Tetrasaccharide iteration synthesis of a heparin-like dodecasaccharide and radiolabelling for in vivo tissue distribution studies

Abstract

Heparin-like oligosaccharides mediate numerous important biological interactions, of which many are implicated in various diseases. Synthetic improvements are central to the development of such oligosaccharides as therapeutics and, in addition, there are no methods to elucidate the pharmacokinetics of structurally defined heparin-like oligosaccharides. Here we report an efficient two-cycle [4+4+4] tetrasaccharide-iteration-based approach for rapid chemical synthesis of a structurally defined heparin-related dodecasaccharide, combined with the incorporation of a latent aldehyde tag, unmasked in the final step of chemical synthesis, providing a generic end group for labelling/conjugation. We exploit this latent aldehyde tag for ³ H radiolabelling to provide the first example of this kind of agent for monitoring in vivo tissue distribution and in vivo stability of a biologically active, structurally defined heparin related dodecasaccharide. Such studies are critical for the development of related saccharide therapeutics, and the data here establish that a biologically active, synthetic, heparin-like dodecasaccharide provides good organ distribution, and serum lifetimes relevant to developing future oligosaccharide therapeutics.

ARTICLE

Received 13 Oct 2012 | Accepted 16 May 2013 | Published 5 Jul 2013

DOI: 10.1038/ncomms3016 OPEN

Tetrasaccharide iteration synthesis of a heparin-like dodecasaccharide and radiolabelling for in vivo tissue distribution studies

Steen U. Hansen1,*, Gavin J. Miller1,*, Claire Cole2, Graham Rushton2, Egle Avizienyte2, Gordon C. Jayson2

& John M. Gardiner1

Heparin-like oligosaccharides mediate numerous important biological interactions, of which many are implicated in various diseases. Synthetic improvements are central to the development of such oligosaccharides as therapeutics and, in addition, there are no methods to elucidate the pharmacokinetics of structurally dened heparin-like oligosaccharides. Here we report an efcient two-cycle [4 4 4] tetrasaccharide-iteration-based approach for rapid

chemical synthesis of a structurally dened heparin-related dodecasaccharide, combined with the incorporation of a latent aldehyde tag, unmasked in the nal step of chemical synthesis, providing a generic end group for labelling/conjugation. We exploit this latent aldehyde tag for 3H radiolabelling to provide the rst example of this kind of agent for monitoring in vivo tissue distribution and in vivo stability of a biologically active, structurally dened heparin related dodecasaccharide. Such studies are critical for the development of related saccharide therapeutics, and the data here establish that a biologically active, synthetic, heparin-like dodecasaccharide provides good organ distribution, and serum lifetimes relevant to developing future oligosaccharide therapeutics.

1 Faculty of EPS, School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

2 School of Cancer and Enabling Sciences, The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.M.G. (email: mailto:[email protected]

Web End [email protected] ).

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Heparin and heparan sulphate (H/HS) are ubiquitous linear polysulphated oligosaccharides of the glycosaminoglycan (GAG) family, comprising a repeating disaccharide unit.

Because of its structural heterogeneity, H/HS is crucially involved in regulating a myriad of cell signalling pathways through modulation of interactions between cytokines and their receptors. This is typied through its involvement in the mediation of broblast growth factor (FGF)-regulated cell phenotypes, such as proliferation, adhesion, motility and angiogenesis17.

Although methods for the isolation of natural H/HS samples from biochemical degradation processes are well-established8,9, synthetic access to structurally dened H/HS mimetics has also received very signicant attention1036. Synthetic access is essential to provide structurally dened H/HS oligosaccharide sequences to interrogate the chemical biology of H/HS-mediated processes, a better understanding of which also offers the potential to aid development of new disease treatments3741. The potential development of such oligosaccharides as therapeutics is also dependent on developing tools to determine the pharmacokinetics, distribution and organ availability of these synthetic species. To date, the limitations of synthetic access to suitable tools has precluded such developments for bioactive lead oligosaccharide structures. This presents the need to develop an efcient procedure for the synthesis of longer bioactive heparin-like oligosaccharides, which also provide efcient access to derivatization/conjugation of structurally dened, biologically signicant synthetic H/HS sequences4245.

Our previous in vitro and in vivo anti-angiogenic assessments of size-fractionated digests4651 and subsequent evaluation of a matrix of structurally dened synthetic oligosaccharides52 indicated that longer [GlcNS-IdoA2S]-containing species were more effective inhibitors of FGF2, and identied the methyl glycoside analogue of dodecasaccharide 1 ([GlcNS-IdoA2S]6-

OMe) as the optimum lead FGF2 and vascular endothelial growth factor antagonist52 (ex vivo evaluation of the synthetic dodecasaccharide [GlcNS-IdoA2S]6-OMe conrmed that at biologically active concentrations inhibiting FGF signalling, there was no statistically signicant impact on anticoagulation, an important feature required for potential development of therapeutic synthetic saccharides of this type).

Here we report a powerful addition to the eld of synthetic heparanoid chemistry, which demonstrates an efcient chemical synthesis of this structurally dened [GlcNS-IdoA2S]6 heparin-like lead dodecasaccharide 1, bearing a terminal latent aldehyde tag (LAT), in just two iterative cycles and four steps from a precursor tetrasaccharide. Concomitant LAT release in a nal-step modication of the oligosaccharide is applied to 3H radiolabelling of dodecasaccharide 1 with minimal structural impact. This demonstrates the viability of the approach for rapid, iterative synthetic access to large oligosaccharides on useful scales, which are suitable for biological conjugations and labelling. The tritium radiolabelled analogue 1, which provides a new tool to determine the pharmacokinetics of the synthetic oligosaccharide and establish the organ distribution and in vivo lifetime of the lead dodecasaccharide 1, critical factors for the potential development of HS oligosaccharide therapeutics.

ResultsStrategy and end labelling. The end modication of oligosaccharides (via ring opening of the terminal acetal unit, for example, for attaching uorophores) is an established method for labelling native heparin and related GAGs to facilitate their separation or analysis53,54. A number of examples have also employed amide derivatization of the uronic carboxylates to introduce uorescent or spin labels, or to attach conjugates55,56.

There are also a range of uses of modied O-glycosides recently employed for conjugation, surface and nanoparticle/dendrimer attachments4245, including applications of click chemistry, in particular the Huisgen reaction.

However, an alternative approach was needed to ensure compatibility with the deprotection/labelling conditions during synthesis and introducing minimal change to the polarity/ functional groups of the oligosaccharide. Thus, a 1,2-diol moiety at the reducing end was incorporated as an LAT. Having an additional O4-sulphate at the non-reducing end of the deprotected dodecasaccharide would allow complete selectivity in a nal-stage periodate-mediated cleavage of the LAT to liberate a reactive aldehyde tag (RAT) directly on the nal oligosaccharide, thus allowing facile reductive labelling or facilitating other conjugations. Our approach was to develop this LAT incorporation concurrently with the aforementioned tetrasaccharide iteration strategy (Fig. 1).

Synthesis of tetrasaccharide building blocks. The synthetic strategy envisaged using one precursor disaccharide building block, 2, to provide a single tetrasaccharide unit, 7, which would function both as an iterative donor (as its O4-trichloroacetyl derivative 8) and as an immediate precursor to an LAT-bearing tetrasaccharide, serving as the initial acceptor tetrasaccharide.

G-I-G-I

1

a

4

HO LAT

4

HO

X

HO LAT

G-I-G-I

1

O

4

Deprotect

X=SPh

PO

G-I-G-I

1

X

2 cycles

Couple

12

9

8

4

PO

G-I-G-I

O

G-I-G-I

5

O LAT

G-I-G-I

1

O

Deprotect, O/N sulfate, release RAT

12

SO RAT

G-I-G-I

O

G-I-G-I

O

G-I-G-I

1

O

(NS)

(OS) (OS) (OS)

(OS)

(NS)

(NS)

(OS)

12

SO

G-I-G-I

O

G-I-G-I

O

G-I-G-I

1

O

H

(NS) (NS) (NS)

b

OH

O

NaO3SO

HO

NaSO3HN

O OSO3Na

NaO2C

OH

O

OH

O

O

HO

NaSO3HN

1

O OSO3Na

NaO2C

OH

O

OH

O

3H

3

Figure 1 | Strategy for synthesis of end-labelled dodecassacharide.(a) Iterative [4 4 4] oligosaccharide synthesis strategy with nal step

RAT release and labelling. G, Glucosamine unit; I, iduronate unit; P, trichloroacetyl; S, SO3Na; LAT, latent aldehyde tag; RAT, reactive aldehyde tag. (b) Structure of radiolabelled dodecasaccharide.

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Disaccharide 2 was prepared as reported previously, exploiting our scalable iduronate thioglycoside acceptor capabilities for constructing such reagents57,58, and is a reverse of the common IdoA-GlcN disaccharide repeat unit seen in several previous heparin-related syntheses1036. Disaccharide 2 was divergently elaborated into trichloroacetimidate donor 4 (see Supplementary Figs S5S12) via free sugar disaccharide 3 (see Supplementary Figs S1S4) and acceptor 5, with these two building blocks then efciently coupled to afford tetrasaccharide 6 (Fig. 2; see Supplementary Figs S13S17). Deprotection at the non-reducing end terminus of 6 then provided the required tetrasaccharide 7 (see Supplementary Figs S18S22), which was also protected as its O4-TCA derivative 8 (see Supplementary Figs S23S31), thereby providing two potential tetrasaccharide donor modules (6 and 8), differing only in their non-reducing terminal O4-protecting group.

Synthesis of tetrasaccharide 6 was similarly efcient whether thioglycoside acceptor a-5 or b-5 was employed, providing access to either tetrasaccharide a-7 or b-7, respectively, both of which can function in the subsequent iterative homologations to effect the same a-selective glycosylations of the desired oligosaccharide acceptor. These tetrasaccharides can readily be accessed on hundreds of milligram to multigram scale.

Notably, and further enhancing the overall synthetic efciency, although 7 has both acceptor (4-OH) and donor (1-SPh) functionality, this material could be directly glycosylated at the reducing terminus without the need for protection at O4. The installation of the required LAT was thus effected using a dibenzylated glycerol unit, designed for its ultimate deprotection

to the required diol concomitant with the penultimate debenzylation of the protected heparin oligosaccharide. Hence, 7 was glycosylated with (S)-2,3-dibenzyloxy propanol affording the reducing end-modied tetrasaccharide acceptor 9 (see Supplementary Figs S32S36) directly, with the non-reducing terminus already in place as an acceptor for the rst tetrasaccharide homologation (Fig. 3).

Coupling of acceptor 9 and tetrasaccharide donors 6 or 8, hence, generated the octasaccharides 10 (see Supplementary Figs S37S39) and 11 (see Supplementary Figs S40S44), respectively, with complete a-anomeric selectivity. This now further establishes that the iduronate donor-terminated tetrasaccharides can function as efcient oligosaccharide homologation building blocks using longer acceptors (a previous GlcN-GlcA-GlcN-IdoA tetrasaccharide had been shown to be effective as a donor for monosaccharide acceptors)24 and compliments such a capability exploited using GlcN donor systems24,44. This also thereby underpins a capability to now access other long [GlcN-IdoA]n-

based sequences using such an accelerated iterative strategy.

Synthesis of oligosaccharide via [4 4 4] two-cycle iteration.

During the deprotection of O4 of octasaccharide 10, it was found that the ceric ammonium nitrate mediated p-methoxybenzyl removal gave a mixture of products, although the TCA group of analogue 11 could be removed in excellent yield using novel mild conditions. Combined with the higher glycosylation yields obtained, this led to selection of the O4-TCA tetrasaccharide 8 for further iterations. Deprotection of octasaccharide 11 provided

OBn

OBn

PMBO

i ii

OH O

O

PMBO

O

BnO

BnO

N

N

O

OBz

OBn

OBn

O

OBn

87% 91%

NH

iv

O

RO

O

MeO C

OBn

O

OBz

PMBO

MeO C

OBn

O

O CCl

BnO

BnO

PMBO

BnO

N

N

N

3

4

O

O

O

OBz

OBn

MeO C

OBn

O

OBz

OBn

MeO C

OBn

O

OBz

iii

MeO C

OBn

O SPh

O

O

O

O

OBn

85% 67%

BnO

BnO

N

2

N

HO

O

O

OBz

O

OBz

BnO

6

N

iii

OBz

MeO C

OBn

MeO C

OBn

O

O SPh

O SPh

90%

MeO C

OBn

O SPh

v

7 (R = -H)

8 (R = -COCCl , 94%)

5

Figure 2 | Synthesis of core tetrasaccharide donor/acceptor modules. (i) NBS, acetone. (ii) CCl3CN, DBU, DCM. (iii) CAN, CH3CN/H2O. (iv) TMSOTf, DCM. (v) CCl3COCl, pyridine, DCM.

O

OBn

OBn

OBn

OBn

HO

O

HO

O

O

Cl C

O

BnO

BnO

RO

O

BnO

O

N

BnO

N

N

N

O

O

O

O

i

MeO C

OBn

O

OBz

OBn

MeO C

OBn

OBn

ii

MeO C

OBn

O

OBz

OBn

ii

MeO C

OBn

O

OBz

OBn

O

O

82% 78%

SPh

O

O

O

O

BnO

BnO

O

BnO

O

N

BnO

N

N

N

O

O

O

O

7

9

O

OBz

13

MeO C

OBn

O

OBz

MeO C

OBn

O

OBz

OBn

MeO C

OBn

O

OBz

OBn

MeO C

OBn

O

OBz

OBn

OBn

O

OBn

O

OBn

2

3

10 (R = -PMB, 81%)11 (R = -COCCl , 92%)

iii 12 (R = -H, 91%)

Figure 3 | Iterative [4 4 4] dodecasaccharide synthesis. (i) (S)-2,3-bis(benzyloxy)propanol, NIS, AgOTf (cat.), DCM. (ii) 6 or 8, NIS, AgOTf (cat.),

DCM. (iii) MeOH/pyridine (5:2).

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acceptor octasaccharide 12 (see Supplementary Figs S45S49) as a substrate, enabling a second cycle of iterative coupling with donor module 8, providing thereby protected dodecasaccharide 13 (see Supplementary Figs S50S54) and completing the efcient and rapid two-cycle iteration. The overall four-step yield from tetrasaccharide 7 to protected dodecasaccharide 13 was 54%, yielding around 300 mg of this oligosaccharide, a signicant scale for such rapid dodecasaccharide assembly.

From protected dodecasaccharide 13, concurrent saponication, O-sulphation, debenzylation/azide reduction and nally N-sulphation provided the fully deprotected and regiospecically sulphated dodecasaccharide 14 (see Supplementary Figs S59S64) bearing the free 1,2-diol LAT unit at the reducing terminus (Fig. 4).

Nuclear magnetic resonance provides unambiguous denition of the complete N-sulphation, evidenced by the clear difference in shift of the H-2 protons on converting the 2-amino to 2-NS functionality (Fig. 5). Differentiation of the NS of the

non-reducing terminal glucosamine 2-NS is evident (the only ring with O4-S) and conrmed by clear correlation spectroscopy correlations (see Supplementary Fig. S60). The clear resolution of signals for the reducing terminal iduronate is clear, and on oxidative cleavage the spectrum for the RAT-terminated dodecasaccharide shows a clear set of two doublets (1.3:1) for the RAT methylene. These are not mutually coupled and the non-equivalent integration would also be consistent with these arising from acetal formation, transiently retaining the LAT aldehyde in a seven-membered ring hemiacetal. This is also consistent with changes in the shifts of H5 and H2 of the reducing terminal iduronate. In addition, the H-5 of ring A is well separated and the small coupling constant for those protons, also evident for the other overlapping H5 protons, shows that these long sulphated oligosaccharides do sit largely in the ido 1C4 conformation.

Oxidative cleavage was effected in near-quantitative yield with sodium periodate, unveiling the target-reducing end aldehyde in the form of reactive conjugate, 15 (see Supplementary Figs S65S67).

OBn

OH

OH

OH

Cl C

O

O

O

NaO SO

O

NaO SO

O

NaO SO

O

BnO

HO

NaSO HN

HO

NaSO HN

HO

NaSO HN

N

O MeO C

OBn

O NaO C

OH

O NaO C

OH

O NaO C

OH

O

OSO Na

OH

O

OBz

OBn

i-iv

O

OSO Na

OH

v

vi or vii

O

OSO Na

OH

O

O

O

O

57% 97% 98%

O

O

O

O

BnO

HO

NaSO HN

HO

NaSO HN

HO

NaSO HN

N

O MeO C

OBn

O NaO C

OH

O NaO C

OH

O NaO C

OH

O

OSO Na

O

OSO Na

O

O

OSO Na

O

OBz

OH

OH

OBn

O

O

O

OBn

O

OH

X

13

3

14

3

16 (X = 1H) 1 (X = 3H)

15

3

3

Figure 4 | Dodecasaccharide deprotections and end labelling. (i) LiOH, tetrahydrofuran (THF)/MeOH/H2O, 74%. (ii) Py.SO3 complex, pyridine, 80%.(iii) H2, Pd(OH)2/C, MeOH/THF/H2O 2:1:1, 96%. (iv) Py.SO3 complex, NaHCO3, H2O, 100%. (v) NaIO4, H2O. (vi) NaBH4, H2O. (vii) NaB3H4, H2O.

a

Ido

H + H

H + H

H5

Glc

H4

H

b

Glucosamine H2 sulpated

Glucosamine H2

Ido

H2

H1 H5

H2

H2

c

Ido

H2

H + H1

H2

H2

H5

H

5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2. 8 2. 7

Figure 5 | Nuclear magnetic resonance conrmation of dodecasaccharide N-sulphation and comparisons of diol LAT and oxidatively cleave RAT. (a) Intermediate amino containing dodecasaccharide. (b) N-sulphated dodecasaccharide. (c) Oxidatively cleaved RAT-terminated oligosaccharide.

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To introduce the tritium label, 15 was then treated with NaB3H4 under basic conditions at 45 C (following cold-label method evaluations on a disaccharide model and the unlabelled reduction of 15 to 16 (see Supplementary Figs S68S70)).

After ensuring the reaction was complete by addition of excess NaBH4 and quenching, the sample was desalted on Sephadex

G-25 to remove excess reducing agent. Radiolabelled 1 was then further puried by size-exclusion chromatography (Fig. 6) and

900

800

700

600

500

c.p.m. 3 H

400

300

200

100

Vo

Vt

0 10 15 20 25 30 35 40 45

Figure 6 | HPLC size-exclusion chromatogram of 1 on Superdex. To conrm the oligomer size of 3H-1 HPLC size-exclusion chromatography on Superdex 75 indicated the 3H-1 elutes (red arrow) at position identical to de-6-O-sulphated dodecasaccharide heparin standard. Vo, excluded volume; Vt, total column volume.

Fraction number

20

60

Brain Lungs Heart Spleen Kidney Liver Plasma

(g) Oligosaccharide per ml tissue

a

Brain Lungs Heart Spleen Kidney Liver Plasma

(g) Oligosaccharide per ml tissue

20 mg kg1

b

40 mg kg1

15

45

10

30

5

15

0 15 30 60 120 240 480 960

0 15 30 60 120 240 480 960

Mins

Mins

100

Brain Lungs Heart Spleen Kidney Liver Plasma

c

(g) Oligosaccharide per ml tissue

80 mg kg1

75

50

25

0 15 30 60 120 240 480 960

Mins

Figure 7 | Tissue localisation of 1. Mice (two) were injected with 20 (a), 40 (b) or 80 mg kg 1 (c) of 1. Tissue quantities are presolublization. Error bars represent the s.e.m. of c.p.m. converted to oligo weight using specic activity.

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300

250

200

c.p.m. 3 H

150

100

Vo

50

Vt

0 10 15 20 25 30 35 40 45 Fraction number

Figure 8 | HPLC SEC of 1 injected s.c. into mice and then extracted and puried from kidneys 4 h after injection. Radiolabelled oligosaccharide 1 was injected s.c. into mice and then extracted and puried from kidneys 4 h after injection (see Methods). Radiolabelled oligosaccharide 1 was subjected to HPLC chromatography on a Superdex 75 size-exclusion column. Red arrow shows elution of 3H-1 (see Fig. 5). Vo, excluded volume; Vt, total column volume.

was eluted from the column in good accordance with an established de-6-O-sulphated dodecasaccharide heparin standard. The specic activity of 1 was determined to be 5.5

106 c.p.m. mg 1 of oligosaccharide, suitable for the required in vivo evaluations.

Applications of radiolabelled heparin dodecasaccharide 1. Radiolabelled heparin-mimetic 1 was thus employed to determine its in vivo clearance and tissue distribution in mice (the biological efcacy of the OMe analogue of which we had previously established in vitro)52.

The mice were dosed subcutaneously (s.c.) with 20, 40 and 80 mg kg 1 of oligosaccharide spiked with 140,000 c.p.m. of 3H-12-mer 1 as a radiotracer. Tissue concentrations were determined from the level of radiolabelled oligosaccharide in tissue samples of known mass during a 16-h period (Fig. 7). A maximum plasma concentration of 18 mg ml 1 was observed 15 min after dosing mice with 20 mg kg 1 of oligosaccharide (Fig. 6a). When dosed with 40 or 80 mg kg 1, maximum plasma concentrations of 44.8 and 84.0 mg ml 1, respectively, were observed after 60 min (Fig. 6b,c). Critically, these data demonstrate that the plasma concentration of oligosaccharide in vivo was sufcient to inhibit the biological activity of FGF2 based on in vitro data52.

All tissues, except lungs, showed a time-dependent accumulation of 1 at all doses, and the maximum concentrations increased with a higher initial dose (Fig. 6). The highest concentration of 1 was detected in tissues when mice were treated with 80 mg kg 1.

In addition, all tissues, except the liver, attained maximal levels of 1 by 120 min. The tissue distribution data described here show that at 40 and 80 mg kg 1, biologically active concentrations52 were achieved in the liver, lungs and spleen within a 2-h period.

The half-life of oligosaccharides in mice was estimated to be B2 h. As murine clearance is so rapid, this result is particularly encouraging for the development of oligosaccharide therapeutics.

From the results shown in Fig. 6, there is good evidence for sustained plasma concentrations of 1 up to around 1 h at the two higher dose levels and that the oligosaccharide is well retained in the plasma at these concentrations. At lower doses, there is a more even distribution among the examined tissues, suggesting that higher doses would be needed to sustain sufcient oligosaccharide concentration in plasma.

To assess the metabolic stability of 1 in vivo, we extracted and puried 3H-12-mer 1 from mouse kidney after a 4-h treatment, to determine the extent of any degradation or metabolism. The majority of the material eluted in a single peak of 3,500 Da, which corresponds to a mass of dodecasaccharide 1 (Fig. 8).

DiscussionThis [4 4 4] iterative oligosaccharide approach thus provides an

efcient two-cycle synthesis of a structurally dened HS dodecasaccharide, illustrating that iduronate donor tetrasaccharides function as effective and selective glycosyl donors with extended saccharide acceptors. The reduction here in the number of synthetic steps taken to assemble these longer heparanoids signicantly enhances their accessibility, and the inclusion of the end label via glycosylation (avoiding O4 protection) to directly afford an end-tagged tetrasaccharide acceptor also adds to the abbreviation of the synthetic route. Notably, homologation here using 4-mer and 8-mer acceptors with this longer donor is shown to perform with an efciency that remains high, even for a [4 8] coupling. The

efciency of this tetrasaccharide-iteration-based synthetic route also facilities the viability of new opportunities for sequence versatility and applications to other diverse conjugation targets.

The synthesis and inclusion of the LAT described here offers a unique strategy for discrete end labelling of this heparin-like structure, such that the label will not interfere with the ligand-binding potential of the molecule, and the small structural change would be anticipated to minimize effects on pharmacokinetics. The end-terminal latent tag offers generality for incorporation into other heparanoids.

Utility of this end group for radiolabelling provides the rst example of using a structurally dened heparin-like dodecasaccharide to quantify in vivo tissue distribution and metabolic stability. Conventional pharmacological development of heparin has relied on its anticoagulant properties that can be measured in patients using universally available tests of the clotting cascade. However, the lack of anticoagulant activity of structurally dened synthetic HS oligosaccharides, although important for nonanticoagulant drugs, also presents the problem of how to measure the pharmacokinetics and metabolism of such novel synthetic heparin-like oligosaccharides in vivo. Here we report for the rst time a novel solution to this problem that should greatly assist the

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preclinical and clinical development of potential oligosaccharide therapeutics. In this dodecasaccharide case, this study has shown that the dodecasaccharide has good in vivo stability, and strongly indicates that oligosaccharide drugs of this type have a high prospect of effective dose distribution and stability on therapeutically valid timescales.

Methods

Synthesis of dodecasaccharide 12. Octasaccharide 11 (346 mg, 0.10 mmol) was dissolved in a mixture of MeOH/pyridine (5 ml per 2 ml) and heated to 50 C for 4 h. The solvents were evaporated and coevaporated with toluene (2 20 ml). The crude

product was puried using ash column chromatography (EtOAc/hexane 1:2 and 3:5). This yielded 12 (300 mg, 91%) as a white foam, along with recovered starting material (20 mg, 5%). Rf 0.10 (EtOAc/hexane 1:2). [a]D20 9.8 (c 0.32, CH2Cl2).

Mass spectrometry (MS) matrix-assisted laser desorption/ionizationtime of ight: m/z: calcd for C181H184N12NaO47 [M Na] : 3300.2; found: 3300.2. Elemental

analysis calcd (%) for C181H184N12O47: C 66.29, H 5.66, N 5.13; found C 66.57,

H 5.98, N 4.96 (see Supplementary Methods for further characterization data).

Synthesis of dodecasaccharide 13. Acceptor 12 (252 mg, 0.077 mmol) and donor 8 (175 mg, 0.100 mmol) were dissolved in dry dichloromethane (DCM) (4 ml) under N2. Freshly activated 4 powdered molecular sieves (222 mg) were added and the solution cooled to 0 C in an ice bath. After 10 min NIS (47 mg, 0.21 mmol) was added, and after another 10 min AgOTf (catalytic amount) was added. The suspension changed colour from pale yellow to deep red and was stirred for a further 35 min. The reaction was quenched into a separating funnel containing a mixture of DCM (50 ml), saturated aqueous NaHCO3 (50 ml) and Na2S2O3 (5 ml, 10% aqueous). After shaking until the iodine colour was removed, the suspension was ltered through a short pad of Celite washing with water and DCM. The layers were separated and the aqueous layer extracted with DCM (10 ml). The organic layers were combined, dried (MgSO4) and the solvent removed in vacuo. The crude product was puried by silica gel ash column chromatography (EtOAc/hexane 7:13) to yield 13 (295 mg, 78%) as a white foam (recovered acceptor (37 mg, 15%)). [a]D20 18.1 (c 0.68, CH2Cl2). MS matrix-assisted laser desorption/ionization

time of ight: m/z: calcd for C265H265Cl3N18NaO70 [M Na] : 4946.7; found:

4946.6. Elemental analysis calcd (%) for C265H265Cl3N18O70: C 64.58, H 5.42,

N 5.12; found C 63.91, H 5.41, N 5.06 (see Supplementary Methods for further characterization data).

Synthesis of dodecasaccharide sodium salt 14. Saponications. Dodecasaccharide 13 (257 mg, 0.052 mmol) was dissolved in tetrahydrofuran (5 ml) and MeOH (1.5 ml), and then cooled to 0 C in an ice bath. Then, LiOH.H2O (55 mg,1.30 mmol) dissolved in 1 ml water was added dropwise over 10 min. The solution was stirred for 5 h at 0 C, and then at room temperature for another 19 h. The solution was then extracted with EtOAc (2 50 ml) and HCl (0.2 M, 40 ml), dried

(MgSO4), ltered and evaporated. The crude product was puried using ash column chromatography (DCM/MeOH 30:1). This yielded the carboxylic acid dodecasaccharide intermediate product A (174 mg, 74%) as a white solid. Rf 0.18 (DCM/MeOH 20:1), then used directly in the next step.

Sulphation of hydroxyls. The dodecasaccharide intermediate A (170 mg,0.042 mmol) was dissolved in dry pyridine (5 ml), pyridine sulphurtrioxide complex (140 mg, 0.88 mmol) added and then heated to 50 C in anoil bath for 8 h. The solution was stirred at room temperature for another 12 h. The reaction was quenched with MeOH and then evaporated. The crude productwas redissolved in MeOH/DCM (10 ml/5 ml), stirred with Amberlite IR-120 Na resin (1.3 g) for 8 h, ltered, resin washed with MeOH (2 5 ml) and the

ltrate evaporated. This residue was then puried using ash column chromatography (DCM/MeOH 20:1). This yielded 2-O-sulphated dodecasaccharide intermediate B (160 mg, 80%) as a white solid.

Hydrogenolysis of benzyls and azides. The IdoA2S benzylated 2-azido-containing dodecasaccharide intermediate B (132 mg, 0.027 mmol) was dissolved in a mixture of MeOH/tetrahydrofuran (4 ml/2 ml), and NaHCO3 (14 mg, 0.165 mmol)

dissolved in 2 ml of water was added, atmosphere exchanged for nitrogen and Pd(OH)2/C (120 mg, 1020%) added, and again ushed with nitrogen.

The nitrogen balloon was replaced with a hydrogen balloon and atmosphere replaced with hydrogen. The reaction was heated to 40 C in an oil bath for 48 h with vigorous stirring. The product mixture was ltered through Celite, washed with MeOH/water (3 3 ml) and water (3 3 ml). The combined ltrate was then

evaporated to give dodecasaccharide amine intermediate C (78 mg, 96%) as a glassy solid (see Supplementary Methods).

Sulphation of amines. The dodecasaccharide amine (65 mg, 0.022 mmol)was dissolved in water (4 ml), NaHCO3 (108 mg, 1.29 mmol) and pyridine sulphur trioxide complex (97 mg, 0.61 mmol) was added with vigorous stirring.

This procedure was repeated after 1.30, 3.30, 5.30, 17.30 and 19.30 h (NaHCO3:

109, 121, 113, 110 and 110 mg; Py.SO3: 92, 88, 111, 100 and 70 mg). After 24 h, the mixture was evaporated. The crude containing Na2SO4 salts was redissolved in minimum amount of water and puried by passage through a Sephadex G-25 column (40 ml) by eluting with water. The fractions containing oligosaccharide were pooled and evaporated to yield 14 (78 mg, 100%) of as a glassy solid. High-resolution MS (Fourier transform MS): m/z: calcd for C75H111N6Na4O102S13

[M-15Na 8H] 7: 462.4234; found: 462.4244 (see Supplementary Methods

for structures of intermediates A, B and C, and further characterization data for B, C and 14, and Supplementary Figs 5659 for spectra of AC).

Synthesis of dodecasaccharide aldehyde 15. The dodecasaccharide 14 (61 mg,0.017 mmol) was dissolved in water (1 ml) and sodium periodate (3.9 mg,0.018 mmol) was added and stirred for 6 h. The crude was puried by passage through a Sephadex G-25 column by eluting with water. The fractions containing oligosaccharide were pooled and evaporated to yield 58 mg (97%) of 15 as a glassy solid (see Supplementary Methods for characterization data).

Synthesis of 3H-labelled dodecasaccharide 1. Three micrograms of 3H-labelled sodium borohydride (1 mCi) was reacted with 1.2 mg of aldehyde-bearing dodecasaccharide 15 in 20 ml of 50 mM NaOH in a sealed reinforced glass Reacti-Vial at 45 C for 2 h. To ensure that all aldehyde was reduced, an excess of unlabelled 1 M sodium borohydride was then added and sample incubated for a further 2 h at 45 C. Reaction was halted by the addition of 5 ml of 1 M sulphuric acid. Tritium-labelled oligosaccharide was then desalted on PD-10 (G-25) column that was preequilibrated with water and 0.5 ml fractions were collected. Labelled oligosaccharide 1, which eluted in fractions 1014, was collected and freeze-dried. To conrm the size of radiolabelled material, the 3H-dodecasaccharide 1 was subjected to HPLC size-exclusion chromatography on an Agilent 1200 HPLC system. The sample was run on a Superdex 75 column (10 mm 300 mm; GE Healthcare) in

PBS at 0.5 ml min 1. Aliquots from 0.5 ml fractions were taken, mixed with 2 ml of Hisafe scintillation uid (Perkin-Elmer) and 3H level was counted on a Wallac 1400 scintillation counter. An unlabelled de-6-O-sulphated heparin dodecasaccharide (Iduron), which is approximately the same size as synthetic dodecasaccharide 1, was used to calibrate the column and was monitored at 232 nm by an in-line ultraviolet detector. Fractions containing 3H-dodecasaccharide 1 were collected, desalted, freeze-dried and weighed. Specic activity was determined as5.5 106 c.p.m. mg 1 of oligosaccharide.

Pharmacokinetic study of 3H-labelled dodecasaccharide 1 in mice. 3H-Labelled dodecasaccharide 1 was administered to SCID-bg female mice s.c. as a single dose at 20, 40 and 80 mg kg 1 and animals were culled at 0.25, 0.5, 1, 2, 4, 8 and 16 h after dosing. Two animals per each treatment group were used. At the time of culling, blood was collected by cardiac puncture. The brain, kidney, liver, spleen, heart and lungs were removed and their weight was measured. Samples were incubated overnight at 60 C in 4 ml of Soluene 350 (Perkin-Elmer). Aliquots of 500 ml of the resultant tissue solution were taken for scintillation counting. Plasma was obtained by centrifuging mouse blood at 1,500 r.p.m. in a bench top Eppendorf microcentrifuge and collecting the supernatant. Plasma (100 ml) was added to 2 ml of scintillation uid and 3H levels were determined by scintillation counting. Concentration of oligosaccharide in plasma and tissues was derived from the specic activity of the radiolabel (see above).

Extraction and purication of 3H-12-mer dodecasaccharide 1 from mouse kidney. To assess the stability of 3H-dodecasaccharide 1 in vivo, one animal was dosed with 1 at 20 mg kg 1 for 4 h, and HS was extracted from mouse kidney post mortem using a routine method for HS extraction59. The kidneys were dissolved for 16 h at 60 C in 4 M guanidine HCl/8 M urea/1% Triton in 50 mM Tris, pH 8.0. The extract was diluted 1:100 with water and applied to a 1 ml DEAE-Sephacel (Sigma) ion exchange column pre-equilibrated with PBS. The resin was then washed with 5 ml of 100 mM phosphate buffer with 0.25 M NaCl to remove hyaluronan and non-GAG material. The oligosaccharide was eluted with 1 M NaCl in phosphate buffer, concentrated to 1 ml and subjected to size-exclusion HPLC chromatography on a Superdex 75 column in PBS. Fractions (0.5 ml) were collected and counted.

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Acknowledgements

We thank the CRUK (C2075/A9106), MRC (G0601746 and G902173) and Holt Foundation for project grant funding; EPSRC for NMR instrumentation (GR/L52246); and we also thank the EPSRC National Mass Spectrometry Service, Swansea, for MS analyses.

Author contributions

S.U.H. and G.J.M. jointly contributed to the development of iteration and LAT strategies, wrote and nalized manuscript with J.M.G. G.R. conducted the preparation, analysis and purication of tritiated saccharides. C.C. helped in conducting biological experiments. G.C.J. gave overall contribution to project aims. J.M.G. and G.C.J. contributed equally to supervision and planning of this work. E.A., G.R. and G.J. contributed to manuscript biology. J.M.G. supervised synthesis and project planning, and helped in writing and nalizing the manuscript.

Additional Information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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How to cite this article: Hansen, S. U. et al. Tetrasaccharide iteration synthesis of a heparin-like dodecasaccharide and radiolabelling for in vivo tissue distribution studies. Nat. Commun. 4:2016 doi: 10.1038/ncomms3016 (2013).

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