** (541 mg, 0.441 mmol) in MeOH (10 mL) was added 1 M NaOMe in MeOH (0.2 mL, 0.2 mmol) and the mixture was stirred at 30 °C for 4 h. The reaction mixture was then neutralised with Amberlite resin (IR-120, H⁺ form) at room temperature, and the mixture was filtered and concentrated. The resultant residue was purified by flash chromatography (MeOH:CHCl₃, 1:24) to give the tetrol **34** as a white foam (432 mg, 93%). Rf 0.58 (MeOH:CHCl₃, 1:9); ¹H NMR (400 MHz, CD₃OD): δ 7.44–7.39 (m, 2H), 7.36–7.20 (m, 23H), 7.18–7.09 (m, 5H), 5.28 (d, *J* = 3.5 Hz, 1H), 4.93 (d, *J* = 11.2 Hz, 1H), 4.83–4.67 (m, 5H), 4.61 (d, *J* = 10.9 Hz, 1H), 4.56 (d, *J* = 7.7 Hz, 1H), 4.49 (d, *J* = 11.9 Hz, 1H), 4.43–4.37 (m, 2H), 4.36–4.30 (m, 2H), 4.24 (d, *J* = 12.0 Hz, 1H), 4.18 (dd, *J* = 10.2, 3.5 Hz, 1H), 4.13 (d, *J* = 7.6 Hz, 1H), 4.06–4.02 (m, 1H), 3.99 (dd, *J* = 10.2, 2.8 Hz, 1H), 3.96 (d, *J* = 2.6 Hz, 1H), 3.85–3.79 (m, 2H), 3.76–3.58 (m, 4H), 3.56–3.49 (m, 5H), 3.46 (s, 3H), 3.45–3.39 (m, 2H); ¹³C NMR (100.6 MHz, CD₃OD): δ 140.2, 140.1, 140.0, 139.60, 139.56, 139.5, 129.6, 129.42, 129.35, 129.34, 129.30, 129.28, 129.2, 129.12, 129.07, 129.0, 128.9, 128.8, 128.7, 128.6, 128.5, 128.44, 128.36, 106.5, 105.4, 96.4, 79.6, 79.5, 78.4, 77.9, 76.7, 76.1, 76.04, 75.96, 75.6, 75.0, 74.5, 74.3, 74.2, 73.7, 73.4, 71.1, 70.1, 70.0, 69.9, 62.2, 57.4; FT-IR (ATR): v 3425 (br) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₆₁H₇₀NaO₁₆⁺: 1081.4556, found: 1081.4543.

620

621

4.2.25 Methyl 3-O-[4-O-(β-D-galactopyranosyl)-α-D-galactopyranosyl]-β-D-galactopyranoside **4**

622

623

624

625

626

627

To a solution of the tetrol **34** (418 mg, 0.395 mmol) in MeOH (20 mL) was added 10% w/w Pd/C (314 mg) and the mixture was stirred at 40 °C under an atmosphere of H₂. At 3 d the reaction mixture was filtered through Celite and concentrated. The resultant residue was purified by flash chromatography (CHCl₃:MeOH:H₂O, 5:4:1) to give the title compound **4** as a white foam (125 mg, 61%). The remaining residue of the reaction was treated under the same conditions to give additional mass (47 mg, 23%). Rf 0.18 (CHCl₃:MeOH:H₂O, 5:4:1); ¹H NMR (400 MHz, D₂O): δ 5.17 (d, *J* = 3.9
628 Hz, 1H), 4.62 (d, *J* = 7.8 Hz, 1H), 4.39 (d, *J* = 7.9 Hz, 1H), 4.30–4.22 (m, 2H), 4.19 (d, *J* = 3.2 Hz, 1H),
629 4.07 (dd, *J* = 10.4, 3.1 Hz, 1H), 3.96 (dd, *J* = 10.4, 3.9 Hz, 1H), 3.92 (dd, *J* = 3.4, 0.7 Hz, 1H), 3.89–
630 3.57 (m, 15H); <sup>13</sup>C NMR (100.6 MHz, D<sub>2</sub>O): δ 105.1, 104.3, 96.1, 78.9, 78.2, 75.8, 75.5, 73.5, 72.1,
631 70.8, 70.4, 69.8, 69.31, 69.26, 65.6, 61.64, 61.61, 61.1, 57.8; FT-IR (ATR): ν 3340 (br) cm<sup>-1</sup>; HR-MS
632 (ESI) *m/z* [M+Na]<sup>+</sup> calculated for C<sub>19</sub>H<sub>34</sub>NaO<sub>16</sub><sup>+</sup>: 541.1739, found: 541.1738.

633

634 4.2.26 Methyl 3-*O*-{4-*O*-[2,4,6-tri-*O*-acetyl-3-*O*-(*-*(3,4,6-tetra-*O*-acetyl-*α*-D-galactopyranosyl)-*β*-D- galactopyranosyl]-2,3,6-tri-*O*-benzyl-*α*-D-galactopyranosyl}-2,4,6-tri-*O*-benzyl-*β*-D-galactopyranoside

636 **35**

637 To a solution of the trichloroacetimidate **23** (341 mg, 0.437 mmol) and the alcohol **26** (333 mg,
638 0.371 mmol) in CH<sub>2</sub>Cl<sub>2</sub> (7 mL) was added molecular sieves 4 Å (1.0 g) and the mixture was stirred at
639 room temperature for 30 min. To the mixture was then added TMSOTf (11 µL, 0.061 mmol) at -20 °C
640 and the mixture was stirred and allowed to return to 0 °C over 1 hr. The reaction was then quenched
641 with NEt<sub>3</sub>, and the mixture filtered through Celite. The filtrate was washed with water (40 mL), brine
642 (40 mL), dried (MgSO<sub>4</sub>), filtered, and concentrated. The resultant residue was purified by flash
643 chromatography (EtOAc:hexanes, 9:11) to give the tetrasaccharide **35** as a white foam (407 mg, 72%).
644 R<sub>f</sub> 0.20 (EtOAc:hexanes, 2:3); <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>): δ 7.36–7.14 (m, 28H), 7.13–7.07 (m, 2H),
645 5.42 (d, *J* = 3.2 Hz, 1H), 5.31 (d, *J* = 3.2 Hz, 1H), 5.28–5.10 (m, 5H), 4.95 (d, *J* = 11.5 Hz, 1H), 4.87 (d,
646 *J* = 11.2 Hz, 1H), 4.77 (d, *J* = 11.5 Hz, 1H), 4.67–4.60 (m, 3H), 4.58–4.53 (m, 2H), 4.47–4.30 (m, 6H),
647 4.29–4.18 (m, 2H), 4.11 (d, *J* = 6.6 Hz, 1H), 4.07 (dd, *J* = 11.2, 7.3 Hz, 1H), 4.00–3.72 (m, 9H), 3.70–
648 3.40 (m, 9H), 2.14 (s, 3H), 2.13 (s, 3H), 2.06 (s, 3H), 2.02 (s, 3H), 1.94 (s, 3H), 1.93 (s, 3H), 1.87 (s,
649 3H); <sup>13</sup>C NMR (125.8 MHz, CDCl<sub>3</sub>): δ 170.5, 170.4, 170.3, 170.2, 170.1, 170.0, 169.3, 139.3, 139.0,
650 138.9, 138.5, 138.3, 138.2, 128.54, 128.52, 128.50, 128.3, 128.2, 128.14, 128.09, 128.08, 128.0, 127.93,
651 127.85, 127.81, 127.78, 127.39, 127.35, 127.3, 127.2, 105.3, 102.2, 95.1, 93.8, 78.6, 78.4, 77.2, 76.9,
652 76.3, 74.9, 74.6, 74.5, 73.50, 73.46, 73.4, 73.2, 72.7, 72.6, 70.5, 70.4, 70.1, 69.0, 68.8, 67.7, 67.4, 66.8,
653 66.7, 65.0, 61.5, 61.2, 57.1, 20.91, 20.89, 20.87, 20.84, 20.75, 20.72, 20.70; FT-IR (ATR): ν 1745 (s)
654 cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₈₁H₉₄NaO₂₈⁺: 1537.5824, found: 1537.5815.

655
656 4.2.27 Methyl 2,4,6-tri-O-benzyl-3-O-{2,3,6-tri-O-benzyl-4-O-[3-O-(α-D-galactopyranosyl)-β-D-galactopyranosyl]-α-D-galactopyranosyl}-β-D-galactopyranoside 36
657

658 To a solution of the tetrasaccharide **35** (396 mg, 0.261 mmol) in MeOH (8 mL) was added 1 M NaOMe
659 in MeOH (0.2 mL, 0.2 mmol) at 0 °C and the mixture was stirred at 30 °C for 10 d. The reaction
660 mixture was then neutralised with Amberlite resin (IR-120, H⁺ form) at room temperature and the
661 mixture was filtered and concentrated. The resultant residue was purified by flash chromatography
662 (MeOH:CHCl₃, 1:9) to give the polyol **36** as a white foam (271 mg, 85%). Rf 0.27 (MeOH:CHCl₃, 1:9);
663 ¹H NMR (500 MHz, CD₃OD): δ 7.44–7.40 (m, 2H), 7.35–7.08 (m, 28H), 5.28 (d, *J* = 3.4 Hz, 1H), 5.05–
664 5.01 (m, 1H), 4.92 (d, *J* = 11.2 Hz, 1H), 4.81 (d, *J* = 11.3 Hz, 1H), 4.77 (d, *J* = 10.9 Hz, 1H), 4.74–4.71
665 (m, 2H), 4.69 (d, *J* = 11.3 Hz, 1H), 4.62–4.57 (m, 2H), 4.49 (d, *J* = 11.9 Hz, 1H), 4.43–4.37 (m, 2H),
666 4.36–4.26 (m, 3H), 4.23 (d, *J* = 11.9 Hz, 1H), 4.16 (dd, *J* = 10.2, 3.4 Hz, 1H), 4.13 (d, *J* = 7.6 Hz, 1H),
667 4.06–4.03 (m, 2H), 4.00 (dd, *J* = 10.2, 2.7 Hz, 1H), 3.95 (d, *J* = 2.7 Hz, 1H), 3.94–3.92 (m, 1H), 3.85–
668 3.80 (m, 3H), 3.78–3.59 (m, 8H), 3.55–3.49 (m, 4H), 3.46 (s, 3H), 3.45–3.41 (m, 1H); ¹³C NMR
669 (125.8 MHz, CD₃OD): δ 140.1, 140.0, 139.6, 139.54, 139.49, 129.6, 129.42, 129.37, 129.35, 129.33,
670 129.30, 129.28, 129.26, 129.2, 129.1, 128.99, 128.96, 128.8, 128.7, 128.58, 128.55, 128.43, 128.36,
671 106.5, 105.4, 97.6, 96.4, 79.7, 79.51, 79.47, 78.4, 78.0, 76.3, 76.2, 76.03, 75.97, 75.7, 74.5, 74.34,
672 74.28, 73.8, 73.5, 72.0, 71.9, 71.4, 71.3, 71.0, 70.2, 70.0, 69.9, 66.4, 62.9, 62.2, 57.4; FT-IR (ATR): ν
673 3401 (br) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₆₇H₈₀NaO₂₁⁺: 1243.5084, found: 1243.5085.

674
675 4.2.28 Methyl 3-O-{4-O-[3-O-(α-D-galactopyranosyl)-β-D-galactopyranosyl]-α-D-galactopyranosyl}-β-D-galactopyranoside **6**
676

677 To a solution of the polyol **36** (259 mg, 0.212 mmol) in MeOH (15 mL) was added 10% w/w Pd/C
678 (195 mg) and the mixture was stirred at 40 °C under an atmosphere of H₂. A further addition of 10%
679 w/w Pd/C (56 mg) was made at 40 h. At 3 d the reaction mixture was filtered through Celite and concentrated. The resultant residue was purified by flash chromatography (CHCl₃:MeOH:H₂O, 8:9:3) to give the title compound **6** as a white foam (21 mg, 15%). The remaining residue of the reaction was treated under the same conditions to give additional mass (77 mg, 53%). R<sub>f</sub> 0.13 (CHCl₃:MeOH:H₂O, 5:4:1); ¹H NMR (400 MHz, D₂O): δ 5.19–5.14 (m, 2H), 4.68 (d, *J* = 7.8 Hz, 1H), 4.39 (d, *J* = 7.9 Hz, 1H), 4.29 (d, *J* = 2.8 Hz, 1H), 4.28–4.16 (m, 4H), 4.06 (dd, *J* = 10.4, 3.1 Hz, 1H), 4.03 (d, *J* = 3.3 Hz, 1H), 4.00–3.92 (m, 2H), 3.90–3.68 (m, 14H), 3.65 (dd, *J* = 9.8, 7.9 Hz, 1H), 3.60 (s, 3H); ¹³C NMR (100.6 MHz, D₂O): δ 105.0, 104.3, 96.1, 96.0, 79.3, 78.2, 78.0, 75.6, 75.5, 71.6, 70.8, 70.7, 70.5, 70.0, 69.8, 69.4, 68.9, 65.6, 65.5, 61.7, 61.6, 61.1, 57.8; FT-IR (ATR): ν 3325 (br) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₂₅H₄₄NaO₂₁⁺: 703.2267, found: 703.2273.

689
4.2.29 Methyl 4-O-(2,4,6-tri-O-acetyl-3-O-chloroacetyl-β-D-galactopyranosyl)-2,3,6-tri-O-benzyl-α-D-galactopyranoside **38**

690
To a solution of the trichloroacetimidate **37** (1.59 g, 3.02 mmol) and the alcohol **9** (1.16 g, 2.50 mmol) in CH₂Cl₂ (25 mL) was added molecular sieves 4 Å (4.0 g) and the mixture was stirred at room temperature for 30 min. To the mixture was then added TMSOTf (39 µL, 0.22 mmol) at -20 °C and the mixture was stirred and allowed to return to 0 °C over 1 hr. The reaction was then quenched with NEt₃, and the mixture filtered through Celite. The filtrate was washed with water (100 mL), brine (100 mL), dried (MgSO₄), filtered, and concentrated. The resultant residue was purified by flash chromatography (EtOAc:hexanes, 7:13) to give the disaccharide **38** as a white foam (1.63 g, 79%). R<sub>f</sub> 0.37 (EtOAc:hexanes, 2:3); ¹H NMR (400 MHz, CDCl₃): δ 7.39–7.24 (m, 15H), 5.34 (dd, *J* = 3.4, 1.0 Hz, 1H), 5.20 (dd, *J* = 10.4, 7.8 Hz, 1H), 5.04 (dd, *J* = 10.4, 3.4 Hz, 1H), 4.83 (d, *J* = 11.7 Hz, 1H), 4.76 (d, *J* = 11.9 Hz, 1H), 4.70 (d, *J* = 7.8 Hz, 1H), 4.66 (d, *J* = 11.7 Hz, 1H), 4.62 (d, *J* = 3.6 Hz, 1H), 4.59 (d, *J* = 11.9 Hz, 1H), 4.57 (d, *J* = 12.0 Hz, 1H), 4.51 (d, *J* = 12.0 Hz, 1H), 4.09 (dd, *J* = 11.1, 7.6 Hz, 1H), 4.03–3.98 (m, 2H), 3.97 (s, 2H), 3.92–3.84 (m, 2H), 3.80–3.74 (m, 2H), 3.68 (dd, *J* = 10.4, 5.1 Hz, 1H), 3.60 (dd, *J* = 10.4, 6.7 Hz, 1H), 3.37 (s, 3H), 2.14 (s, 3H), 2.00 (s, 3H), 1.81 (s, 3H); ¹³C NMR
(100.6 MHz, CDCl₃): δ 170.7, 170.4, 169.8, 166.9, 138.6, 138.51, 138.50, 128.63, 128.58, 128.5, 128.1,
127.99, 127.97, 127.8, 127.7, 127.6, 102.1, 98.6, 78.3, 76.91, 76.89, 73.9, 73.8, 73.5, 72.8, 70.3, 70.0,
69.2, 69.0, 66.7, 61.1, 55.5, 40.6, 20.9, 20.83, 20.76; FT-IR (ATR): ν 1745 (s) cm⁻¹; HR-MS (ESI) *m/z*
[M+Na]⁺ calculated for C₄₂H₄₉³⁵ClNaO₁₅⁺: 851.2652, found: 851.2651.

709

710 4.2.30 Methyl 4-O-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-benzyl-α-D-galactopyranoside
711 **39**

712 To a solution of the disaccharide **38** (1.62 g, 1.95 mmol) in THF (50 mL) were added H₂NCSNH₂
713 (892 mg, 11.7 mmol), NaHCO₃ (576 mg, 6.86 mmol), and Bu₄NI (721 mg, 1.95 mmol) and the mixture
714 was stirred at 55 °C for 2 h. The reaction mixture was then concentrated. The resultant residue was
715 purified by flash chromatography (EtOAc:hexanes, 1:1) to give the alcohol **39** as a white foam (1.39 g,
716 95%). R_f 0.27 (EtOAc:hexanes, 1:1); ¹H NMR (400 MHz, CDCl₃): δ 7.40–7.23 (m, 15H), 5.28 (dd, J =
717 3.7, 1.0 Hz, 1H), 4.94 (dd, *J* = 10.0, 7.8 Hz, 1H), 4.85 (d, *J* = 11.8 Hz, 1H), 4.76 (d, *J* = 12.0 Hz, 1H),
718 4.70 (d, *J* = 7.8 Hz, 1H), 4.67 (d, *J* = 11.8 Hz, 1H), 4.67 (d, *J* = 3.5 Hz, 1H), 4.61 (d, *J* = 12.0 Hz, 1H),
719 4.57 (d, *J* = 12.0 Hz, 1H), 4.51 (d, *J* = 12.0 Hz, 1H), 4.12 (dd, *J* = 11.3, 6.7 Hz, 1H), 4.04 (d, *J* = 2.8 Hz,
720 1H), 3.96 (dd, *J* = 11.3, 6.5 Hz, 1H), 3.93–3.85 (m, 2H), 3.84–3.75 (m, 2H), 3.72–3.65 (m, 2H), 3.61
721 (dd, *J* = 10.5, 6.8 Hz, 1H), 3.38 (s, 3H), 2.41 (d, *J* = 6.0 Hz, 1H), 2.16 (s, 3H), 2.01 (s, 3H), 1.89 (s, 3H);
722 ¹³C NMR (100.6 MHz, CDCl₃): δ 171.2, 171.1, 170.5, 138.8, 138.6, 138.5, 128.6, 128.53, 128.51,
723 128.0, 127.9, 127.8, 127.7, 127.64, 127.60, 101.6, 98.5, 78.4, 77.0, 76.1, 73.7, 73.6, 73.1, 71.4, 70.7,
724 70.2, 69.6, 69.4, 61.9, 55.5, 21.1, 20.9, 20.8; FT-IR (ATR): ν 3467 (br), 1742 (s) cm⁻¹; HR-MS (ESI)
725 *m/z* [M+Na]⁺ calculated for C₄₀H₄₈NaO₁₄⁺: 775.2936, found: 775.2932.

726

727 4.2.31 Methyl 4-O-(2,4,6-tri-O-acetyl-3-O-{2,3-di-O-benzoyl-4,6-O-[bis(tert-butyl)silylene]-α-D-galactopyranosyl}-β-D-galactopyranosyl)-2,3,6-tri-O-benzyl-α-D-galactopyranoside **40**

729 To a solution of the trichloroacetimidate **8** (1.48 mg, 2.20 mmol) and the alcohol **39** (1.38 g, 1.83 mmol)
730 and in CH₂Cl₂ (25 mL) was added molecular sieves 4 Å (3.5 g) and the mixture was stirred at room
temperature for 30 min. To the mixture was then added TMSOTf (44 µL, 0.24 mmol) at -20 °C and the
mixture was stirred and allowed to return to 0 °C over 1 hr. The reaction was then quenched with NEt₃,
and the mixture filtered through Celite. The filtrate was washed with water (100 mL), brine (100 mL),
dried (MgSO₄), filtered, and concentrated. The resultant residue was purified by flash chromatography
(EtOAc:hexanes, 3:7) to give the trisaccharide **40** as a white foam (1.34 g, 58%). Rf 0.45
(EtOAc:hexanes, 2:3); ¹H NMR (400 MHz, CD₃OD): δ 8.02-7.97 (m, 2H), 7.95-7.90 (m, 2H), 7.59-
7.50 (m, 2H), 7.46-7.28 (m, 18H), 7.26-7.21 (m, 1H), 5.82 (dd, *J* = 10.7, 3.6 Hz, 1H), 5.41 (d, *J* = 3.6
Hz, 1H), 5.40-5.35 (m, 2H), 5.04 (dd, *J* = 10.2, 7.9 Hz, 1H), 4.88 (d, *J* = 3.1 Hz, 1H), 4.74 (d, *J* = 11.7
Hz, 1H), 4.71-4.68 (m, 3H), 4.66 (d, *J* = 7.9 Hz, 1H), 4.64 (d, *J* = 11.7 Hz, 1H), 4.55 (d, *J* = 12.0 Hz,
1H), 4.50 (d, *J* = 12.0 Hz, 1H), 4.31 (dd, *J* = 12.6, 2.0 Hz, 1H), 4.21 (dd, *J* = 12.6, 1.5 Hz, 1H), 4.10 (d,
*J* = 2.9 Hz, 1H), 4.06 (dd, *J* = 10.2, 3.4 Hz, 1H), 4.02 (dd, *J* = 10.0, 5.6 Hz, 1H), 3.97-3.94 (m, 1H),
3.93-3.82 (m, 4H), 3.78 (dd, *J* = 10.1, 3.5 Hz, 1H), 3.65 (dd, *J* = 10.9, 4.2 Hz, 1H), 3.60 (dd, *J* = 10.9,
7.4 Hz, 1H), 3.36 (s, 3H), 1.96 (s, 3H), 1.79 (s, 3H), 1.55 (s, 3H), 1.43 (s, 9H), 0.99 (s, 9H); ¹³C NMR
(100.6 MHz, CD₃OD): δ 172.0, 171.8, 171.6, 167.4, 167.3, 140.0, 139.9, 139.8, 134.54, 134.53, 131.0,
130.94, 130.90, 130.5, 129.61, 129.60, 129.5, 129.41, 129.40, 129.1, 128.9, 128.7, 128.6, 103.6, 99.6,
95.7, 79.1, 78.2, 78.1, 74.9, 74.6, 74.3, 74.2, 72.6, 72.14, 72.05, 71.8, 71.6, 70.5, 69.0, 68.9, 67.9, 67.1,
62.7, 55.6, 28.0, 27.9, 24.1, 21.7, 21.6, 20.7, 20.1; FT-IR (ATR): ν 1748 (s), 1723 (s) cm⁻¹; HR-MS
(ESI) *m/z* [M+Na]⁺ calculated for C₆₈H₈₂NaO₂₁Si⁺: 1285.5010, found: 1285.5018.

749

4.2.32 Methyl 4-O-[2,4,6-tri-O-acetyl-3-O-(2,3-di-O-benzoyl-α-D-galactopyranosyl)-β-D-galactopyranosyl]-2,3,6-tri-O-benzyl-α-D-galactopyranoside **41**

To a solution of the trisaccharide **40** (1.34 g, 1.06 mmol) in THF (20 mL) was added 70% w/w hydrogen fluoride in pyridine (0.42 mL, 16 mmol) at 0 °C and the mixture was stirred at room temperature for 1 h.
The reaction was then quenched with sat. aq. NaHCO₃ at 0 °C and the resultant mixture was extracted with EtOAc (40 mL). The organic extract was washed with water (20 mL), brine (20 mL), dried (MgSO₄), filtered, and concentrated, co-evaporating with toluene (3 x 20 mL). The resultant residue was
purified by flash chromatography (EtOAc:hexanes, 3:2) to give the diol **41** as a white foam (858 mg, 757
758 72%). R<sub>f</sub> 0.31 (EtOAc:hexanes, 3:2); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 8.03–7.98 (m, 2H), 7.96–7.91 (m,
759 2H), 7.54–7.47 (m, 2H), 7.43–7.22 (m, 19H), 5.76 (dd, J = 10.7, 3.7 Hz, 1H), 5.50 (d, J = 3.7 Hz, 1H),
760 5.46 (dd, J = 10.7, 3.1 Hz, 1H), 5.34 (d, J = 3.1 Hz, 1H), 5.20 (dd, J = 10.1, 7.8 Hz, 1H), 4.81 (d, J =
761 11.5 Hz, 1H), 4.80 (d, J = 11.9 Hz, 1H), 4.65 (d, J = 11.5 Hz, 1H), 4.63–4.58 (m, 3H), 4.54 (d, J = 12.0
762 Hz, 1H), 4.48 (d, J = 12.0 Hz, 1H), 4.47–4.44 (m, 1H), 4.10–4.06 (m, 1H), 4.01–3.84 (m, 8H), 3.82 (dd,
763 J = 10.1, 3.3 Hz, 1H), 3.70–3.64 (m, 2H), 3.59 (dd, J = 10.4, 6.8 Hz, 1H), 3.36 (s, 3H), 2.72 (d, J = 2.8
764 Hz, 1H), 2.35 (dd, J = 7.0, 5.4 Hz, 1H), 1.95 (s, 3H), 1.87 (s, 3H), 1.50 (s, 3H); <sup>13</sup>C NMR (125.8 MHz,
765 CDCl<sub>3</sub>): δ 170.5, 170.0, 169.9, 166.0, 165.7, 138.8, 138.6, 138.5, 133.6, 133.4, 130.1, 129.9, 129.6,
766 129.3, 128.63, 128.60, 128.55, 128.53, 128.50, 128.2, 128.1, 127.97, 127.96, 127.7, 127.6, 102.5, 98.6,
767 93.9, 78.4, 77.2, 77.1, 74.0, 73.8, 73.5, 73.3, 70.9, 70.7, 70.5, 70.1, 69.8, 69.7, 69.3, 67.7, 64.9, 63.2,
768 61.4, 55.5, 21.1, 20.8, 19.9; FT-IR (ATR): ν 3494 (br), 1745 (s), 1723 (s) cm<sup>-1</sup>; HR-MS (ESI) m/z
769 [M+Na]<sup>+</sup> calculated for C<sub>60</sub>H<sub>66</sub>NaO<sub>21</sub><sup>+</sup>: 1145.3989, found: 1145.3971.

770

4.2.33 Methyl 4-O-[2,4,6-tri-O-acetyl-3-O-(2,3-di-O-benzoyl-4,6-O-benzylidene-α-D-galactopyranosyl)-β-D-galactopyranosyl]-2,3,6-tri-O-benzyl-α-D-galactopyranoside **42**

To a solution of the diol **41** (748 mg, 0.667 mmol) in MeCN (6 mL) were added PhCH(OMe)<sub>2</sub> (0.15 mL, 1.0 mmol) and CSA (12 mg, 0.052 mmol) and the mixture was stirred at room temperature for 1 h. The reaction was then quenched with NEt<sub>3</sub> at 0 °C and the reaction mixture was concentrated.
The resultant residue was purified by flash chromatography (EtOAc:hexanes, 2:3) to give the trisaccharide **42** as a white foam (710 mg, 88%). R<sub>f</sub> 0.30 (EtOAc:hexanes, 2:3); <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>): δ 8.06–8.02 (m, 2H), 7.97–7.92 (m, 2H), 7.54–7.45 (m, 4H), 7.43–7.22 (m, 22H), 5.83 (dd, J = 10.9, 3.5 Hz, 1H), 5.55 (d, J = 3.5 Hz, 1H), 5.53 (s, 1H), 5.51 (dd, J = 10.9, 3.4 Hz, 1H), 5.34 (d, J = 3.3 Hz, 1H), 5.20 (dd, J = 10.0, 7.9 Hz, 1H), 4.85–4.78 (m, 2H), 4.67–4.59 (m, 5H), 4.55 (d, J = 12.0 Hz, 1H), 4.49 (d, J = 12.0 Hz, 1H), 4.30 (d, J = 12.7 Hz, 1H), 4.07–3.96 (m, 3H), 3.94–3.80 (m, 6H), 3.70–3.64 (m, 2H), 3.60 (dd, J = 10.3, 6.7 Hz, 1H), 3.36 (s, 3H), 1.95 (s, 3H), 1.86 (s, 3H), 1.60 (s, 3H); <sup>13</sup>C
783 NMR (125.8 MHz, CDCl₃): δ 170.4, 169.9, 169.6, 166.2, 165.9, 138.8, 138.6, 138.5, 137.5, 133.4,
784 130.1, 129.9, 129.7, 129.6, 129.2, 128.6, 128.53, 128.51, 128.46, 128.3, 128.1, 127.99, 127.97, 127.7,
785 127.6, 126.3, 102.5, 100.9, 98.6, 95.2, 78.3, 77.1, 77.0, 74.4, 74.1, 73.9, 73.8, 73.5, 70.8, 70.5, 70.1,
786 69.3, 69.21, 69.18, 67.9, 65.3, 63.0, 61.3, 55.5, 21.2, 20.8, 20.0; FT-IR (ATR): ν 1747 (s), 1723 (s) cm⁻¹;
787 HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₆₇H₇₀NaO₂₁⁺: 1233.4302, found: 1233.4280.

788
789 4.2.34 Methyl 2,3,6-tri-O-benzyl-4-O-[3-O-(4,6-O-benzylidene-α-D-galactopyranosyl)-β-D-galactopyranosyl]-α-D-galactopyranoside 43

791 To a solution of the trisaccharide **42** (782 mg, 0.646 mmol) in MeOH (15 mL) was added 1 M NaOMe
792 in MeOH (0.4 mL, 0.4 mmol) at room temperature and the mixture was stirred at 40 °C for 5 d. The
793 reaction mixture was then neutralised with Amberlite resin (IR-120, H⁺ form) at room temperature, and
794 the mixture was filtered and concentrated. The resultant residue was purified by flash chromatography
795 (MeOH:CHCl₃, 1:19) to give the polyol **43** as a white foam (436 mg, 77%). Rf 0.51 (MeOH:CHCl₃,
796 1:9); ¹H NMR (400 MHz, CD₃OD): δ 7.56-7.51 (m, 2H), 7.44-7.24 (m, 18H), 5.60 (s, 1H), 5.08 (d, J =
797 3.5 Hz, 1H), 4.78-4.72 (m, 4H), 4.67 (d, J = 11.7 Hz, 1H), 4.61-4.54 (m, 3H), 4.31 (d, J = 2.8 Hz, 1H),
798 4.24 (d, J = 3.6 Hz, 1H), 4.22-4.19 (m, 1H), 4.10-3.97 (m, 5H), 3.96-3.88 (m, 3H), 3.81 (dd, J = 10.5,
799 5.0 Hz, 1H), 3.78-3.69 (m, 2H), 3.67-3.59 (m, 3H), 3.46-3.41 (m, 1H), 3.37 (s, 3H); ¹³C NMR
800 (100.6 MHz, CD₃OD): δ 139.82, 139.80, 139.78, 139.5, 129.8, 129.5, 129.41, 129.37, 129.22, 129.19,
801 129.0, 128.93, 128.85, 128.8, 128.6, 127.5, 105.7, 102.3, 99.8, 98.2, 79.8, 79.7, 78.2, 77.9, 76.7, 76.4,
802 74.5, 74.4, 74.2, 71.8, 71.1, 70.5, 70.1, 70.0, 66.5, 64.4, 62.3, 55.6; FT-IR (ATR): ν 3410 (br) cm⁻¹; HR-
803 MS (ESI) *m/z* [M+Na]⁺ calculated for C₄₇H₅₆NaO₁₆⁺: 899.3461, found: 899.3452.

804
805 4.2.35 Methyl 2,3,6-tri-O-benzyl-4-O-[2,4,6-tri-O-benzyl-3-O-(2,3-di-O-benzyl-4,6-O-benzylidene-α-D-galactopyranosyl)-β-D-galactopyranosyl]-α-D-galactopyranoside **44**

807 To a solution of the polyol **43** (418 mg, 0.477 mmol) in DMF (5 mL) were added NaH 60% w/w
dispersion in oil (156 mg, 3.9 mmol) and BnBr (0.32 mL, 2.7 mmol) at room temperature and the
mixture was stirred for 1.5 h. The reaction was then quenched with MeOH at 0 °C and the mixture was concentrated. The residue was taken up in EtOAc (50 mL) and was washed with water (30 mL), brine (30 mL), dried (MgSO₄), filtered, and concentrated. The resultant residue was purified by flash chromatography (EtOAc:hexanes, 1:3) to give the trisaccharide **44** as a white foam (577 mg, 91%). R<sub>f</sub> 0.37 (EtOAc:hexanes, 3:7); ¹H NMR (400 MHz, CDCl<sub>3</sub>): δ 7.49–6.99 (m, 45H), 5.18 (s, 1H), 5.17–5.09 (m, 3H), 4.86 (d, *J* = 11.4 Hz, 1H), 4.84 (d, *J* = 11.4 Hz, 1H), 4.80–4.64 (m, 5H), 4.60–4.54 (m, 3H), 4.46 (d, *J* = 12.0 Hz, 1H), 4.37–4.27 (m, 5H), 4.23 (d, *J* = 2.9 Hz, 1H), 4.10 (dd, *J* = 10.1, 3.3 Hz, 1H), 3.99–3.87 (m, 3H), 3.85–3.79 (m, 2H), 3.78–3.58 (m, 7H), 3.54–3.34 (m, 6H), 3.00 (dd, *J* = 12.5, 1.5 Hz, 1H); ¹³C NMR (125.8 MHz, CDCl<sub>3</sub>): δ 139.6, 139.2, 138.93, 138.90, 138.7, 138.6, 138.5, 138.2, 138.1, 128.9, 128.6, 128.49, 128.48, 128.44, 128.38, 128.34, 128.32, 128.2, 128.1, 128.0, 127.93, 127.91, 127.82, 127.81, 127.76, 127.72, 127.68, 127.66, 127.63, 127.60, 127.55, 127.5, 127.1, 126.5, 103.7, 100.9, 98.8, 95.9, 78.8, 78.4, 77.6, 77.0, 76.0, 75.8, 74.8, 74.7, 74.6, 74.4, 73.7, 73.6, 73.5, 73.4, 73.1, 72.9, 71.7, 70.5, 69.7, 69.2, 68.8, 62.3, 55.5; FT-IR (ATR): ν 2907 (w), 2861 (w) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C<sub>82</sub>H<sub>86</sub>NaO<sub>16</sub>⁺: 1349.5808, found: 1349.5796.

4.2.36 Methyl 2,3,6-tri-O-benzyl-4-O-[2,4,6-tri-O-benzyl-3-O-(2,3,6-tri-O-benzyl-α-D-galactopyranosyl)-β-D-galactopyranosyl]-α-D-galactopyranoside **45**

To a solution of the trisaccharide **44** (568 mg, 0.428 mmol) in THF (7 mL) were added molecular sieves 4 Å (0.6 g) and 95% w/w NaCNBH₃ (285 mg, 4.3 mmol) and the mixture was stirred at room temperature. To the mixture was then added dropwise 2 M HCl in Et₂O (2 mL, 4 mmol) and the mixture was stirred for 30 min. The reaction mixture was then diluted with CH₂Cl₂ (6 mL), neutralised with NEt₃, and filtered through Celite. The filtrate was washed with water (2 x 40 mL), sat. aq. NaHCO₃ (2 x 40 mL), brine (40 mL), dried (MgSO₄), filtered, and concentrated. The resultant residue was purified by flash chromatography (EtOAc:hexanes, 3:7) to give the alcohol **45** as a colourless oil (480 mg, 84%). R<sub>f</sub> 0.54 (EtOAc:hexanes, 2:3); ¹H NMR (600 MHz, CDCl₃): δ 7.40–7.03 (m, 45H), 5.14 (d, *J* = 3.5 Hz, 1H), 5.10 (d, *J* = 11.7 Hz, 1H), 5.07 (d, *J* = 10.8 Hz, 1H), 4.85–4.78 (m, 3H), 4.71–4.65
835 (m, 4H), 4.60–4.54 (m, 3H), 4.47–4.39 (m, 3H), 4.36–4.26 (m, 5H), 4.23 (d, *J* = 2.3 Hz, 1H), 4.11–4.08
836 (m, 1H), 4.02 (dd, *J* = 9.9, 3.4 Hz, 1H), 3.93–3.87 (m, 2H), 3.86–3.82 (m, 2H), 3.80 (dd, *J* = 9.9, 3.0 Hz, 1H), 3.74–3.70 (m, 3H), 3.68 (dd, *J* = 10.6, 4.7 Hz, 1H), 3.61 (dd, *J* = 10.6, 7.1 Hz, 1H), 3.51–3.47 (m, 1H), 3.46–3.42 (m, 1H), 3.40–3.33 (m, 5H), 3.16–3.14 (m, 1H), 3.06 (dd, *J* = 10.5, 4.5 Hz, 1H); ¹³C NMR (150.9 MHz, CDCl₃): δ 139.5, 139.2, 139.0, 138.7, 138.6, 138.5, 138.2, 137.9, 128.6, 128.49, 128.46, 128.4, 128.3, 128.23, 128.20, 128.1, 128.0, 127.89, 127.86, 127.84, 127.80, 127.79, 127.7, 127.64, 127.62, 127.60, 127.51, 127.47, 127.1, 103.4, 98.8, 95.6, 78.8, 78.6, 77.7, 77.5, 77.0, 75.9, 74.9, 74.6, 74.5, 73.7, 73.6, 73.50, 73.49, 73.4, 73.0, 72.9, 72.8, 71.9, 70.6, 70.4, 69.7, 69.0, 68.8, 67.8, 55.4; FT-IR (ATR): ν 3486 (br) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₈₂H₈₈NaO₁₆⁺: 1351.5965, found: 1351.5945.

845
846 4.2.37 Methyl 4-O-{3-O-[4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-benzyl-α-D-galactopyranosyl]-2,4,6-tri-O-benzyl-β-D-galactopyranosyl}-2,3,6-tri-O-benzyl-α-D-galactopyranoside
847
848 **46**

849 To a solution of the trichloroacetimidate **7** (206 mg, 0.418 mmol) and the alcohol **45** (460 mg, 0.346 mmol) in CH₂Cl₂ (7 mL) was added molecular sieves 4 Å (0.9 g) and the mixture was stirred at 850 room temperature for 30 min. To the mixture was then added TMSOTf (11 µL, 0.061 mmol) at -20 °C 851 and the mixture was stirred and allowed to return to 0 °C over 1 hr. The reaction was then quenched 852 with NEt₃, and the mixture filtered through Celite. The filtrate was washed with water (40 mL), brine 853 (40 mL), dried (MgSO₄), filtered, and concentrated. The resultant residue was purified by flash 854 chromatography (EtOAc:hexanes, 3:7) to give the tetrasaccharide **46** as a white foam (433 mg, 75%). Rf 855 0.49 (EtOAc:hexanes, 2:3); ¹H NMR (600 MHz, CDCl₃): δ 7.35–7.04 (m, 45H), 5.31 (dd, *J* = 3.4, 0.9 856 Hz, 1H), 5.18 (d, *J* = 3.2 Hz, 1H), 5.14 (dd, *J* = 10.4, 7.8 Hz, 1H), 5.05 (d, *J* = 11.4 Hz, 1H), 4.99 (d, *J* = 857 11.6 Hz, 1H), 4.94 (dd, *J* = 10.5, 3.5 Hz, 1H), 4.87 (d, *J* = 6.7 Hz, 1H), 4.75–4.69 (m, 2H), 4.67–4.57 858 (m, 6H), 4.55–4.49 (m, 3H), 4.44 (d, *J* = 12.0 Hz, 1H), 4.37–4.21 (m, 8H), 4.03 (dd, *J* = 11.1, 7.7 Hz, 859 1H), 3.91–3.79 (m, 7H), 3.78–3.58 (m, 6H), 3.50–3.40 (m, 4H), 3.39–3.33 (m, 4H), 2.12 (s, 3H), 1.99 860
(s, 3H), 1.97 (s, 3H), 1.79 (s, 3H); ¹³C NMR (150.9 MHz, CDCl₃): δ 170.5, 170.4, 170.3, 169.8, 139.6, 139.4, 139.0, 138.8, 138.74, 138.69, 138.5, 138.4, 138.2, 128.6, 128.54, 128.47, 128.44, 128.39, 128.34, 128.29, 128.2, 128.1, 127.92, 127.90, 127.83, 127.78, 127.76, 127.74, 127.72, 127.60, 127.57, 127.53, 127.48, 127.45, 127.4, 127.2, 103.0, 102.1, 98.8, 95.4, 78.78, 78.75, 78.5, 77.6, 77.0, 76.2, 74.6, 74.42, 74.37, 73.8, 73.5, 73.4, 73.14, 73.08, 73.05, 73.0, 72.9, 72.2, 71.1, 70.4, 70.3, 69.7, 69.4, 69.3, 68.81, 68.79, 66.9, 61.1, 55.3, 20.9, 20.80, 20.78, 20.76; FT-IR (ATR): ν 1747 (s) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₉₆H₁₀₆NaO₂₅⁺: 1681.6915, found: 1681.6876.

4.2.38 Methyl 2,3,6-tri-*O*-benzyl-4-*O*-{2,4,6-tri-*O*-benzyl-3-*O*-[2,3,6-tri-*O*-benzyl-4-*O*-(*β*-D-galactopyranosyl)-*α*-D-galactopyranosyl]-*β*-D-galactopyranosyl}-*α*-D-galactopyranoside **47**

To a solution of the tetrasaccharide **46** (425 mg, 0.256 mmol) in MeOH (8 mL) was added 1 M NaOMe in MeOH (0.15 mL, 0.15 mmol) at 0 °C and the mixture was stirred at room temperature for 21 h. The reaction mixture was then neutralised with Amberlite resin (IR-120, H⁺ form) and the mixture was filtered and concentrated. The resultant residue was purified by flash chromatography (MeOH:CHCl₃, 3:97) to give the tetrol **47** as a white foam (336 mg, 88%). R<sub>f</sub> 0.32 (MeOH:CHCl₃, 1:19); ¹H NMR (400 MHz, CD₃OD): δ 7.41–7.05 (m, 45H), 5.28 (d, *J* = 3.4 Hz, 1H), 4.99 (d, *J* = 11.1 Hz, 1H), 4.94 (d, *J* = 11.3 Hz, 1H), 4.89–4.85 (m, 1H), 4.79 (d, *J* = 11.3 Hz, 1H), 4.71–4.59 (m, 7H), 4.54–4.19 (m, 12H), 4.15 (dd, *J* = 10.1, 3.3 Hz, 1H), 4.03–3.77 (m, 8H), 3.72–3.36 (m, 13H), 3.34 (s, 3H); ¹³C NMR (150.9 MHz, CD₃OD): δ 140.5, 140.4, 140.1, 139.81, 139.79, 139.7, 139.64, 139.57, 139.5, 129.6, 129.44, 129.38, 129.35, 129.34, 129.33, 129.27, 129.2, 129.14, 129.12, 129.08, 128.93, 128.87, 128.82, 128.81, 128.78, 128.7, 128.6, 128.53, 128.48, 128.3, 105.4, 104.2, 99.8, 96.6, 79.9, 79.8, 79.5, 78.7, 78.1, 78.0, 76.7, 76.1, 75.9, 75.7, 75.5, 75.0, 74.6, 74.44, 74.36, 74.3, 73.7, 73.6, 73.5, 73.1, 71.5, 70.9, 70.5, 70.2, 70.0, 62.1, 55.6; FT-IR (ATR): ν 3433 (br) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₈₈H₉₈NaO₂₁⁺: 1513.6493, found: 1513.6444.
4.2.39 Methyl 4-O-{3-O-[4-O-(β-D-galactopyranosyl)-α-D-galactopyranosyl]-β-D-galactopyranosyl}-α-D-galactopyranoside 5

To a solution of the tetrol 47 (329 mg, 0.221 mmol) in MeOH (30 mL) was added 10% w/w Pd/C (285 mg) and the mixture was stirred at 40 °C under an atmosphere of H₂. At 4 d the reaction mixture was filtered through Celite and concentrated. The resultant residue was purified by flash chromatography (CHCl₃:MeOH:H₂O, 5:4:1) to give the title compound 5 as a white foam (56 mg, 37%). The remaining residue of the reaction was treated under the same conditions to give additional mass (60 mg, 40%). R_f 0.12 (CHCl₃:MeOH:H₂O, 5:4:1); ¹H NMR (600 MHz, D₂O): δ 5.16 (d, J = 3.9 Hz, 1H), 4.88-4.86 (m, 1H), 4.66 (d, J = 7.9 Hz, 1H), 4.61 (d, J = 7.9 Hz, 1H), 4.30-4.23 (m, 3H), 4.16 (d, J = 3.2 Hz, 1H), 4.06 (dd, J = 10.4, 3.1 Hz, 1H), 3.99-3.94 (m, 2H), 3.93-3.89 (m, 3H), 3.87-3.66 (m, 13H), 3.60 (dd, J = 9.9, 7.9 Hz, 1H), 3.43 (s, 3H); ¹³C NMR (150.9 MHz, D₂O): δ 105.1, 104.9, 100.1, 96.2, 79.2, 78.9, 78.3, 75.8, 75.5, 73.5, 72.1, 70.8, 70.74, 70.67, 70.6, 70.4, 69.4, 69.3, 69.2, 65.6, 61.63, 61.57, 61.1, 55.8; FT-IR (ATR): v 3339 (br) cm⁻¹; HR-MS (ESI) *m/z* [M+Na]⁺ calculated for C₂₅H₄₄NaO₂₁⁺: 703.2267, found: 703.2258.

900

901

CRediT authorship contribution statement

902 **Fayliesha Spyker**: Writing – review & editing, Writing – original draft, Methodology, Formal analysis, 903 Data curation. **Elizabeth Ficko-Blean**: Writing – review & editing, Writing – original draft, Project 904 administration, Funding acquisition, Conceptualisation. **Keith A Stubbs**: Supervision, Writing – review 905 & editing, Writing – original draft, Project administration, Investigation, Formal analysis, 906 Conceptualisation.

907

908

Declaration of competing interest

909 The authors have no conflicts to declare.

910
911 Acknowledgements

912 FS and KAS acknowledge the facilities, and the scientific and technical assistance of Microscopy

913 Australia at the Centre for Microscopy, Characterisation and Analysis, The University of Western

914 Australia, a facility funded by the University, State and Federal Commonwealth Governments. FS is

915 supported by a Research Training Program Scholarship provided by the Australian Federal Government

916 and The University of Western Australia. FS is also the holder of the Hackett Postgraduate Research

917 Scholarship. EF-B acknowledges support from the Agence National de la Recherche (ANR) through the

918 ANR Mirror Mirror (ANR-22-CE11-0025-01).

919

920 Appendix A. Supplementary data

921 Supplementary data to this article can be found online at [Insert Doi here].

922

923 Data availability

924 The data supporting this article have been included as part of the Supplementary Information.

925

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