research communications\(\def\hfill{\hskip 5em}\def\hfil{\hskip 3em}\def\eqno#1{\hfil {#1}}\)

Journal logoCRYSTALLOGRAPHIC
COMMUNICATIONS
ISSN: 2056-9890
Volume 71| Part 3| March 2015| Pages 254-257

Crystal structure of N-{N-[N-acetyl-(S)-leuc­yl]-(S)-leuc­yl}norleucinal (ALLN), an inhibitor of proteasome

CROSSMARK_Color_square_no_text.svg

aPeptides International, Inc., 11621 Electron Drive, Louisville, KY 40299, USA, bLeidos Biomedical Research Inc., Basic Science Program, Argonne National Laboratory, Argonne, IL 60439, USA, and cSynchrotron Radiation Research Section, MCL, National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439, USA
*Correspondence e-mail: dauter@anl.gov

Edited by M. Gdaniec, Adam Mickiewicz University, Poland (Received 25 January 2015; accepted 30 January 2015; online 7 February 2015)

The title compound, C20H37N3O4, also known by the acronym ALLN, is a tripeptidic inhibitor of the proteolytic activity of the proteasomes, enzyme complexes implicated in several neurodegenerative diseases and other disorders, including cancer. The crystal structure of ALLN, solved from synchrotron radiation diffraction data, revealed the mol­ecules in extended conformation of the backbone and engaging all peptide N and O atoms in inter­molecular hydrogen bonds forming an infinite anti­parallel β-sheet.

1. Chemical context

Proteasomes are high-mol­ecular-mass multicatalytic enzyme complexes localized in the nucleus and cytosol of all eukaryotic cells. As a part of the ubiquitin–proteasome pathway, the complex executes a remarkable set of functions, ranging from the complete destruction of abnormal and misfolded proteins to the specific proteolytic activation of crucial signaling mol­ecules (Adams, 2003[Adams, J. (2003). Cancer Treat. Rev. 29 (Suppl. 1), 3-9.]; Groll & Potts, 2011[Groll, M. & Potts, B. C. (2011). Curr. Top. Med. Chem. 11, 2850-2878.]). The ubiquitin–proteasome pathway has been implicated in several forms of malignancy, in the pathogenesis of some autoimmune disorders, the aging process related cardiac dysfunction, diabetic complications, and neurodegenerative diseases (e.g. Alzheimer's, Parkinson's, Huntington's) (Dahlmann, 2007[Dahlmann, B. (2007). BMC Biochem. 8 (Suppl. 1), S3.]; Paul, 2008[Paul, S. (2008). BioEssays, 30, 1172-1184.]; Jankowska et al., 2013[Jankowska, E., Stoj, J., Karpowicz, P., Osmulski, P. A. & Gaczynska, M. (2013). Curr. Pharm. Des. 19, 1010-1028.]). Therefore, study of proteasome functions and the design and development of proteasome inhibitors is being pursued in many laboratories (Bennett & Kirk, 2008[Bennett, M. K. & Kirk, C. J. (2008). Curr. Opin. Drug Disc. 11, 616-625.]). A great amount of effort has been expended to explore proteasome inhibition as a novel targeted approach in cancer therapy. The first success came with FDA approval of Bortezomid for the treatment of multiple myeloma (Kane et al., 2006[Kane, R. C., Farrell, A. T., Sridhara, R. & Pazdur, R. (2006). Clin. Cancer Res. 12, 2955-2960.]; Goldberg, 2012[Goldberg, A. L. (2012). J. Cell Biol. 199, 583-588.]). Since then, numerous compounds have been reported to inhibit the components of the ubiquitin–proteasome system, and several new drug candidates undergoing clinical trials have emerged (Genin et al., 2010[Genin, E., Reboud-Ravaux, M. & Vidal, J. (2010). Curr. Top. Med. Chem. 10, 232-256.]; Tsukamoto & Yokosawa, 2010[Tsukamoto, S. & Yokosawa, H. (2010). Planta Med. 76, 1064-1074.]; Frankland-Searby & Bhaumik, 2012[Frankland-Searby, S. & Bhaumik, S. R. (2012). Biochim. Biophys. Acta, 1825, 64-76.]; Jankowska et al., 2013[Jankowska, E., Stoj, J., Karpowicz, P., Osmulski, P. A. & Gaczynska, M. (2013). Curr. Pharm. Des. 19, 1010-1028.]). Peptide aldehydes were the first inhibitors designed to target the proteasome, and are still the most commonly used and best characterized group of such inhibitors (Kisselev et al., 2012[Kisselev, A. F., van der Linden, W. A. & Overkleeft, H. S. (2012). Chem. Biol. 19, 99-115.]). A notable one among them, Ac-Leu-Leu-Nle-H (ALLN, MG101), is also a potent inhibitor of nonproteasomal cysteine protease calpain I (Pietsch et al., 2010[Pietsch, M., Chua, K. C. & Abell, A. D. (2010). Curr. Top. Med. Chem. 10, 270-293.]). ALLN, a cell-permeable tripeptide aldehyde reversible inhibitor of chymotripsin-like proteolytic activity of the proteasomes, was the first to be crystallized in a complex with an eukaryotic proteasome (Groll et al., 1997[Groll, M., Ditzel, L., Lowe, J., Stock, D., Bochtler, M., Bartunik, H. D. & Huber, R. (1997). Nature, 386, 463-471.]). Crystallographic analysis of the complex at 2.4 Å resolution revealed a structural organization of the proteasome and how the inhibitor binds to its active site. ALLN, as well as other peptide aldehydes, do it via reversible hemiacetal formation with the involvement of N-terminal threonine hy­droxy group in the proteasome β-subunits (Borissenko & Groll, 2007[Borissenko, L. & Groll, M. (2007). Chem. Rev. 107, 687-717.]). The aldehyde structure derived from the crystal complex coordinates was used in mol­ecular modeling of inhibitor-proteasome inter­actions (Zhang et al., 2009[Zhang, S., Shi, Y., Jin, H., Liu, Z., Zhang, L. & Zhang, L. (2009). J. Mol. Model. 15, 1481-1490.]). High resolution structural data from this study may provide better accuracy in future modeling of the inhibitor inter­actions with proteasome and other potential intra­cellular targets.

[Scheme 1]

2. Structural commentary

We report here the crystal structure of ALLN refined against 0.65 Å resolution diffraction data measured with synchrotron radiation. The mol­ecule adopts an extended conformation of the backbone chain (Fig. 1[link]) with the φ,ψ-torsion angles residing in the β region of the Ramachandran plot (Ramakrishnan & Ramachandran, 1965[Ramakrishnan, C. & Ramachandran, G. N. (1965). Biophys. J. 5, 909-933.]). All three consecutive peptide residues are in trans conformation and their ω angles are −179.42 (9), 173.77 (8), and 177.72 (10)°. The side chains of the two leucine and one norleucine residues have unstrained conformations, and do not deviate by more than 7° from either trans or gauche rotamers along the consecutive C—C bonds.

[Figure 1]
Figure 1
The mol­ecule of ALLN, showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 50% probability level.

3. Supra­molecular features

All of the peptide ALLN N and O atoms are engaged in inter­molecular hydrogen bonds (Table 1[link]) between mol­ecules related by the crystallographic 21 axis, forming an infinite anti­parallel β-sheet throughout the crystal (Fig. 2[link]). The inter­actions between the sheets are mainly by the hydro­phobic contacts of the aliphatic amino acid side chains. The arrangement of ALLN molecules in the ac plane, interacting through their aliphatic side chains, is illustrated in Fig. 3[link].

Table 1
Hydrogen-bond geometry (Å, °)

D—H⋯A D—H H⋯A DA D—H⋯A
N20—H201⋯O31i 0.88 2.05 2.897 (3) 161
N30—H301⋯O21ii 0.88 1.99 2.863 (3) 171
N40—H401⋯O12i 0.88 1.96 2.827 (3) 169
Symmetry codes: (i) [-x+1, y+{\script{1\over 2}}, -z+1]; (ii) [-x+1, y-{\script{1\over 2}}, -z+1].
[Figure 2]
Figure 2
Backbones of three neighboring mol­ecules of ALLN, forming a fragment of an anti­parallel β-sheet extending through the crystal. The amino acid side chains are not shown for clarity.
[Figure 3]
Figure 3
Arrangement of ALLN mol­ecules in the ac plane of the crystal, inter­acting through their aliphatic side chains.

4. Synthesis and crystallization

The title aldehyde was prepared according to the general synthetic procedure reported by Schaschke et al. (1996[Schaschke, N., Musiol, H. J., Assfalg-Machleidt, I., Machleidt, W., Rudolph-Bohner, S. & Moroder, L. (1996). FEBS Lett. 391, 297-301.]), and a 45% overall yield was obtained. The product was crystallized from aceto­nitrile.

5. Refinement details

Crystal data, data collection and structure refinement details are summarized in Table 2[link]. A needle-like crystal elongated in the a direction was selected, picked up in the rayon loop and then quickly cryo-cooled in a stream of cold nitro­gen gas at the single-axis goniostat of the SER-CAT synchrotron station ID19 at the Advanced Photon Source, Argonne National Laboratory, USA. Diffraction images were collected with the use of MAR300 CCD detector in two passes differing in the effective exposure and resolution limits in order to adequately measure the weakest high-resolution reflections, as well as the strongest low-angle reflections without overloading detector pixels. All 38117 measured intensities from both passes were integrated, scaled and merged by HKL-2000 (Otwinowski & Minor, 1997[Otwinowski, Z. & Minor, W. (1997). Methods in Enzymology, Vol. 276, Macromolecular Crystallography, Part A, edited by C. W. Carter Jr & R. M. Sweet, pp. 307-326. New York: Academic Press.]) into the set of 4561 unique reflections with the overall Rmerge factor of 0.049. The data set is rather strong, with the I/σ(I) ratio equal to 25 at the highest resolution of 0.65 Å. H atoms were located in a difference synthesis and refined as riding on their parent atoms in geometrically idealized positions. Because of the short wavelength of synchrotron radiation, all Friedel mates were averaged during data processing. The chirality of the mol­ecule was deduced from the known chiral centres in the substrates used in chemical synthesis.

Table 2
Experimental details

Crystal data
Chemical formula C20H37N3O4
Mr 383.59
Crystal system, space group Monoclinic, P21
Temperature (K) 100
a, b, c (Å) 10.85 (1), 9.510 (9), 11.200 (11)
β (°) 94.85 (2)
V3) 1152 (2)
Z 2
Radiation type Synchrotron, λ = 0.6199 Å
μ (mm−1) 0.09
Crystal size (mm) 0.30 × 0.05 × 0.02
 
Data collection
Diffractometer MAR300 CCD
Absorption correction Multi-scan (SCALEPACK; Otwinowski et al., 2003[Otwinowski, Z., Borek, D., Majewski, W. & Minor, W. (2003). Acta Cryst. A59, 228-234.])
Tmin, Tmax 0.974, 0.999
No. of measured, independent and observed [I > 2σ(I)] reflections 4561, 4561, 4492
Rint 0.049
(sin θ/λ)max−1) 0.767
 
Refinement
R[F2 > 2σ(F2)], wR(F2), S 0.041, 0.115, 1.07
No. of reflections 4561
No. of parameters 244
No. of restraints 1
H-atom treatment H-atom parameters constrained
Δρmax, Δρmin (e Å−3) 0.44, −0.29
Computer programs: HKL-2000 (Otwinowski & Minor, 1997[Otwinowski, Z. & Minor, W. (1997). Methods in Enzymology, Vol. 276, Macromolecular Crystallography, Part A, edited by C. W. Carter Jr & R. M. Sweet, pp. 307-326. New York: Academic Press.]), SHELXD and SHELXL97 (Sheldrick, 2008[Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122.]), ORTEP-3 for Windwows (Farrugia, 2012[Farrugia, L. J. (2012). J. Appl. Cryst. 45, 849-854.]) and pyMOL (DeLano, 2002[DeLano, W. L. (2002). The pyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA, USA.]).

Supporting information


Chemical context top

Proteasomes are high-molecular-mass multicatalytic enzyme complexes localized in the nucleus and cytosol of all eukaryotic cells. As a part of the ubiquitin–proteasome pathway, the complex executes a remarkable set of functions, ranging from the complete destruction of abnormal and misfolded proteins to the specific proteolytic activation of crucial signaling molecules (Adams, 2003; Groll & Potts, 2011). The ubiquitin–proteasome pathway has been implicated in several forms of malignancy, in the pathogenesis of some autoimmune disorders, the aging process related cardiac dysfunction, diabetic complications, and neurodegenerative diseases (e.g. Alzheimer's, Parkinson's, Huntington's) (Dahlmann, 2007; Paul, 2008; Jankowska et al., 2013). Therefore, study of proteasome functions and the design and development of proteasome inhibitors is being pursued in many laboratories (Bennett & Kirk, 2008). A great amount of effort has been expended to explore proteasome inhibition as a novel targeted approach in cancer therapy. The first success came with FDA approval of Bortezomid for the treatment of multiple myeloma (Kane et al., 2006; Goldberg, 2012). Since then, numerous compounds have been reported to inhibit the components of the ubiquitin–proteasome system, and several new drug candidates undergoing clinical trials have emerged (Genin et al., 2010; Tsukamoto & Yokosawa, 2010; Frankland-Searby & Bhaumik, 2012; Jankowska et al., 2013). Peptide aldehydes were the first inhibitors designed to target the proteasome, and are still the most commonly used and best characterized group of such inhibitors (Kisselev et al., 2012). A notable one among them, Ac-Leu-Leu-Nle-H (ALLN, MG101), is also a potent inhibitor of nonproteasomal cysteine protease calpain I (Pietsch et al., 2010). ALLN, a cell-permeable tripeptide aldehyde reversible inhibitor of chymotripsin-like proteolytic activity of the proteasomes, was the first to be crystallized in a complex with an eukaryotic proteasome (Groll et al., 1997). Crystallographic analysis of the complex at 2.4 Å resolution revealed a structural organization of the proteasome and how the inhibitor binds to its active site. ALLN, as well as other peptide aldehydes, do it via reversible hemiacetal formation with the involvement of N-terminal threonine hy­droxy group in the proteasome β-subunits (Borissenko & Groll, 2007). The aldehyde structure derived from the crystal complex coordinates was used in molecular modeling of inhibitor-proteasome inter­actions (Zhang et al., 2009). High resolution structural data from this study may provide better accuracy in future modeling of the inhibitor inter­actions with proteasome and other potential intra­cellular targets.

Structural commentary top

We report here the crystal structure of ALLN refined against 0.65 Å resolution diffraction data measured with synchrotron radiation. The molecule adopts an extended conformation of the backbone chain (Fig. 1) with the ϕ,ψ-torsion angles residing in the β region of the Ramachandran plot (Ramakrishnan & Ramachandran, 1965). All three consecutive peptide residues are in trans conformation and their ω angles are -179.42 (9), 173.77 (8), and 177.72 (10)°. The side chains of the two leucine and one norleucine residues have unstrained conformations, and do not deviate by more than 7° from either trans or gauche rotamers along the consecutive C—C bonds.

Supra­molecular features top

All of the peptide ALLN N and O atoms are engaged in inter­molecular hydrogen bonds between molecules related by the crystallographic 21 axis, forming an infinite anti­parallel β-sheet throughout the crystal (Fig. 2). The inter­actions between the sheets are mainly by the hydro­phobic contacts of the aliphatic amino acid side chains.

Synthesis and crystallization top

The title aldehyde was prepared according to the general synthetic procedure reported by Schaschke et al. (1996), and a 45% overall yield was obtained. The product was crystallized from aceto­nitrile.

Refinement details top

Crystal data, data collection and structure refinement details are summarized in Table 2. A needle-like crystal elongated in the a direction was selected, picked up in the rayon loop and then quickly cryo-cooled in a stream of cold nitro­gen gas at the single-axis goniostat of the SER-CAT synchrotron station ID19 at the Advanced Photon Source, Argonne National Laboratory, USA. Diffraction images were collected with the use of MAR300 CCD detector in two passes differing in the effective exposure and resolution limits in order to adequately measure the weakest high-resolution reflections, as well as the strongest low-angle reflections without overloading detector pixels. All 38117 measured intensities from both passes were integrated, scaled and merged by HKL-2000 (Otwinowski & Minor, 1997) into the set of 4561 unique reflections with the overall Rmerge factor of 0.049. The data set is rather strong, with the I/σ(I) ratio equal to 25 at the highest resolution of 0.65 Å. H atoms were located in a difference synthesis (Fig. 3a) and refined as riding on their parent atoms in geometrically idealized positions. Because of the short wavelength of synchrotron radiation, all Friedel mates were averaged during data processing. The chirality of the molecule was deduced from the known chiral centres in the substrates used in chemical synthesis.

Computing details top

Data collection: sergui, SER-CAT APS beamline software; cell refinement: HKL-2000 (Otwinowski & Minor, 1997); data reduction: HKL-2000 (Otwinowski & Minor, 1997); program(s) used to solve structure: SHELXD (Sheldrick, 2008); program(s) used to refine structure: SHELXL (Sheldrick, 2008); molecular graphics: ORTEP-3 for Windwows (Farrugia, 2012) and pyMOL (DeLano, 2002); software used to prepare material for publication: SHELXL97 (Sheldrick, 2008).

Figures top
[Figure 1] Fig. 1. The molecule of ALLN, showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 50% probability level.
[Figure 2] Fig. 2. Backbones of three neighboring molecules of ALLN, forming a fragment of an antiparallel β-sheet extending through the crystal. The amino acid side chains are not shown for clarity.
[Figure 3] Fig. 3. Arrangement of ALLN molecules in the ac plane of the crystal, interacting through their aliphatic side chains.
N-{N-[N-Acetyl-(S)-leucyl]-(S)-leucyl}norleucinal top
Crystal data top
C20H37N3O4F(000) = 460
Mr = 383.59Dx = 1.110 Mg m3
Monoclinic, P21Synchrotron radiation, λ = 0.6199 Å
a = 10.85 (1) Åθ = 1.5–28.4°
b = 9.510 (9) ŵ = 0.09 mm1
c = 11.200 (11) ÅT = 100 K
β = 94.85 (2)°Needle, colourless
V = 1152 (2) Å30.30 × 0.05 × 0.02 mm
Z = 2
Data collection top
MAR300 CCD
diffractometer
4561 independent reflections
Radiation source: SER-CAT 22ID synchrotron beamline, APS, USA4492 reflections with I > 2σ(I)
Si111 double crystal monochromatorRint = 0.049
ω scansθmax = 28.4°, θmin = 1.5°
Absorption correction: multi-scan
(SCALEPACK; Otwinowski et al., 2003)
h = 016
Tmin = 0.974, Tmax = 0.999k = 014
4561 measured reflectionsl = 1717
Refinement top
Refinement on F2Primary atom site location: structure-invariant direct methods
Least-squares matrix: fullSecondary atom site location: difference Fourier map
R[F2 > 2σ(F2)] = 0.041Hydrogen site location: inferred from neighbouring sites
wR(F2) = 0.115H-atom parameters constrained
S = 1.07 w = 1/[σ2(Fo2) + (0.081P)2 + 0.1533P]
where P = (Fo2 + 2Fc2)/3
4561 reflections(Δ/σ)max < 0.001
244 parametersΔρmax = 0.44 e Å3
1 restraintΔρmin = 0.29 e Å3
Crystal data top
C20H37N3O4V = 1152 (2) Å3
Mr = 383.59Z = 2
Monoclinic, P21Synchrotron radiation, λ = 0.6199 Å
a = 10.85 (1) ŵ = 0.09 mm1
b = 9.510 (9) ÅT = 100 K
c = 11.200 (11) Å0.30 × 0.05 × 0.02 mm
β = 94.85 (2)°
Data collection top
MAR300 CCD
diffractometer
4561 independent reflections
Absorption correction: multi-scan
(SCALEPACK; Otwinowski et al., 2003)
4492 reflections with I > 2σ(I)
Tmin = 0.974, Tmax = 0.999Rint = 0.049
4561 measured reflections
Refinement top
R[F2 > 2σ(F2)] = 0.0411 restraint
wR(F2) = 0.115H-atom parameters constrained
S = 1.07Δρmax = 0.44 e Å3
4561 reflectionsΔρmin = 0.29 e Å3
244 parameters
Special details top

Experimental. diffraction data were measured at the station 22ID of the APS synchrotron by rotation method a in three sweeps of different exposure and all data were scaled and merged into one data set

Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes.

Refinement. Refinement of F2 against all reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on all data will be even larger.

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) top
xyzUiso*/Ueq
C100.26159 (10)0.47871 (12)0.27033 (9)0.01223 (18)
C110.14253 (11)0.53384 (15)0.20793 (12)0.0195 (2)
H1010.14860.63590.19810.029*
H1020.07370.51200.25620.029*
H1030.12800.48930.12910.029*
O120.27570 (9)0.35222 (10)0.29248 (11)0.02159 (19)
N200.34966 (8)0.57472 (10)0.29951 (8)0.01103 (15)
H2010.33360.66330.28180.013*
C210.48033 (9)0.60494 (11)0.48414 (8)0.00972 (16)
O210.46004 (9)0.73127 (9)0.49827 (7)0.01513 (16)
C220.47046 (9)0.54014 (11)0.35897 (8)0.00941 (16)
H2210.48040.43580.36480.011*
C230.57151 (10)0.60292 (12)0.28663 (10)0.01324 (18)
H2310.56860.55420.20830.016*
H2320.55150.70310.27050.016*
C240.70443 (11)0.59414 (15)0.34574 (12)0.0197 (2)
H2410.70630.63980.42630.024*
C250.79082 (15)0.6765 (2)0.2697 (2)0.0362 (4)
H2510.76060.77310.25890.054*
H2520.79270.63120.19130.054*
H2530.87440.67770.31030.054*
C260.75042 (14)0.44426 (18)0.36297 (19)0.0322 (3)
H2610.69470.39220.41140.048*
H2620.83390.44500.40400.048*
H2630.75240.39870.28470.048*
N300.51541 (8)0.51775 (10)0.57466 (8)0.01027 (15)
H3010.52370.42740.56060.012*
C310.65117 (9)0.49218 (11)0.75420 (9)0.01002 (17)
O310.64628 (8)0.36532 (9)0.77767 (8)0.01517 (16)
C320.53994 (9)0.57155 (11)0.69646 (8)0.00945 (16)
H3210.56070.67380.69280.011*
C330.42768 (10)0.55287 (12)0.76899 (9)0.01255 (18)
H3310.40340.45250.76650.015*
H3320.35780.60750.72990.015*
C340.44800 (10)0.59874 (13)0.90039 (9)0.01353 (18)
H3410.51670.54080.94000.016*
C350.48372 (15)0.75302 (16)0.91469 (12)0.0239 (3)
H3510.49560.77651.00010.036*
H3520.41780.81180.87590.036*
H3530.56080.77010.87730.036*
C360.33127 (12)0.56946 (18)0.96323 (11)0.0233 (3)
H3610.34420.59881.04720.035*
H3620.31280.46860.95940.035*
H3630.26180.62210.92350.035*
N400.75393 (9)0.56824 (11)0.77879 (9)0.01460 (17)
H4010.75560.65780.75930.018*
C410.83380 (14)0.45232 (19)0.96234 (14)0.0283 (3)
H4110.76270.48980.99490.034*
O410.89574 (15)0.3697 (2)1.02126 (16)0.0518 (5)
C420.86236 (11)0.50104 (14)0.83793 (12)0.0187 (2)
H4210.88490.41770.78980.022*
C430.97094 (12)0.60363 (17)0.84929 (13)0.0235 (2)
H4311.04160.55830.89600.028*
H4320.94710.68740.89450.028*
C441.01242 (12)0.65110 (17)0.72874 (14)0.0244 (3)
H4410.94260.69960.68330.029*
H4421.08050.72000.74320.029*
C451.05617 (19)0.5319 (2)0.65273 (18)0.0373 (4)
H4510.98550.46860.63070.045*
H4521.11960.47700.70130.045*
C461.1102 (2)0.5807 (3)0.53884 (19)0.0458 (5)
H4611.13630.49870.49430.069*
H4621.18180.64140.55970.069*
H4631.04740.63330.48910.069*
Atomic displacement parameters (Å2) top
U11U22U33U12U13U23
C100.0124 (4)0.0105 (4)0.0138 (4)0.0002 (3)0.0009 (3)0.0023 (3)
C110.0134 (4)0.0200 (6)0.0243 (5)0.0018 (4)0.0028 (4)0.0005 (4)
O120.0181 (4)0.0080 (4)0.0378 (5)0.0016 (3)0.0023 (3)0.0013 (4)
N200.0141 (3)0.0063 (3)0.0121 (3)0.0006 (3)0.0030 (3)0.0008 (3)
C210.0143 (4)0.0062 (4)0.0083 (4)0.0003 (3)0.0016 (3)0.0008 (3)
O210.0277 (4)0.0051 (3)0.0119 (3)0.0031 (3)0.0024 (3)0.0008 (3)
C220.0135 (4)0.0060 (4)0.0082 (3)0.0008 (3)0.0019 (3)0.0008 (3)
C230.0150 (4)0.0110 (4)0.0139 (4)0.0012 (3)0.0020 (3)0.0001 (3)
C240.0141 (4)0.0181 (5)0.0267 (5)0.0019 (4)0.0008 (4)0.0001 (4)
C250.0208 (6)0.0296 (8)0.0591 (11)0.0023 (6)0.0089 (6)0.0118 (8)
C260.0195 (5)0.0219 (7)0.0548 (10)0.0055 (5)0.0012 (6)0.0079 (7)
N300.0172 (4)0.0056 (3)0.0075 (3)0.0006 (3)0.0020 (3)0.0005 (3)
C310.0132 (4)0.0072 (4)0.0092 (3)0.0003 (3)0.0015 (3)0.0003 (3)
O310.0204 (4)0.0062 (3)0.0180 (3)0.0007 (3)0.0035 (3)0.0011 (3)
C320.0137 (4)0.0065 (4)0.0077 (3)0.0000 (3)0.0015 (3)0.0008 (3)
C330.0133 (4)0.0132 (4)0.0109 (4)0.0011 (3)0.0002 (3)0.0013 (3)
C340.0167 (4)0.0143 (5)0.0096 (4)0.0008 (4)0.0013 (3)0.0005 (3)
C350.0375 (7)0.0162 (6)0.0185 (5)0.0037 (5)0.0052 (5)0.0075 (4)
C360.0211 (5)0.0331 (7)0.0164 (5)0.0007 (5)0.0065 (4)0.0021 (5)
N400.0135 (4)0.0083 (4)0.0209 (4)0.0013 (3)0.0051 (3)0.0030 (3)
C410.0235 (6)0.0324 (8)0.0273 (6)0.0049 (5)0.0078 (5)0.0109 (6)
O410.0423 (7)0.0570 (11)0.0533 (9)0.0014 (7)0.0120 (6)0.0360 (8)
C420.0142 (4)0.0163 (5)0.0242 (5)0.0006 (4)0.0061 (4)0.0056 (4)
C430.0170 (5)0.0249 (6)0.0276 (6)0.0069 (5)0.0039 (4)0.0006 (5)
C440.0181 (5)0.0226 (6)0.0323 (6)0.0030 (5)0.0002 (4)0.0042 (5)
C450.0417 (9)0.0321 (9)0.0394 (8)0.0022 (7)0.0114 (7)0.0022 (7)
C460.0419 (9)0.0611 (15)0.0356 (8)0.0080 (10)0.0094 (7)0.0023 (9)
Geometric parameters (Å, º) top
C10—O121.2351 (19)C33—C341.533 (2)
C10—N201.3420 (17)C33—H3310.9900
C10—C111.5097 (19)C33—H3320.9900
C11—H1010.9800C34—C351.522 (2)
C11—H1020.9800C34—C361.526 (2)
C11—H1030.9800C34—H3411.0000
N20—C221.4567 (17)C35—H3510.9800
N20—H2010.8800C35—H3520.9800
C21—O211.2340 (18)C35—H3530.9800
C21—N301.3398 (16)C36—H3610.9800
C21—C221.5268 (19)C36—H3620.9800
C22—C231.5380 (18)C36—H3630.9800
C22—H2211.0000N40—C421.4489 (17)
C23—C241.537 (2)N40—H4010.8800
C23—H2310.9900C41—O411.196 (2)
C23—H2320.9900C41—C421.525 (2)
C24—C261.517 (3)C41—H4110.9500
C24—C251.534 (2)C42—C431.527 (2)
C24—H2411.0000C42—H4211.0000
C25—H2510.9800C43—C441.527 (2)
C25—H2520.9800C43—H4310.9900
C25—H2530.9800C43—H4320.9900
C26—H2610.9800C44—C451.517 (3)
C26—H2620.9800C44—H4410.9900
C26—H2630.9800C44—H4420.9900
N30—C321.4602 (18)C45—C461.521 (3)
N30—H3010.8800C45—H4510.9900
C31—O311.2369 (18)C45—H4520.9900
C31—N401.3380 (16)C46—H4610.9800
C31—C321.5207 (17)C46—H4620.9800
C32—C331.5305 (18)C46—H4630.9800
C32—H3211.0000
O12—C10—N20122.69 (12)C34—C33—H331108.6
O12—C10—C11121.19 (11)C32—C33—H332108.6
N20—C10—C11116.12 (12)C34—C33—H332108.6
C10—C11—H101109.5H331—C33—H332107.5
C10—C11—H102109.5C35—C34—C36109.95 (11)
H101—C11—H102109.5C35—C34—C33112.95 (10)
C10—C11—H103109.5C36—C34—C33109.47 (10)
H101—C11—H103109.5C35—C34—H341108.1
H102—C11—H103109.5C36—C34—H341108.1
C10—N20—C22123.53 (11)C33—C34—H341108.1
C10—N20—H201118.2C34—C35—H351109.5
C22—N20—H201118.2C34—C35—H352109.5
O21—C21—N30123.22 (11)H351—C35—H352109.5
O21—C21—C22120.75 (9)C34—C35—H353109.5
N30—C21—C22116.00 (11)H351—C35—H353109.5
N20—C22—C21108.61 (9)H352—C35—H353109.5
N20—C22—C23108.99 (10)C34—C36—H361109.5
C21—C22—C23109.22 (10)C34—C36—H362109.5
N20—C22—H221110.0H361—C36—H362109.5
C21—C22—H221110.0C34—C36—H363109.5
C23—C22—H221110.0H361—C36—H363109.5
C24—C23—C22115.88 (11)H362—C36—H363109.5
C24—C23—H231108.3C31—N40—C42119.07 (12)
C22—C23—H231108.3C31—N40—H401120.5
C24—C23—H232108.3C42—N40—H401120.5
C22—C23—H232108.3O41—C41—C42123.77 (18)
H231—C23—H232107.4O41—C41—H411118.1
C26—C24—C25109.90 (13)C42—C41—H411118.1
C26—C24—C23113.10 (11)N40—C42—C41109.39 (12)
C25—C24—C23109.16 (13)N40—C42—C43110.32 (13)
C26—C24—H241108.2C41—C42—C43109.45 (11)
C25—C24—H241108.2N40—C42—H421109.2
C23—C24—H241108.2C41—C42—H421109.2
C24—C25—H251109.5C43—C42—H421109.2
C24—C25—H252109.5C42—C43—C44113.48 (12)
H251—C25—H252109.5C42—C43—H431108.9
C24—C25—H253109.5C44—C43—H431108.9
H251—C25—H253109.5C42—C43—H432108.9
H252—C25—H253109.5C44—C43—H432108.9
C24—C26—H261109.5H431—C43—H432107.7
C24—C26—H262109.5C45—C44—C43113.88 (15)
H261—C26—H262109.5C45—C44—H441108.8
C24—C26—H263109.5C43—C44—H441108.8
H261—C26—H263109.5C45—C44—H442108.8
H262—C26—H263109.5C43—C44—H442108.8
C21—N30—C32120.51 (11)H441—C44—H442107.7
C21—N30—H301119.7C44—C45—C46113.8 (2)
C32—N30—H301119.7C44—C45—H451108.8
O31—C31—N40122.24 (11)C46—C45—H451108.8
O31—C31—C32121.86 (10)C44—C45—H452108.8
N40—C31—C32115.90 (11)C46—C45—H452108.8
N30—C32—C31107.38 (9)H451—C45—H452107.7
N30—C32—C33111.39 (9)C45—C46—H461109.5
C31—C32—C33110.81 (10)C45—C46—H462109.5
N30—C32—H321109.1H461—C46—H462109.5
C31—C32—H321109.1C45—C46—H463109.5
C33—C32—H321109.1H461—C46—H463109.5
C32—C33—C34114.85 (10)H462—C46—H463109.5
C32—C33—H331108.6
O12—C10—N20—C220.51 (17)N40—C31—C32—N30112.91 (11)
C11—C10—N20—C22179.42 (9)O31—C31—C32—C3354.24 (13)
C10—N20—C22—C21113.52 (11)N40—C31—C32—C33125.23 (10)
C10—N20—C22—C23127.58 (11)N30—C32—C33—C34176.58 (9)
O21—C21—C22—N2053.20 (13)C31—C32—C33—C3457.10 (13)
N30—C21—C22—N20128.72 (9)C32—C33—C34—C3559.28 (13)
O21—C21—C22—C2365.55 (14)C32—C33—C34—C36177.85 (10)
N30—C21—C22—C23112.52 (11)O31—C31—N40—C421.75 (17)
N20—C22—C23—C24171.82 (10)C32—C31—N40—C42177.72 (10)
C21—C22—C23—C2453.30 (13)C31—N40—C42—C4163.52 (15)
C22—C23—C24—C2664.09 (15)C31—N40—C42—C43176.03 (11)
C22—C23—C24—C25173.23 (12)O41—C41—C42—N40164.16 (18)
O21—C21—N30—C324.26 (16)O41—C41—C42—C4374.9 (2)
C22—C21—N30—C32173.77 (9)N40—C42—C43—C4463.46 (16)
C21—N30—C32—C31141.31 (10)C41—C42—C43—C44176.13 (13)
C21—N30—C32—C3397.19 (12)C42—C43—C44—C4560.78 (18)
O31—C31—C32—N3067.62 (13)C43—C44—C45—C46173.82 (15)
Hydrogen-bond geometry (Å, º) top
D—H···AD—HH···AD···AD—H···A
N20—H201···O31i0.882.052.897 (3)161
N30—H301···O21ii0.881.992.863 (3)171
N40—H401···O12i0.881.962.827 (3)169
Symmetry codes: (i) x+1, y+1/2, z+1; (ii) x+1, y1/2, z+1.
Hydrogen-bond geometry (Å, º) top
D—H···AD—HH···AD···AD—H···A
N20—H201···O31i0.882.052.897 (3)161.1
N30—H301···O21ii0.881.992.863 (3)171.0
N40—H401···O12i0.881.962.827 (3)168.6
Symmetry codes: (i) x+1, y+1/2, z+1; (ii) x+1, y1/2, z+1.

Experimental details

Crystal data
Chemical formulaC20H37N3O4
Mr383.59
Crystal system, space groupMonoclinic, P21
Temperature (K)100
a, b, c (Å)10.85 (1), 9.510 (9), 11.200 (11)
β (°) 94.85 (2)
V3)1152 (2)
Z2
Radiation typeSynchrotron, λ = 0.6199 Å
µ (mm1)0.09
Crystal size (mm)0.30 × 0.05 × 0.02
Data collection
DiffractometerMAR300 CCD
diffractometer
Absorption correctionMulti-scan
(SCALEPACK; Otwinowski et al., 2003)
Tmin, Tmax0.974, 0.999
No. of measured, independent and
observed [I > 2σ(I)] reflections
4561, 4561, 4492
Rint0.049
(sin θ/λ)max1)0.767
Refinement
R[F2 > 2σ(F2)], wR(F2), S 0.041, 0.115, 1.07
No. of reflections4561
No. of parameters244
No. of restraints1
H-atom treatmentH-atom parameters constrained
Δρmax, Δρmin (e Å3)0.44, 0.29

Computer programs: sergui, SER-CAT APS beamline software, HKL-2000 (Otwinowski & Minor, 1997), SHELXD (Sheldrick, 2008), SHELXL (Sheldrick, 2008), ORTEP-3 for Windwows (Farrugia, 2012) and pyMOL (DeLano, 2002), SHELXL97 (Sheldrick, 2008).

 

Acknowledgements

This work was in part supported with Federal funds from the National Cancer Institute, under contract No. HHSN261200800E. X-ray data were collected at the SERCAT 19ID beamline of the Advanced Photon Source, Argonne National Laboratory. Use of the APS was supported by the US Department of Energy under contract No. W-31–109-Eng-38.

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Volume 71| Part 3| March 2015| Pages 254-257
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