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. Author manuscript; available in PMC: 2008 Sep 9.
Published in final edited form as: Org Lett. 2006 Oct 26;8(22):5093–5096. doi: 10.1021/ol062026m

S-Adenosylhomocysteine Analogues with the Carbon-5' and Sulfur Atoms Replaced by a "Vinyl Unit"

Daniela Andrei 1, Stanislaw F Wnuk 1,*
PMCID: PMC2532837  NIHMSID: NIHMS63825  PMID: 17048851

Abstract

Cross-metathesis of suitably protected 5'-deoxy-5'-methyleneadenosines with racemic and chiral N-Boc protected six-carbon amino acids bearing a terminal double bond in the presence of the Hoveyda-Grubbs catalyst gave adenosylhomocysteine analogues with the C5'–C6' double bond. Bromination with pyridinium tribromide and dehydrobromination with DBU followed by standard deprotections yielded the 5'-(bromo)vinyl analogue.

The enzyme S-adenosyl-L-homocysteine (AdoHcy) hydrolase (EC 3.3.1.1) effects hydrolytic cleavage of AdoHcy to adenosine (Ado) and L-homocysteine (Hcy).1 The cellular levels of AdoHcy and Hcy are critical because AdoHcy is a potent feedback inhibitor of crucial transmethylation enzymes.1,2 Also elevated plasma levels of Hcy in humans have been shown to be a risk factor in coronary artery disease.3

The X-ray crystal analysis of human AdoHcy hydrolase inactivated with 9-(dihydroxycyclopentene)-adenine4a and neplanocin A,4b as well as AdoHcy hydrolase from rat liver4c established the presence of a water molecule in the active site of the enzyme. This observation made it a high priority to prepare analogues of AdoHcy that closely resemble the natural substrate that bind tightly to the enzyme. Such compounds should form "stable" complexes with the enzyme that would help to identify key binding groups at the active site of the enzyme that interact with the Hcy moiety and participate in subsequent elimination and 'hydrolytic' activity steps.

Based on the previous finding that AdoHcy hydrolase is able to add the enzyme-sequestered water molecule across the 5',6'-double bond of 5'-deoxy-5'-(halo or dihalomethylene)adenosines causing covalent binding inhibition,5,6 we now describe the synthesis of AdoHcy analogues A (X = H) with the 5',6'-olefin motif incorporated in place of the carbon-5' and sulfur atoms (Figure 1). The analogues A or B (X = halogen) should be substrates for the oxidative activity of the enzyme, and the resulting 3'-keto products might be substrates for the 'hydrolytic' activity. Enzyme-mediated addition of water might occur at C5' or C6' of A or B to generate new species with hydroxyl or keto (after β-elimination of HBr) binding sites within the enzyme. X-Ray structures of such 'oxidation' and/or 'hydrolytic' activity-bound products might provide important information regarding key residues in the protein and their interactions with substrates (Hcy unit) and/or the sequestered water molecule.

Figure 1.

Figure 1

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S-Adenosyl-L-homocysteine and analogues with sulfur atom replaced by the "vinyl unit".

Retrosynthetic analysis indicates that AdoHcy analogue A (X = H) can be prepared by construction of a new C5'-C6' double bond via Wittig or metathesis reactions. For example, condensation of adenosine 5'-aldehyde with Wittig-type reagent or cross-metathesis between 5'-deoxy-5'-methyleneadenosine and the appropriate amino acid-derived terminal alkenes should give A. Since nucleoside 5'-aldehydes are unstable in the presence of strong bases required for the generation of non-stabilized phosphorane-Wittig reagents,7 we decided to target an AdoHcy analogue of type A via the cross-metathesis reaction. Another possibility is Pd-catalyzed cross-coupling approaches between sp2-sp3 hybridized carbons to form a new C6'–C7' single bond as a key step.8,9

Alkylation of protected glycine 1 with 4-bromo-1-butene followed by hydrolysis of the resulting Schiff base derivative10 2 yielded racemic 2-amino-5-hexenoate 3 (Scheme 1). Attempted cross-metathesis11 between 5'-deoxy-2',3'-O-isopropylidene-5'-methyleneadenosine 9a5a,7 with N-benzoyl 4 or N-Boc 5 protected amino acids bearing terminal double bond in the presence of 1st and 2nd (2-imidazolidinylidene-Ru) generation Grubbs catalysts11c,e failed to give desired products 10a or 11a (Scheme 2). Also, metathesis of the 6-N-benzoyl adenosine substrate 9b with 4 or 5 was unsuccessful. It is noteworthy that metathesis between 5'-deoxy-2',3'-O-isopropylidene-5'-methyleneuridine12 and 4 (CH2Cl2/2nd generation Grubbs catalyst) afforded the desired product of type 10 (i.e., B = U; 62%)13 in addition to two dimers resulting from the self-metathesis of nucleoside14 and amino acid15 (e.g, 17) substrates.

Scheme 1.

Scheme 1

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Scheme 2.

Scheme 2

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We found however that treatment of 9b with 4 in the presence of Hoveyda-Grubb's catalyst16 (o-isopropoxy-phenylmethylene-Ru) led to the formation of metathesis product 10b (51%) in addition to dimer 17 (11%) while self-metathesis of 9b was not observed. Metathesis of the 6-N,N-dibenzoyl 9c with 4 gave 10c in 60% yield in addition to dimer 17 (18%). The protection of 6-amino group of the adenine ring seems to be necessary because metathesis between 9a and 4 or 5 in the presence of Hoveyda-Grubbs catalyst did not yield the corresponding product 10a or 11a.

Metathesis of 9b and 9c with N-Boc protected 5 gave 11b (61%) and 11c (76%) in higher isolated yields. Moreover, by-products of the self-metathesis of amino acid or nucleoside substrates were not isolated. The cross-metathesis products 10 and 11 were found to be predominantly the trans isomers.17 Purification on a silica gel column afforded 10 and 11 as 5'E isomers of a ~1:1 mixture of 9'R/S diastereomers. The E stereochemistry for 10 and 11 was established from 1H NMR spectra based on the magnitude of JH5'–H6'. For example, the 5' proton in 11c appears at δ 5.58 (dd, JH5'–H4' = 7.3 Hz and JH5'–H6' = 15.2 Hz) while the 6' proton resonates at δ 5.73 (dt, JH6'–H7'/7" = 6.5 Hz and JH5'–H6' = 15.2 Hz).

Deprotection of 10 or 11 turned out to be more challenging than we expected. Thus, treatment of 11c (or 11b) with a 1:1 mixture of saturated (at ~0 °C) methanolic ammonia solution and methanol for 48 h at ~5 °C removed the 6-N-benzoyl group(s) and produced a partially separable mixture of methyl 12 and ethyl 13 esters (~3:2, ~92% total yield). Using diluted NH3/MeOH minimized formation of the amidation byproducts (~5%). Acid-catalyzed deprotection of 12 and 13 with an aqueous solution of trifluoroacetic acid (TFA) effected the removal of both Boc and the isopropylidene protection groups to give 14 and 15 in high yields. It is important to perform debenzoylation of 11c (or 11b) as initial deprotection step, because treatment of 11c (or 11b) with TFA/H2O resulted in the substantial cleavage of the glycosylic bond. Saponification of 14 and 15 with NaOH in H2O/MeOH solution and purification on RP-HPLC afforded the sodium salt of 16 [67%; E, 9'R/S (~1:1)].

Since separation of 9'R/S diastereomers in products 10–16 was difficult, we attempted the synthesis of analogue A with 9'S configuration employing a chiral amino acid precursor e.g., (S)-homoallylglycine. Given that the methods available for the preparation of enantiomerically pure unnatural amino acids usually require multistep synthesis,18 we chose the enantioselective hydrolysis of racemic 5 as a way to provide chiral (S)-homoallylglycine. Thus, treatment of 5 with α-chymotrypsin in phosphate buffer (24 h, 37 °C)19 gave the unreacted (R)-ester 5 (~50%) and (S)-acid 6 (~50%, Scheme 1). Enantiomeric purity of the 5-R was established using the Mosher test.20a Thus, treatment of 5-R with TFA/H2O followed by acylation with (R)-2-methoxy-2-trifluoromethyl-2-phenylacetyl chloride (MTPA-Cl)20 gave 8-R/S. (Note that the absolute configuration at the chiral carbon in Mosher reagent is the same but the R/S descriptors change owing to the change in Cahn-Ingold-Prelog priority.) Analysis of the 19F NMR spectra [δ -69.16 (s, 0.02F) and -69.55 (s, 0.98F)] established the stereochemistry for 5 as R (ee 96%) in agreement with Mosher's empirical formula.20a Since metathesis of the "free" carboxylic acid precursor 6-S with 9b or 9c in the presence of Hoveyda-Grubbs catalyst failed, the 6-S was converted into the methyl ester 7-S with diazomethane.

Cross-metathesis of 9c with 7-S afforded 18-S (77%; Scheme 3). Sequential deprotections of 18-S with NH3/MeOH (to give 12-S, 91%) and TFA/H2O gave the enantiomerically pure 14-S (90%) as the single E isomer (Scheme 3). On the other hand metathesis of 9c with 5-R gave ethyl ester 11c-R. Contrary to products 10–16 obtained from racemic homoallylglycine, the 13C NMR spectra for the products obtained from (S)- and (R)- homoallylglycine substrates showed a single set of peaks.

Scheme 3.

Scheme 3

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Finally, we attempted the synthesis of bromovinyl analogue B by the bromination-dehydrobromination strategy. Treatment of 11c with pyridinium tribromide21 gave the 5',6'-dibromo diastereomers 19 which were dehydrobrominated with 1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) to yield 20 as a single isomer (one of the 6-N-benzoyl protective group was also partially cleaved) in 70% yield (Scheme 4). Standard deprotections with NH3/MeOH and TFA/H2O followed by saponification with NaOH and HPLC purification gave 23 (E, 54% overall).

Scheme 4.

Scheme 4

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The regioselectivity of HBr elimination and the position of bromine (at 5') were assigned based on the presence of a triplet signal for the olefinic hydrogen (H6') in 1H NMR spectra [δ 6.40 (t, J6'–7'/7" = 7.6 Hz) for 23]. This assignment was also supported by COSY experiment. The product E configuration is expected from a specific anti-addition in the pyridinium tribromide bromination of the E alkene 11c followed by an E2 (anti elimination) process. This was also supported by NOESY analysis of 23 in which the cross-peaks between H4' and H7'/7" were observed.

In summary, we have developed a synthesis of AdoHcy analogues in which the carbon-5' and sulfur atoms is replaced by a "vinyl unit" utilizing cross-metathesis reactions between 5'-deoxy-5'-methyleneadenosine analogues and homoallylglycine in the presence of Hoveyda-Grubbs catalyst. The 5'-(bromo)vinyl AdoHcy analogue has been prepared via the bromination-dehydrobromination strategy. Enzymatic studies with AdoHcy hydrolase and our attempts to synthesize 6'-(halo)vinyl analogues B via cross-coupling approaches will be published elsewhere.

Supplementary Material

1si20060920_02. Supporting Information Available.

Experimental procedures and characterization data for all compounds (PDF). This material is available free of charge via Internet at http://pubs.acs.org.

2si20060920_02

Acknowledgment

We thank NIH/NIGMS program (S06 GM08205) for supporting this research, US ARO (W911NF-04-1-0022) for financial help to purchase 600 MHz NMR spectrometer and Materia Company for a gift of Hoveyda-Grubbs catalyst.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1si20060920_02. Supporting Information Available.

Experimental procedures and characterization data for all compounds (PDF). This material is available free of charge via Internet at http://pubs.acs.org.

NIHMS63825-supplement-1si20060920_02.pdf (166.2KB, pdf)
2si20060920_02
NIHMS63825-supplement-2si20060920_02.pdf (671.7KB, pdf)

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