A simple microscale method for determining the relative stereochemistry of statine units

Article abstract of DOI:10.1021/jo8012429

(Chemical Equation Presented) A simple method to determine the relative stereochemistry of statine amino acids (γ-amino-β-hydroxyacids) by using ¹H NMR spectroscopy is described. Configurational assignment of statine units within complex natural products is possible without degradation or derivatization as the syn and anti diastereomers can be distinguished by using a combination of chemical shift and coupling constant information derived from the α-methylene ABX system. Seventy-three examples are provided, demonstrating the scope and limitations of the methodology. These examples range in complexity from simple statine units to cyclic depsipeptides, such as tamandarin B.

Full text of DOI:10.1021/jo8012429

A Simple Microscale Method for Determining the Relative
Stereochemistry of Statine Units
Alejandro Preciado and Philip G. Williams*
Department of Chemistry, UniVersity of Hawaii at Manoa, Honolulu, Hawaii 96822
philipwi@hawaii.edu
ReceiVed June 25, 2008
A simple method to determine the relative stereochemistry of statine amino acids (γ-amino-ꢀ-hydroxyacids)
by using 1H NMR spectroscopy is described. Configurational assignment of statine units within complex
natural products is possible without degradation or derivatization as the syn and anti diastereomers can
be distinguished by using a combination of chemical shift and coupling constant information derived
from the R-methylene ABX system. Seventy-three examples are provided, demonstrating the scope and
limitations of the methodology. These examples range in complexity from simple statine units to cyclic
depsipeptides, such as tamandarin B.
Introduction
becoming more challenging as the isolation of submicromolar
quantities of metabolites becomes increasingly more common.6
Statine-containing peptides exemplify these challenges. Sta-
tine amino acids (1), γ-amino-ꢀ-hydroxy acids, are present in
several compounds including the anticancer agents didemnin
B,7 tamandarin B,8 and hapalosin.9 This isostere is often
associated with improved pharmacokinetic properties and
increased potency.10,11 While the planar moiety is easily
Modern structure determination of small molecules remains
a challenging problem. The number of structural reassignments
reported each year is testimony to the myriad of potential
pitfalls.1 While classical approaches to structure determination
rely heavily on chemical degradation, most modern approaches
use nondestructive techniques to provide the same connectivity
information.2 Any structure determination encompasses three
discrete assignments: planar, relative, and absolute. Advances
in NMR instrumentation and NMR pulse sequences have greatly
simplified the assignment of the planar structure, i.e., the
constitutional connectivites between the various nuclei.3 Con-
versely, relative4 and absolute stereochemical assignments5 are
(5) For a review on determining absolute configurations by NMR see: Seco,
J. M.; Quin˜oa´, E.; Riguera, R. Chem. ReV. 2004, 1, 17–117.
(6) (a) Wolfender, J.-L.; Queiroz, E. F.; Hostettmann, K. Magn. Reson. Chem.
2005, 43, 697–709. (b) Bugni, T. S.; Richards, B.; Bhoite, L.; Cimbora, D.;
Harper, M. K.; Ireland, C. M. J. Nat. Prod. 2008, 71, 1095–1098.
(7) (a) Rinehart, K. L., Jr.; Gloer, J. B.; Hughes, R. G., Jr.; Renis, H. E.;
McGovren, J. P.; Swynenberg, E. B.; Stringfellow, D. A.; Kuentzel, S. L.; Li,
L. H. Science 1981, 212, 933–935. (b) Rinehart, K. L., Jr.; Gloer, J. B.; Cook,
J. C., Jr.; Mizsak, S. A.; Scahill, T. A. J. Am. Chem. Soc. 1981, 103, 1857–
1859.
(8) Vervoort, H.; Fenical, W.; de Epifanio, R. J. Org. Chem. 2000, 65, 782–
792.
(9) Stratmann, K.; Burgoyne, D. L.; Moore, R. E.; Patterson, G. M. L.; Smith,
C. D. J. Org. Chem. 1994, 59, 7219–7226.
* To whom correspondence should be addressed. Phone: 808-956-5720. Fax:
808-956-5908.
(1) Nicolaou, K. C.; Snyder, S. A. Angew. Chem., Int. Ed. 2005, 44, 1012–
1044.
(2) Crews, P.; Rodriquez, J.; Jaspars, M. Organic Structure Analysis. Oxford
University Press: Oxford, 1998.
(3) Reynolds, W. F.; Enriquez, R. G. J. Nat. Prod. 2002, 65, 221–244.
(4) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana, K.
J. Org. Chem. 1999, 64, 866–876.
(10) Zuo, Z.; Luo, X.; Zhu, W.; Shen, J.; Shen, X.; Jiang, H.; Chen, K. Bioorg.
Med. Chem. 2005, 13, 2121–2131.
(11) Fehrentz, J. A.; Chromier, B.; Bignon, E.; Venaud, S.; Chemann, J. C.;
Nisato, D. Biochem. Biophys. Res. Commun. 1992, 188, 865–872.
9228 J. Org. Chem. 2008, 73, 9228–9234
10.1021/jo8012429 CCC: $40.75 2008 American Chemical Society
Published on Web 11/07/2008

Determining RelatiVe Stereochemistry of Statine Units
SCHEME 1. Synthesis of Statine Units
identified by conventional NMR spectroscopy, there is no simple
method to determine the relative configuration of the vicinal
chiral centers bearing the amine and the alcohol. Traditional
solutions to this problem have involved either hydrolysis and
subsequent HPLC/GC comparison with synthetic standards or
conversion to the oxazolidine/oxazolidinone derivative.
Ideally, a simple NMR-based method could be used to
determine the relative configuration of the statine unit within
the intact peptide. This would be analogous to the Universal
NMR databases developed by Kishi for configurational assign-
ment of polyketides, which utilizes the distinctive 13C NMR
chemical shift patterns of the diastereomers.12 Several years ago
we noted that it might be possible to distinguish the two
diastereomers of 4-amino-3-hydroxy-5-phenylpentanoic acid
using the proton signals for H2-2,13 but little supporting evidence
was available at that time for the generality of the method.
SCHEME 2. Conversion to the Oxazolidine Derivatives
Results and Discussion
To explore these issues, we synthesized a variety of γ-amino-
ꢀ-hydroxy acids derived from the proteogenic amino acids using
a simple procedure (Scheme 1). The imidazole-activated Boc-
amino acid was coupled with the enolate derived from tert-
butyl acetate or allyl acetate. Subsequent reduction with lithium
or sodium borohydride produced a mixture of C-3 diastereo-
mers,14 which were separated by normal phase chromatography
to yield the protected statine units 4 and 5. As expected, the
syn diastereomer, formed via a chelation-controlled reduction,14
was the major product in all cases.15 This stereochemical
outcome was confirmed by converting representative compounds
11, 12, 16, 21, and 22 to the oxazolidine derivatives (Scheme
2)16 and analysis of the vicinal proton-proton coupling constant
between the two chiral centers. The syn and anti derivatives
mers.20 These results are likely due to the conformational
flexibility between the two chiral centers C-3 and C-4 and are
consistent with the trends observed for 1,2-diols21 and for R,γ-
dimethyl-ꢀ-hydroxycarbonyls.22
A more detailed analysis of the NMR spectroscopic data for
the diastereomeric statine derivatives revealed a diagnostic
pattern associated with the methylene protons (H2-2) though.
When NMR spectra were recorded in CDCl3, the ABX patterns
of the R-methylene protons of the two diastereomers were
essentially mirror images. For the syn diastereomer, the down-
field proton signal of the methylene pair was consistently a
doublet of doublets with a small vicinal coupling to H-3.
Conversely, for the anti diastereomer, the downfield methylene
proton signal displayed a large vicinal coupling to H-3. The
upfield protons of these pairs showed the opposite pattern, i.e.,
the syn and anti diastereomers had large and small vicinal
couplings to H-3, respectively. Figure 2 shows the AB portion
of the 1H NMR spectra of 8a and 8b where this trend is clearly
observed. On average, the magnitude of the 3J values between
the downfield resonance of H2-2 and H-3 was 10 Hz for the
anti diastereomer and 2 Hz for the syn diastereomer (Figure 3).
Technically in a second-order ABX system, such as shown
in Figure 2, only JAB and the sum JAX + JBX can be determined
directly from the spectrum. In other words, exact values for
JAX and JBX, specifically in this case 3JH3,H2d and 3JH3,H2u, cannot
be determined by simple subtraction of the line frequencies.2
In practice outside a small community, this issue is largely
ignored and coupling constants in second-order ABX systems
are extracted as if these are first order. We have chosen to
3
displayed JH4,H3 values of approximately 6 and 2 Hz,
respectively.17,18
A comparison of the spectral data for the diastereomers
revealed no predictive clustering of carbon or proton chemical
shifts that could be used to assign the relative configuration of
an unknown statine unit (Figure 1).19 Likewise, the magnitude
of the 3J values between H-3 and H-4 did not provide a
consistent means of distinguishing the respective diastereo-
(12) Lee, J.; Kobayashi, Y.; Tezuka, K.; Kishi, Y. Org. Lett. 1999, 1, 2181–
2184.
(13) Williams, P. G.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J. J. Nat. Prod.
2003, 66, 1006–1009.
(14) Harris, B. D.; Bhat, K. L.; Joullie, M. M. Tetrahedron Lett. 1987, 28,
2837–2840.
(15) In general, the anti diastereomer eluted first with normal phase HPLC,
except for the threonine- and proline-derived statine units. While the order of
elution was reversed for these two pairs of derivatives, the syn reduction product
depicted dominates.
(16) Ori, M.; Toda, N.; Takami, K.; Tago, K.; Kogen, H. Tetrahedron 2005,
61, 2075–2104.
(17) Harris, B. D.; Joullie, M. M. Tetrahedron 1988, 44, 3489–3500.
(18) The stereochemistry of 24a was determined by conversion to the
oxazolidinone derivative through treatment with triphosgene.The stereochemistry
was then assigned via analysis of the JH3,H4 coupling constant. See: Wang, M.;
Chen, Y.; Lou, L.; Tang, W.; Wang, X.; Shen, J. Tetrahedron Lett. 2005, 46,
5309–5312.
(19) A comparison of carbon and proton chemical shifts with the nature of
the side chain (polar, nonpolar, aromatic, branched, etc) provided no predictive
trend either.
(20) There are reports of using 3JH3,H4 values for the relative stereochemical
assignment of an Ala-Sta derivative (4-methylcarnitine: Comber, R. N.;
Brouillette, W. J. J. Org. Chem. 1988 53 1121-1122), but based on our data
(see the Supporting Information) this is not a general trend
(21) Higashibayashi, S.; Kishi, Y. Tetrahedron 2004, 60, 11977–11982.
(22) Heathcock, C. H.; Pirrung, M. C.; Sohn, J. E. J. Org. Chem. 1979, 44,
4294–4299.
J. Org. Chem. Vol. 73, No. 23, 2008 9229

Preciado and Williams
FIGURE 1. (a) Chemical shifts (ppm) for H-3 and H-4 of the syn (blue diamond) and anti (red circle) statine diastereomers. (b) Chemical shifts
for C-3 and C-4 of the syn (blue diamond) and anti (red circle) statine diastereomers.
FIGURE 2. Expansions of the H2-2 ABX systems in the 1H NMR spectra of two diastereomers (syn and anti). These patterns are essentially mirror
images allowing assignment of the relative configuration.
FIGURE 3. Comparison of the vicinal proton-proton coupling
constants between H-2D and H-3 for the anti (blue diamond) and syn
(red circle) diastereomers for compounds 6-24.
analyze all the ABX systems in this manner as it provides a
simple way of designating “large” and “small” for this technique.
1
FIGURE 4. Expansion of the AB pattern for H2-2 in the H NMR
spectra: (a) 10b at 300 MHz, (b) 10b at 800 MHz, (c) 16b at 300
MHz, and (d) 16b at 800 MHz.
Table 1 shows the complete data set for 39 synthetic statine
derivatives in CDCl3. In all cases, the proton signals for H2-2
were unambiguously identified by either simple inspection of
2. Since second-order systems occur when ∆J/(∆δ in Hz) < 6,
increasing the spectrometer frequency provided one simple
solution. Figure 4 shows expansions of the ABX systems for
the two derivatives that displayed the largest second-order
distortions at 300 MHz and the corresponding spectra at 800
MHz.23 In both cases, the higher field strength resolves the
signals into a pattern consistent with the rest of the data set.
Coupling constants in these second-order systems could also
be obtained by comparison of the AB systems of experimental
and simulated NMR spectrum at 300 MHz (Figure 5).24
Coupling constants and chemical shifts in Table 1 for the anti-
Ala (10a), syn-Pro (24b), and syn-Glu-Sta (23b) were deter-
mined in this manner.
1
the H NMR spectra or through a combination of 1D TOCSY
and selective decoupling experiments. The anti derivatives
clearly showed the diagnostic pattern, with the downfield
methylene resonance displaying a large vicinal coupling to H-3,
while the upfield proton had a small vicinal coupling. These
could be easily distinguished from the corresponding syn
derivatives based on the pattern shown in Figure 2.
It is worth noting that in some cases, the proton resonances
for the ABX system were strongly second order at 300 MHz.
The statine units derived from Ala (10a,b) displayed such a
large distortion that the magnitude of the coupling constants
could not be directly extracted at 300 MHz. In general, this
effect was more prevalent with the syn diastereomers as lysine
(16b), glutamate (23b), methionine (19b), and proline (24b)
derivatives all had strongly distorted ABX spin systems for H2-
(23) At 500 MHz, the 1H NMR spectra for these compounds were second
order.
9230 J. Org. Chem. Vol. 73, No. 23, 2008

Determining RelatiVe Stereochemistry of Statine Units
TABLE 1. 1H NMR Data for H2-2 ABX System of Statine Derivatives
anti compd
parent AA
δHa (JAX
)
δHb (JBX
)
syn compd
δHa (JAX
)
δHb (JBX)
6ad
7a
8a
leucine
valine
2.47 (8.2)
2.66 (9.8)
2.45 (0.4)
2.54 (3.0)
2.39 (2.8)
2.31 (2.2)
2.53 (0.7)d
2.27 (2.4)
2.41 (2.5)
2.28 (2.6)
2.25 (2.6)
2.43 (4.7)
2.36 (3.1)
2.26 (1)a
2.40 (2.6)
2.53 (3.0)
2.31 (4.0)
2.28 (1.6)
2.43 (3.3)
2.30 (3.3)
2.53 (2.2)
2.33 (2.5)
6bd
7b
8b
2.29 (3.7)
2.63 (2.6)
2.48 (2.9)
2.43 (1)a
2.54 (2.2)
2.51 (2.4)
2.61 (2.2)
2.51 (2.5)
2.54 (1)a
2.55 (2.0)
2.45 (0.7)
2.48 (3.0)
2.53 (1.9)
2.58 (1.4)
2.46 (3.7)
2.50 (3.2)
2.28 (10.0)
2.50 (8.9)
2.35 (8.8)
2.31 (8.7)
2.51 (9.4)
2.41 (8.9)
2.52 (8.2)
2.41 (9.0)
2.46 (8.3)
2.51 (8.6)
2.41 (9.3)
2.35 (9.0)
2.46 (9.0)
2.54 (9.4)
2.40 (8.5)
2.33 (8.7)
isoleucine
isoleucine
alanine
phenylalanine
phenylalanine
tyrosine
tryptophan
histidine
lysine
2.48 (10.1)
2.60 (9.9)
2.56 (11.0)d
2.51 (10.4)
2.61 (10.2)
2.51 (10.4)
2.52 (10.5)
2.58 (8.2)
2.48 (9.8)
2.47(10.2)
2.47 (10.0)
2.60 (10.2)
2.43 (9.2)
2.39 (9.0)
2.53 (9.8)
2.40 (9.4)
2.60 (10.2)
2.36 (9.7)
9a
9b
10a
11a
12a
13a
14a
15a
16a
17a
18ac
19a
20a
21a
21c
22a
23a
24ad
10bb
11b
12b
13b
14b
15b
16bb
17b
18b
19b
20b
21b
cysteine
aspartate
methionine
serine
threonine
threonine
arginine
22b
23bd
24b
2.37 (3.4)
2.56 (3.5)
2.54 (3.5)
2.28 (8.9)
2.53 (8.9)
2.44 (8.5)
glutamate
proline
a broad proton signal with no discernible coupling to Hx that for illustrative purposes is considered approximately a 1 Hz coupling. b Data recorded at
800 MHz. c Coupling constants determined though a combination of 1D-TOCSY and selective homonuclear decoupling at 500 MHz. d Coupling
constants and chemical shifts determined by comparison of simulated and experimental NMR spectra.
TABLE 2. Relevant Literature Examples
anti compd
parent AA
δHa (JAX
)
δHb (JBX
)
syn compd
δHa (JAX
)
δHb (JBX
)
ref
25a
26a
27a
28a
29a
30a
31a
32a
33a
34a
35a
36a
37a
38a
39a
40a
leucine
valine
valine
isoleucine
2.34 (7.9)
NA
NA
NA
NA
2.40 (9.5)
2.59 (9.9)
2.57 (10.3)
2.54 (9.8)
2.38 (9.0)
2.55 (9.5)
NA
2.27 (3.4)
NA
NA
NA
NA
2.04 (2.4)
2.37 (2.6)
2.38 (3.0)
2.28 (3.1)
2.19 (4.0)
2.32 (3.0)
NA
25b
26b
27b
27b
29b
30b
31b
32b
33b
34b
35b
36b
37b
38b
39b
40b
41b
42b
41b
NA
2.48 (2.9)
2.58 (2.9)
2.64 (2.1)
2.60 (2.7)
2.60 (2.7)
2.63 (2.5)
NA
2.29 (10.0)
2.47 (9.1)
2.32 (10.6)
2.48 (9.1)
2.42 (8.7)
2.22 (10.1)
NA
NA
NA
NA
NA
28
29
28
30
30
28
31
31
32
32
32
8
isoleucine
phenylalanine
phenylalanine
phenylalanine
phenylalanine
phenylalanine
phenylalanine
isoleucine
NA
NA
NA
NA
3.25 (1)a
3.25 (2.7)
2.74 (3.7)
NA
2.44 (8.1)
2.48 (7.5)
2.69 (10.5)
NA
isoleucine
NA
NA
8
hydroxyglutamic acid
hydroxyglutamic acid
hydroxyglutamic acid
phenylalanine
tryptophan
2.62 (8.8)
2.65 (9.2)
NA
NA
NA
2.54 (5.0)
2.55 (4.6)
NA
NA
NA
33
33
33
34
35
36
37
2.62, m
2.65 (3.6)
2.35 (3.7)
2.38 (3.7)
NA
41a
2.57 (5.6)
2.28 (9.1)
2.29 (9.5)
NA
42a (DMSO)
43a (DMSO)
44a (MeOH)
tyrosine
leucine
NA
2.36 (7.2)
NA
2.27 (6.7)
a Broad proton signal with no discernible coupling to Hx that for illustrative purposes is considered approximately a 1 Hz coupling.
units containing 3° amines can be assigned by using this technique.
The generality of the trend is further exemplified by the linear di-
and tripeptides 33a, 34a, and 35a, which contain additional chiral
centers.
(24) Spin simulations were done with MestreC 4.9.8.0 in an iterative fashion
comparing with the experimental spectra until identical patterns were obtained.
Experimental data were used as a constraint in these simulations when possible,
FIGURE 5. (a) 300 MHz spectrum of 16b in CDCl3 at 300 MHz. (b)
Simulated spectrum at 300 MHz (δHa 2.426, δHb 2.385, 2JAB ) -15.84,
2
3
3
i.e., δH3, JH2u,H2d, JH3,H2d + JH3,H2u, ca. δH2
.
3
3JAX ) 0.7 Hz, JBX ) 9.3 Hz)
(25) A Scifinder Scholar search for the basic carbon skeleton yielded
approximately 10 000 compounds, but the utility of this literature NMR data
varied greatly. For example, H2-2 was frequently reported as a multiplet spanning
a considerable chemical shift range. This indicated the methylene protons were
probably diastereotopic (otherwise H2-2 would be a simple doublet), but provided
no coupling constant information for comparison.
A number of statine-containing compounds have been de-
scribed.25 Table 2 summarizes some additional reports consistent
with our observations. The diagnostic trend was observed
with statine derivatives when the amine was protected as the tert-
butoxycarbonyl, acetyl, and dibenzyl derivatives and with a variety
of different protecting groups on the C-1 ester. These latter
examples (25a, 25b, 30a, 30b) suggest the configuration of statine
(26) Sakai, R.; Stroh, J. G.; Sullins, D. W.; Rinehart, K. L. J. Am. Chem.
Soc. 1995, 117, 3734–3748.
(27) Zhang et al. have recently reported a new cyclic thiazolyl peptide
Phillipmycin containing a statine unit, whose configuration can be correctly
predicted with this method. See: Zhang, C.; et al. J. Am. Chem. Soc. 2008, 130,
12102-12110.
J. Org. Chem. Vol. 73, No. 23, 2008 9231

Preciado and Williams
Natural products of significant structural complexity can also
be assigned by using this approach. For example, the config-
uration of the statine unit within the didemnin26/tamandarin and
hapalosin series of cyclic natural products (36b, 37b, 41b) can
be correctly predicted as syn based on analysis of the reported
NMR data. Likewise, the epimeric derivatives belonging to
the Kutzneride class of compounds (38-40) can also be
correctly assigned with the syn derivatives displaying the
smaller three-bond proton-proton coupling to the downfield
signal of H2-2.27
Natural products (42-44) in solvents other than CDCl3 show
the same diagnostic patterns, and while it would be tempting
to apply this methodology, our data indicate that caution must
be exercised. As illustrated in Table 3 there is a clear solvent
(33) Broberg, A.; Menkis, A.; Vasiliauskas, R. J. Nat. Prod. 2006, 69, 97–
102.
(34) Okuno, T.; Ohmori, K.; Nishiyama, S.; Yamamura, S.; Nakamura, K.;
Houk, K. N.; Okamoto, K. Tetrahedron 1996, 52, 14723–14734.
(35) Ishida, K.; Murakami, M. J. Org. Chem. 2000, 65, 5898–5900, The
vicinal coupling constants for the methylene proton signals (δH 2.35 and 2.28)
in Table 1 of this reference are transposed. We thank Drs. Ishida, Murakami,
and Okada for providing copies of the original 1H NMR spectrum so that we
could check these data. The NMR spectra is included in our Supporting
Information.
(28) Andre´s, J. M.; Pedrosa, R.; Pe´rez, A.; Pe´rez-Encabo, A. Tetrahedron
2001, 57, 8521–8530. This reference also includes two Pro-Stat derivatives in
which the ABX pattern appears to be reversed. We have synthesized one of
these compounds, the Pro-Stat protected as a tert-butyl ester, and find the NMR
data to be consistent with the rest of our data set.
(29) Liang, B.; Richard, D. J.; Portonovo, P. S.; Joullie, M. M. J. Am. Chem.
Soc. 2001, 123, 4469–4474.
(30) Rinehart, K. L.; Sakai, R.; Kishore, V.; Sullins, D. W.; Li, K. M. J.
Org. Chem. 1992, 57, 3007–3013.
(31) Doi, T.; Kokubo, M.; Yamamoto, K.; Takahashi, T. J. Org. Chem. 1998,
63, 428–429.
(32) Palomo, C.; Miranda, J. I.; Linden, A. J. Org. Chem. 1996, 61, 9196–
9201.
(36) Isolation: Nakatani, S.; Kamata, K.; Sato, M.; Onuki, H.; Hirota, H.;
Matsumoto, J.; Ishibashi, M. Tetrahedron Lett. 2005, 46, 267–271. Synthesis:
Hanazawa, S.; Arai, M.; Li, X.; Ishibashi, M. Bioorg. Med. Chem. Lett. 2008,
18, 95-98.
(37) Isolation: Nakao, Y.; Fujita, M.; Warabi, K.; Matsunaga, S.; Fusetani,
N. J. Am. Chem. Soc. 2000, 122, 10462–10463. Synthesis: Konno, H.; Kubo,
K.; Makabe, H.; Toshiro, E.; Hinoda, N.; Nosaka, K.; Akaji, K. Tetrahedron
2007, 63, 9502–9513.
9232 J. Org. Chem. Vol. 73, No. 23, 2008

Determining RelatiVe Stereochemistry of Statine Units
TABLE 3. Solvent Effect
anti compd
solvent
δHa (JAX
)
δHb (JBX
)
syn compd
δHa (JAX
)
δHb (JBX)
6a
7a
6a
11a
6a
7a
10a
CDCl3
2.47 (10.2)
2nd order
2.20 d (2H, 7.0)
2.33 (4.2)
2.27 d (2H, 6.8)
2.47 (2 H, 6.6)
2.50 (4.2)
2.34 (2.4)
2.16 (8.8)
2.43 (8.8)
6b
7b
6b
11b
6b
7b
10b
2.47 (2.5)
2.36 (2.5)
2.39 (3.2)
2.39 (3.9)
2.39 (3.2)
2.60 (2.6)
2.57 (3.5)
2.34 (8.9)
2.16 (9.6)
2.15 (9.3)
2.16 (9.3)
2.15 (9.3)
2.35 (9.1)
2.39 (9.3)
DMSO-d6
DMSO-d6
DMSO-d6
CD3OD
acetone-d6
acetone-d6
effect, which is more pronounced for the anti derivatives than
the syn. In some cases, such as 6a and 7a in DMSO and MeOH-
d4, the methylene protons become magnetically equivalent.38
In other cases, the diastereomers of the pairs 10a/10b and 11a/
11b are indistinguishable in acetone-d6 and DMSO-d6, respec-
tively, as in both cases, the downfield methylene proton displays
the small vicinal proton coupling. Application of this methodol-
ogy in these instances would lead to an erroneous assignment
of the anti diastereomer as syn. Thus, CDCl3 insoluble com-
pounds should be converted to a soluble derivative prior to
analysis.39
FIGURE 6. Proposed Conformational Model.
Conformational preferences for 6a and 6b are known based
on conformational computations.41 Analysis of these data
suggests one possible explanation for the observed NMR
patterns as shown in Figure 6. In both diastereomers, the
carbonyls are in an eclipsed conformation with the C-2/C-3 bond
coplanar to the carbonyl, but with the hydroxy C-O oxygen
bond (+)-gauche and (-)-gauche to the carbonyl in the anti
and syn diastereomers, respectively. Stabilization of these
conformations by hydrogen bonding, analogous to that proposed
in the Stiles-House model for R-alkyl-ꢀ-hydroxycarbonyls42
or Roush’s method for ꢀ-hydroxy ketones with alkyl substituents
in the γ-position,40 is suggested based on the work of Rich41
and Nisato,43 although not required.44 If these conformers
predominate in CDCl3 then the pro-S methylene proton is
deshielded in both diastereomers relative to the pro-R. This
deshielding of the pro-S methylene proton may be due to the
amide attached to C-4, in a fashion analogous to that reported
by Whelton et al. for substituted cyclohexamines.45 It should
be noted that for simplicities sake, we have chosen to depict
only a single rotatmer between the vicinal chiral centers C-3/
C-4, although at least two rotatmers exist in solution based on
coupling constant analysis. A reversal of the relative population
of the rotamers around the C-3/C-4 bond may ultimately be
responsible for the solvent dependency. A more detailed
conformational analysis of the factors responsible for the
observed NMR patterns is underway.
This approach is not without precedent as a similar technique
was proposed by Roush et al. to assign the relative stereochem-
istry of ꢀ-hydroxy ketones with alkyl substituents in the
γ-position.40 Two differences should be noted. First in Roush’s
method, varying the solvent had no noticeable effect on the
pattern in the 1H NMR spectra for the two compounds examined.
Second, with those substrates C-5 must be branched, e.g.,
analogous to 7a, for the two diastereomers to be dis-
tinguishable.40a The latter is not the case for statine moieties as
is illustrated by examples 6, 10-20, and 22-23 in Table 1.
These compounds are not branched at C-5, yet are distinguish-
able.
(38) Occasionally, H2-2 appeared as a simple doublet in the initial proton
spectra of 6-24 immediately after silica chromatography. After 24 h in CDCl3,
these proton signals were generally resolved.
The trend noted here provides the simplest methodology for
the relative configurational assignment of CDCl3-soluble statine-
containing peptides. The methodology requires no degradation
or derivatization, and is amenable to micromolar concentrations
of analytes given the sensitivity limits of modern NMR
spectrometers. Several examples above suggest this methodology
is applicable to statine units within cyclic peptides/depsipeptides,
although until a more complete understanding of the underlying
factors responsible is available, we would urge caution in these
constrained systems. While we have not found any examples,
conformational constraints in cyclic systems could induce a
(39) Because of this requirement of CDCl3 solubility, data for the deprotected
compounds 6-24 were generally not included.
(40) (a) Roush, W. R.; Bannister, T. D.; Wendt, M. D.; Van Nieuwenhze,
M. S.; Gustin, D. J.; Dilley, G. J.; Lane, G. C.; Scheidt, K. A.; Smith, W. J., III.
J. Org. Chem. 2002, 67, 4284–4289. (b) Dias, L. C.; Aguilar, A. M., Jr.; Steil,
L. J.; Roush, W. R. J. Org. Chem. 2005, 70, 10461–10465.
(41) Rich, D. H.; Terada, Y.; Kawai, M. Int. J. Pept. Prot. Res. 1983, 22,
325–332.
(42) (a) Stiles, M.; Winkler, R. R.; Chang, Y.-L.; Traynor, L. J. Am. Chem.
Soc. 1964, 86, 3337–3342. (b) House, H. O.; Crumrine, D. S.; Teranishi, A. Y.;
Olmstead, H. D. J. Am. Chem. Soc. 1973, 95, 3310–3324.
(43) Toniolo, C.; Valle, G.; Bonora, G. M.; Lelj, F.; Barone, V.; Fraternali,
F.; Callet, G.; Wagnon, J.; Nisato, D. Int. J. Pept. Prot. Res. 1987, 30, 583–595.
(44) Kitamura, M.; Nakano, K.; Miki, T.; Okada, M.; Noyori, R. J. Am. Chem.
Soc. 2001, 123, 8939–8950.
1
reversal of the H NMR trend similar to that illustrated with
solvents other than chloroform. For cyclic depsipeptides,
(45) Whelton, B. D.; Lowry, B. R.; Carr, J. B.; Huitric, A. C. J. Pharm. Sci.
1973, 62, 728–737.
J. Org. Chem. Vol. 73, No. 23, 2008 9233

Preciado and Williams
room temperature over 15 min. This aqueous solution was extracted
with 60 mL of Et2O (3 × 20 mL) and then dried over MgSO4.
Purification over silica eluting with hexane/EtOAc provided a
mixture of diastereomers. HPLC purification on silica provided pure
diastereomers.
conversion to a linear derivative would be advisable. Another
fundamental limitation of the technique is obtaining sufficient
dispersion in the ABX NMR spin system. Potential solutions
to this issue include increasing the NMR field strength, although
most of our data were acquired at 300 MHz, or simulation of
the observed spin system to extract coupling constants through
comparison with experimental spectra. As such, this methodol-
ogy should be broadly applicable to the assignment of statine
units within both natural products and synthetic derivatives using
1H NMR spectroscopy.
Allyl 4(S)-[(tert-butoxycarbonyl)amino]-5-methyl-3(S)-hydroxy-
hexanoate (7a): amorphous powder; [R]27 -1.7 (c 1.0, CHCl3);
D
UV (i-PrOH) λmax (log ε) 248 nm (8.10); IR (film) νmax 3375, 1715,
1506, 1261, 1172 cm-1; 1H NMR (CDCl3 at room temperature) δ
5.90 (ddt, J ) 17.2, 10.4 Hz, 1H), 5.33 (dq, J ) 17.2, 1.5 Hz, 1H),
5.26 (dd, J )10.4, 1.5 Hz, 1H), 4.83 (d, J ) 9.8 Hz, 1H), 4.60 (dt,
J ) 5.8, 1.5 Hz, 2H), 4.20 (br. d, J ) 9.4 Hz, 1H), 3.20 (d, J ) 2.6
Hz, 1H), 3.15 (td, J ) 10.5, 1.7 Hz, 1H), 2.60 (dd, J ) 16.9, 9.8
Hz, 1H), 2.51 (dd, J ) 16.9, 3.2 Hz, 1H), 1.86 (dm, 1H), 1.43 (m,
9H), 0.99 (d, J ) 6.7 Hz, 3H), 0.96 (d, J ) 6.8 Hz, 3H); 13C NMR
δ 173.3, 156.4, 131.7, 118.7, 79.2, 67.0, 65.5, 59.6, 39.0, 30.3,
29.7, 28.4, 19.8, 19.5; HRESI-TOF m/z 324.1767 [M + Na]+ [calcd
for C15H27NO5Na+ 324.1787, -4.5 ppm error].
Experimental Section
General Procedure for Synthesis of ꢀ-Keto Esters.15 One gram
of the protected amino acid (1 equiv) was dissolved in 20 mL of
dry THF. To this solution was added 1.1 equiv of 1,1′-carbonyl-
diimidazole (recrystallized from THF) with stirring under dry
nitrogen at room temperature. Butyl lithium (2.2 M hexane solution,
3.3 equiv) under nitrogen was diluted with 20 mL of THF and
cooled to 0 °C with an ice bath. To this was added dropwise
diisopropylamine (3.6 equiv). After being stirred for 10 min at 0
°C, the solution was diluted with 70 mL of THF and cooled to
-78 °C. To the LDA solution was added either tert-butyl acetate
or allyl acetate (3.5 equiv). After 10 min, the Boc-amino acid/
imidazole solution was cooled to -78 °C and cannulated into the
enolate solution under nitrogen. The reaction was allowed to stir
for 30 min at -78 °C then quenched with 50 mL of 10% citric
acid and allowed to warm to room temperature. The aqueous
solution was extracted with 200 mL of EtOAc (4 × 50 mL) and
washed with 100 mL of saturated NaHCO3 (2 × 50 mL) and 50
mL of brine and dried over MgSO4. The solvent was evaporated
and the crude material purified on silica eluting with mixtures of
hexane/EtOAc.
Allyl 4(S)-[(tert-butoxycarbonyl)amino]-5-methyl-3(R)-hydroxy-
hexanoate (7b): [R]24D +7.9 (c 1.0, CHCl3); UV (i-PrOH) λmax (log
ε) 203 (7.56), 205 nm (5.54); IR (film) νmax 3456, 3369, 1696 cm-1
;
1H NMR (CDCl3 at room temperature) δ 5.98 (ddt, J ) 17.2, 10.4,
5.8 Hz, 1H), 5.33 (ddt, J ) 17.2, 2.7, 1.5 Hz, 1H), 5.24 (dd, J )
10.4, 1.1 Hz, 1H), 4.57 (dt, J ) 5.8, 1.3 Hz, 2H), 4.50 (d, J ) 9.6
Hz, 1H), 3.89 (td, J ) 8.4, 2.9 Hz, 1H), 3.49 (m, 1H), δ 2.58 (dd,
J ) 16.5, 2.9 Hz, 1H), 2.46 (dd, J ) 16.5, 8.8 Hz, 1H), 2.09 (m,
1H), 1.43 (m, 9H), 0.94 (d, J ) 6.9 Hz, 3H), 0.88 (d, J ) 6.8 Hz,
3H); 13C NMR δ 172.9, 156.4, 131.8, 118.6, 79.6, 69.2, 65.5, 58.8,
38.3, 28.3, 27.5, 20.1, 16.3; HRESI-TOF m/z 302.1955 [M + H]+
+
[calcd for C15H28NO5 302.1962, 2.4 ppm error].
Acknowledgment. This work was funded by grants from the
Victoria S. and Bradley L. Geist Foundation (20070461), the
National Science Foundation (OCE04-32479), and the National
Institute of Environmental Health Sciences (P50 ES012740).
Funds for the upgrades of the NMR instrumentation were
provided by the CRIF program of the National Science
Foundation (CH E9974921) and the Elsa Pardee Foundation.
The purchase of the Agilent LC-MS was funded by grant
W911NF-04-1-0344 from the Department of Defense. We thank
P. Moeller, NOAA, and W. Yoshida, UH Manoa, for the 800
and 500 MHz NMR data, respectively. B. Wong and S. Yuen
aided in the collection of the physical data.
Allyl 4(S)-[(tert-butoxycarbonyl)amino]-5-methyl-3-oxohexano-
ate (4b): amorphous powder; [R]27 +8.5 (c 5.4, CHCl3); UV (i-
D
PrOH) λmax (log ε) 248 nm (8.10); IR (film) νmax 3369, 1749, 1246,
1
1168 cm-1; H NMR (CDCl3 at room temperature) δ 5.90 (ddt, J
) 17.2, 10.4, 5.6 Hz, 1H), 5.33 (dd, J ) 17.2, 1.5 Hz, 1H), 5.28
(dd, J ) 10.4, 1.5 Hz, 1H), 5.06 (d, J ) 8.3 Hz, 1H), 4.63 (d, J
)5.6 Hz, 2H), 4.32 (dd, J ) 8.7, 4.1 Hz, 1H), 3.57 (s, 2H), 2.24
(m, 1H), 1.48 (s, 9H), 1.01 (d, J ) 6.8 Hz, 3H), 0.82 (d, J ) 6.8
Hz, 3H); 13C NMR δ 202.0, 166.2, 155.7, 131.5, 80.0, 66.0, 64.3,
47.0, 29.5, 28.2, 19.8, 16.7; HRESI-MS m/z 322.1636 [M + Na]+
[calcd for C15H25NO5Na+ 322.1631, -3.8 ppm error].
General Procedure for Reduction.15 To a flame-dried flask was
added 0.5 g (1 equiv) of BOC-ꢀ-keto-γ-amino tert-butyl ester
dissolved in 20 mL of dry THF then the mixture was cooled to
-78 °C under nitrogen. To this solution was added 1.3 equiv of
LiBH4 (recrystallized from Et2O) with stirring at -78 °C until the
reaction reached completion as determined by TLC. The reaction
was quenched by the addition of 1 N HCl and allowed to warm to
Supporting Information Available: General experimental
procedures for the preparation of and spectral data for com-
pounds 6-24. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO8012429
9234 J. Org. Chem. Vol. 73, No. 23, 2008