A facile synthesis of α-N-ribosyl-asparagine and α-N-ribosyl- glutamine building blocks

Article abstract of DOI:10.3390/molecules18088779

Adenosine diphosphate ribosylation (ADP-ribosylation) is a widely occurring post-translational modification of proteins at nucleophilic side chain of amino acid residues. Elucidation of ADP-ribosylation events would benefit greatly from the availability of well-defined ADP-ribosylated peptides and analogues thereof. In this paper we present a novel approach to the chemical synthesis of ribosylated amino acid building blocks using traceless Staudinger ligation. We describe an efficient and stereoselective synthesis of α-N-ribosyl- asparagine (α-N-ribosyl-Asn) and α-N-ribosyl-glutamine (α-Nribosyl-Gln) building blocks starting from 5-tert-butyldiphenylsilyl- β-D-ribofuranosyl azide. The N-glycosyl aminoacids are produced in good yields as pure α-anomers, suitably protected for peptide synthesis.

Full text of DOI:10.3390/molecules18088779

Molecules 2013, 18, 8779-8785; doi:10.3390/molecules18088779
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
A Facile Synthesis of α-N-Ribosyl-Asparagine and
α-N-Ribosyl-Glutamine Building Blocks
Gaetano Speciale, Anna Bernardi and Filippo Nisic *
Universita’ degli Studi di Milano, Dipartimento di Chimica, via Golgi 19, 20133 Milano, Italy;
E-Mails: g.speciale@student.unimelb.edu.au (G.S.); anna.bernardi@unimi.it (A.B.)
* Author to whom correspondence should be addressed; E-Mail: filippo.nisic@unimi.it;
Tel.: +39-02-5031-4092, Fax: +39-02-5031-4072.
Received: 2 July 2013; in revised form: 16 July 2013 / Accepted: 19 July 2013 /
Published: 24 July 2013
Abstract: Adenosine diphosphate ribosylation (ADP-ribosylation) is a widely occurring
post-translational modification of proteins at nucleophilic side chain of amino acid
residues. Elucidation of ADP-ribosylation events would benefit greatly from the
availability of well-defined ADP-ribosylated peptides and analogues thereof. In this paper
we present a novel approach to the chemical synthesis of ribosylated amino acid building
blocks using traceless Staudinger ligation. We describe an efficient and stereoselective
synthesis of α-N-ribosyl-asparagine (α-N-ribosyl-Asn) and α-N-ribosyl-glutamine (α-N-
ribosyl-Gln) building blocks starting from 5-tert-butyldiphenylsilyl-β-D-ribofuranosyl
azide. The N-glycosyl aminoacids are produced in good yields as pure α-anomers, suitably
protected for peptide synthesis.
Keywords: ADP-ribosylation; glycoconjugates; ribofuranosyl aminoacids; Staudinger
ligation; stereoselective synthesis
1. Introduction
Glycosylated peptides play a fundamental role in biological systems: in fact, more than half of all
proteins carry carbohydrate moieties, generating different glycoforms whose exact composition often
controls protein function and distribution in biological systems [1–4]. The details of glycan regulation
of protein activity and stability are still under intense scrutiny and the synthesis of well-defined
glycopeptides is therefore an important target [5,6], which still presents many challenges to organic

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chemistry. One of the processes that is currently being elucidated is adenosine diphosphate
ribosylation (ADP-ribosylation), a wide-occurring post-translational modification effected by enzymes
that transfer ADP-ribose from NAD+ to Asn, Glu, Asp, Arg or Cys residues of proteins, so altering
their function [7]. An important contribution for the clarification of the role of ADP-ribosylation
events came in 2010, when Filippov and coworkers [8] reported the synthesis of ribosylated
oligopeptides using α-N-ribosyl-asparagine (α-N-ribosyl-Asn) and α-N-ribosyl-glutamine (α-N-ribosyl-
Gln) building blocks. Despite the relevance of this work, the approach suffers from a poorly selective
synthesis of the required ribosylated building blocks 4α and 5α (Scheme 1), which were prepared by
PtO2 reduction of azide 3, followed by EDC-mediated coupling of the resulting mixture of epimeric
amines with Z-Asp-OBn or Z-Glu-OBn, respectively. The N-ribosyl-aminoacids were obtained as a
3:1 α: β anomeric mixtures, from which the desired α-anomers were to be chromatographically
isolated. Acetyl transfer to the anomeric nitrogen from the 2-O-acetate also occurs during the reaction,
further reducing the process yields, particularly for the asparagine derivative 4α.
Scheme 1. Filippov’s synthesis of ribosylated amino acids (from ref. [8]).
We have recently developed a protocol for the stereoselective synthesis of either α- or
β-N-glycofuranosyl amides using the Staudinger traceless ligation [9,10]. In this reaction, a starting
anomeric glycosyl azide is partially reduced by an appropriate phosphine and then intramolecularly
acylated in order to afford the amide (Scheme 2) [11]. We have shown that for furanosyl azides the
outcome of the process is controlled by the configuration and the protection state of the contiguous
hydroxyl group, so that 1,2-cis amides (α-, in the ribo series) are obtained when this group is
unprotected and 1,2-trans amides (β-, in the ribo series) when it is acetylated. For unprotected
furanoses, the anomeric configuration of the product amides appears to be dictated by O-P
coordination, as supported by 31P-NMR studies [10].
Scheme 2. Traceless Staudinger ligation of furanosyl azides with functionalized
phosphines (from ref [9]).
RO
RO
RO
R= OH
RO
RO
1,2-cis
O
1
O
RO
NHCOR'
N3
+
RO
PPh2
RO
RO
O
R'
1,2-trans
O
O
R= OAc
NHCOR'
R' = C4H9

Molecules 2013, 18
8781
As an application of this methodology, we here report the facile stereoselective synthesis of 4α and
5α that could be obtained in good yields and with full stereoselectivity with a single synthetic
operation starting from β-5-O-tert-butyldiphenylsilyl-ribosylazide 2.
2. Results and Discussion
Previous research in our laboratories had shown that transfer of aminoacid chains in Staudinger
ligations of glycosyl azides was particularly effective using fluorinated phosphines 6 [12]. Thus we
envisaged that phosphines 6a and 6b (Scheme 3), functionalized with Z-Asp-OBn and Z-Glu-OBn
acyl chains, respectively, would be the reagent of choice to prepare the target α-N-ribosyl-Asn and
α-N-ribosyl-Gln building blocks. Phosphines 6a and 6b were synthesized in excellent yields by
EDC-mediated acylation of 2-diphenylphosphanyl-5-fluorophenol (7) [13] and purified by filtration on
short pads of silica.
Scheme 3. Synthesis of functionalized phosphines 6a and 6b.
An early trial of ligation was conducted using the unprotected β-D-ribofuranosyl azide 1 [14] with
phosphines 6a and 6b for 20 h at 70 °C in a 98:2 DMA:DMPU [15] solution, followed by water
quenching (Scheme 4). In both ligation reactions 45% of a 3:2 mixture of α-ribofuranosyl amide
(8α or 9α) and β-ribopyranosyl amide (10 or 11) was obtained. The isomer distribution was determined
1
by H-NMR analysis of the crude reaction mixtures: the α-ribofuranosyl amide (8α or 9α) was
characterized by the presence of the H1 proton signal at 5.65 ppm and by the high chemical shift of C4
(84.3 ppm), which is diagnostic for the furanose form. The structures of 10 and 11 were assigned on
the basis of the vicinal coupling constant of the anomeric protons (J1,2 = 8 Hz, H1 = 5.12 ppm) and of
the low chemical shift value of C4 (68.8 ppm), that confirms the pyranose structure. The
β-glycopyranosyl isomers 10 and 11 obtained in these reactions must derive from ring-expansion
occurring after a ring-opening process.
Scheme 4. Ligation of 1 with phosphines 6a and 6b.
Reagents and Conditions: DMA/DMPU 98:2, 70 °C, 20 h; (b) H2O, 2 h, 70 °C.

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To avoid ring expansion, the 5-hydroxy group of 1 was protected as a tert-butyldiphenylsilyl ether
and the 5-tert-butyldiphenylsilyl-β-D-ribofuranosyl azide 2 was synthesized in good yields as described
by Filippov [8]. Ligation of 2 with 6a or 6b was carried out as above (20 h at 70 °C in a 98:2 DMA:
DMPU mixture) (Scheme 5). In both cases, 400 MHz 1H-NMR analysis of the crude showed only one
signal in the furanose anomeric region, implying that no pyranose was formed, as expected, and that
the α/β ratio of the resulting ribofuranosyl amides 12α and 13α was > 99:1. The two compounds were
isolated by flash chromatography, which afforded the desired building blocks in 60% and 76% yields,
respectively, as single α isomers. After acetylation (Ac2O, cat. DMAP, CH2Cl2, quant.), 4α and 5α were
obtained. Their spectroscopic data were fully consistent with the already published characterization [8].
Scheme 5. Synthesis of ribofuranosyl amides 4α and 5α.
Reagents and Conditions: DMA/DMPU 98:2, 70 °C, 20 h; (b) H2O, 2 h, 70 °C; (c) Ac2O, cat. DMAP, CH2Cl2.
3. Experimental
Solvents were dried by standard procedures: dichloromethane and N,N-diisopropylethylamine were
dried over calcium hydride; N,N-dimethylacetamide (DMA), 1,3-dimethyltetrahydro-2(1H)-
pyrimidinone (DMPU), chloroform and pyridine were dried over activated molecular sieves. Reactions
1
13
31
requiring anhydrous conditions were performed under nitrogen. H-, C- and P-NMR spectra were
1
recorded at 400 MHz on a Bruker AVANCE-400 instrument. Chemical shifts (δ) for H and 13C
spectra are expressed in ppm relative to internal Me4Si as standard. Chemical shifts (δ) for 31P are
expressed in ppm relative to internal H3PO4 as standard. Signals were abbreviated as s, singlet; bs,
broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Mass spectra were obtained with a Bruker
ion-trap Esquire 3000 apparatus (ESI ionization) or FT-ICR Mass Spectrometer APEX II & Xmass
software (Bruker Daltonics)—4.7 Magnet and Autospec Fission Spectrometer (FAB ionization). Thin
layer chromatography (TLC) was carried out with pre-coated Merck F254 silica gel plates. Flash
chromatography (FC) was carried out with Macherey-Nagel silica gel 60 (230–400 mesh). The typical
scale used for the ligation reactions in this paper was 20 mg (0.1 mmol) of unprotected glycosyl azide.
General Procedure for the Synthesis of 2-(diphenylphosphanyl)-4-fluorophenyl Esters 6
A solution of the o-diphenylphosphinophenol (7, 1 equiv.), the commercially available
N-carbobenzyloxy-L-aminoacid-1-benzyl ester (Z-Asp-OBn or Z-Glu-OBn, 1.2 equiv.) and
N,N-dimethylaminopyridine (0.1 equiv.) in dry CH2Cl2 (0.1 M) were added, at room temperature and
under nitrogen, to a suspension of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride
(EDC, 1.4 equiv.) and dry N,N-diisopropylethylamine (1.4 equiv.) in dry CH2Cl2. The mixture was

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stirred at RT for 2 h, monitoring by TLC (60:40 hexane/AcOEt). The reaction mixture was diluted with
CH2Cl2 and washed with 5% aqueous HCl and water: the organic layer was dried over Na2SO4 and
concentrated. The crude product obtained was purified by flash chromatography, as indicated in each case.
1-Benzyl 5-[2-(Diphenylphosphanyl)-4-fluorophenyl] N-(Benzyloxycarbonyl)-L-aspartate (6a). The
1
crude product was purified by flash chromatography (hexane/AcOEt 60:40). yield = 85%. H-NMR
(CDCl3, 25 °C): δ = 7.37–7.23 (m, 20H, Ph), 7.02–6.84 (m, 2H, H-2, H-3), 6.49 (m, 1H, H-1), 5.69
(d, JNH-CH = 8.6 Hz, 1H, NH), 5.17 (s, 2H, CH2O), 5.13 (s, 2H, CH2O), 4.62 (m, JCH-CH2 = 4.4 Hz, 1H,
CH), 3.00 (dt, J = 4.9 Hz, J = 17.4 Hz, 1H, Ha, CH2), 2.67 (dd, J = 4.3 Hz, J = 17.4 Hz, 1H, Hb, CH2).
13C-NMR (CDCl3, 25 °C): δ = 170.4, 169.2, 161.9, 159.4 (CO(Cbz)), 134.6, 134.0, 132.0, 131.7, 129.0,
128.9, 128.7, 128.6, 128.4, 128.3, 128.2 (CAr), 124.0, 116.9, 116.6, 67.8 (CH2O), 67.3 (CH2O), 50.4
,
(CH), 36.5 (CH2). 31P-NMR (161 MHz, CDCl3 25 °C): δ = −14.3 ppm. FAB-MS: m/z 636 (M+1).
1-Benzyl 5-[2-(Diphenylphosphanyl)-4-fluorophenyl] N-(Benzyloxycarbonyl)-L-glutamate (6b). The
1
crude product was purified by flash chromatography (hexane/AcOEt 70:30). yield = 85%. H-NMR
(CDCl3, 25 °C): δ = 7.37–7.24 (m, 20 H, Ph), 7.05 (m, 1H, H-2), 6.99 (m, 1H, H-3), 6.45 (m, 1H, H-1),
5.37 (d, JNH-CH = 8.1 Hz, 1H, NH), 5.16 (s, 2H, CH2-O), 5.07 (s, 2H, CH2-O), 4.38 (m, 1H, CH), 2.26
13
(m, 2H, CH2-CO), 2.05 (m, 1H, Ha, CH2-CH), 1.80 (m, 1H, Hb, CH2-CH). C-NMR (CDCl3, 25 °C):
δ = 170.7, 161.7, 159.3, 156.1 (CO(Cbz)), 134.4, 134.3, 134.2, 134.0, 129.1, 129.0, 128.8, 128.5, 128.4
(CAr), 124.2, 124.1, 120.0, 119.8, 116.8, 116.5, 67.6 (CH2-O), 67.4 (CH2-O), 53.5 (CH), 30.1
(CH2-CO), 27.4 (CH2). 31P-NMR (161 MHz, CDCl3, 25 °C): δ = −14.3 ppm. FAB-MS: m/z 650 (M+1).
General Procedure for the Staudinger Traceless Ligation of Ribofuranosyl Azides
Phosphine 6a or 6b (2 equiv.) was added, at room temperature, to a 0.1 M solution of ribofuranosyl
azide 2 (1 equiv.) in 98: 2 N,N-dimethylacetamide and DMPU. The solution was stirred for 20 h at 70 °C,
then 10% water was added and the mixture was stirred for an additional 2 h at the same temperature.
The solvent was evaporated under reduced pressure, and the residue was purified by flash
chromatography as indicated in each case.
General Procedure for the Acetylation of the Ribofuranosyl Amides
Ac2O (6 equiv.), pyridine (6 equiv.) and a catalytic amount of N,N-dimethylaminopyridine were
added, at room temperature, to a solution of substrate (1 equiv.) in dry CH2Cl2 (0.1 M). The solution
was stirred for 24 h and then was concentrated in vacuo. The residue was dissolved in AcOEt and
washed with aqueous 5% HCl, aqueous 5% NaHCO3 and water. The organic layer was dried over
Na2SO4 and concentrated. The crude was then purified by flash chromatography (hexane:AcOEt,
80:20 to 50:50). Purification afforded products with spectroscopic data coherent with the fully
characterized compounds published by Filippov and coworkers [8].
Nδ-(5-tert-Butyldiphenylsilyl-α-D-ribosyl)-Nα-benzyloxycarbonyl asparagine benzyl ester (12α). The
1
crude product was purified by flash chromatography (CHCl3/MeOH 95:5). yield = 60%. H-NMR
(CD3OD, 25 °C): δ = 7.71–7.64, 7.42–7.24 (3 × m, 20H, Arom.), 5.73 (d, J1,2 = 4.8 Hz, 1H, H-1), 5.15,
5.07 (2 × s, 2H, CH2Bn, CH2Cbz), 4.65 (m, 1H, CH α-Asn), 4.25 (t, J = 5 Hz, 1H, H-3), 4.15 (t, J = 4.8 Hz,

Molecules 2013, 18
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1H, H-2), 3.97 (m, 1H, H-4), 3.72 (dd, J4,5 = 3.2 Hz, Jgem = 11.3 Hz, 2H, H-5 H-5’), 2.84 (m, 2H, CH2
13
β-Asn), 1.04 (s, 9H, t-Bu). C-NMR (CD3OD, 25 °C): 172.9, 172.4 (CO α-Asn, CO γ-Asn), 158.6
(CO Cbz), 138.2 (Cq Arom.), 137.3 (Cq Arom.), 136.9, 136.8 (Arom.), 134.6, 134.4 (Cq Arom.),
131.1, 131.0, 129.7, 129.6, 129.5, 129.5, 129.3, 129.3, 129.0, 128.8, 128.4, 128.1 (Arom.), 84.7 (C-4),
81.9 (C-1), 72.8 (C-3), 72.1 (C-2), 68.4, 67.9 (CH2 Bn, CH2 Cbz), 65.3 (C-5), 52.4 (CH,
α-Asn), 38.9 (CH2, β-Asn), 27.4 (CH3, t-Bu), 20.2 (Cq, t-Bu). FT-ICR (ESI) calcd. for C40H46N2O9Si
[M+Na]+ 749.29726; found 749.29721.
Nδ-(5-tert-Butyldiphenylsilyl-α-D-ribosyl)-Nα-benzyloxycarbonyl glutamine benzyl ester (13α). The
1
crude product was purified by flash chromatography (CHCl3/MeOH 95:5). yield = 76%. H-NMR
(CD3OD, 25 °C): δ = 7.71–7.64, 7.43–7.23 (3 x m, 20H, Arom.), 5.72 (d, J1,2 = 4.6 Hz, 1H, H-1), 5.15,
5.06 (2 × s, 2H, CH2Bn, CH2Cbz), 4.25 (m, 1H, CH α-Gln), 4.23 (t, J = 4.9 Hz, 1H, H-3), 4.15 (t,
J = 4.8 Hz, 1H, H-2), 3.95 (m, 1H, H-4), 3.70 (dd, J4,5 = 3.2 Hz, Jgem= 10.2 Hz, 2H, H-5 H-5’), 2.35
(m, 2H, CH2 γ-Asn), 2.13 (m, 1H, CH2 β-Asn), 1.95 (m, 1H, CH2 β-Asn), 1.02 (s, 9H, t-Bu). 13C-NMR
(CD3OD, 25 °C): 136.7, 136.6 (Arom.), 134.6, 134.4 (Cq Arom.), 130.8, 130.8, 129.7, 129.5, 129.4,
129.1, 128.9, 128.8, 128.7 (Arom.),84.5 (C-4), 81.6 (C-1), 72.7 (C-3), 71.9 (C-2), 67.9, 67.6 (CH2 Bn,
CH2 Cbz), 65.0 (C-5), 55.1 (CH α-Gln), 33.3 (CH2 β-Gln), 28.0 (CH2 γ-Gln), 27.3 (t-Bu), 22.9 (Cq
t-Bu). FT-ICR (ESI) calcd. for C41H48N2O9Si [M+Na]+ 763.31291; found 763.31298.
4. Conclusions
In conclusion, we have described an improved synthesis of the building blocks 4α and 5α:
application of the Staudinger Traceless Ligation protocol allowed to obtain the ribosylated amino acids
from 2 in good yields and with excellent selectivity for the α anomer. This represents a major
advantage over pre-existing synthesis of these glycosyl aminoacids [8,16,17] and should stimulate
further research in the field of ADP-ribosylation.
Acknowledgments
This work was supported by the University of Milan (PUR) and Ministero dell’Universita’ e della
Ricerca (PRIN). Exact mass were obtained from CIGA.
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1, 6 and 7 are available from the authors.
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