Design and synthesis of substrates for newborn screening of Maroteaux-Lamy and Morquio A syndromes

Article abstract of DOI:10.1016/j.bmcl.2010.08.080

In continued efforts to develop enzymatic assays for lysosomal storage diseases appropriate for newborn screening laboratories we have synthesized novel and specific enzyme substrates for Maroteaux-Lamy (MPS VI) and Morquio A (MPS IVA) diseases. The sulfated monosaccharide derivatives were found to be converted to product by the respective enzyme in blood from healthy patients but not by blood from patients with the relevant lysosomal storage disease. The latter result shows that the designed substrates are highly selective for the respective enzymes.

Full text of DOI:10.1016/j.bmcl.2010.08.080

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5994–5996
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of substrates for newborn screening of Maroteaux–Lamy
and Morquio A syndromes
Trisha A. Duffey a, Tanvir Khaliq a, C. Ronald Scott b, Frantisek Turecek a, Michael H. Gelb a,c,
⇑
a Dept. of Chemistry, University of Washington, Seattle, WA 98195, United States
b Dept. of Medicine, University of Washington, Seattle, WA 98195, United States
c Dept. of Biochemistry, University of Washington, Seattle, WA 98195, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
In continued efforts to develop enzymatic assays for lysosomal storage diseases appropriate for newborn
screening laboratories we have synthesized novel and specific enzyme substrates for Maroteaux–Lamy
(MPS VI) and Morquio A (MPS IVA) diseases. The sulfated monosaccharide derivatives were found to
be converted to product by the respective enzyme in blood from healthy patients but not by blood from
patients with the relevant lysosomal storage disease. The latter result shows that the designed substrates
are highly selective for the respective enzymes.
Received 25 June 2010
Revised 13 August 2010
Accepted 17 August 2010
Available online 25 August 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Lysosomal storage disease
Newborn screening
Tandem mass spectrometry
Enzyme assay
Clinical chemistry
The mucopolysaccharidoses (MPSs) are a class of lysosomal stor-
age diseases caused by the deficiency in an enzyme in glycosamino-
glycan catabolism. These syndromes result in non-degraded or
partially degraded glycosaminoglycans amassing in the lysosome
resulting in irreversible multisystemic organ damage.1–4 Recently,
treatments have become available for some of the MPS syndromes;
however optimal benefits from these treatments would require
commencement of treatment prior to the onset of the irreversible
symptoms. In continuance of our efforts on neonatal diagnosis of
lysosomal storage diseases by tandem mass spectrometry,5,6 we car-
ried out the synthesis of two novel substrates that can be used to as-
say N-acetyl galactosamine 4-sulfatase, the enzyme deficient in
Maroteaux–Lamy syndrome (MPS VI) and galactose 6-sulfatase,
the enzyme deficient in Morquio A syndrome (MPS IVA).
ose 6-sulfate. The anomeric carbon of these sugars was coupled to
an umberferryl moiety, which could be used for fluorescence assays
in laboratories lacking tandem mass spectrometers. A carbon chain
with a fragmentable N-tert-butyl carbamate was attached to direct
the fragmentation of the parent ion in the mass spectrometer along
a single reaction pathway, which increases the sensitivity of the
The assays require substrates that are readily prepared, specific
for the enzymes, and tagged for detection in the newborn screening
laboratory. Natural substrates for the sulfatases are oligosaccha-
rides containing a sulfate on the terminal sugar. While these oligo-
saccharides would be selective substrates for the enzymes, they are
difficult to prepare on the scale needed for worldwide newborn
screening (ꢀ10 g/year). In contrast, aryl sulfates are readily avail-
able but show low specificity between the sulfatase enzymes. We
based our substrate design (Fig. 1) on the terminal sugar of the nat-
ural substrates, that is, N-acetylgalactosamine 4-sulfate and galact-
⇑
Corresponding author.
E-mail address: gelb@chem.washington.edu (M.H. Gelb).
Figure 1. Maroteaux–Lamy substrate 1 and internal standard 3 and Morquio A
substrate 2 and internal standard 4.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.080

T. A. Duffey et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5994–5996
5995
Scheme 1. Preparation of MPS IVA substrate 2 and internal standards 3 and 4. Reagents and conditions: (a) N-Boc-1,6-hexanediamine, N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide hydrochloride, DMF, CH2Cl2, overnight, 52%; (b) N-Boc-1,5-pentanediamine, N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride, DMF,
CH2Cl2, overnight, 62%; (c) glycosyl chloride (7a, 7b) or glycosyl bromide (9), tetrabutylammonium hydrogensulfate, aq NaOH (1 M), DCM, 30–60 min; (d) sodium methoxide,
MeOH, CHCl3, 4 h, 8 (from 6a and 7a): 53% over two steps; 3 (from 6b and 7b): 50% over two steps; 10 (from 6b and 9): 28% over two steps, 4 (from 6a and 9): 35% over two
steps; (e) sulfur trioxide–pyridine complex, pyridine, 5 h; DOWEX 50WX8 (Na+ form), 45%.
Scheme 2. Preparation of MPS VI substrate 1. Reagents and conditions: (a) benzoyl chloride (2.1 equiv), pyridine, 0 °C, 3 h, 58%; (b) triflic anhydride, pyridine, DCM, ꢁ20 °C,
3 h; (c) sodium nitrite, DMF, overnight, 51% over two steps; (d) sulfur trioxide–pyridine complex, pyridine, 3 h; DOWEX 50WX8 (Na+ form), 50%; (e) sodium methoxide,
MeOH, 5 d, 80%.
tandem mass spectrometry assay. The linker chain lengths were
chosen such that the mass of the products and internal standards
do not coincide with the masses of any of the other neonatal assays
developed earlier in our laboratory, thereby allowing the assays to
be multiplexed in the mass spectrometry analysis.
to afford 10. Finally, sulfation of the free hydroxyl followed by
cleavage of the benzoate protecting groups gave the desired MPS
VI substrate (1).
Full synthetic details are provided as Supplementary data.
With the two substrates and two internal standards in hand,
enzymatic activity was studied. The enzymatic activity was mea-
sured by incubating a solution of substrate and internal standard
with a 2 mm diameter dried blood spot punch for 16 h. The amount
of product was quantified by tandem mass spectrometer using the
internal standards. For MPS VI, the range of activity measured with
The synthesis of both the compounds (Schemes 1 and 2) started
with the amide coupling of 7-hydroxycoumarinyl-4-acetic acid with
either mono-Boc protected 1,6-hexanediamine or 1,5-pentanedi-
amine. The resulting substituted coumarins were glycosylated with
two different glycosyl halide donors under phase transfer catalysis
to afford the b-glycosides.7 The acetate esters were then deprotected
to afford glycosides 8, 3, 4 and 10. Glycosides 3 and 4 are the desired
internal standards for the enzymatic assays and were used without
further elaboration. The MPS IVA substrate 2 was readily synthe-
sized from glycoside 10 by selective sulfation of the primary 6-hy-
droxyl group over the secondary 2-, 3- and 4-hydroxyl groups as
revealed by the downfield shift of H-6 in the sulfate relative to the
non-sulfate.8
The MPS VI substrate (1) required further synthetic manipula-
tions in order to install the sulfate at the more hindered 4-hydroxy
of the sugar. Therefore, the less hindered 3- and 6-hydroxyls were
selectively benzoylated to afford dibenzoate 9. The glucosamine
was then converted to a galactosamine by inversion of the 4-hydro-
xyl by formation of the triflate and displacement with sodium nitrite
10 dried blood spots from healthy individuals was 1.6–10.5
hꢁ1 (L blood)ꢁ1 compared to 0.08 mol hꢁ1 (L blood)ꢁ1 using a dried
blood spot from an MPS VI patient. For MPV IVA, the values are
0.021–0.35
mol hꢁ1 (L blood)ꢁ1 for 30 dried blood spots from
healthy patients and 0.00039–0.00043
mol hꢁ1 (L blood)ꢁ1 from
lmol
l
l
l
six patients with MPS IVA. These results serve to illustrate that the
substrates are highly specific for the respective enzymes. Full details
of the assay including assay statistics and performance on a large
number of dried blood spots will be reported elsewhere.
In conclusion, we have synthesized substrates for two lyso-
somal sulfatases, N-acetylgalactosamine 4-sulfatase and galactose
6-sulfatase, they are highly specific for their respective enzymes.
The syntheses developed can be readily scaled to prepare the
ꢀ10 g/year of material needed to support worldwide newborn

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T. A. Duffey et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5994–5996
screening. Further evaluation of these reagents is under investiga-
tion in newborn screening laboratories.
References and notes
1. Dorfman, A.; Matalon, R. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 630.
2. Legum, C. P.; Schorr, S.; Berman, E. R. Adv. Pediatr. 1976, 22, 305.
3. Kresse, H.; Paschke, E.; Von Figura, K.; Gilberg, W.; Fuchs, W. Proc. Natl. Acad. Sci.
U.S.A. 1980, 77, 6822.
Acknowledgements
4. Klein, U.; Kresse, H.; Von Figura, K. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 5185.
5. Wang, D.; Wood, T.; Sadilek, M.; Scott, C. R.; Turecek, F.; Gelb, M. H. Clin. Chem.
2007, 53, 137.
We are grateful to Biomarin Pharmaceutical Inc. Novato CA and
to the NIH (grant R01DK067859) for financial support.
6. Blanchard, S.; Sadilek, M.; Scott, C. R.; Turecek, F.; Gelb, M. H. Clin. Chem. 2008,
54, 2067.
7. Carriere, D.; Meunier, S. J.; Tropper, F. D.; Cao, S.; Roy, R. J. Mol. Catal. A: Chem.
2000, 154, 9.
Supplementary data
Supplementary data (experimental procedure for the synthesis
and spectral data) associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.080.
8. Sawada, T.; Fujii, S.; Nakano, H.; Ohtake, S.; Kimata, K.; Habuchi, O. Carbohydr.
Res. 2005, 340, 1983.