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Facile synthesis of N-6 adenosine modified analogue toward S-adenosyl methionine derived probe for protein arginine methyltransferases
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Chinese Chemical Letters 22 (2011) 1439–1442 www.elsevier.com/locate/cclet
Facile synthesis of N-6 adenosine modified analogue toward S-adenosyl methionine derived probe for protein arginine methyltransferases Wei Hong a, James Dowden b,* a
School of Chemistry and Chemical Engineering, Beifang University of Nationalities, Yinchuan 750021, China b School of Chemistry, University of Nottingham, Nottingham NG7 2RD, UK Received 24 May 2011 Available online 10 October 2011
Abstract Chemically modified cellular co-factors that provide function, such as immobilization or incorporation of fluorescent dyes, are valuable probes of biological activity. A convenient route to obtain S-adenosyl methionine (AdoMet) analogues modified at N-6 adenosine to feature a linker terminating in azide functionality is described herein. Subsequent decoration of such AdoMet analogues with guanidinium terminated linkers leads to novel potential bisubstrate inhibitors for protein arginine methyltransferases, PRMTs. # 2011 James Dowden. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Protein arginine methyltransferases; S-Adenosyl methionine; Nucleosides
Protein arginine methyltransferases (PRMTs, EC 2.1.1.125) utilize S-adenosyl methionine (AdoMet, Fig. 1) to catalyze post-translational methylation of arginine residues of a variety of substrate proteins, leading to S-adenosyl homocysteine (AdoHcy, Fig. 1) and monomethylarginine (mMA). Further methylation often follows, with Type I PRMTs delivering asymmetrical dimethylarginine (aDMA) and Type II PRMTs symmetrical dimethylarginine (sDMA), respectively [1]. At least nine PRMTs have been identified in mammals and an important roles in governing cellular processes, such as including gene regulation, RNA splicing and transport, or signal transduction are emerging [2]. There is a significant interest in developing potential drugs for individual PRMTs, especially for the treatment of hormone dependent cancer [3]. Functional chemical probes may be valuable for exploring inhibition, and potential immobilization of specific PRMTs. So far, we have reported AdoMet analogue 1 (Fig. 1) as a potent inhibitor of PRMT1 (IC50 6 mmol/L), that features structure intended to target both AdoMet and arginine subsites of the enzyme [4]. Modification of AdoMet analogue 1 to facilitate labeling or immobilization of this enzyme for functional studies would be highly desirable. We therefore designed compound 2 (Fig. 1) to featuring N-6 modification of adenosine to incorporate azide functionality via a long tether. We speculated that the flexible linker might occupy a channel pointing away from the adenosine
* Corresponding author. E-mail address: [email protected] (J. Dowden). 1001-8417/$ – see front matter # 2011 James Dowden. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2011.09.007
1440
W. Hong, J. Dowden / Chinese Chemical Letters 22 (2011) 1439–1442 N
NH2 HO
O
X O
NH2
N
HO
N
O
N
N
R
N N OH
HO HN H2N
R
N
O
OH HO AdoMet - X = S, R = CH3 AdoHcy - X = S, R = H AzaAdoMet - X = N, R = CH3
H N
N
NH2
NH2 Cl
1-R=H 2-R=
N3
O
3
Fig. 1. Structure of AdoMet and related compounds.
binding site, thus presenting the reactive azide functionality in a remote position available for modification using bioorthogonal reactions [5]. Herein, we report convenient synthesis of this N-6 modified AdoMet analogue 2. AzaAdoMet (Fig. 1) has been reported as a potent but general methyltransferase inhibitor that has generally been synthesized by various alkylation strategies in moderate overall yields [6]. We have reported that sequential reductive amination focussed on 50 -amino-50 -deoxyadenosine [7] offered a flexible alternative route to deliver the target tertiary amines [4]. Our initial task was to synthesize an equivalent N-6 modified adenosine 9 (Scheme 1) and investigate its incorporation into this route toward PRMT inhibitors. Azide-tethered amine 5 [8] was incorporated at the N-6 position of adenosine was attempted using two approaches shown in Scheme 1. Vorbru¨ggen glycosylation of 6-chloropurine with ribofuranose tetraacetate 3 was achieved using tin tetrachloride to give the protected 6-chloride adenosine analogue 4 in 78% yield [9]. The purinyl chloride was then substituted by the azide-tethered amine 5 in the presence of triethylamine to form the desired compound 6 in a disappointing yield of 43%. Finally, acetate protecting groups were cleaved using sodium methoxide and the crude product directly reacted with 2, 2-dimethoxylpropane in the presence of toluenesulfonic acid and acetone to give the N-6 modified adenosine 9 in 51% yield. Superior overall yields could be achieved by subjecting inosine 7 to familiar conditions to deliver acetonide-protected inosine 8 in 63% yield [10]. The azide-tethered amine 5 was then installed by in situ activation of using benzotriazole-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate (PyBOP) in the presence of N, N0 -diisopropylethylamine (DIPEA) and subsequent displacement with amine 5 to furnish the compound 9 in 75% yield [11]. This two-step process was clearly more efficient and could be easily scaled up.
Cl
N OAc
O
AcO AcO
3
N
4
H2N
OAc
N3
O 5
N N
AcO
3
N
O
AcO
b
N AcO
OAc
N
O
AcO
a
R
N
OAc
6 c
O
HO
N
NH N
OH
HO 7
O
N
O
N
d
N
O
HO
NH
e
O
H2N
O
N3
5 8
N N
O
O
3
N
O
HO
N O
R
N
9 R=
HN
O
N3 3
Scheme 1. Reagent and conditions: (a) SnCl4, 6-chloropurine, CH3CN, rt, 78%; (b) 5, Et3N, dry DMF, rt to 60 8C, 43%; (c) NaOCH3, dry MeOH, rt, and then TsOH, 2, 2-dimethoxypropane, acetone, rt, 51%; (d) TsOH, 2, 2-dimethoxylpropane, acetone, rt, 63%; (e) 5, PyBOP, DIPEA, dry DMF, rt, 75%.
W. Hong, J. Dowden / Chinese Chemical Letters 22 (2011) 1439–1442 R
N N
O
HO
a
N
O
O
N
O
N
N
N
O
b
NHBoc t-BuO
O
d
N N BocHN
O
N
O
O
NHBoc 14 13
R
N
NHBoc t-BuO
N
O
O
O 12
R
N N H
O
O 11
NHBoc t-BuO
N N
10
c
N
O
H2N
O
O
R
N
N
9
N
O
N
HO e
15 NHBoc
OH
HO
H2N
N N
O 2
HN
N
N
O
N
O
O
R
N
NH2
N N
O
BocHN
R
N
O
1441
NH2 Cl
R=
HN
O
N3 3
Scheme 2. Reagents and conditions: (a) phthalimide, DIAD, Ph3P, dry THF, rt, 76%; (b) hydrazine hydrate, ethanol, reflux, 82%; (c) 12, NaBH(OAc)3, DCE, rt, 75%; (d) 14, NaBH(OAc)3, DCE, rt, 99%; (e) 98% TFA, rt, 87%.
We next investigated incorporation of the N-6 modified adenosine analogue 9 into our route to deliver putative PRMT inhibitors via overall Gabriel synthesis and sequential reductive aminations and deprotection (Scheme 2). Compound 9 was first converted to the phthalimide 10 using standard Mitsunobu conditions in 76% yield and then deprotected by refluxing with hydrazine monohydrate in ethanol to deliver the azide-tethered 50 -amino-50 -deoxyadenosine 11 in 82% yield [7]. Condensation of compound 11 with aldehyde 12 (derived from aspartic acid) [12] by reductive amination with sodium triacetoxyborohydride delivered the amine analogue of AdoHcy 13, a flexible precursor to a range of potential AdoMet analogues, in 75% yield. Further reductive amination with guanidine bearing aldehyde 14, gave precursor 15 in good yield (99%), which was purified by silica column chromatography. Finally, deprotection using aqueous TFA solution removed all protecting groups and the resulting trifluoroacetate salt subject to anion exchange chromatography using Amberlyst IRA-400 (Cl) to deliver the target N-6 modified AdoMet analogue 2 as its hydrochloride salts as hygroscopic solid [13]. The in vitro bio-evaluation of N-6 modified AdoMet analogue 2 is in progress. Preliminary evaluation was carried using human recombinant PRMT1, [S-3H3C]-SAM and substrate recombinant Sam68, and the methyl transfer was measured by scintillation counting [14]. The preliminary result showed that compound 2 has no significant inhibition for PRMT1, but compound 1 can inhibit PRMT1 with IC50 6 mmol/L [4]. Through comparing the conformations of compounds 1 and 2, it was presumed that there is no enough space on PRMT1 binding pocket for the long azide tether on N-6 position of compound 2, which makes it difficult to bind at there. The further bio-evaluation will be carried out and molecular modeling will be used to analyze the results. In summary, we describe facile synthesis of an azide-tethered 50 -amino-50 -deoxy adenosine derivative 11, a flexible precursor to functional probes of a range of AdoMet analogues. Decoration of this compound with guanidine functionally via a propyl linker gave an analogue of our previously described PRMT1 inhibitor. Acknowledgments This work was financially supported by the Medical Research Council Grant (No. G0700840) and the University of Nottingham (Studentship to Wei Hong).
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W. Hong, J. Dowden / Chinese Chemical Letters 22 (2011) 1439–1442
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
M.T. Bedford, S. Richard, Mol. Cell 18 (2005) 263. M.T. Bedford, J. Cell Sci. 120 (2007) 4243. R.A. Copeland, M.E. Solomon, V.M. Richon, Nat. Rev. Drug Discov. 8 (2009) 724. J. Dowden, W. Hong, R.V. Parry, et al. Bioorg. Med. Chem. Lett. 20 (2010) 2103. N.J. Agard, J.M. Baskin, J.A. Prescher, et al. ACS Chem. Biol. 1 (2006) 644. M.J. Thompson, A. Mekhalfia, D.P. Hornby, et al. J. Org. Chem. 64 (1999) 7467. A.M. Reeve, C.A. Townsend, Tetrahedron 54 (1998) 15959. S.S. Iyer, A.S. Anderson, S. Reed, et al. Tetrahedron Lett. 45 (2004) 4285. M. Hocek, P. Silhar, I.H. Shih, et al. Bioorg. Med. Chem. Lett. 16 (2006) 5290. R.J. Middleton, S.J. Briddon, Y. Cordeaux, et al. J. Med. Chem. 50 (2007) 782. Z.K. Wan, E. Binnun, D.P. Wilson, et al. Org. Lett. 7 (2005) 5877. R.M. Werner, O. Shokek, J.T. Davis, J. Org. Chem. 62 (1997) 8243. 1 Spectral data of 2: slightly yellow hygroscopic solid, ½a23 D +20.59 (c 1.02, MeOH); y (max/cm ) 2108 (N3), 1737 (C O), 1670 (C C, aromatic); 1H NMR (400 MHz, D2O): d 8.35 (s, 2H, ArH), 6.07 (d, 1H, J = 3.2, 10 -H), 4.71 (m, 1H, 20 -H), 4.39 (m, 2H, 30 -H and 40 -H), 4.02 (m, 1H, CHCH2), 3.75 (m, 2H, 50 -CH2), 3.72 (m, 2H, adenosine-NHCH2), 3.65–3.37 (m, 14H, OCH2 6 and CH2N3), 3.33 (m, 2H, CH2NH), 3.27 (m, 2H, CHCH2CH2N), 3.12 (m, 2H, CH2CH2CH2N), 2.33 (m, 1H, CHCHaHb), 2.21 (m, 1H, CHCHaHb), 1.89 (m, 2H, CH2CH2CH2); 13C NMR (100 MHz, D2O): d 170.6 (C), 156.6 (C), 149.0 (C), 146.7 (C), 144.2 (CH), 143.1, (CH), 119.7 (C), 90.1 (CH), 78.0 (CH), 73.0 (CH), 71.5 (CH), 70.4–68.2 (CH2 5), 65.1 (CH2), 54.9 (CH2 2), 51.0 (CH2), 50.4 (CH), 50.1 (CH2), 42.4 (CH2), 38.0 (CH2), 24.3 (CH2), 22.6 (CH2); ESI-MS 668.4 (M+H+); HRMS calcd. for C26H46N13O8+ 668.3587, found 668.3611. [14] D.H. Cheng, N. Yadav, R.W. King, et al. J. Biochem. 279 (2004) 23892.
Chinese Chemical Letters 22 (2011) 1439–1442 www.elsevier.com/locate/cclet
Facile synthesis of N-6 adenosine modified analogue toward S-adenosyl methionine derived probe for protein arginine methyltransferases Wei Hong a, James Dowden b,* a
School of Chemistry and Chemical Engineering, Beifang University of Nationalities, Yinchuan 750021, China b School of Chemistry, University of Nottingham, Nottingham NG7 2RD, UK Received 24 May 2011 Available online 10 October 2011
Abstract Chemically modified cellular co-factors that provide function, such as immobilization or incorporation of fluorescent dyes, are valuable probes of biological activity. A convenient route to obtain S-adenosyl methionine (AdoMet) analogues modified at N-6 adenosine to feature a linker terminating in azide functionality is described herein. Subsequent decoration of such AdoMet analogues with guanidinium terminated linkers leads to novel potential bisubstrate inhibitors for protein arginine methyltransferases, PRMTs. # 2011 James Dowden. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Protein arginine methyltransferases; S-Adenosyl methionine; Nucleosides
Protein arginine methyltransferases (PRMTs, EC 2.1.1.125) utilize S-adenosyl methionine (AdoMet, Fig. 1) to catalyze post-translational methylation of arginine residues of a variety of substrate proteins, leading to S-adenosyl homocysteine (AdoHcy, Fig. 1) and monomethylarginine (mMA). Further methylation often follows, with Type I PRMTs delivering asymmetrical dimethylarginine (aDMA) and Type II PRMTs symmetrical dimethylarginine (sDMA), respectively [1]. At least nine PRMTs have been identified in mammals and an important roles in governing cellular processes, such as including gene regulation, RNA splicing and transport, or signal transduction are emerging [2]. There is a significant interest in developing potential drugs for individual PRMTs, especially for the treatment of hormone dependent cancer [3]. Functional chemical probes may be valuable for exploring inhibition, and potential immobilization of specific PRMTs. So far, we have reported AdoMet analogue 1 (Fig. 1) as a potent inhibitor of PRMT1 (IC50 6 mmol/L), that features structure intended to target both AdoMet and arginine subsites of the enzyme [4]. Modification of AdoMet analogue 1 to facilitate labeling or immobilization of this enzyme for functional studies would be highly desirable. We therefore designed compound 2 (Fig. 1) to featuring N-6 modification of adenosine to incorporate azide functionality via a long tether. We speculated that the flexible linker might occupy a channel pointing away from the adenosine
* Corresponding author. E-mail address: [email protected] (J. Dowden). 1001-8417/$ – see front matter # 2011 James Dowden. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2011.09.007
1440
W. Hong, J. Dowden / Chinese Chemical Letters 22 (2011) 1439–1442 N
NH2 HO
O
X O
NH2
N
HO
N
O
N
N
R
N N OH
HO HN H2N
R
N
O
OH HO AdoMet - X = S, R = CH3 AdoHcy - X = S, R = H AzaAdoMet - X = N, R = CH3
H N
N
NH2
NH2 Cl
1-R=H 2-R=
N3
O
3
Fig. 1. Structure of AdoMet and related compounds.
binding site, thus presenting the reactive azide functionality in a remote position available for modification using bioorthogonal reactions [5]. Herein, we report convenient synthesis of this N-6 modified AdoMet analogue 2. AzaAdoMet (Fig. 1) has been reported as a potent but general methyltransferase inhibitor that has generally been synthesized by various alkylation strategies in moderate overall yields [6]. We have reported that sequential reductive amination focussed on 50 -amino-50 -deoxyadenosine [7] offered a flexible alternative route to deliver the target tertiary amines [4]. Our initial task was to synthesize an equivalent N-6 modified adenosine 9 (Scheme 1) and investigate its incorporation into this route toward PRMT inhibitors. Azide-tethered amine 5 [8] was incorporated at the N-6 position of adenosine was attempted using two approaches shown in Scheme 1. Vorbru¨ggen glycosylation of 6-chloropurine with ribofuranose tetraacetate 3 was achieved using tin tetrachloride to give the protected 6-chloride adenosine analogue 4 in 78% yield [9]. The purinyl chloride was then substituted by the azide-tethered amine 5 in the presence of triethylamine to form the desired compound 6 in a disappointing yield of 43%. Finally, acetate protecting groups were cleaved using sodium methoxide and the crude product directly reacted with 2, 2-dimethoxylpropane in the presence of toluenesulfonic acid and acetone to give the N-6 modified adenosine 9 in 51% yield. Superior overall yields could be achieved by subjecting inosine 7 to familiar conditions to deliver acetonide-protected inosine 8 in 63% yield [10]. The azide-tethered amine 5 was then installed by in situ activation of using benzotriazole-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate (PyBOP) in the presence of N, N0 -diisopropylethylamine (DIPEA) and subsequent displacement with amine 5 to furnish the compound 9 in 75% yield [11]. This two-step process was clearly more efficient and could be easily scaled up.
Cl
N OAc
O
AcO AcO
3
N
4
H2N
OAc
N3
O 5
N N
AcO
3
N
O
AcO
b
N AcO
OAc
N
O
AcO
a
R
N
OAc
6 c
O
HO
N
NH N
OH
HO 7
O
N
O
N
d
N
O
HO
NH
e
O
H2N
O
N3
5 8
N N
O
O
3
N
O
HO
N O
R
N
9 R=
HN
O
N3 3
Scheme 1. Reagent and conditions: (a) SnCl4, 6-chloropurine, CH3CN, rt, 78%; (b) 5, Et3N, dry DMF, rt to 60 8C, 43%; (c) NaOCH3, dry MeOH, rt, and then TsOH, 2, 2-dimethoxypropane, acetone, rt, 51%; (d) TsOH, 2, 2-dimethoxylpropane, acetone, rt, 63%; (e) 5, PyBOP, DIPEA, dry DMF, rt, 75%.
W. Hong, J. Dowden / Chinese Chemical Letters 22 (2011) 1439–1442 R
N N
O
HO
a
N
O
O
N
O
N
N
N
O
b
NHBoc t-BuO
O
d
N N BocHN
O
N
O
O
NHBoc 14 13
R
N
NHBoc t-BuO
N
O
O
O 12
R
N N H
O
O 11
NHBoc t-BuO
N N
10
c
N
O
H2N
O
O
R
N
N
9
N
O
N
HO e
15 NHBoc
OH
HO
H2N
N N
O 2
HN
N
N
O
N
O
O
R
N
NH2
N N
O
BocHN
R
N
O
1441
NH2 Cl
R=
HN
O
N3 3
Scheme 2. Reagents and conditions: (a) phthalimide, DIAD, Ph3P, dry THF, rt, 76%; (b) hydrazine hydrate, ethanol, reflux, 82%; (c) 12, NaBH(OAc)3, DCE, rt, 75%; (d) 14, NaBH(OAc)3, DCE, rt, 99%; (e) 98% TFA, rt, 87%.
We next investigated incorporation of the N-6 modified adenosine analogue 9 into our route to deliver putative PRMT inhibitors via overall Gabriel synthesis and sequential reductive aminations and deprotection (Scheme 2). Compound 9 was first converted to the phthalimide 10 using standard Mitsunobu conditions in 76% yield and then deprotected by refluxing with hydrazine monohydrate in ethanol to deliver the azide-tethered 50 -amino-50 -deoxyadenosine 11 in 82% yield [7]. Condensation of compound 11 with aldehyde 12 (derived from aspartic acid) [12] by reductive amination with sodium triacetoxyborohydride delivered the amine analogue of AdoHcy 13, a flexible precursor to a range of potential AdoMet analogues, in 75% yield. Further reductive amination with guanidine bearing aldehyde 14, gave precursor 15 in good yield (99%), which was purified by silica column chromatography. Finally, deprotection using aqueous TFA solution removed all protecting groups and the resulting trifluoroacetate salt subject to anion exchange chromatography using Amberlyst IRA-400 (Cl) to deliver the target N-6 modified AdoMet analogue 2 as its hydrochloride salts as hygroscopic solid [13]. The in vitro bio-evaluation of N-6 modified AdoMet analogue 2 is in progress. Preliminary evaluation was carried using human recombinant PRMT1, [S-3H3C]-SAM and substrate recombinant Sam68, and the methyl transfer was measured by scintillation counting [14]. The preliminary result showed that compound 2 has no significant inhibition for PRMT1, but compound 1 can inhibit PRMT1 with IC50 6 mmol/L [4]. Through comparing the conformations of compounds 1 and 2, it was presumed that there is no enough space on PRMT1 binding pocket for the long azide tether on N-6 position of compound 2, which makes it difficult to bind at there. The further bio-evaluation will be carried out and molecular modeling will be used to analyze the results. In summary, we describe facile synthesis of an azide-tethered 50 -amino-50 -deoxy adenosine derivative 11, a flexible precursor to functional probes of a range of AdoMet analogues. Decoration of this compound with guanidine functionally via a propyl linker gave an analogue of our previously described PRMT1 inhibitor. Acknowledgments This work was financially supported by the Medical Research Council Grant (No. G0700840) and the University of Nottingham (Studentship to Wei Hong).
1442
W. Hong, J. Dowden / Chinese Chemical Letters 22 (2011) 1439–1442
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
M.T. Bedford, S. Richard, Mol. Cell 18 (2005) 263. M.T. Bedford, J. Cell Sci. 120 (2007) 4243. R.A. Copeland, M.E. Solomon, V.M. Richon, Nat. Rev. Drug Discov. 8 (2009) 724. J. Dowden, W. Hong, R.V. Parry, et al. Bioorg. Med. Chem. Lett. 20 (2010) 2103. N.J. Agard, J.M. Baskin, J.A. Prescher, et al. ACS Chem. Biol. 1 (2006) 644. M.J. Thompson, A. Mekhalfia, D.P. Hornby, et al. J. Org. Chem. 64 (1999) 7467. A.M. Reeve, C.A. Townsend, Tetrahedron 54 (1998) 15959. S.S. Iyer, A.S. Anderson, S. Reed, et al. Tetrahedron Lett. 45 (2004) 4285. M. Hocek, P. Silhar, I.H. Shih, et al. Bioorg. Med. Chem. Lett. 16 (2006) 5290. R.J. Middleton, S.J. Briddon, Y. Cordeaux, et al. J. Med. Chem. 50 (2007) 782. Z.K. Wan, E. Binnun, D.P. Wilson, et al. Org. Lett. 7 (2005) 5877. R.M. Werner, O. Shokek, J.T. Davis, J. Org. Chem. 62 (1997) 8243. 1 Spectral data of 2: slightly yellow hygroscopic solid, ½a23 D +20.59 (c 1.02, MeOH); y (max/cm ) 2108 (N3), 1737 (C O), 1670 (C C, aromatic); 1H NMR (400 MHz, D2O): d 8.35 (s, 2H, ArH), 6.07 (d, 1H, J = 3.2, 10 -H), 4.71 (m, 1H, 20 -H), 4.39 (m, 2H, 30 -H and 40 -H), 4.02 (m, 1H, CHCH2), 3.75 (m, 2H, 50 -CH2), 3.72 (m, 2H, adenosine-NHCH2), 3.65–3.37 (m, 14H, OCH2 6 and CH2N3), 3.33 (m, 2H, CH2NH), 3.27 (m, 2H, CHCH2CH2N), 3.12 (m, 2H, CH2CH2CH2N), 2.33 (m, 1H, CHCHaHb), 2.21 (m, 1H, CHCHaHb), 1.89 (m, 2H, CH2CH2CH2); 13C NMR (100 MHz, D2O): d 170.6 (C), 156.6 (C), 149.0 (C), 146.7 (C), 144.2 (CH), 143.1, (CH), 119.7 (C), 90.1 (CH), 78.0 (CH), 73.0 (CH), 71.5 (CH), 70.4–68.2 (CH2 5), 65.1 (CH2), 54.9 (CH2 2), 51.0 (CH2), 50.4 (CH), 50.1 (CH2), 42.4 (CH2), 38.0 (CH2), 24.3 (CH2), 22.6 (CH2); ESI-MS 668.4 (M+H+); HRMS calcd. for C26H46N13O8+ 668.3587, found 668.3611. [14] D.H. Cheng, N. Yadav, R.W. King, et al. J. Biochem. 279 (2004) 23892.