A Chemoenzymatic Route to Prepare Acyclic Nucleoside Analogues

Article abstract of DOI:10.1002/ejoc.201501412

Acyclic nucleosides are potential antiviral and antitumor agents. In this work, their preparation through a novel chemoenzymatic procedure involving the N-alkylation of a nucleobase and subsequent aldol condensation catalyzed by a dihydroxyacetone phosphate-dependant aldolase (Rabbit Muscle Aldolase, RAMA) is described.

Full text of DOI:10.1002/ejoc.201501412

DOI: 10.1002/ejoc.201501412
Communication
Acyclic Nucleoside Synthesis
A Chemoenzymatic Route To Prepare Acyclic Nucleoside
Analogues
Martín A. Palazzolo,[a,b] Mariano J. Nigro,[a] Adolfo M. Iribarren,[a,c] and
Elizabeth Sandra Lewkowicz*[a]
Abstract: Acyclic nucleosides are potential antiviral and anti- cleobase and subsequent aldol condensation catalyzed by a di-
tumor agents. In this work, their preparation through a novel hydroxyacetone phosphate-dependant aldolase (Rabbit Muscle
chemoenzymatic procedure involving the N-alkylation of a nu- Aldolase, RAMA) is described.
Introduction
Nucleoside analogues have are a valuable source of antiviral
and antitumor agents.[1] The discovery of the antiviral activity
of two acyclic analogues of guanosine and adenosine in 1978,
9-(2-hydroxyethoxymethyl)guanine (acyclovir)[2] and S-9-(2,3-di-
hydroxypropyl)adenine (S-DHPA),[3] encouraged the study on
this family of nucleosides (Scheme 1). Acyclic nucleoside (AN)
analogues are used as antiviral agents due to the ability of the
corresponding triphosphate derivatives to function as chain ter-
minators during DNA or RNA biosynthesis and because of their
enhanced chemical and metabolic stability.[4] Among them,
acyclovir and ganciclovir are potent antiherpetic drugs, whereas
thymine (phosphonomethoxy) alkyl derivatives such as 1-[2-
(phosphonomethoxy)ethyl]thymine (PMET), 1-[3-hydroxy-2-
(phosphonomethoxy)propyl]thymine (HPMPT) and 1-[3-fluoro-
2-(phosphonomethoxy)propyl]thymine (FPMPT) were found as
inhibitors of thymidine phosphorylase from SD-lymphoma.[5]
AN phosphonates also exhibit antiviral and cytostatic activ-
ity. Adefovir and tenofovir are useful compounds against HBV
and HIV infections[6] while cidofovir has been approved for
treatment of CMV retinitis in AIDS patients and in other viral
diseases like papilloma virus-induced warts.[7] As a general
statement, ANs possess a branched or linear alkyl chain, substi-
Scheme 1. Acyclic nucleoside analogues.
tuted with at least one hydroxy group, attached to the N atom
of the nucleobase that mimics the pentofuranose ring in natural
nucleosides. Moreover, the flexibility of the alkyl chain enables
the acyclic analogues to adopt a suitable conformation to fit in
the active site of the enzymes involved in their metabolism.[8]
[a] Laboratorio de Biocatálisis y Biotransformaciones, Departamento de
These characteristics, combined with the need of new active
compounds against drug resistant viruses, have led to the con-
stant development of new AN structures and consequently,
methodologies for their synthesis.
Ciencia y Tecnología
Universidad Nacional de Quilmes
Roque Sáenz Peña 352, 1876 Bernal, Buenos Aires, Argentina
E-mail: elewko@unq.edu.ar
[b] Dr. M. Palazzolo. Present address
Efforts have also been focused on the preparation of differ-
ent derivatives of ANs with the aim of tuning characteristics
like hydrophilicity, solubility, electronic properties and specific
binding, as in the case of phosphonates, that provide improved
Area de Química Orgánica, Facultad de Química, Bioquímica y Farmacia
Universidad Nacional de San Luis, INTEQUI-CONICET
San Luis 5700, Argentina
[c] Laboratorio de Química de Ácidos Nucleicos
INGEBI-CONICET
phosphatase resistance,[9,10] or
L-valine esters prodrugs with in-
Vuelta de Obligado 2490, 1428 C.A.B.A, Argentina
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejoc.201501412.
creased bioavailability.[11] The stereochemistry of the ANs often
plays an important role in determining their biological activity
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Communication
and selectivity. For instance, the (R)-enantiomer of tenofovir, is are readily cleaved by endogenous phosphatases releasing rap-
about 50 times more effective against HIV than its (S)-counter- idly the pharmacologically active component. To this group of
part.[9]
derivatives belongs fludarabine monophosphate (9-ꢀ-D-arab-
inofuranosyl-2-fluoroadenine-5′-monophosphate), a hydrophilic
prodrug widely used in the treatment of chronic lymphocytic
leukaemia therapies.[22]
A number of synthetic routes to prepare acyclic nucleoside
analogues in enantiomerically pure form have been reported,
starting from optically active compounds like carbohydrates, di-
ethyl
L-tartrate,
L-ascorbic acid,
D
-lactose or nucleosides.[12]
In the present communication, we report the production of
Acyclic pyrimidine nucleoside analogues were successfully syn- AN monophosphates by means of a novel chemoenzymatic
thesized by coupling a commercially available acyclic moiety route that comprises the stereoselective formation of a specific
with 6-azapyrimidines using the Vorbrüggen procedure.[13] Spe- C–C bond catalyzed by RAMA.
cial efforts have been directed towards the synthesis of acyclic
purine analogues, since the preparation of such derivatives
comprises low-yield multistep procedures.[6] Recently, we re-
Results and Discussion
ported an efficient organocatalysed strategy for the synthesis
of novel ANs, though racemic mixtures were obtained.[14]
The traditional strategy for the synthesis of ANs consists of the
cumbersome stereoselective preparation of the acyclic chain
followed by regioselective coupling to the nucleobase. On the
contrary, the chemoenzymatic route herein proposed takes ad-
vantage of the benefits provided by biocatalyzed reactions like
simplicity, chemo-, regio-, and stereoselectivity and sustainabil-
ity[23] allowing the easy and stereoselective building of the
acyclic chain. However, enzyme specificity is still a concern and
only particular kinds of acyclic moieties can be generated.
As a first attempt, we explored the synthesis of an acyclic
One of the most explored biocatalyzed routes that provided
a wide platform of nucleoside analogues involves the use of
nucleoside phosphorylases as biocatalysts.[15] Nevertheless, the
synthesis of acyclic derivatives of nucleosides by means of bio-
catalysis has not been explored so far.
Aldolases catalyze the reversible formation of C–C bonds by
the aldol addition of a nucleophilic donor, usually a ketone
enolate, onto an electrophilic aldehyde acceptor. These en-
zymes can usually accept a wide variety of aldehydes as accept-
ors, but the donor compound is often structurally invariable.[16] derivative of thymine (Scheme 2). A key intermediate, the pro-
For this reason, aldolases can be classified into different classes tected aldehyde derivative of the nucleobase, was prepared by
according to their donor specificity. The most studied group adapting the procedure reported by Tanaka et al.[24] for the
utilizes dihydroxyacetone phosphate (DHAP) as donor, being its synthesis of CMP-sialic acid analogues (CMP = cytidine mono-
best known member fructose 1,6-bisphosphate (FDP) aldolase. phosphate). In this way, a mixture of thymine (1a) and K2CO3
The rabbit muscle aldolase (RAMA), the most versatile and use- in DMF was stirred in the presence of a suitable alkylating
ful enzyme among the available FDP aldolases, displays a broad agent, such as 2-bromo-1,1-dimethoxyethane. After 24 h, the
synthetic scope, which includes the preparation of sugars, crude mixture was filtered and the solvent removed under re-
deoxysugars, fluorosugars, iminocyclitols, macrocycles, macro- duced pressure to obtain compound 2a in 38 % yield after puri-
lide antibiotics, pheromones, terpenoids, and alkaloids.[17] fication by silica gel column chromatography.
Wong and co-workers explored the use of RAMA in the synthe-
sis of adenosine analogues carrying modified sugar moieties,
Once quantitative acid hydrolysis of the acetal 2a was
achieved, the resulting reaction mixture containing the alde-
hyde 3a was neutralized and incorporated into a buffered solu-
tion comprising DHAP and RAMA, and then stirred at room
temperature. The consumption of 3a was monitored by silica
gel TLC whereas the formation of 5-(1-thymidyl)-3,4-dihydroxy-
2-oxopentyl phosphate 4a was quantified by HPLC-UV (86 %
describing the production of 6-adenyl-6-deoxy-
D-fructose and
6-adenyl-6-deoxy-
L
-sorbose.[18] Apart from RAMA, the utility of
a glycine-dependent aldolase was also demonstrated in the
synthesis of the precursors for the preparation of a short-chain
RNA mimic reported by Miura et al.[19]
The production of monophosphorylated nucleoside deriva- conversion) and its molecular weight confirmed by HPLC-MS.
tives as prodrugs is highly desired.[20] The presence of a phos- Finally, the product 4a was isolated by reverse phase chroma-
phate group increases the solubility of the parent drug by sev- tography (70 % yield) and its structure analyzed by NMR spec-
eral orders of magnitude[21] and furthermore, phosphate esters troscopy. The overall yield of the reaction was 26.6 %, but it
Scheme 2. Schematic representation of the chemoenzymatic synthesis of an acyclic nucleoside analogue.
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Communication
filtrates purified by C18 reverse-phase silica column chromatogra-
phy. The purity was determined by HPLC and the identity was con-
firmed by NMR spectroscopy.
could be improved since the first chemical step was not opti-
mized.
It is well established that aldolases have a high level of
stereocontrol of the configuration at the formed chiral cen-
ters.[25] Therefore, the stereochemistry of the reaction is virtually
independent of the structure of the acceptor substrate and thus
the stereochemical outcome is highly predictable.[26]
In particular, in the active site of RAMA, the nucleophilic at-
tack of the enamine intermediate formed between DHAP and
the enzyme occurs on the si-face of the acceptor aldehyde, thus
affording the product with (3S, 4R) stereochemistry with respect
to the carbonyl group[16] as was previously stated by
Schoewaart et al.[27] The coupling constant between H9 and
H10 (J:1.89 Hz) confirms the syn configuration of the hydroxy
groups in the stereogenic centers of 4a.
Acknowledgments
This work was supported by Universidad Nacional de Quilmes,
CONICET and Secretaría de Ciencia y Técnica de la Nación, Ar-
gentina. E. S. L. and A. M. I. are research members of CONICET;
M. A. P. and M. J. N. are CONICET and ANPCyT fellows respec-
tively.
Keywords: Nucleosides · Enzyme catalysis · Lyases · Aldol
reactions · Antiviral agents
The structural arrangement of compound 4a yields an AN
that mimics arabinothymidine monophosphate. Arabinonucleo-
sides display antiviral and antitumoral activity and in particular,
vidarabine – the corresponding adenine analogue – is an active
nucleoside analogue currently used against viral infections
caused by herpes simplex and varicella zoster viruses.[28]
After preparing the respective aldehydes, other nucleobases,
such as adenine (3b), uracil (3c), and cytosine (3d), were evalu-
ated as acceptors in RAMA-catalyzed reactions employing the
developed chemoenzymatic strategy. HPLC analysis of the un-
purified mixtures revealed similar conversions compared to the
thymine-based molecule (4b: 88 %, 4c: 83 %, 4d: 88 %).
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Experimental Section
General Procedure for the Preparation of Aldehydes 3a–d
Mixtures of 1a–1d (1 equiv.) and K2CO3 (2 equiv.) in DMF (10 mL)
were stirred at 90 °C in the presence of 2-bromo-1,1-dimethoxy-
ethane (2 equiv.). After 24 h, the reactions were filtered and the
solvent was removed under reduced pressure. The crude mixtures
were purified by column chromatography and characterized by 1D
and 2D NMR spectroscopy. Aldehydes 3a–3d were obtained after
hydrolysis of the corresponding acetals 2a–2d in HCl (1 N) at 90 °C
for 1 h and neutralization with NaOH (10 N).
Biocatalyzed Aldol Condensation
The neutral reaction mixture containing 3a–3d (0.08 mmol), DHAP
(0.04 mmol) and commercially available rabbit muscle aldolase
(RAMA, EC 4.1.2.13, 2 U) in phosphate buffer pH 7.5 (50 mM, 2 mL)
was stirred at 200 rpm and 25 °C. After 3 h an additional portion
of RAMA (2 U) was added. The crude biotransformation mixtures
were filtered through a celite pad to remove the enzyme and the
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Received: November 6, 2015
Published Online: January 29, 2016
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