Tricarbocyanine cholesteryl laurates labeled LDL: new near infrared fluorescent probes (NIRFs) for monitoring tumors and gene therapy of familial hypercholesterolemia.

Article abstract of DOI:10.1016/S0960-894X(02)00193-2

For monitoring low-density lipoprotein receptors (LDLr) in tumors and in livers of patients with familial hypercholesterolemia (FH) treated with gene therapy, a series of tricarbocyanine cholesteryl laurates were synthesized with the cholesteryl laurate moiety serving as the lipid-chelating anchor for low-density lipoprotein (LDL). One of these conjugates, TCL17, was successfully used to label LDL to give a new NIRF, TCL17-LDL. Ex vivo biological studies on an LDLr overexpressing tumor model, human hepatoblastoma G(2) (HepG(2)), confirmed that this NIRF were internalized selectively by the tumor and detected with high sensitivity by a low-temperature 3-D redox scanner.

Full text of DOI:10.1016/S0960-894X(02)00193-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1485–1488
Tricarbocyanine Cholesteryl Laurates Labeled LDL:
New Near Infrared Fluorescent Probes (NIRFs) for
Monitoring Tumors and Gene Therapy of
Familial hypercholesterolemia
Gang Zheng,a,* Hui Li,a Kathy Yang,a Dana Blessington,b Kai Licha,c
a
Sissel Lund-Katz,d Britton Chanceb and Jerry D.Glickson
aDepartment of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA
bDepartment of Biochemistry/Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA
cInstitut fur Diagnostikforschung GmbH an der Freien Universitat Berlin, 14050, Germany
¨
¨
dDepartment of Pediatric GINutrition, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
Received 14 December 2001; accepted 11 March 2002
Abstract—For monitoring low-density lipoprotein receptors (LDLr) in tumors and in livers of patients with familial hypercholes-
terolemia (FH) treated with gene therapy, a series of tricarbocyanine cholesteryl laurates were synthesized with the cholesteryl lau-
rate moiety serving as the lipid-chelating anchor for low-density lipoprotein (LDL).One of these conjugates, TCL17, was
successfully used to label LDL to give a new NIRF, TCL17-LDL.Ex vivo biological studies on an LDLr overexpressing tumor
model, human hepatoblastoma G2 (HepG2), confirmed that this NIRF were internalized selectively by the tumor and detected with
high sensitivity by a low-temperature 3-D redox scanner. # 2002 Elsevier Science Ltd.All rights reserved.
LDL plays a central role in the etiology of athero-
sclerosis1 and is also a key vehicle for selective delivery
of therapeutic agents to a number of tumors that over-
express LDL receptors (LDLr).2ꢀ4 Familial hyper-
cholesterolemia (FH) is a genetic disorder caused by
mutations in the LDLr gene, resulting in elevated con-
centrations of LDL-bound cholesterol in blood, leading
to premature coronary heart disease.1 Currently, the
only effective treatments for homozygous FH are liver
transplantation,5 removal of LDL by plasma exchange6
and LDL apheresis.7 However, these treatments are
laborious and in the case of liver transplantation con-
tain risks with both operation and tissue rejection.In
recent years gene therapy by viral delivery and expres-
sion of the normal LDLr gene in the liver has become a
promising new alternative for treating this disease.8
Both diagnosis of malignancies that overexpress LDLr
and monitoring FH patients treated with liver gene
therapy require a robust, inexpensive and noninvasive
assay system.To date only a few radioactive imaging
agents (125I, 111In or 68Ga labeled LDL and 14C labeled
cholesterol) fit this category.1,9,10 In most cases, probes
were directly or indirectly affixed to protein residues of
Apolipoprotein B-100, the binding ligand to the LDLr.
Therefore, the probe/LDL ratio was generally kept low
to avoid disrupting the 3-D structure of the recognition
protein.As an alternative approach, Urizzi et al.
labeled the LDL with indium via a lipid-chelating agent
incorporated in the LDL phospholipid monolayer.
10
Near infrared (NIR) optical imaging is a new, safe, and
inexpensive cancer detection modality, which permits
noninvasive differentiation of tumor and normal (heal-
thy) tissue based on differences in tissue absorption or
fluorescence.11 Because tissue is relatively transparent to
NIR light, target specific NIRF can overcome the small
intrinsic contrast between tumors and normal tissue and
thus achieve high sensitivity/specificity.Tricarbocyanine
dyes have many of the ideal properties of NIR fluoro-
phore, namely long wavelength absorption (750–
850 nm), high extinction coefficients (>100,000) and
quantum yields.12 They have proven to be promising
contrasting agents for the in vivo NIR imaging of
tumors13 and have been successfully applied as NIRFs
for a number of target-specific carriers.14
*Corresponding author.Te:l. +1-215-898-3105; fax: +1-215-573-
2113; e-mail: zheng@rad.upenn.edu
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd.All rights reserved.
PII: S0960-894X(02)00193-2

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G. Zheng et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1485–1488
Figure 1. Selected tricarbocyanine dyes containing isothiocyanate,
succinimide ester and carboxylic acid groups.
Our strategy for monitoring LDLr in tumors over-
expressing LDLr is to incorporate tricarbocyanine cho-
lesteryl laurate within the lipid region of LDL, which
could be internalized by tumor cells via LDLr-mediated
endocytosis.Accumulation of the NIRF in the target
cells provides a facile mechanism for amplification of
the NIR detectable signal.Herein, we report the syn-
thesis and preliminary biological evaluation of novel
tricarbocyanine cholesteryl laurates as target specific
NIRFs for LDLr.
Figure 2. Synthesized tricarbocyanine dye labeled cholesteryl laurates.
extremely unstable16 and this led to very low yields in
the synthesis of tricarbocyanine conjugates.17 These
efforts, however, produced three desired conjugates,
TCL10–12 (Fig.2). 18 Nevertheless, due to the low yield,
it is not practical for undertaking systematic in vivo
biological evaluation of these new compounds.
For our studies, three tricarbocyanine dyes with iso-
thiocyanate, succinimide ester and carboxylic acid liners
15
were selected as NIR fluorophores (Fig.1).
To enable a directed and uniform conjugation of the
above three dyes, we prepared a cholesteryl laurate with
a primary amine group as the common substrate.As
shown in Scheme 1, the 3b-hydroxyl group of pregneo-
lone 4 was first protected as its THP ether analogue by
reaction with dihydropyran.The THP ether 5 was then
subjected to methylenation by Wittig reaction to form
the diene 6.Upon the removal of THP group of 6 with
p-TSA as the catalyst, 3b-hydroxypregna-5,20(22)-diene
7 was formed and converted to laurate 8 with lauroyl
chloride in pyridine.The in situ hydroboration amina-
tion of 8 gave the final product 22-amino-5-pregnen-
3b-yl laurate 9.
In order to solve this problem, we developed an alter-
native pathway (Scheme 2) for synthesis of a steric hin-
dered and thus more stable cholesteryl laurate amine.
As shown in Scheme 2, a commercially available cho-
lesteryl amine 13, 5-androsten-17b-amino-3b-ol (Ster-
aloid Inc., Newport, RI), was first Boc-protected (14)
and esterified with lauroyl chloride to give its corre-
sponding cholesteryl laurate amine 15.Deprotection of
15 at the C-17 gave the corresponding cholesteryl laurate
Though the coupling reactions between isothiocyanate,
succinimide ester, carboxylic acid and primary amine
were well known, we soon found that amine 9 was
Scheme 1. Synthesis of cholesteryl laurate with a primary amine
group.
Scheme 2. Synthesis of a stable cholesteryl laurate amine 13 and its
corresponding tricarbocyanine dye conjugate, TCL17.

G. Zheng et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1485–1488
1487
for consultation; and to Mr.David Nelson for tech-
nical assistance.This research was supported by NIH
contract BAA NO1-CM97065–02 and an Oncologic
Foundation of Buffalo award.Partial support by NIH
grants P20 CA86255–01 and HL56083 are also
acknowledged.
References and Notes
1. Goldstein, J. L.; Hobbs, H. H.; Brown, M. S. In The
Metabolic and Molecular Bases of Inherited Disease, 7th ed.;
Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle. D., Eds.;
McGraw-Hill, Inc.: New York, 1995; Vol. II, p 1981.
2.Lundberg, B. Cancer Res. 1987, 47, 4105.
3.Firestone, R.A. Bioconjugate Chem. 1994, 5, 105.
4. Shaw, J. M.; Shaw, K. V.; Yanovich, S.; Iwanik, M.; Futch,
W. S.; Rosowsky, A.; Schook, L. B. Ann. N.Y. Acad. Sci.
1987, 507, 252.
5. Bilheimer, D. W.; Goldstein, J. L.; Grundy, S. M.; Starzl,
T.W;. Brown, M.S. N. Engl. J. Med. 1984, 311, 1658.
6. Thompson, G. R.; Lowenthal, R.; Myant, N. B. Lancet
1975, 1, 1208.
Figure 3. The redox image of HepG2 tumor tissue after TCL17-LDL
tail vein injection.The color scale bar reflects the relative fluorescent
intensity.
amine 16 in 60% overall yield.Coupling of this amine
to the tricarbocyanine dye 1 yielded a new dye con-
7. Zwiener, R. J.; Uauy, R.; Petruska, M. L.; Huet, B. A.
J. Pediatr. 1995, 126, 728.
1
jugate TCL17 in good yield (40%). H NMR and mass
spectroscopy studies confirmed its structure.18 Sub-
sequently, following the method described by Pitas et
al.,19 TCL17 was successfully used to label LDL and
gave a new NIRF, namely TCL17-LDL.20
8. Grossman, M.; Raper, S. E.; Kozarsky, K.; Stein, E. A.;
Engelhardt, J. F.; Muller, D.; Lupien, P. J.; Wilson, J. M. Nat.
Genet. 1994, 6, 335.
9. Moerlein, S. M.; Daugherty, A.; Sobel, B. E.; Welch, M. J.
J. Nuclear Med. 1991, 32, 300.
10. Urizzi, P.; Souchard, J.-P.; Palevody, C.; Ratovo, G.;
Hollande, E.; Nepveu, F. Intl. J. Cancer 1997, 70, 315.
11. Sevick-Muraca, E. M.; Lopez, G.; Reynold, J. S.; Troy,
T.L;. Hutchinson, C.L. Photochem. Photobiol. 1997, 66, 55.
12.(a) Narayanan, N;. Patonay, G. J. Org. Chem. 1995, 60,
2391. (b) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.;
Waggoner, A.S. Cytometry 1989, 10, 11.
To confirm the selective delivery of this new NIRF to
tumors via LDLr-mediated endocytosis, a low-temperature
3-D redox scanner21 was used as our NIR fluorescent
imager on a LDLr overexpressed tumor model, HepG2.
The redox scanner functions by means of automated
scanning of surface fluorescence state of the frozen tis-
sue.The scanning process is fully computerized and
programs have been developed which allow 3-D recon-
struction of the data in terms of ‘redox ratio models’.
Figure 3 shows a redox image of HepG2 tumor tissue
intravenously injected with TCL17-LDL.22 As shown in
this figure, strong fluorescent signal (red region of the
image) of this NIRF was detected only inside the tumor
tissue, demonstrating that TCL17-LDL was selective
internalized into the tumor.
13. Ntziachristos, V.; Yodh, A. G.; Schnall, M.; Chance, B.
Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2767.
14. (a) Weissleder, R.; Tung, C.-H.; Mahmood, U.; Bogna-
nov, A. Nat. Biotech. 1999, 17, 375.(b) Becker, A;. Hessenius,
C.; Licha, K.; Ebert, B.; Sukowski, U.; Semmler, W.; Wie-
denmann, B.; Grotzinger, C. Nat. Biotech. 2001, 19, 327.
15.Dye 1 (IRD41) and 3 (IRD80) were purchased from
LICOR (Lincoln, NB).Dye 2 (NIR97235-NHS ester) was a
gift from Shering AG, Germany.
16.Amine 9 was found to be unstable, it decomposed even in
very weak basic conditions: (a) pH 9.3 buffer, 2 h; (b) Et3N in
CH2Cl2, DMF, THF, CH3CN and toluene; (c) DMAP,
NMM, 4-picoline and 2,6-di-tert-butylpyridine in DMF.
However, it is relatively stable with diisopropylethyl amine
(DIPEA) in DMF.
17.For labeling the isothiocyanate functionality, a slight
excess of dye 1 was reacted with amine 9 in DMF with DIPEA
for 48 h, yielding the labeled conjugate 10 in 10% yield.Under
similar conditions, succinimide ester containing dye 2 reacted
with 9 gave small amount of dye conjugate 12.The coupling
reaction between carboxylic acid containing dye 3 and amine 9
using 1:1:1 proportions DCC/HOBT/DIPEA in DMF gave
dye conjugate 11 in 10% yield.
In summary, efforts were made to synthesize a choles-
teryl laurate with a primary amine functional group that
was stable enough to form its corresponding tricarbo-
cyanine conjugate in a reasonable yield.By using the
low-temperature 3-D redox scanner, we have confirmed
in a HepG2 tumor model that the selective delivery of
TCL17-LDL to tumor tissue via LDLr pathway was
achieved.Because of its target specificity and its high
sensitivity, this new NIRF should be useful for mon-
itoring LDLr in tumors overexpressing LDLr and in
livers of FH patients treated with gene therapy.
18.Spectroscopic data and characterization for selected com-
pounds.
Pregneolone 3-THP ether (5): Anal.calcd for C 26H40O3: C,
77.95; H, 10.06. Found: C, 77.68; H, 9.88; 1H NMR (CDCl3) d
5.35 (1H, 6-H), 4.72 (1H, 20-H), 3.92 (m, 1H, 60H), 3.52 (m,
1H, 3-H), 3.51 (m, 1H, 60-H), 2.12 (s, 3H, 21-H), 1.01 (s, 3H,
19-H), 0.63 (s, 3H, 18-H); 13C NMR (CDCl3) d 209.4 (C-20),
141.1 (C-5), 121.2 (C-6), 96.9 (C-10), 31.8 (C-21), 19.3 (C-19),
13.2 (C-18).
Acknowledgements
We are grateful to Dr.Ponzy Lu of Chemistry Depart-
ment for housing our organic lab; to Drs.Hank Kung
and Madeleine Joullie of PENN, Nara Narayanan of
MWG Biotech and Zhe Wang of DuPont Pharmaceutical

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G. Zheng et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1485–1488
3ꢀ(Tetrahydropyan-20-yl)oxypregna-5,20(22)-diene (6): Anal.
calcd for C27H42O2: C, 81.35; H, 10.70. Found: C, 81.95; H,
5-Androsten-17ꢀ-Boc-amino-3ꢀ-yl laurate (15): Exact mass
calcd for C36H61NO4: 571.46; Found by ESI-MS: 572.3
1
1
10.72; H NMR (CDCl3) d 5.34 (1H, 6-H), 4.82 (1H, 22-H),
(MH+); H NMR (CDCl3) d 5.36 (m, 1H, 6-H), 4.61 (m, 1H,
4.69 (2H, 22-H, 20-H), 3.89 (m, 1H, 60-H), 3.50 (m, 1H, 3-H),
3.48 (m, 1H, 60-H), 1.74 (s, 3H, 21-H), 0.99 (s, 3H, 19-H), 0.56
(s, 3H, 18-H); 13C NMR (CDCl3) d 145.7 (C-20), 140.9 (C-5),
121.3 (C-6), 110.6 (C-22), 96.9 (C-1), 75.9 (C-3).
3-H), 4.39 (brs, 1H, N-H), 3.54 (m, 1H, 17-H), 1.02 (s, 3H, 19-
H), 0.85 (t, 3H, CH3 of lauroyl), 0.67 (s, 3H, 18-H); 13C NMR
(CDCl3) d 173.5 (CO2), 140.1 (C-5), 122.4 (C-6), 73.8 (C-3),
14.3 (C-19), 12.0 (C-18).
3ꢀ-Hydroxypregna-5,20(22)-diene (7): Anal.calcd for
C22H34O: C, 84.02; H, 10.90. Found: C, 82.42; H, 10.82; H
5-Androsten-17ꢀ-amino-3ꢀ-yl laurate (16): Exact mass calcd
1
1
for C31H53NO2: 471.41; Found by ESI-MS: 472.5 (MH+); H
NMR (CDCl3) d 5.38 (1H, 6-H), 4.87 (1H, 22-H), 4.73 (1H,
22-H), 3.54 (m, 1H, 3-H), 1.78 (s, 3H, 21-H), 1.03 (s, 3H,
19-H), 0.61 (s, 3H, 18-H); 13C NMR (CDCl3) d 145.5 (C-20)
140.8 (C-5), 121.5 (C-6), 110.6 (C-22), 71.7 (C-3).
NMR (CDCl3) d 5.40 (m, 1H, 6-H), 4.61 (m, 1H, 3-H), 2.71
(m, 1H, 17-H), 1.02 (s, 3H, 19-H), 0.87 (t, 3H, CH3 of lauroyl),
0.68 (s, 3H, 18-H); 13C NMR (CDCl3) d 173.5 (CO2), 140.0
(C-5), 122.5 (C-6), 73.8 (C-3), 14.3 (C-19), 11.3 (C-18).
Tricarbocyanine cholesteryl laurate 17 (TCL17) (coupling of
dye 1 and amine 16): Exact mass calcd for C79H104N4O6S2:
1268.74; Found by ESI-MS: 1269.6 (MH+) and 1291.6
(M+Na+).
Pregna-5,20(22)-diene-3ꢀ-yl laurate (8): Anal.calcd for
1
C34H56O2: C, 82.20; H, 11.36. Found: C, 82.43; H, 11.82; H
NMR (CDCl3) d 5.40 (1H, 6-H), 4.87 (1H, 22-H), 4.86 (1H,
22-H), 4.73 (m, 1H, 3-H), 1.78 (s, 3H, 21-H), 1.05 (s, 3H,
19-H), 0.60 (s, 3H, 18-H); 13C NMR (CDCl3) d 173.2 (C-10),
145.5 (C-20), 139.7 (C-5), 122.4 (C-6), 110.6 (C-22), 73.5 (C-3).
22-Amino-5-pregnen-3ꢀ-yl laurate (9): Exact mass calcd for
C34H59NO2: 513.4; Found by ESI-MS: 514.4 (MH+); 1H
NMR (CDCl3) d 5.36 (1H, 6-H), 4.59 (1H, 3-H), 3.54 (2H,
22H), 1.02 (s, 3H, 19-H), 0.64 (s, 3H, 18-H); 13C NMR (CDCl3)
d 173.3 (CO), 139.7 (C-5), 122.5 (C-6), 73.7 (C-3), 70.6 (C-22).
Tricarbocyanine cholesteryl laurate 10 (TCL10) (coupling of
isothiocyanate of dye 1 and amine 9): Exact mass calcd for
C82H110N4O6S2: 1310.79; Found by ESI-MS: 1311.2 (MH+).
Tricarbocyanine cholesteryl laurate 11 (TCL11) (coupling of
carboxylic acid of dye 3 and amine 9): Exact mass calcd for
C81H113N3O10S2: 1351.79; Found by ESI-MS: 1352.4 (MH+).
Tricarbocyanine cholesteryl laurate 12 (TCL12) (coupling of
succinimide ester of dye 2 and amine 9): Exact mass calcd for
C67H95N3O6S: 1069.69; Found by ESI-MS: 1070.5 (MH+).
5-Androsten-17ꢀ-Boc-amino-3ꢀ-ol (14): Exact mass calcd
19. Pitas, R. E.; Innerarity, T. L.; Weinstein, J. N.; Mahley,
R.W. Arteriosclerosis 1981, 1, 177.
20.Tricarbocyanine dyes including IRD41 are sensitive to
photobleaching.Therefore, aluminum foil was frequently used
to exclude ambient light in reactions and LDL reconstitution
process to avoid photodegradation of the dye.The resulting
TCL17-LDL can be stored at 4 ꢁC in dark for up to four
weeks without detectable change in fluorescence and absorp-
tion.Meanwhile, the laser power used in ex vivo detection is
small (5 mw), we found that photobleaching is relatively
insignificant in this case.Chemically, TCL17 is quite stable.It
can be easily purified by preparative TLC with 10% methanol
in dichloromethane.
21. Quistorff, B.; Haselgrove, J. C.; Chance, B. Anal. Biochem.
1985, 148, 389.
22.HepG tumor was inoculated subcutaneously on nude
2
mice with 10ꢂ106 cells.After the development of the tumor to
about 1 cm in diameter, the mice received an intravenously
injection of TCL17-LDL.Two hours after the injection, the
mice were frozen in the liquid nitrogen under the anesthetiza-
tion and the tumor tissue was used for the redox scanning.
1
for C24H39NO3: 389.29; Found by ESI-MS: 390.4 (MH+); H
NMR (CDCl3) d 5.34 (m, 1H, 6-H), 4.40 (brs, 1H, N-H), 3.53
(m, 2H, 3-H+17-H), 1.43 (s, 9H, Boc-H), 1.00 (s, 3H, 19-H),
0.67 (s, 3H, 18-H); 13C NMR (CDCl3) d 141.1 (C-5), 121.5 (C-
6), 71.9 (C-3), 19.6 (C-19), 12.0 (C-18).