Retinoylserine and retinoylalanine, natural products of the moth Trichoplusia ni

Article abstract of DOI:10.1021/np0496791

Insect cells convert vitamin A into a number of retinoids that are evolutionary conserved with those of mammalian cells. However, insect cells also produce additional natural retinoids. Namely, two retinoic acid peptides, N-trans-retinoylserine (1) and N-

Full text of DOI:10.1021/np0496791

1536
J. Nat. Prod. 2005, 68, 1536-1540
Notes
Retinoylserine and Retinoylalanine, Natural Products of the Moth
Trichoplusia ni
Barbara Rogge,†,‡,§ Yasuhiro Itagaki, Nathan Fishkin, Ester Levi,† Ralph Ru¨hl,| San-San Yi,∇
Koji Nakanishi, and Ulrich Hammerling*,†
Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, Department of Chemistry,
Columbia University, New York, New York 10027, Department of Biochemistry and Molecular Biology, University of Debrecen,
Debrecen, Hungary, Institute of Nutritional Science, University of Potsdam, Potsdam, Germany, and Molecular Biology
Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Received September 30, 2004
Insect cells convert vitamin A into a number of retinoids that are evolutionarily conserved with those of
mammalian cells. However, insect cells also produce additional natural retinoids. Namely, two retinoic
acid peptides, N-trans-retinoylserine (1) and N-trans-retinoylalanine (2), have been isolated from a cell
line of the common cabbage looper, Trichoplusia ni. These are the first examples of naturally occurring
retinoic acid linked to amino acids through an amide bond; the amino acid moieties are depicted in the
more common L-configuration, although the absolute configuration was not determined due to the
minuscule sample amount.
In this report, we describe the characterization and
synthesis of N-trans-retinoylserine (1) and N-trans-retin-
oylalanine (2) isolated from insect cells (Figure 1). Vitamin
A is used in higher animals as a precursor for retinalde-
hyde, the universal visual pigment, and for retinoic acid
(3), which serves as an important transcriptional activator
in development and homeostasis. Furthermore, hydroxy-
lated forms of vitamin A, and indeed vitamin A itself, play
a broad role in signal transduction. Insect cells convert
vitamin A into a similar, evolutionarily conserved spectrum
of retinoids. However, the usage of vitamin A or all-trans-
retinol (4) in insects is an underexplored area. With the
exception of retinal, which represents the universal pig-
ment of all eyes1 for both invertebrates and vertebrates,
the importance of retinol for insects is not known. Yet
indirect signs of systemic vitamin A usage have been noted.
These include observations that retinoids have profound
effects on the development of invertebrates and vertebrates
alike.2 Since the RAR (retinoic acid receptor) and RXR
(retinoid X receptor) classes of retinoid receptors3,4 do not
make an appearance in evolution until the vertebrate
boundary, mechanisms other than retinoic acid-mediated
gene regulation are implicated for invertebrates.5 In fact,
biochemical evidence in flies and moths shows that vitamin
A is consistently metabolized to hydroxylated forms: 14-
hydroxy-retro-retinol (5) (14-HRR, enantiomeric mixture)
and 13,14-dihydroxyretinol (6) (DHR, configuration un-
known).6,7 Additionally, the rare metabolite anhydroretinol
(7) (AR), first encountered in mammals,8 was found in
Drosophila cell lines, while the enzyme retinol dehydratase,
used in AR biogenesis, was isolated and characterized from
Spodoptera frugiperda cells.9 Finally, retinol-binding sites
on the conserved zinc-finger fold of the Drosophila kinases,
cRaf (serine/threonine Raf kinase family member C) and
PKC (protein kinase C), have been identified.10,11 Although
perhaps no more than fossil footprints, they hint at a
primordial function of vitamin A in signal transduction.
These observations suggest a picture of widespread vitamin
A metabolism and usage in insects.
* To whom correspondence should be addressed. Tel: (212) 639-7523.
Fax: (212) 794-4019. E-mail: u-hammerling@ski.mskcc.org.
† Immunology Program, Memorial Sloan-Kettering Cancer Center.
‡ University of Potsdam.
While surveying the natural vitamin derivatives of a cell
line derived from the moth Trichoplusia ni (Walker, 1858),
we encountered retinoyl amides of the amino acids serine
and alanine. Although retinoyl esters of a number of
mammalian proteins were reported,12,13 amino acid deriva-
tives of retinoic acid as natural products have not been
§ Present address: Institute of Pathobiochemistry, University of Mainz,
Mainz, Germany.
Columbia University.
University of Debrecen.
|
∇ Molecular Biology Program, Memorial Sloan-Kettering Cancer Center.
10.1021/np0496791 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/17/2005

Notes
Journal of Natural Products, 2005, Vol. 68, No. 10 1537
Figure 1. Contour plot chromatogram, C18 HPLC, of Trichoplusia ni Hi-5 retinoids. Cells were grown for 24 h in the presence of (A) retinoic acid
(3) or (B) all-trans-retinol (4); inset C shows UV spectra in MeOH. HPLC peaks denote the following: peak 1, N-trans-retinoylserine (1), λmax 350
nm (broken line in inset C); peak 2, N-trans-retinoylalanine (2), λmax 336 nm (dashed line in inset C); peak 3, all-trans-retinoic acid (3), λmax 336
nm (solid line in inset C); peak 4, butylated hydroxytoluene; peak 5, 14-HRR (5); peak 6, 13-cis-retinol (8); peak 7, all-trans-retinol (4). The shifts
in UVmax of 1, 2, and 3 cannot be fully rationalized at this stage. The OH group in 1 could be contributing to the 14 nm red shift relative to 2 and
3. It is possible that H-bonding partially reduces the bond order of the amide carbonyl, thereby increasing polyene conjugation with the amide
nitrogen.
identified previously. Synthetic N-retinoyl amino acids
were previously tested, though, for efficacy in controlling
differentiation of epithelial cell lines.14,15
2, increased prominently over 24 h. The fact that RA (3)
also increased in quantity with extended incubation at the
expense of retinol suggested the genesis of a derivative of
RA (3). This was confirmed by substituting RA (3) for
retinol in the cell culture medium, upon which no com-
pound other than RA (3) and the two unknown retinoids 1
and 2 emerged in the extract (Figure 1B). Incubation of
control cell lines with retinol, including a large number of
mammalian lymphoblast and fibroblast cells lines as well
as Drosophila S2M3 cells, yielded neither compound 1 nor
2,8 indicating that the two do not represent chemical
artifacts but are specific for Hi-5 cells and are produced
by biochemical conversions. Although insects are known
to metabolize vitamin A, it was not known whether the
bioconversion products include compounds 1 and 2.
Upon scaling up the incubation and purifying the retinoic
acid derivatives to homogeneity, protonated molecular ions
were obtained at m/z 388 for compound 1 and m/z 372 for
compound 2 by ESIMS. These protonated molecular ions
were observed whether the incubations were carried out
with retinol (4) or RA (3) as the substrate. The molecular
ion m/z 387.2410 observed by HRFABMS corresponded to
a molecular formula of C23H33O4N (calcd 387.2409). The
UV of compound 1 (Figure 1C) suggested that the retinoid
contained the full polyene chain of RA (3) and that the
remaining mass was likely to be due to the presence of the
amino acid serine. As the compound was resistant to alkali
Cultured cells are normally supplied with vitamin A by
fetal bovine serum. To create defined culture conditions,
the Hi-5 insect cells were grown in the presence of 10 µM
retinol. The retinoids produced by metabolic conversion of
vitamin A were extracted with BuOH and separated by
HPLC on a C18 column, using a H2O/MeOH/CHCl3 gradi-
ent. A contour plot chromatogram of Hi-5 retinol metabo-
lites revealed, in addition to peaks 1 and 2, the following
known retinoids: peak 3 (RA, all-trans-retinoic acid, 3),
peak 5 (14-HRR, 5), peak 6 (13-cis-retinol, 8), and peak 7
(trans-retinol, 4) (Figure 1A). These were identified by their
UV absorption spectra and comparison of HPLC retention
times with reference standards.6 DHR (6) was also observed
in some instances.7 The peak eluting at 18 min represents
the antioxidant butylated-hydroxytoluene (BHT).
Peaks 1 and 2 represented two unknown compounds (1
and 2) with UV absorption maxima at 350 and 336 nm
(Figure 1C) and retention times of 14.9 and 16.0 min,
respectively. While retention times differed between HPLC
runs, the retention time relative to the reference RA (3)
was reproducible. Compound 1 preceded RA (3) (peak 3)
by 2.2 min. Whether incubated with retinol for 3 or 24 h,
the composition did not change qualitatively, but the
amount of the hitherto unknown retinoids, notably 1 and

1538 Journal of Natural Products, 2005, Vol. 68, No. 10
Notes
Figure 2. Synthesis of N-trans-retinoylserine (1) by condensation of retinoic acid (3) with resin-bound trityl L-serine.
Figure 3. Protonated molecular ion ESIMS/MS spectra of natural (A) and synthetic (B) N-trans-retinoylserine (1).
treatment, attachment of serine to RA (3) was presumed
to be through an amide, as opposed to an anhydride or ester
bond.
Thus, N-trans-retinoylserine (1) was synthesized by
condensation of RA (3) with trityl-protected, resin-bound
2-chlorotritylserine (9), as shown in Figure 2. The ESIMS/
MS spectrum of both the natural product and synthetic
N-retinoylserine showed the same fragmentation pattern
(Figure 3).
The HRFABMS elemental composition of compound 2
was determined as C23H33O3N (obsd 371.2464, calcd
371.2461), corresponding to the molecular ion of N-trans-
retinoylalanine, which was then synthesized by condensa-
tion of RA (3) with L-alanine methyl ester, followed by ester
hydrolysis (Figure 4).
The identical ESIMS/MS spectra of natural and syn-
thetic products established the structure of compound 2
as N-retinoylalanine (Figure 5). The absolute configuration

Notes
Journal of Natural Products, 2005, Vol. 68, No. 10 1539
Figure 4. Synthesis of N-trans-retinoylalanine (2) by condensation of retinoic acid (3) with L-alanine methyl ester.
Figure 5. Protonated molecular ion ESIMS/MS spectra of natural (A) and synthetic (B) N-trans-retinoylalanine (2).
The collision gas was He. The 1H NMR spectra were recorded
on a Bruker 400 MHz NMR spectrometer.
of the amino acid moieties could not be determined due to
the miniscule amounts of the natural products isolated, but
they are depicted with the more common L-configurations.
While the biological roles of N-trans-retinoylserine (1)
and N-trans-retinoylalanine (2) are not known, as is indeed
the case for most other retinoids found in insect cells, these
may represent the prototypes of retinoyl peptides. In any
event, the occurrence of these vitamin A derivatives, along
with retinaldehyde, RA (3), 14-HRR (5), DHR (6), and AR
(7),6-9 bears witness to the richness of retinoid metabolism
in invertebrates. The biochemistry of these metabolites
represents a largely unexplored area of research that will
yield important information regarding the unconventional,
non-nuclear functions of vitamin A in the mammalian
species16 since invertebrates are likely to have vitamin A
pathways that are conserved in higher organisms.
Isolation of N-trans-Retinoylamino Acids. The Trichop-
lusia ni cell line Hi-5 (Granados, R.R. US 5 300 435, 1994) was
purchased from Invitrogen (Carlsbad, CA) and grown as
suspension culture in serum-free Express Five medium at 27
°C to a density of 2 million cells per mL. All-trans-retinol (4),
or alternatively RA (3), was added at a final concentration of
10 µM, and culturing was continued from 3 to 24 h. Control
cultures of Hi-5 cells without added retinol yielded no discern-
ible retinol metabolites. Cells were harvested, washed repeat-
edly with phosphate buffered saline, and extracted with
n-BuOH in the presence of added BHT by the method of
McClean et al.17 Retinoids were resolved on a C18 column by
HPLC using a H2O/MeOH/CHCl3 gradient as described.6
A
photodiode array UV detector was used to identify eluted
retinoids. The C18 column was calibrated with authentic
retinoids RA (3), all-trans-retinol (4), 14-HRR (5), and DHR
(6) as standards. The molecular formulas of compound 1 and
2 were determined to be C23H3304N (387.2419, calcd 387.2409)
and C23H3303N (371.2464, calcd 371.2461), respectively, by
HRFABMS.
Experimental Section
General Experimental Procedures. HPLC was per-
formed using an ODS column (Vydac, Hesperia CA, C18 0.46
mm × 250 mm) and Waters Model 600E HPLC equipped with
a Model 996 UV photodiode array detector. LR- and HR-
FABMS were measured on a JEOL HX110A/110A tandem
mass spectrometer using m-nitrobenzyl alcohol (NBA) as the
matrix. ESI and ESIMS/MS were measured on a Micromass
Q-TOF equipped with a Waters 2690 HPLC. Needle voltage
was 3000 V, and the collision energy for MS/MS was 30 V.
Synthesis of N-trans-Retinoylserine (1) (Figure 2).
N-trans-Retinoylserine (1) was synthesized by the scheme
shown in Figure 2: 0.25 mmol of L-Ser(Trt)-2-Cl-Trt resin (9)
(AnaSpec, San Jose, CA) was swollen in 10 mL of CH2Cl2 for
10 min, drained, and reacted with 1 mmol of RA (3) dissolved
in 4 mL of DMF in the presence of 1 mL of DIC (N,N-
diisopropylcarbodiimide, Acros, Pittsburgh, PA), 20 mg of

1540 Journal of Natural Products, 2005, Vol. 68, No. 10
Notes
DMAP ((dimethylamino)pyridine), and 135 mg of N-hydroxy-
benzotriazole hydrate (HOBt) (Novabiochem, San Diego, CA).
A coupling efficiency close to 100% was achieved after 18 h.
The resin was washed with DMF, and the reaction product
(10) was cleaved from the resin by 1:2:7 mixtures of HOAc,
2,2,2-trifluoroethanol, and CH2Cl2 for 90 min at room tem-
perature. Product 1 (161 mg of crude peptide; 43% yield) was
purified by HPLC on a C4 column (Vydac, Hesperia, CA), using
a 0.1% TFA in H2O/MeCN gradient.
br m, H-8, H-10, NH), 5.78 (1H, s, H-14), 4.66 (1H, m, alanine
CR-H), 2.42 (3H, s, H-20), 2.10 (2H, m, H-4), 2.08 (3H, s, H-19),
1.78 (3H, s, H-18), 1.69 (2H, m, H-3), 1.55 (5H, m, H-2 and
alanine CH3), 1.11 (6H, s, H-16 and H-17); HRFABMS m/z M+
371.2465 (calcd for C23H33O3N, 371.2461).
Acknowledgment. This work was supported by grants
from the National Institutes of Health (CA49933, CA89362,
CA08748, and GM36564). We are also grateful for an NIH
Vision Training grant, EY13933-3 (to N.F.).
1H NMR (400 MHz, CDCl3) δ 6.95 (1H, dd, J ) 11 Hz, 15
Hz, 11-H), 6.71 (1H, br s, NH), 6.29-6.14 (4H, m, H-7, H-12,
H-8, and H-10), 5.75 (1H, s, H-14), 4.67 (1H, m, H-a), 4.17 (1H,
d, J ) 9 Hz, -CH2-OH), 3.82 (1H, d, J ) 9 Hz, -CH2-OH),
2.36 (3H, s, H-20), 2.06 (2H, m, H-4), 1.99 (3H, s, H-19), 1.74
(3H, s, H-18), 1.61 (2H, m, H-3), 1.48 (2H, m, H-2), 1.03 (6H,
s, H-16 and H-17); HRFABMS m/z M+ 387.2413 (calcd for
C21H3304N, 387.2409).
References and Notes
(1) Wald, G. Science 1968, 162, 230-232.
(2) Maden, M. Acta Biotheoretica 1993, 41, 425-445.
(3) Evans, R. M.; Hollenberg, S. M. Cell 1988, 52, 1-3.
(4) Petkovitch, M.; Brand, N. J.; Krust, A.; Chambon, P. Nature 1987,
330, 444-450.
(5) Blaner, W. S.; Olson, J. A. In The Retinoids: Biology, Chemistry and
Medicine, 2nd ed.; Sporn, M. B., Roberts, A. B., Goodman, D. W., Eds.;
Raven Press: Ltd: New York, 1994; pp 209-286.
(6) Buck, J.; Derguini, F.; Levi, E.; Nakanishi, K.; Hammerling, U.
Science 1991, 254, 1654-1656.
(7) Derguini, F.; Nakanishi, K.; Hammerling, U.; Chua, R.; Eppinger,
T.; Levi, E.; Buck, J. J. Biol. Chem. 1995, 270, 18875-18880.
(8) Buck, J.; Grun, F.; Derguini, F.; Chen, Y.; Kimura, S.; Noy, N.;
Hammerling, U. J. Exp. Med. 1993, 178, 675-680.
(9) Grun, F.; Noy, N.; Hammerling, U.; Buck, J. J. Biol. Chem. 1996,
271, 16135-16138.
Synthesis of N-trans-Retinoylalanine (2) (Figure 4). RA
(3) (20 mg, 0.066 mmol), EDC-HCl (37.8 mg, 0.198 mmol), and
the free base of L-alanine methyl ester (6.8 mg, 0.066 mmol)
were dissolved in 3 mL of dry CH2Cl2 and stirred for 20 min
at room temperature and then treated with DMAP (1.6 mg,
0.0132 mmol). The reaction was stirred for 4 h and then
quenched with saturated aqueous NH4Cl. The organic layer
was washed once with saturated aqueous NaHCO3 and once
with H2O and then dried over Na2SO4 and concentrated in
vacuo. The residue was purified by flash column chromatog-
raphy (SiO2, 4:1 hexanes-EtOAc). The isolated N-trans-
retinoylalanine methyl ester (11) was redissolved in 4 mL of
absolute EtOH and treated with 50 mg of KOH for 3 h. The
mixture was diluted with H2O, the aqueous solution was
acidified to pH 3 with 6 N HCl, and the precipitate was
extracted with ether (3 × 10 mL). The ether layer was washed
with brine and then dried over Na2SO4. The solvent was
removed in vacuo, and the yellow solid was purified by flash
column chromatography (SiO2, 10:1 CHCl3-MeOH) to give
10.3 mg of N-trans-retinoylalanine (2) (42% yield) as yellow
crystals: 1H NMR (400 MHz, CDCl3) δ 6.98 (1H, dd, J ) 11,
15 Hz, H-11), 6.32 (1H, m, H-7), 6.25 (1H, m, H-12), 6.18 (3H,
(10) Hoyos, B.; Imam, A.; Chua, R.; Swenson, C.; Tong, G. C.; Levi, E.;
Noy, N.; Hammerling, U. J. Exp. Med. 2000, 192, 835-846.
(11) Imam, A.; Hoyos, B.; Swenson, C.; Levi, E.; Chua, R.; Viriya, E.;
Hammerling, U. FASEB J. 2001, 15, 28-30.
(12) Breitman, T. R.; Takahashi, N. Biochem. Soc. Trans. 1996, 24, 723-
727.
(13) Myhre, A. M.; Takahashi, N.; Blomhoff, R.; Breitman, T. R.; Norum,
K. R. J. Lipid Res. 1996, 37, 1971-1977.
(14) Shealy, F. Y.; Frye, J. L.; Schiff, L. J. J. Med. Chem. 1988, 31, 190-
196.
(15) Wille, J. J.; Chopra, D. P. Cancer Lett. 1988, 40, 235-246.
(16) Hoyos, B.; Imam, A.; Korichneva, I.; Levi, E.; Chua, R.; Hammerling,
U. J. Biol. Chem. 2002, 277, 23949-23957.
(17) McClean, S. W.; Ruddel, M. E.; Gross, E. G.; DeGiovanni, J. J.; Peck,
G. L. Clin. Chem. 1982, 28, 693-696.
NP0496791