Synthesis and structural characterization of carboxyethylpyrrole-modified proteins: Mediators of age-related macular degeneration

Bioorganic & Medicinal Chemistry 17 (2009) 7548–7561

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Synthesis and structural characterization of carboxyethylpyrrole-modiﬁed

proteins: mediators of age-related macular degeneration

Liang Lu ^a, Xiaorong Gu ^b, Li Hong ^a, James Laird ^a, Keeve Jaffe ^c, Jaewoo Choi ^a,

John Crabb ^b, Robert G. Salomon ^a,

*

^aDepartment of Chemistry, Case Western Reserve University, Cleveland, OH 44106-7078, United States

^bCole Eye Institute of the Cleveland Clinic Foundation, Cleveland, OH 44195, United States

^cFrantz Biomarkers, LLC, Mentor, OH 44060, United States

a r t i c l e i n f o

a b s t r a c t

Article history:

Protein modiﬁcations in which the e-amino group of lysyl residues is incorporated into a 2-(x-carboxy-

Received 31 August 2009

Revised 4 September 2009

Accepted 5 September 2009

Available online 13 September 2009

ethyl)pyrrole (CEP) are mediators of age-related macular degeneration (AMD). They promote both angi-

ogenesis into the retina (‘wet AMD’) and geographic retinal atrophy (‘dry AMD’). Blood levels of CEPs are

biomarkers for clinical prognosis of the disease. To enable mechanistic studies of their role in promoting

AMD, for example, through the activation of B- and T-cells, interaction with receptors, or binding with

complement proteins, we developed an efﬁcient synthesis of CEP derivatives, that is especially effective

for proteins. The structures of tryptic peptides derived from CEP-modiﬁed proteins were also determined.

A key ﬁnding is that 4,7-dioxoheptanoic acid 9-ﬂuorenylmethyl ester reacts with primary amines to

provide 9-ﬂuorenylmethyl esters of CEP-modiﬁed proteins that can be deprotected in situ with 1,8-diaza-

bicyclo[5.4.0]undec-7-ene without causing protein denaturation. The introduction of multiple CEP-

modiﬁcations with a wide variety of CEP:protein ratios is readily achieved using this strategy.

Keywords:

Age-related macular degeneration

Carboxyethylpyrrole

Organic synthesis

Protein modiﬁcation

Lipid oxidation

Angiogenesis

Biomarkers

1. Introduction

owing to increases in human life span, AMD is expected to nearly

double in the next 25 years.⁴Proteomic characterization of drusen,

Although it is relatively scarce in most tissues, docosahexaenoic

acid (DHA) is essential for the growth, functional development and

maintenance of the brain and is most abundant in photoreceptor

cell membranes in the retina. Owing to the presence of the six

homoconjugated C@C bonds in DHA, it is exquisitely sensitive to

oxidative damage. Oxidative cleavage of phospholipids containing

DHA produces reactive electrophilic phospholipid fragments, for

example, 4-hydroxy-7-oxohept-5-enoates, that convert the pri-

extracellular deposits that accumulate between the retina and the

blood bearing choriocapillaris, revealed that CEP adducts are more

abundant in AMD than in normal eyes.⁵CEPs are also elevated in

the blood of individuals with AMD.⁶CEPs are not simply benign

markers of oxidative damage, but also promote the growth of cap-

illaries (neovascularization), and possibly contribute to choroidal

neovacularization, also known as ‘wet’ AMD.⁷Such neovasculariza-

tion is responsible for ꢀ90% of the loss of vision associated with

AMD. Remarkably, CEPs initiate an autoimmune response that

may contribute to retinal degeneration. It had been observed that

immunoglobins and complement components accumulate in sub-

retinal neovascular membranes from AMD patients^8,9and that

autoantibodies against CEPs are elevated in the blood of individu-

als with AMD⁶and in rodents exposed to intense light.¹⁰Recently

it was shown that mice immunized with CEP-modiﬁed serum albu-

min also develop autoantibodies to this hapten, ﬁx complement

component-3 in Bruch’s membrane, accumulate drusen below

the retinal pigment epithelium during aging, and develop lesions

in the retinal pigment epithelium mimicking geographic atrophy,

the blinding end-stage condition characteristic of the ‘dry’ form

of AMD.¹¹Apparently, these mice are sensitized to the generation

of CEP adducts in the outer retina, where DHA is abundant and

conditions for oxidative damage are permissive.

mary amino group of protein lysyl residues into 2-(x-carboxy-

ethyl)pyrrole (CEP) derivatives.¹CEPs are especially abundant in

ocular tissues from individuals with age-related macular degener-

ation (AMD), a slow, progressive disease²that is the major cause of

untreatable loss of vision among the elderly in developed coun-

tries.³Roughly 11% of people in the United States have AMD, and

Abbreviations: AMD, age-related macular degeneration; BSA, bovine serum

albumin; CEP, carboxyethylpyrrole; CEPH, 6-(2-carboxyethyl-1-pyrrolyl)-hexanoyl;

COA, chicken ovalbumin; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DHA, docosa-

hexaenoic acid; DMF, N,N-dimethylformamide; DOHA, 4,7-dioxoheptanoic acid;

ELISA, enzyme-linked immunosorbent assay; FmOH, 9-ﬂorenylmethanol; GPDH,

glyceraldehyde phosphate dehydrogenase; HSA, human serum albumin; MSA,

mouse serum albumin; PBS, phosphate buffered saline; THF, tetrahydrofuran.

* Corresponding author. Tel.: +1 216 368 2592; fax: +1 216 368 3006.

E-mail address: rgs@case.edu (R.G. Salomon).

doi:10.1016/j.bmc.2009.09.009

L. Lu et al. / Bioorg. Med. Chem. 17 (2009) 7548–7561

7549

Generating proteins with various levels of CEP-modiﬁcation has

proven difﬁcult, especially for higher CEP to protein molar ratios.

We now report an efﬁcient synthesis of CEP-modiﬁed proteins that

was used to create the above mouse model of AMD. This synthesis

and the characterization of tryptic peptides derived from the CEP-

modiﬁed proteins will also enable mechanistic studies of their role

in promoting AMD, for example through possible activation of B-

and T-cells or interaction with complement proteins and as ligands

for CEP receptors. A key contribution of the present report is that

the 9-ﬂuorenylmethyl ester of 4,7-dioxoheptanoic acid reacts with

we coupled this Grignard reagent with 3-carbomethoxypropionyl

chloride and obtained the methyl ester 1a (Scheme 2). The desired

9H-ﬂuoren-9-ylmethyl ester 4,7-dioxo-heptanoic acid (DOHA-Fm,

4) was then obtained through saponiﬁcation to afford the carbox-

ylic acid 2a, esteriﬁcation with 9-ﬂuorenylmethanol (FmOH), fol-

lowed by hydrolysis of ethylene ketal in 3a.

Subsequently we determined that the Grignard reagent from 2-

(2-bromoethyl)-1,3-dioxane¹⁶shows similar selectivity toward

reaction with the acyl chloride but not the carbomethoxy group

of 3-carbomethoxypropionylchloride to deliver 1b in good yield.

In addition, puriﬁcation of the corresponding carboxylic acid 2b

is facilitated by the fact that it readily crystallizes. The keto alde-

hyde 3b is also a crystalline solid. Initially, we used a large excess

of FmOH (4 equiv) and allowed the esteriﬁcation to proceed for

3 days. However, the excess FmOH interfered with puriﬁcation,

requiring multiple column chromatographies. A more practical

procedure, using 1.2 equiv of FmOH and a 19 h reaction time,

delivered 3b in 85% yield after a single column chromatography

on silica gel. This procedure was successfully and reproducibly ap-

plied on a multi-gram scale. Hydrolysis of the propylene acetal 3b

occurs more readily than the corresponding ethylene acetal 3a,

allowing shorter reaction times, and the isolation of pure DOHA-

Fm (4) is more readily achieved from the acetal 3b than from the

corresponding ethylene acetal 3a.

lysyl e-amino groups to provide esters of CEPs that can be depro-

tected with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) without

causing protein denaturation. The introduction of multiple CEPs

into proteins is readily achieved using this strategy. In addition,

the preparation of CEPs tethered to proteins through an

x-amino

hexanoate linker and their strong binding with anti-CEP antibodies

is described. To characterize the CEP-modiﬁed proteins, the struc-

tures of tryptic peptides derived from CEP-modiﬁed proteins were

also determined.

2. Results

2.1. Paal–Knoor synthesis using 4,7-dioxo-heptanoic acid is

ineffective for the preparation of CEPs

2.3. Synthesis of CEP–peptide and CEP–protein adducts by

Paal–Knoor synthesis with DOHA-Fm

The reaction of

c-keto aldehydes with primary amines, the

Paal–Knoor synthesis,¹²is generally an efﬁcient method for the

preparation of pyrroles. We previously successfully applied this

reaction to the generation of carboxyheptylpyrrole and carboxy-

propylpyrrole derivatives through the reactions of 9,12-dioxodo-

decanoic or 5,8-dioxooctanoic acid with proteins.¹³However,

attempts at preparing the corresponding carboxyethylpyrrole

derivatives of proteins by treatment with 4,7-dioxoheptanoic acid

(DOHA) generally caused precipitation, and in the few instances

that precipitation did not occur, the ratio of pyrrole to protein,

for example, 1.6:1 for human serum albumin,⁶was much lower

than we had obtained previously for the longer chain carboxyalkyl-

pyrroles. Another distinguishing feature of DOHA was the nearly

complete absence of a signal for the aldehydic hydrogen in its ¹H

NMR spectrum. We postulated that the unusual ¹H NMR spectrum

and aberrant reactivity of DOHA are consequences of the proximity

While the angiogenecity of CEPs was ﬁrst detected in protein

derivatives, the CEP dipeptide 2-(2-acetylamino-acetylamino)-6-

[2-(2-carboxy-ethyl)-pyrrol-1-yl]-hexanoic acid methyl ester (6)

was also found to be potently angiogenic.⁷Therefore, we examined

the applicability of the new synthetic method to prepare 6. Paal–

Knoor synthesis of the Fm ester 5 of a CEP dipeptide was readily

achieved in 80% yield by the condensation of methyl 6-amino-2-

((2-acetylamino)acetyl)amino)hexanoate (Ac-Gly-Lys-OMe) with

1 equiv of DOHA-Fm (Scheme 3). For peptide synthesis, the depro-

tection of 9-ﬂuorenylmethyl esters is normally accomplished with

piperidine in DMF. Piperidine serves both as a base to fragment the

Fm group and as a scavenger to trap the dibenzofulvene released.¹⁷

The removal of an Fm group from an ester bound to a protein has

not been reported. Piperidine was unsatisfactory, vide infra. This

was readily determined by the persistence of a UV absorbtion at

of the carboxyl group to the c-ketoaldehyde array and that DOHA

exists in equilibrium with the corresponding spiroacylal hemiace-

tal (Scheme 1). To obviate complications engendered by the car-

boxyl group, we sought a masked derivative that could be

deprotected under conditions that would not lead to denaturation

and consequent precipitation of proteins. This excluded acidic con-

ditions. Therefore, we opted for a 9-ﬂuorenylmethyl ester.

O

Br

(CH₂)

O

n

1) Mg, THF

O

2)

ClCO(CH₂)₂COOMe

THF, -78 ºC

O

2.2. Synthesis of a 9-ﬂuorenylmethyl (Fm) ester of DOHA

OR

(CH₂)

n

O

A Grignard reagent derived from 2-(2-bromoethyl)-1,3-dioxo-

lane¹⁴was previously acylated with 8-carbomethoxyoctanoyl

chloride to selectively deliver a ketone.¹⁵Following this precedent,

1a, n = 2, R = CH₃(60%)

2a, n = 2, R = H (90%)

3a, n = 2, R = Fm (95%)

or

NaOH, H₂O/MeOH/THF

FmOH, DCC, DMAP, CH₂Cl₂

1b, n = 3, R = CH₃(78%)

2b, n = 3, R = H (92%)

3b, n = 3, R = Fm (85%)

NaOH, H₂O/MeOH/THF

OH

FmOH, DCC, DMAP, CH₂Cl₂

O

N

O

OH

O

Protein

O

CEP

88-90%

O

3a or 3b

O

DOHA

HOAc:H₂O

(3:1, v/v)

50 ºC

NH₂

Protein

HO

O

DOHA-Fm (4)

O

Scheme 1.

Scheme 2.

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L. Lu et al. / Bioorg. Med. Chem. 17 (2009) 7548–7561

tio of 1.6:1 for CEP-HSA,⁶the new synthetic method using DOHA-

O

H

N

H

N

Fm provided CEP-HSA (8a) with a much higher pyrrole to protein

ratio, 7.6 1.1 to 1, and provided CEP-MSA (8b) with a pyrrole to

protein ratio of 5.2 1.0 to 1. To enable studies on the inﬂuence

of pyrrole to protein ratio on biological activity, we incubated var-

ious amounts of the DOHA-Fm reagent with BSA to prepare CEP–

BSA (8c) with pyrrole to protein ratios of 10.1 1.0, 7.0 1.1,

5.1 1.0 and and 3.7 0.8. The similar preparation of CEP-modiﬁed

chicken egg albumin, rabbit myoglobin, and glycerol-3-phosphate

dehydrogenase are detailed in the experimental procedures.

N

H

OMe

N

H

OMe

80%

O

N

DOHA-Fm

methanol

NH₂

Ac-Gly-Lys-OMe

COOR

5, R = Fm

6, R = H

DBU,THF

86%

2.4. Characterization of CEP-modiﬁed proteins

Scheme 3.

To characterize the location of CEP modiﬁcations in proteins fol-

lowing reaction with DOHAFm and deprotection with DBU, the pro-

teins were digested with trypsin (protein/trypsin = 50:1, w/w) and

then analyzed by liquid chromatography tandem MS (LC–MS/MS)

with a CapLC system (Micromass, Beverly, MA) and a quadrupole

time-of-ﬂight mass spectrometer (QTOF2, Micromass).²¹Peptides

265 nm that is characteristic of the ﬂuorene group. Therefore, we

examined the efﬁcacy of a stronger base. Previously, a catalytic

1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)/octanethiol cocktail in

THF was found effective for removal of ﬂourenemethoxycarbonyl

protecting groups from

a

-amino acids.¹⁷We found that DBU suc-

cessfully removed all Fm groups from protein adducts, vide infra,

and this reagent was also applied to deprotection of the dipeptide

Fm ester 5. Removal of the Fm protecting group by treatment of 5

with DBU delivered the CEP dipeptide 2-(2-acetylamino-acetyl-

amino)-6-[2-(2-carboxy-ethyl)-pyrrol-1-yl]-hexanoic acid methyl

ester (6) in 86% yield.

were separated on a 75

l

m ꢁ 5 cm Biobasic C18 column (New

Objective, Cambridge, MA) by using aqueous formic acid/ acetoni-

trile solvents, a ﬂow rate of 250 nL/min, and a gradient of 5–40% ace-

tonitrile over 57 min followed by 80% acetonitrile for 2 min. Protein

identiﬁcations from MS/MS data was accomplished with MASSLYNX

3.5 software (Waters), the Mascot search engine (Matrix Science)

and the Swiss-protein and National Center for Biotechnology

information protein sequence databases.^22,23MS/MS analysis of

modiﬁcations at speciﬁc m/z revealed peptide sequences with

unambiguous CEP adducts on lysyl residues of the CEP–proteins.

No CEPFm modiﬁcations were found after deprotection. We also

analyzed CEPFm modiﬁed HSA before deprotection. We identiﬁed

3 CEPFm modiﬁcations at m/z 725.281, 660.289 and 776.820 on lysyl

CEP-modiﬁed proteins are needed for immunoassays that mea-

sure levels of CEPs or anti-CEP autoantibodies in vivo.⁶CEP–protein

adducts are also essential for the recently described mouse model

of AMD.¹¹CEP-MSA is used as an antigen to immunize mice and

elicit immune responses against CEP–protein adducts generated

in the retina. We now ﬁnd that the production of CEP–protein ad-

ducts can be readily accomplished by incubation of DOHA-Fm (4)

with protein in 30% DMF/phosphate-buffered saline (PBS) solution

for 5 days at 37 °C followed by deprotection by addition of DBU to

the reaction mixture and stirring for an additional 9 h. One equiv-

alent of DOHA-Fm was used for each lysine group present in hu-

man serum albumin (HSA) or mouse serum albumin (MSA)

(Scheme 4). Low molecular weight contaminants were removed

by dialysis (Mr cutoff 14,000) of the reaction mixture against 20%

DMF in 10 mM PBS. An especially important feature of the use of

Fm esters of DOHA is the ease with which residual Fm groups

can be detected and their complete removal assured by UV spec-

troscopy, that is, monitoring absorption at 265 nm. The ﬁnal pro-

tein concentration was determined by Pierce bicinchoninic acid

(BCA) protein assay¹⁸or Bio-rad protein assay.¹⁹The pyrrole con-

centration was determined by the generation of a characteristic

chromophore through reaction with 4-(dimethylamino)benzalde-

hyde, the Ehrlich reagent,²⁰using the CEP dipeptide 6 as a quanti-

tative standard. In contrast with the preparation of CEP-HSA by

direct treatment with DOHA, that delivered a pyrrole to protein ra-

residues in the LC–MS/MS of HSA peptides ²⁵DAHKSEVAHR³⁴

,

²³⁴AFKAWAVAR²⁴², and ²⁴⁷FPKAEFAEVSK²⁵⁷in CEPFmHSA. We

identiﬁed six CEP modiﬁcations at m/z 589.266, 571.260, 674.762,

881.401, 687.784, 636.252 on lysyl residues in the LC–MS/MS of

HSA peptides ¹⁶¹KYLYEIAR¹⁶⁸

,

²³⁴AFKAWAVAR²⁴²

,

³⁵FKDLGEENFK⁴⁴

,

and ²⁵DAHKSE-

,

⁴³⁸KVPQVSTPTLVEVSR⁴⁵²

,

²⁴⁷FPKAEFAEVSK²⁵⁷

VAHR³⁴, respectively. The similar LC–MS/MS analyses of CEP-modi-

ﬁed chicken egg albumin, rabbit myoglobin, and glycerol-3-

phosphate dehydrogenase are detailed in the Experimental Section.

2.5. CEP linked to proteins with an

x-aminohexanoyl tether

Direct coupling of DOHA-Fm to proteins results in a high yield

of CEP modiﬁcations of lysyl residues. As an alternative approach

for anchoring CEP haptens to proteins for use as coating agents

to capture anti-CEP antibodies, we examined the utility of CEPs an-

chored to proteins through hexanoyl amides of protein lysyl resi-

dues. An Fm masked 2-carboxyethylpyrrole 9 was generated

through the reaction of DOHA-Fm with 6-aminocaproic acid. After

puriﬁcation, 9 was activated by conversion into an N-hydroxy-

succinimide ester 10. Incubation of the active ester 10 with bovine

serum albumin (BSA) followed by deprotection in situ by addition

of DBU to the reaction mixture, delivered a 6-(2-carboxyethyl-1-

pyrrolyl)hexanoyl amide derivative of BSA, CEPH-BSA (11, Scheme

5). Low molecular weight impurities were readily removed by dial-

ysis (Mr cutoff 14,000) with 20% DMF in 10 mM PBS 2 ꢁ 12 h and

then with 10 mM PBS 2 ꢁ 12 h. The protein concentration was

determined by a modiﬁed Lowry protein assay²⁴using the Lowry

protein assay reagent and Folin-Ciocalteu reagent. The pyrrole con-

centration was determined using Ehrlich assay. The pyrrole to BSA

ratio in CEPH-BSA (11) was 5.4 0.9 to 1.

NH₂

O

N

protein

DOHA-Fm (4)

O

DMF, PBS

7a, protein = HSA

7b, protein = MSA

7c, protein = BSA

COOH

N

DBU,DMF, PBS

8a, protein = HSA

8b, protein = MSA

8c, protein = BSA

protein

The antibody binding afﬁnity of CEPH-BSA (11) was determined

by competitive enzyme-linked immunosorbant assay (ELISA)²⁵

using an anti-CEP-KLH polyclonal antibody (Fig. 1). CEP-HSA was

Scheme 4.

L. Lu et al. / Bioorg. Med. Chem. 17 (2009) 7548–7561

7551

O

S

HOOC(CH₂)₅NH₂

HOOC(CH₂)₅N

80%

H₂O

MeOH

H

+

NH

N

O-t-Bu

80%

OPhNO₂

DOHA-Fm (4)

H₂N(CH₂)₄

COO-Fm

O

9

O

12

(CH₂)₄

90%

HOSu

DCC

DMF, PBS

OOC(CH₂)₅N

N

NH₂

O

BSA

CH₂Cl₂

COO-Fm

O

NH

10

O

90%

H

N

(CH₂)₄

O-t-Bu

HN

BSA

(CH₂)₅N

HN

BSA

(CH₂)₅N

(CH₂)₄

O

CF₃COOH

CH₂Cl₂

N

H

S

DBU

DMF

PBS

O

13

COOH

COO-Fm

CEPH-BSA (11)

NH

Scheme 5.

84%

O

DOHA-Fm

MeOH

(CH₂)₄NH₂

ROOC

(CH₂)₄

N

H

S

14

O

100

80

60

40

20

0

NH

O

(CH₂)₄

N

(CH₂)₄

N

H

S

15, R = Fm

IC₅₀= 3.02

DBU, THF

16, R = H

85%

IC₅₀= 1.93

Scheme 6.

the retina. Because levels of PEs are strictly regulated, they are be-

lieved to have unique functional importance.²⁶In view of the reac-

tivity of the primary amino group of PEs and the abundance of DHA

in brain and retina, we anticipate that DHA-derived oxidatively

truncated phospholipids containing reactive electrophilic 4-hydro-

xy-7-oxohept-5-enoates convert the primary amino group of PEs

into CEP-PE derivatives. We synthesized PE-CEPs to facilitate their

detection and identiﬁcation in vivo. Reaction of DOHA-Fm with 1-

palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or

1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (lys-

10^-2

10^-1

10⁰

10¹

10²

10³

Inhibitor (pmol/ml)

Figure 1. Inhibition curves showing cross-reactivity of the anti-CEP-KLH antibody

for CEP-HSA (d) and CEPH-BSA (Ç) against CEP-HSA as coating agent.

used as a coating agent and standard whose binding was inhibited

by CEPH-BSA. The IC₅₀of CEPH-BSA (1.93 pmol/mL) is lower than

the IC₅₀of CEP-HSA (3.02 pmol/mL) indicating that CEPH-BSA has

a slightly higher afﬁnity than CEP-HSA for binding anti-CEP-KLH

antibody.

O

2.6. Synthesis of biotinylated CEP derivatives

OMe

O

87%

H₂O

HOAc

OMe

O

1

17

A biotinylated CEP Fm ester (15) was prepared from DOHA-Fm

and 4-amino butylbiotin (Scheme 6). Deprotection of the interme-

diate 3-(1-{4-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-

pentanoylamino]-butyl}-1H-pyrrol-2-yl)-propionic acid 9H-ﬂuo-

ren-9-ylmethyl ester 15 by treatment with DBU in THF generated

3-(1-{4-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-penta-

noylamino]-butyl}-1H-pyrrol-2-yl)-propionic acid (16).

All of the functionality in biotinylated CEP derivative 16 sur-

vived treatment with sodium hydroxide ethanol solution. This

observation suggested the feasibility of simpler synthesis for pre-

paring CEP derivatives of substrates that are stable to strong base.

Thus, we prepared 3-(1-{4-[5-(2-oxo-hexahydro-thieno[3,4-d]imi-

dazol-4-yl)-pentanoylamino]-hexyl}-1H-pyrrol-2-yl)-propionic acid

(20) by reacting 4,7-dioxo-heptanoic acid methyl ester (DOHA-

Me, 17) with biotinyl-1,6-diaminohexane (18) followed by hydro-

lysis of the methyl ester 19 with ethanolic sodium hydroxide

(Scheme 7).

O

NH

90%

O

H₂N(CH₂)₆NH₂

pyridine

OPhNO₂

S

O

H₂O

NH

O

NH(CH₂)₆NH₂

S

18

O

S

NH

17

+

18

O

75%

MeOH

N

NH(CH₂)₆

O

OR

19, R = CH₃

20, R = H

2.7. Synthesis of ethanolamine phospholipid CEP derivatives

NaOH, EtOH

84%

Phosphatidylethanolamines (PEs) are major components of cer-

tain membranes in the brain cells and in the photoreceptor cells of

Scheme 7.

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L. Lu et al. / Bioorg. Med. Chem. 17 (2009) 7548–7561

oPE) followed by deprotection of the intermediate Fm esters 21 or

23 with DBU (Scheme 8) delivered the CEP-PEs 22 and 24.

with MHC class I proteins. MS/MS analysis consistently detected

modiﬁcation on the ALKAWSVAR sequence of CEP–BSA in prepara-

tions generated with different CEP ratios, implying that this se-

quence is readily accessed and covalently altered by an aldehyde

electrophile. Figure 2 shows a ball-and-stick model providing 3D

visualization of HSA using ^PYMOLv0.99. X-ray crystallography of

HSA reveals a dimer structure with two identical units. All lysines

are shown as space-ﬁlling structures in the left unit while only the

lysines that become incorporated into CEPs are shown as space-ﬁll-

ing structures in the right unit. The lysine residue K236 in the

²³⁴AFKAWAVAR²⁴²sequence is marked. The 3D structure shows

that this lysine is located at and protrudes from the surface of

HSA. The physical environment and chemical characteristics of this

speciﬁc lysyl residue probably facilitate access by DOHA-Fm and

covalent adduction through Schiff base formation with its terminal

aldehyde group leading to pyrrole formation. In vivo, pyrrole for-

mation involves an analogous interaction between a protein and

the aldehyde group of an oxidatively truncated phospholipid

sn2-fatty acyl chain protruding like a whisker²⁸from a membrane

bilayer or the exterior of a lipoprotein particle. Based on the tryptic

peptides identiﬁed above, further studies are underway to synthe-

size MHC-bound CEP modiﬁed peptides and to test their ability to

elicit an immune response by antigen speciﬁc T-cells.

2.8. Synthesis of an active pentaﬂuorophenyl ester of a lysyl CEP

Pentaﬂuorophenyl esters of protected amino acids are widely

used in peptide synthesis. We are interested in testing the ability

of CEP modiﬁed peptides bound to major histocompatibility pro-

teins to elicit an immune response to antigen speciﬁc T-cells. Fur-

thermore, complexes of CEP modiﬁed peptides bound to constructs

called ‘dimer X’, that have major histocompatibility proteins fused

to immunoglobin Fc constant regions, can be used to ﬂuorescently

label antigen speciﬁc T-cells, and consequently enable their quan-

titation by ﬂuorescence activated cell sorting.²⁷The pentaﬂuor-

ophenyl ester 26 of a CEP-Fm modiﬁed lysine was synthesized as

a building block for construction of CEP modiﬁed peptides. Reac-

tion of DOHA-Fm with 6-amino-2-(9H-ﬂuoren-9-ylmethoxycar-

bonylamino)-hexanoic acid (Fmoc-Lys-OH) delivered 25. The

latter was then coupled with pentaﬂuorophenol using the tradi-

tional DCC, DMAP method (Scheme 9).

3. Discussion

Immunoglobins and B-cells bind divalently through their vari-

able (Fv) regions with haptens such as CEP. This can cause aggrega-

tion of proteins and/or cells displaying CEP modiﬁcations, and can

activate B-cells. Furthermore, immunoglobins possess a constant

(Fc) effector region that activates immune responses. In contrast,

monoclonal scFv antibodies are monovalent constructs that con-

tain only a single Fv binding region and no constant Fc region,

and are therefore not expected to cause protein aggregation and

deposition. Furthermore, monoclonal scFv antibodies are likely to

favor penetration of the internal limiting membrane and access

the subretinal space when injected intravitreously because of their

relatively low molecular weight.²⁹Therefore, we hypothesize that

a monoclonal anti-CEP scFv antibody may be useful as a competi-

tive inhibitor of CEP induced neovascularization. Speciﬁc human

scFv antibodies can be selected from engineered libraries of phage

that display the antibody protein on their exterior. Selection is

accomplished by using the corresponding hapten as ‘bait’ to catch

phage that produce (with the help of E. coli) and display antigen

speciﬁc scFv on their exterior. One approach can exploit the CEP

hapten described above, anchored through a biotin linker to strep-

tavidin-coated magnetic beads.

The CEP-modiﬁed tryptic peptides that we identiﬁed enabled

determination of the speciﬁc lysyl groups that have been altered

in various CEP-modiﬁed proteins. These tryptic peptides may also

provide clues to the identity of the CEP-modiﬁed peptides involved

in cellular immune responses. For example, major histocompat-

ability (MHC) class I proteins selectively bind peptides of 8–10 res-

idues that have nonpolar ‘anchor residues’ at or near their termini.

Notably, the HSA tryptic nonapeptide ²³⁴AFKAWAVAR²⁴²amino

acid sequence is very similar to the sequence ²³³ALKAWSVAR²⁴¹

in BSA and identical to the ²³⁴AFKAWAVAR²⁴²sequence in MSA

(see Experimental Procedures). This sequence incorporates nonpo-

lar amino acid residues on both ends and is appropriate for binding

O

POPE

or

76%

P

O

COO-Fm

COOH

4 +

O

R

OH

N

lysoPE

TEA, CHCl₃

O

C₁₄H₂₉

21, R = oleoyl

23, R = H

O

P

O

90%

DBU, CHCl₃

O

R

OH

N

O

C₁₄H₂₉

4. Conclusions

22, R = oleoyl

24, R = H

O

The introduction of multiple CEP modiﬁcations of lysyl residues

is readily achieved through reaction of proteins with DOHA-Fm, a

9-ﬂuorenylmethyl ester of 4,7-dioxoheptanoic acid, followed by

deprotection of intermediate Fm esters of CEPs, without causing

protein denaturation, by treatment with DBU. CEP-modiﬁed mouse

serum albumin available through this synthetic method was used

to create a mouse model of AMD.¹¹CEP–proteins and peptides pre-

pared through this new methodology have also been used as bio-

markers for clinical prognosis of AMD⁶, and for studies of their

possible role in promoting pathological angiogenesis in ‘wet’

AMD⁷, as well as mechanistic studies of their antigenicity, and

apparent stimulation of complement accumulation and lesion for-

mation in the ‘dry’ form of AMD.¹¹The medicinal and diagnostic

utility of CEP derivatives described herein are under active investi-

gation in our laboratories. We anticipate that the synthetic

methodology reported above will facilitate studies on the activa-

tion of B- and T-cells by CEP-peptides. It will allow testing of the

hypotheses that CEP derivatives represent a link between lipid oxi-

Scheme 8.

H

COOH

Fmoc

75%

N

Fmoc

N

O

DOHA-Fm

MeOH

NH₂HOAC

OFm

N

25

26

F

90%

O

F

O

C₆F₅OH, DCC

DMAP, CH₂Cl₂

N

F

HN

Fmoc

Scheme 9.

L. Lu et al. / Bioorg. Med. Chem. 17 (2009) 7548–7561

7553

Figure 2. A ball-and-stick model 3D structure for HSA. All the lysines are shown as space-ﬁlling structures in the left unit. Only lysines that become CEP modiﬁed are shown

in the right unit. The lysine (K236) in the sequence, which is common to HSA, BSA and MSA, is marked.

dation and other manifestations of angiogenesis such as tumor

growth or wound healing.

evidence by disappearance of the red-brown I₂color. Then 50 mL

THF was added to the ﬂask. After completion of the addition, more

THF (30 mL) was added. The reaction mixture was stirred for an-

other 1 h and then cooled to ꢂ78 °C followed by slow addition of

3-carbomethoxypropionyl chloride (11.5 g, 74 mmol) dissolved in

40 mL dry THF. The resulting mixture was stirred for another

40 min, then quenched with 200 mL of a saturated aqueous solu-

tion of NH₄Cl, and extracted with EtOAc (4 ꢁ 200 mL). The com-

bined organic phase was washed with brine, dried with MgSO₄,

and evaporated to obtain the crude product. The crude compound

was puriﬁed by distillation to give 13.4 g (78%) of pure 1b. ¹H NMR

(CDCl₃, 400 MHz) d 4.57 (t, J = 5.2 Hz, 1H), 4.05–4.10 (m, 2H), 3.71–

3.78 (m, 2H), 3.67 (s, 3H), 2.74 (t, J = 6.6 Hz, 2H), 2.59 (t, J = 7.0 Hz,

4H), 2.00–2.10 (m, 1H), 1.89–1.92 (m, 2H), 1.30–1.36 (m, 1H). ¹³C

NMR (CDCl₃, 400 MHz) d 208.51 (CO), 173.49 (COO), 101.03 (CH),

67.05 (CH₂), 52.01 (CH₃), 37.32 (CH₂), 36.82 (CH₂), 29.16 (CH₂),

27.96 (CH₂), 25.91(CH₂). HRMS (FAB) (m/z) calcd for C₁₁H₁₉O₅

(MH⁺) 231.1188, found 231.2631.

5. Experimental section

5.1. 6-[1,3]Dioxolan-2-yl-4-oxo-hexanoic acid methyl ester (1a)

A solution of 2-(2-bromoethyl)-1,3-dioxolane (1 g, 5.5 mmol) in

anhydrous THF (2 mL) was added dropwise to a ﬂame-dried

100 mL ﬂask with Mg turnings (150 mg, 6.25 mmol) and 4 mL

THF and a small piece of I₂under argon at room temperature to ini-

tiate the reaction. After adding a few drops, the reaction started as

evidence by disappearance of the red-brown I₂color. Then 5 mL

THF was added to the ﬂask. After completion of the addition, more

THF (15 mL) was added. The reaction mixture was stirred for an-

other 1 h and then cooled to ꢂ78 °C followed by slow addition of

3-carbomethoxypropionyl chloride (710 mg, 4.7 mmol) dissolved

in 2.5 mL dry THF. The resulting mixture was stirred for another

40 min, then quenched with 30 mL of a saturated aqueous solution

of NH₄Cl, and extracted with EtOAc (4 ꢁ 15 mL). The combined or-

ganic phase was washed with brine, dried with MgSO₄, and evap-

orated to obtain the crude product. The crude compound was

puriﬁed by silica gel chromatography (30% ethyl acetate in hexane,

TLC: R_f= 0.3) to give 714 mg (60%) of pure 1. ¹H NMR (CDCl₃,

200 MHz) d 4.85 (t, J = 4.3 Hz, 1H), 3.7–3.90 (4H), 3.62 (s, 3H),

2.67 (m, 2H), 2.56 (t, J = 7.48 Hz, 4H), 1.93 (dt, J = 4.3, 7.48 Hz,

2H). ¹³C NMR (CDCl₃, 50 MHz, APT) d 207.81 (+) (CO), 173.09 (+)

(COO), 103.06 (ꢂ) (CH), 64.83 (+) (CH₂), 51.61 (ꢂ) (CH₃), 36.87

(+) (CH₂), 36.30 (+) (CH₂), 27.62 (+) (CH₂), 27.41 (+) (CH₂). HRMS

(FAB) (m/z) calcd for C₁₀H₁₇O₅(MH⁺) 217.1076, found 217.1081.

5.3. 6-[1,3]Dioxolan-2-yl-4-oxo-hexanoic acid (2a)

Ester 1a (390 mg, 1.8 mmol) in 10 mL of H₂O/MeOH/THF (2:5:3,

v/v/v) was stirred for 3 h with NaOH (367 mg, 9.2 mmol) at room

temperature. The reaction mixture was then acidiﬁed with 3 N

HCl to pH 3.0 and extracted with EtOAc (3 ꢁ 15 mL). The combined

organic phase was washed with brine, dried with MgSO₄, and

evaporated to give acid acetal 2a (360 mg, 90%), bp 129 °C/

0.25 mm Hg. ¹H NMR (CDCl₃, 200 MHz), d 4.85 (t, J = 4.3 Hz, 1H),

3.7–3.90 (4H), 2.5–2.64 (6H), 1.93 (dt, J = 4.3, 7.48 Hz, 2H). ¹³C

NMR (CDCl₃, 100 MHz,) d 207.81 (CO), 178.03 (COOH), 103.17

(CH), 64.97 (CH₂), 36.76 (CH₂), 36.36 (CH₂), 27.71 (CH₂), 27.51

(CH₂). HRMS (FAB) (m/z) calcd for C₉H₁₅O₅(MH⁺) 203.0919, found

203.0917.

5.2. 6-[1,3]Dioxan-2-yl-4-oxo-hexanoic acid methyl ester (1b)

A solution of 2-(2-bromoethyl)-1,3-dioxane (16 g, 84 mmol) in

anhydrous THF (32 mL) was added dropwise to a ﬂame-dried

1000 mL ﬂask with Mg turnings (2.4 g, 100 mmol) and 64 mL

THF and a small piece of I₂under argon at room temperature to ini-

tiate the reaction. After adding a few drops, the reaction started as

5.4. 6-[1,3]Dioxan-2-yl-4-oxo-hexanoic acid (2b)

Ester 1b (3.4 g, 14.8 mmol) in 90 mL of H₂O/MeOH/THF (2:5:3,

v/v/v) was stirred for 3 h with NaOH (3.1 g, 77.5 mmol) at room

7554

L. Lu et al. / Bioorg. Med. Chem. 17 (2009) 7548–7561

temperature. The reaction mixture was then acidiﬁed with 3 N HCl

to pH 3.0 and extracted with EtOAc (3 ꢁ 80 mL). The combined or-

ganic phase was washed with brine, dried with MgSO₄, and evap-

orated to give the acid acetal 2b (2.9 g, 92%) that was crystallized

from CH₂Cl₂/hexane 1:4 to deliver white crystals, mp 98–9 °C. ¹H

NMR (CDCl₃, 400 MHz), d 4.57 (t, J = 5.2 Hz, 1H), 4.05–4.10 (m,

2H), 3.71–3.78 (m, 2H), 2.74 (t, J = 6.6 Hz, 2H), 2.55–2.67 (m, 4H),

2.00–2.10 (m, 1H), 1.89–1.92 (m, 2H), 1.30–1.36 (m, 1H). ¹³C

NMR (CDCl₃, 400 MHz) d 208.44 (CO), 178.79 (COO), 101.01 (CH),

67.05 (CH₂), 37.05 (CH₂), 36.75 (CH₂), 29.16 (CH₂), 28.01(CH₂),

25.91(CH₂). HRMS (FAB) (m/z) calcd for C₁₀H₁₅O₅(MꢂH)

215.1006, found 215.6009.

5.7. 4,7-Dioxo-heptanoic acid 9H-ﬂuoren-9-ylmethyl ester

(DOHA-Fm, 4). Method A

Ester 3a (94 mg, 0.25 mmol) in 10 mL of AcOH/H₂O (3:1, v/v)

was stirred at 50 °C for 5 h. TLC (100% CHCl₃): R_f= 0.5 showed

the completion of the reaction. The solvent was removed by rotary

evaporation. Flash chromatography of the residue (25% ethyl ace-

tate in hexane) gave 4 (73 mg, 88%). ¹H NMR (CDCl₃, 400 MHz), d

9.78 (s, 1H), 7.77 (d, J = 6.74 Hz, 2H), 7.60 (d, J = 7.7 Hz, 2H), 7.42

(dd, J = 6.74, 7.7 Hz, 2H), 7.30 (dd, J = 6.74, 7.7 Hz, 2H), 4.39(d,

J = 7.38 Hz, 2H), 4.20 (t, J = 7.38 Hz, 1H), 2.67–2.79 (8H). ¹³C NMR

(CDCl₃, 100 MHz), d 206.56 (CO), 200.27 (CHO), 172.53 (COO),

143.70 (C), 141.23 (C), 127.73 (CH), 127.07 (CH), 124.98 (CH),

119.96 (CH), 66.47 (CH₂), 46.70 (CH), 37.42 (CH₂), 36.87 (CH₂),

34.53 (CH₂), 27.93 (CH₂). HRMS (FAB) (m/z) calcd for C₂₁H₂₀O₄

(M⁺) 336.1362, found 336.1361. Method B: Ester 3b (94 mg,

0.25 mmol) in 10 mL of AcOH/H₂O (3:1, v/v) was stirred at 50 °C

for 5 h. TLC (100% CHCl₃): R_f= 0.5 showed the completion of the

reaction. The solvent was removed by rotary evaporation. Flash

chromatography of the residue (25% ethyl acetate in hexane) gave

4 (73 mg, 88%).

5.5. 6-[1,3]Dioxolan-2-yl-4-oxo-hexanoic acid 9H-ﬂuoren-9-

ylmethyl ester (3a)

(9H-Fluoren-9-yl)-methanol (373 mg, 1.9 mmol) in 3 mL dry

CH₂Cl₂was slowly added to the solution of dicyclohexylcarbodiim-

ide (DCC, 295 mg, 1.425 mmol), dimethlamino pyridine (DMAP,

58 mg, 0.475 mmol) and the acid 2a (96 mg, 0.475 mmol) in 5 mL

dry CH₂Cl₂. The resulting mixture was stirred for 72 h at room tem-

perature. The solvent was removed. Flash chromatography of the

residue (30% ethyl acetate in hexane, TLC: R_f= 0.3) gave the ester

3a (153 mg, 95%) as a colorless oil. ¹H NMR (CDCl₃, 200 MHz) d

7.77 (d, J = 6.74 Hz, 2H), 7.60 (d, J = 7.7 Hz, 2H), 7.42 (dd, J = 6.74,

7.7 Hz, 2H), 7.29 (dd, J = 6.74, 7.7 Hz, 2H), 4.90 (t, J = 4.3 Hz, 1H),

4.40 (d, J = 7.38 Hz, 2H), 4.21 (t, J = 7.38 Hz, 1H), 3.80–3.96 (4H),

2.62–2.73 (4H), 2.56 (t, J = 7.3 Hz, 2H), 1.98 (td, J = 7.3, 4.3 Hz,

2H). ¹³C NMR (CDCl₃, 50 MHz, APT), d 207.76 (+) (CO), 172.62 (+)

(COO), 143.69 (+) (C), 141.19 (+) (C), 127.69 (ꢂ) (CH), 127.04 (ꢂ)

(CH), 125.00 (ꢂ) (CH), 119.92 (ꢂ) (CH), 103.12 (ꢂ) (CH), 66.45 (+)

(CH₂), 64.90 (+) (CH₂), 46.67 (ꢂ) (CH), 36.89 (+) (CH₂), 36.35 (+)

(CH₂), 27.90 (+) (CH₂), 27.48 (+) (CH₂). HRMS (FAB) (m/z) calcd

for C₂₃H₂₄O₅(M⁺) 380.1624, found 380.1614; calcd for C₂₃H₂₅O₅

(MH⁺) 381.1702, found 381.1711.

5.8. 2-(2-Acetylamino-acetylamino)-6-{2-[2-(9H-ﬂuoren-9-

ylmethoxycarbonyl)-ethyl]-pyrrol-1-yl}-hexanoic acid methyl

ester (5)

Methyl 6-amino-2-((2-acetylamino)acetyl)amino) hexanoate

(Ac-Gly-Lys-OMe, 25.6 mg, 0.08 mmol) in 1 mL methanol was

added dropwise to DOHAFm (27 mg, 0.08 mmol) in 1.5 mL metha-

nol. The solution was stirred for 9 h at room temperature under ar-

gon. The solvent was removed by rotary evaporation. TLC (4%

methanol in chloroform): R_f= 0.34. The crude compound was puri-

ﬁed by silica gel chromatography (4% methanol in chloroform) to

give 35.8 mg (80%) of pure 5. ¹H NMR (CDCl₃, 400 MHz), d 7.77 (d,

J = 7.6 Hz, 2H), 7.60 (d, J = 7.6 Hz, 2H), 7.36–7.32 (dd, J = 7.6, 7.2 Hz,

2H), 7.27 (dd, J = 7.6, 7.2 Hz, 2H), 6.60 (dd, J = 2.4, 1.6 Hz, 1H), 6.54

(m, 1H), 6.28 (s, 1H), 6.09 (dd, J = 3.6, 2.4 Hz, 1H), 5.90 (m, 1H),

4.56–4.61 (m, 1H), 4.40 (d, J = 7.2 Hz, 2H), 4.21 (t, J = 7.2 Hz, 1H),

3.91 (dABq, J = 5.2, 14 Hz, 2H), 3.82 (t, J = 7.2 Hz, 2H), 3.76 (s, 3H),

2.65–2.86 (4H), 2.01 (s, 3H), 1.86 (m, 2H), 1.66 (m, 2H), 1.36 (m,

2H). ¹³C NMR (CDCl₃, 100 MHz), d 172.98 (CO), 172.33 (CO),

170.58 (CO), 168.66 (CO), 143.74 (C), 141.31 (C), 130.73(C), 127.80

(CH), 127.12 (CH), 125.01 (CH), 120.37 (CH), 120.04 (CH), 106.99

(CH), 105.26 (CH), 66.47 (CH₂), 52.53 (CH), 51.86 (CH₃O), 46.78

(CH), 46.09 (CH₂), 43.23 (CH₂), 33.22 (CH₂), 31.89 (CH₂), 30.67

(CH₂), 22.92 (CH₂), 22.40 (CH₂), 21.41 (CH₃). HRMS (FAB) (m/z) calcd

5.6. 6-[1,3]Dioxan-2-yl-4-oxo-hexanoic acid 9H-ﬂuoren-9-

ylmethyl ester (3b)

In a 25 mL ﬂame-dried round-bottomed ﬂask equipped with a

magnetic stirring bar, acid 2b (331 mg, 1.53 mmol) was dissolved

in 4 mL of dry dichloromethane and cooled to ꢂ20 °C. A commer-

cially available solution of dicyclohexylcarbodiimide (Aldrich, Mil-

waukee, 1.0 M in dichloromethane, 2 mL, 2 mmol) was added

dropwise and the solution stirred at temperature for 20 min during

which time a white precipitate formed. A solution of 9-ﬂuorene-

methanol (364 mg, 1.85 mmol) and N,N-dimethylaminopyridine

(36 mg, 0.29 mmol) in dry dichloromethane (3 mL) in a ﬂame-dried

15 mL round-bottomed ﬂask equipped with a magnetic stirring bar

was added dropwise via cannula to the reaction mixture at ꢂ10 °C

and left stirring 18.5 h to slowly warm to room temperature. The

solution was concentrated via rotary evaporation, dissolved in ethyl

acetate, allowed to sit for 2 h at ꢂ20 °C, and then ﬁltered. The ﬁltrate

was concentrated and then puriﬁed by ﬂash chromatography using

20% ethyl acetate/hexanes to give 3b (512 mg, 85%) as a white crys-

talline solid, mp 67.5–69.5 °C, (R_f= 0.10 in 25% ethyl acetate/hex-

anes). ¹H NMR (400 MHz, CDCl₃) d 7.77 (dt, J = 0.9, 7.6 Hz, 5H),

7.60 (ddd, J = 0.8, 1.9, 7.5 Hz, 2H), 7.41 (tdd, J = 0.6, 1.2, 7.5 Hz, 2H),

7.32 (td, J = 1.2, 7.5 Hz, 2H), 4.56 (t, J = 4.9 Hz, 1H), 4.38 (d,

J = 7.2 Hz, 2H), 4.22 (t, J = 7.2 Hz, 1H), 4.10–4.04 (m, 2H), 3.78–3.69

(m, 2H), 2.77–2.64 (4H), 2.57 (t, J = 7.3 Hz, 2H), 2.12–1.97 (m, 1H),

1.90 (td, J = 4.9, 7.3 Hz, 2H), 1.36–1.28 (ddt, J = 1.2, 2.5, 13.4, 1H).

¹³C NMR (100 MHz, CDCl₃) d 208.14, 172.70, 143.74, 141.24,

127.73, 127.08, 125.04, 119.97, 100.77, 77.32, 77.00, 76.68, 66.80,

66.51, 46.71, 37.02, 36.56, 28.93, 27.95, 25.70. HRMS (FAB) (m/z)

calcd for C₂₄H₂₇O₅(MH⁺) 395.1814, found 395.1620.

+

þ

for C₃₂H₃₈N₃O₆(MH ) 560.2755, found 560.2747.

5.9. 2-(2-Acetylamino-acetylamino)-6-[2-(2-carboxy-ethyl)-

pyrrol-1-yl]-hexanoic acid methyl ester (CEP-dipep, 6)

DBU (75 lL) was added to 5 (27 mg, 0.047 mmol) in 2.5 mL THF.

The system was stirred for 6 h under argon. After the removal of

solvent, the crude compound was puriﬁed by silica gel chromatog-

raphy (6% methanol in chloroform) to give 21 mg (86%) of CEP-di-

pep. TLC (10% methanol in chloroform): R_f= 0.3; ¹H NMR (CDCl₃,

400 MHz), d 7.18 (d, J = 8 Hz, 1H), 6.81 (m, 1H), 6.53 (m, 1H),

6.03 (dd, J = 3.2, 3.2 Hz, 1H), 5.87 (m, 1H), 4.53 (dt, J = 1H), 3.95

(dABq, J = 5.2, 13.6 Hz, 2H), 3.81 (t, J = 6.8 Hz, 2H), 3.70 (s, 3H),

2.65–2.86 (4H), 2.01 (s, 3H), 1.8–1.62 (4H), 1.36 (m, 2H). ¹³C

NMR (CDCl₃, 100 MHz), d 176.01 (COOH), 172.40 (COO), 171.35

(CO), 169.28 (CO), 131.40 (C), 119.92 (CH), 107.25 (CH), 105.00

(CH), 52.50 (CH), 52.00 (CH₃O), 45.66 (CH₂), 43.19 (CH₂), 32.98

(CH₂), 31.44 (CH₂), 30.90 (CH₂), 22.94 (CH₂), 22.07 (CH₂), 21.37

(CH₃). HRMS (FAB) (m/z) calcd for C₁₈H₂₈N₃O₆(MH⁺) 382.1978,

þ

found 382.1986.

L. Lu et al. / Bioorg. Med. Chem. 17 (2009) 7548–7561

7555

5.10. Tryptic digestion

unambiguous modiﬁcation on the ALKAWSVAR sequence of CEP–

BSA and the AFKAWAVAR sequence of MSA.

CEP modiﬁed proteins were dissolved in NH₄HCO₃buffer (8 M

urea/2 M NH₄HCO₃) to make a ﬁnal concentration 2–3 pmol/

Trypsin solution (0.1 g/ L) was made by suspending sequencing

grade modiﬁed porcine trypsin (20 g) (Promega, Cat. V511A) in

trypsin resuspension buffer (Promega, Cat. V511A) (200 L). This

trypsin solution was then added to protein solution in a ratio of

1:50 (w/w) enzyme/protein. After incubation at 37 °C for 24 h, the

solution was centrifuged at 3000 rpm for 5 min. The upper layer

l

L.

5.12. Carboxyethylpyrrole-modiﬁed mouse serum albumin

(CEP-MSA, 8b)

l

A solution of DOHAFm (18.5 mg, 0.055 mmol) in 8 mL DMF was

added slowly to the solution of 100 mg mouse serum albumin in

18 mL 10 mM PBS (pH 7.4). The mixture was stirred under argon

for 4 days. DBU (360 lL) was added to the system and stirred over-

(10

l

L) was subjected to LC–MS analysis on a CapLC system (Micro-

night under argon followed by two successive 24 h dialyses (Mr cut-

off 14,000) against 1 L 20% DMF in 10 mM PBS (pH 7.4) and two

additional dialyses (24 h each) against 1 L 10 mM PBS (pH 7.4) at

4 °C. The ﬁnal protein concentration (2.43 mg/mL) was determined

by the Pierce bicinchoninic acid (BCA) protein assay. The pyrrole

mass, Beverly, MA) and a quadrupole time-of-ﬂight mass spectrom-

eter (QTOF2, Micromass) using MassLynx 3.5 software, the Mascot

search engine (Matrix Science) and the Swiss-protein and National

Center for Biotechnology information protein sequence databases.³⁰

concentration (210 lM) was determined by Ehrlich assay. LC–MS/

5.11. Carboxyethylpyrrole-modiﬁed human serum albumin

(CEP-HSA, 8a)

MS revealed 15 CEP modiﬁcations at m/z 650.3296, 872.9611,

571.3492, 849.4302, 612.8407, 769.4069, 694.6888, 1047.0802,

857.4633 on lysyl residues of the MSA peptides ²⁵EAHKSEI AHR³⁴

,

A solution of DOHA-Fm (2 mg, 0.006 mmol) in 750

added to 1.5 mL 0.08 mM solution of HSA in PBS. The mixture was

stirred under argon for 4 days. DBU (200 L) was added to the sys-

lL DMF was

²⁰⁶LDGVKEKALVSSVR²¹⁹(2 CEPs), ²³⁴AFKAWAVAR²⁴²

ADFAEITKLATDLTK^{264 258}LATDLTKVNK^{267 373}LAKKYEATLEK³⁸³

(2 CEPs), ⁴³⁵YTQKAPQVSTPTLVEAAR^{452 544}EKQIKKQTALAELVK⁵⁵⁸

,

²⁴³LSQTFPN-

,

l

,

tem and stirred overnight under argon followed by two successive

12 h dialyses (Mr cutoff 14,000) against 500 mL 20% DMF in 10 mM

PBS (pH 7.4) and two additional dialyses (12 h each) against

500 mL 10 mM PBS (pH 7.4) at 4 °C. The ﬁnal protein concentration

(1.80 mg/mL) was determined by the Pierce bicinchoninic acid

(3 CEPs), ⁵⁵⁰QTALAELVKHKPKATAEQLK⁵⁶⁹(3 CEPs), respectively.

The sequence coverage was 35%. No CEPFm modiﬁcations were

found after deprotection. The tandem MS spectrum of AFKAWAVAR

was shown in Figure 4.

(BCA) protein assay. The pyrrole concentration (187.14

determined by Ehrlich assay. LC–MS/MS revealed six CEP modiﬁca-

l

M) was

5.13. Carboxyethylpyrrole-modiﬁed bovine serum albumin

(CEP–BSA, 8c)

tions at m/z 589.266, 571.260, 674.762, 881.401, 687.784, 636.252

on lysyl residues of the HSA peptides ¹⁶¹KYLYEIAR¹⁶⁸

,

²³⁴AFKAWA-

Eight different samples with CEP:BSA ratios from 0.6 to 10.0 were

prepared. A solutioncontainingvariousamounts (see Table 1) of DO-

HAFm in 8 mL DMF was added dropwise over 30 min to a solution of

100 mg BSA in 18 mL 10 mM PBS (pH 7.4). The mixture was stirred

under argon for 4 days. Various amounts of DBU (see Table 1) were

added and the resulting mixture was stirred overnight under argon

followed by two successive 24 h dialyses (Mr cutoff 14,000) against

2 L of 20% DMF in 10 mM PBS (pH 7.4) and two additional dialyses

(24 h each) against 2 L of 10 mM PBS (pH 7.4) at 4 °C. The ﬁnal pro-

tein concentration was determined by the Pierce bicinchoninic acid

VAR²⁴²

,

³⁵FKDLGEENFK⁴⁴

, ,

⁴³⁸KVPQVSTPTLVEVS-R^{452 247}FPKAE-

FAEVSK²⁵⁷

,

²⁵DAHKSEVAHR³⁴, respectively. No CEPFm modiﬁ-

cations were found after deprotection. Figure 3 shows the tandem

MS spectrum of the doubly charged ion m/z 571.260 from a MS scan

of tryptic digested CEP-HSA. The spectrum shows a series of frag-

ment ions (b ions and y ions) sufﬁcient to identify a CEP modiﬁcation

on Lys²³⁶from HSA residues ²³⁴AFKAWAVAR²⁴². This particular

sequence is very similar with the sequence ²³³ALKAWSVAR²⁴¹in

BSA and is exactly the same as in MSA. MS/MS analysis revealed

Figure 3. Tandem MS characterization of the doubly charged ion m/z 571.260 from a MS scan of tryptic digested CEP modiﬁed HSA shows series of fragment ions sufﬁcient to

unambiguously identify a CEP modiﬁcation on the lysyl residue (K236). Asterisks denote fragment ions with a modiﬁed lysyl residue.

7556

L. Lu et al. / Bioorg. Med. Chem. 17 (2009) 7548–7561

Figure 4. Tandem MS characterization of the doubly charged ion m/z 571.3492 from a MS scan of tryptic digested CEP modiﬁed MSA shows series of fragment ions sufﬁcient

to unambiguously identify a CEP modiﬁcation on the lysyl residue (K236). Asterisks denote fragment ions with a modiﬁed lysyl residue.

Table 1

7.4 PBS (10 mM, 5 mL). The mixture was stirred under argon for

4 days. DBU (140 lL) was added and the resulting mixture was

Generation of various CEP:BSA ratios

stirred overnight under argon followed by two successive 24 h

dialyses (Mr cutoff 3500) against 500 mL 20% DMF in 10 mM pH

7.4 PBS and two more dialyses (24 h each) against 500 mL

10 mM pH 7.4 PBS at 4 °C. The ﬁnal protein concentration

(0.65 mg/mL) was determined by Bio-Rad protein assay. The pyr-

Sample

DOHA-Fm

(mg)

DBU

L)

[BSA]

(mg/mL)

[Pyrrole] (

l

M)

CEP:BSA

(l

(mean SD)^a

1

2

3

4

5

6

7

8

36

18

9

720

360

180

90

3.37

3.14

3.13

2.97

2.80

2.96

2.78

2.99

496

348

248

167

91.1

66.9

43.6

26.1

10.1 1.0

7.0 1.1

5.1 1.0

3.7 0.8

2.2 0.1

1.5 0.3

1.0 0.6

0.6 0.03

5

role concentration (91 lM) was determined by Ehrlich assay. LC–

2.5

1.25

0.6

0.3

45

MS/MS revealed CEP modiﬁcations at m/z 671.2667, 604.9635,

815.2087, 557.4288, 785.6061, 899.6666, on lysyl residue of the

myoglobin peptides ³²LFTGHPETLEKFDKFKHLK⁵⁰(3 CEP modiﬁca-

22.5

10.8

5.4

a

tions), ⁴⁸HLKTEAEMK⁵⁶

,

⁶³KHGTVVLTALGGILK⁷⁷

,

⁷⁹KGHHEAELKPLA

,

¹⁴⁰NDIAAKYKELG

The pyrrole concentration for each sample was measured three times over 1 h

and the CEP:BSA ratio mean SD was calculated.

QSHATK⁹⁶

,

¹¹⁹HPGDFGADAQGAMTKALELFR¹³⁹

FQG¹⁵³(2 CEP modiﬁcations). The peptide sequence coverage

achieved 91%. No CEPFm modiﬁcations were found after

deprotection.

(BCA) protein assay, while the pyrrole concentration was measured

by Ehrlich assay. The ﬁnal protein concentrations, pyrrole concen-

trations, and CEP:BSA ratios are shown in Table 1.

5.16. Carboxyethylpyrrole-modiﬁed GPDH (CEP-GPDH)

5.14. Carboxyethylpyrrole-modiﬁed chicken egg albumin (CEP-

CEO)

GPDH (30 mg) was added in 10 mL of 10 mM PBS (pH 7.4). After

vortexing for 5 min and centrifugation (4 °C) at 3000 rpm for

10 min (GPDH does not dissolve well in PBS), 4 mL of clear solution

was taken and DOHAFm (5.8 mg, 0.017 mmol) in DMF (4 mL) was

added slowly to it. The mixture was stirred under argon for 4 days.

A solution of DOHAFm (18 mg, 0.054 mmol) in DMF (3.2 mL)

was added slowly to the solution of CEO (76 mg) in pH 7.4 PBS

(10 mM, 7 mL). The mixture was stirred under argon for 4 days.

DBU (120 lL) was then added and the resulting mixture was stirred

DBU (140 lL) was added and the resulting mixture was stirred

overnight under argon followed by two successive 24 h dialyses (Mr

cutoff 3500) against 500 mL 20% DMF in 10 mM pH 7.4 PBS and two

more dialyses (24 h each) against 500 mL 10 mM pH 7.4 PBS at 4 °C.

The ﬁnal protein concentration (0.29 mg/mL) was determined by

overnight under argon followed by two successive 24 h dialyses

(Mr cutoff 3500) against 1 L 20% DMF in 10 mM pH 7.4 PBS and

two more dialyses (24 h each) against 1 L 10 mM pH 7.4 PBS at

4 °C. The ﬁnal protein concentration (2.94 mg/mL) was determined

by the Pierce bicinchoninic acid (BCA) protein assay. The pyrrole

the Bio-Rad protein assay. The pyrrole concentration (65 lM) was

determined by Ehrlich assay. LC–MS/MS revealed six CEP modiﬁca-

tions at m/z 655.3343, 669.3289, 923.9847, 639.3413, 740.8362 on

lysyl residues of the GPDH peptides ¹⁰⁵AGAHLKGGAKR¹¹⁵

concentration (93 lM) was determined by Ehrlich assay. LC–MS/

MS revealed eight CEP modiﬁcations at m/z 540.2886, 801.7021,

839.3332, 808.0259, 610.2589, 630.8334, 459.1887 on lysyl resi-

(2 CEP modiﬁcations), ¹⁸⁴TVDGPSGKLWR¹⁹⁴

,

¹⁹⁸GAAQNIIPASTGAA-

dues of the CEO peptides ⁵¹TQINKVVR^{58 85}DILNQITKPNDVYSFS-

,

KAVGK^{216 258}VVKQASEGPLK^{268 321}VVDLMVHMASKE³³², respec-

,

LASR¹⁰⁴

, , ,

¹⁸⁷AFKDEDTQAMPFR^{199 200}VTEQESKPVQMMYQIGLFR²¹⁸

²¹⁹VASMASEKMK²²⁸

,

²⁷⁷KIKVYLPR²⁸⁴(2 CEP modiﬁcations), ²⁸⁵MK

tively. The peptide sequence coverage achieved 45%. No CEPFm

modiﬁcations were found after deprotection.

MEEK²⁹⁰, respectively. The sequence coverage was 46%. No CEPFm

modiﬁcations were found after deprotection.

5.17. 6-{2-[2-(9H-Fluoren-9-ylmethoxycarbonyl)-ethyl]-pyrrol-

1-yl}-hexanoic acid (9)

5.15. Carboxyethylpyrrole-modiﬁed myoglobin (CEP-

myoglobin)

6-Aminocaproic acid (10.8 mg, 0.082 mmol) in 400

was slowly added to DOHA-Fm (21 mg, 0.0625 mmol) in 600

methanol. The solution became cloudy with the addition. The heter-

lL water

lL

A solution of DOHAFm (12 mg, 0.036 mmol) in DMF (3.6 mL)

was added slowly to the solution of myoglobin (32.38 mg) in pH

L. Lu et al. / Bioorg. Med. Chem. 17 (2009) 7548–7561

7557

ogeneous system was stirred for 48 h under argon at room temper-

5.20. 6-(2-Carboxyethyl-1-pyrrolyl)-hexanoyl MSA amide

ature and became homogenous. The solution was extracted with

CH₂Cl₂, washed with brine, dried with Na₂SO₄and evaporated, the

yellowish residue was loaded to the silica gel in a ﬁlter with chloro-

form and washed with 15 mL chloroform, 15 mL 10% ethyl acetate in

hexane, 15 mL 20% ethyl acetate in hexane and 60 mL 50% ethyl ace-

tate in hexane successively. The 50% ethyl acetate/hexane washed

fractions were collected and dried to give 21.8 mg (81%) acid 9.

TLC (ethyl acetate/hexane, 2:3 v/v): R_f= 0.22. ¹H NMR (CDCl₃,

400 MHz), d 7.77 (d, J = 6.74 Hz, 2H), 7.58 (d, J = 7.7 Hz, 2H), 7.42

(dd, J = 7.7, 7.2 Hz, 2H), 7.30 (dd, J = 6.74, 7.2 Hz, 2H), 6.58 (dd,

J = 3.2, 3.6 Hz, 1H), 6.06 (dd, J = 3.2, 3.2 Hz, 1H), 5.87 (m, 1H), 4.42

(d, J = 7.2 Hz, 2H), 4.22 (t, J = 7.2 Hz, 1H), 3.79 (t, J = 7.2 Hz, 2H),

2.72–2.84 (m, 4H). 2.34 (t, J = 7.2 Hz, 2H), 1.75–1.34 (6H). ¹³C NMR

(CDCl₃, 100 MHz), d 177.40 (COOH), 172.90 (COO), 143.73 (C),

141.20 (C), 130.66 (C), 127.78 (CH), 127.10 (CH), 125.00 (CH),

120.34 (CH), 120.04 (CH), 106.88 (CH), 105.20 (CH), 66.40 (CH₂),

46.79 (CH₂), 46.28 (CH), 33.45 (CH₂), 33.20 (CH₂), 31.01 (CH₂),

26.23 (CH₂), 24.26 (CH₂), 21.44 (CH₂). HRMS (FAB) (m/z) calcd for

C₂₇H₃₀NO₄(MH⁺) 432.2175, found 432.2190.

(CEPH-MSA)

A solution of CEPFmSu (630

MSA (2 mg/mL, 1 mL) in PBS (10 mM, pH 7.4). The cloudy solution

became clear with overnight stirring. After 2 days, DBU (25 L) was

added and the resulting mixture was stirred overnight under argon

followed by two successive 12 h dialyses (Mr cutoff 3500) against

500 mL 20% DMF in PBS (10 mM, pH 7.4) and two more dialyses

(12 h each) against 500 mL PBS (10 mM, pH 7.4) at 4 °C. The ﬁnal

protein concentration (2.06 mg/mL) was determined by modiﬁed

lg) in DMF (150

l

L) was added in

l

Lowry protein assay. The pyrrole concentration (69.5 lM) was

determined by Ehrlich assay. LC–MS/MS revealed four CEPH mod-

iﬁcations at m/z 512.2829, 627.8604, 608.8177, 732.3707 on lysyl

residues of the MSA peptides ²⁰⁶LDGVKEK^{212 234}AFKAWAVAR²⁴²

, ,

³⁷⁶KYEATLEK^{383 435}YTQKAPQVSTPTLVEAAR⁴⁵²

, , respectively. No

CEPHFm modiﬁcations were found after deprotection.

5.21. 6-(2-Carboxyethyl-1-pyrrolyl)-hexanoyl CEO amide

(CEPH-CEO)

A solution of CEPFmSu (1.0 mg) in DMF (150 lL) was added in

CEO (4 mg/mL, 1 mL) in PBS (10 mM, pH 7.4). After the coupling

underwent for 2 days, the solution was still not clear until the

5.18. 6-{2-[2-(9H-Fluoren-9-ylmethoxycarbonyl)-ethyl]-pyrrol-

1-yl}-hexanoic acid 2,5-dioxo-pyrrolidin-1-yl ester (CEPFmSu, 10)

addition of DBU (25 lL). The system was stirred overnight under

Acid 9 (15 mg, 0.035 mmol) and N-hydroxysuccinimide (4.5 mg,

0.039 mmol), DCC (7.5 mg, 0.036 mmol) were dissolved in dry

CH₂Cl₂(7.5 mL) under Argon. The clear solution became cloudy

after 15 min. The reaction mixture was stirred for 3.5 h. Solvent

was removed by evaporation. The crude product was puriﬁed by sil-

ica gel chromatography with ethyl acetate/hexane (2:3, v/v) to de-

liver 16.6 mg (90%) active ester 10. TLC (ethyl acetate/hexane, 2:3):

R_f= 0.25. ¹H NMR (CDCl₃, 400 MHz), d 7.77 (d, J = 6.74 Hz, 2H), 7.58

(d, J = 7.7 Hz, 2H), 7.42 (dd, J = 7.7, 7.2 Hz, 2H), 7.30 (dd, J = 6.74,

7.2 Hz, 2H), 6.58 (dd, J = 3.2, 3.6 Hz, 1H), 6.06 (dd, J = 3.2, 3.2 Hz,

1H), 5.87 (m, 1H), 4.42 (d, J = 7.2 Hz, 2H), 4.22 (t, J = 7.2 Hz, 1H),

3.79 (t, J = 7.2 Hz, 2H), 2.72–2.86 (m, 8H), 2.60 (t, J = 7.2 Hz, 2H),

1.81–1.70 (m, 4H), 1.48–1.40 (m, 2H). ¹³C NMR (CDCl₃100 MHz),

d 172.79 (COO), 169.10 (COO), 168.39 (CO), 143.75 (C), 141.29 (C),

130.69 (C), 127.77 (CH), 127.10 (CH), 125.01 (CH), 120.34 (CH),

120.01 (CH), 106.92 (CH), 105.24 (CH), 66.36 (CH₂), 46.79 (CH₂),

46.21 (CH), 33.31 (CH₂), 30.86 (CH₂), 30.72 (CH₂), 25.87 (CH₂),

25.55 (CH₂), 24.25 (CH₂), 21.44 (CH₂). HRMS (FAB) (m/z) calcd for

C₃₁H₃₃N₂O₆(MH⁺) 529.2338, found 529.2340.

argon followed by two successive 12 h dialyses (Mr cutoff

14,000) against 500 mL 20% DMF in PBS (10 mM, pH 7.4) and

two more dialyses (12 h each) against 500 mL PBS (10 mM, pH

7.4) at 4 °C. The ﬁnal protein concentration (3.46 mg/mL) was

determined by modiﬁed Lowry protein assay. The pyrrole concen-

tration (85.4 lM) was determined by Ehrlich assay. LC–MS/MS re-

vealed a CEPH modiﬁcation at m/z 596.8715 on a lysyl residue of

the CEO peptide ⁵¹TQINKVVR⁵⁸. No CEPHFm modiﬁcations were

found after deprotection.

5.22. 6-(2-Carboxyethyl-1-pyrrolyl)-hexanoyl GDPH amide

(CEPH-GPDH)

GPDH (10 mg) was added in PBS (10 mM, 10 mL). After vortexing

for 5 min and centrifugation (4 °C) at 3000 rpm for 10 min, the upper

clear solution (2 ml) was taken and CEPFmSu (770

(300 L) was added. The cloudy solution became clear after 2 days.

Then DBU (25 L) was added and the resulting mixture was stirred

lg) in DMF

l

overnight under argon followed by two successive 12 h dialyses (Mr

cutoff 3500) against 500 mL 20% DMF in PBS (10 mM, pH 7.4) and

two more dialyses (12 h each) against 500 mL PBS (10 mM, pH 7.4)

at 4 °C. The ﬁnal protein concentration (0.32 mg/mL) was determined

by modiﬁed Lowry protein assay. The pyrrole concentration

5.19. 6-(2-Carboxyethyl-1-pyrrolyl)-hexanoyl BSA amide

(CEPH-BSA, 11)

A solution of 1.4 mg CEPFmSu in 150

of 10 mM pH 7.4 PBS containing3 mg/mLof BSA. The cloudysolution

became homogenous with overnight stirring. After 2 days, 25

lL DMF was added to 1 mL

(32.5 lM) was determined by Ehrlich assay. LC–MS/MS revealed

CEPH modiﬁcations at m/z 635.0367, 712.5500, 919.7650, 572.9763,

899.6923, 726.0422, 980.7797, 731.1005, 941.7422, 579.9560,

508.9000, 696.1018, 797.5823 on lysyl residues of the GPDH peptides

lL

DBUwasaddedandtheresultingmixturestirredovernightunderar-

gon followed by two successive 12 h dialysis (Mr cutoff 14,000)

against 500 mL 20% DMF in 10 mM PBS (pH 7.4) and two additional

dialysis (12 h each) against 500 mL 10 mM PBS (pH 7.4) at 4 °C. The

ﬁnal protein concentration (1.34 mg/mL) was determined by modi-

¹VKVGVNGFGR¹⁰

HLKGGAK¹¹⁴

¹⁸⁴TVDGPSGKLWR¹⁹⁴

ELNGK

^{224 217}VIPELNGKLTGMAFR^{231 249}

²⁵⁸VVKQASEGPLK²⁶⁸and ³²¹VVDLMVHMASKE³³²

, , ,

⁵³FHGTVKAENGK^{63 64}LVINGKAITIFQER^{77 105}AGA-

,

¹⁶⁰VIHDHFGIVEGLMTTVHAITATQKTVDGPSGK¹⁹¹

¹⁹⁸GAAQNIIPASTGAAKAVGK^{216 213}AVGKVIP-

AAKYDDIK^{256 252}YDDIKK²⁵⁷

No CEPHFm

,

ﬁed Lowry protein assay. The pyrrole concentration (134 lM) was

,

.

determined by Ehrlich assay. LC–MS/MS revealed nine CEPH modiﬁ-

cations at m/z 594.9934, 618.8188, 541.8060, 937.9577, 526.7835,

612.3604, 689.4319, 765.3984, 964.5088 corresponding to the BSA

modiﬁcations were found after deprotection. The peptide sequence

coverage achieved 68%.

peptides ⁴⁰⁰LKHLVDEPQNLIK⁴¹²

⁴³⁷KVPQVSTPTLVEVSR⁴⁵¹

⁵⁴⁸KQTALVELLK^{557 246}FPKAEFVEVTK²⁵⁶

,

²³³ALKAWSVAR²⁴¹

⁴⁵²SLGKVGTR^{459 490}TPVSEKVTK⁴⁹⁸

²⁴⁹AEFVEVTKLVTDLTK²⁶³

,

²⁴²LSQKFPK²⁴⁸

,

5.23. {4-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-

pentanoylamino]-butyl}-carbamic acid tert-butyl ester (13)

,

respectively and unambiguously conﬁrmed CEPH adducts on

lysyl residues. No CEPHFm modiﬁcations were found after

deprotection.

N-Boc-1,4-butanediamine (20 mg, 0.106 mmol) in 2 mL CH₂Cl₂

was added in d-biotin p-nitrophenyl ester (38 mg, 0.10 mmol) in

2 mL CH₂Cl₂. The system was stirred for 10 h. TLC (30% ethyl ace-

7558

L. Lu et al. / Bioorg. Med. Chem. 17 (2009) 7548–7561

tate in hexane): R_f= 0.25. After removal of the solvent by rotary

evaporation, the crude compound was puriﬁed by ﬂash chroma-

tography (30% ethyl acetate in hexane) to give 28 mg (80%) 13.

¹H NMR (CD₃OD, 400 MHz), d 4.47–4.50 (m, 1H), 4.28–4.31 (m,

1H), 3.2 (m, 1H), 3.16 (t, J = 7.2 Hz, 2H), 3.03 (t, J = 6.8 Hz, 2H),

2.92 (dd, J = 12.4, 4.8 Hz, 1H), 2.68–2.71 (d, J = 12.8 Hz, 1H), 2.19

(t, J = 7.6 Hz, 2H), 1.44–1.7 (10H), 1.42 (s, 9H).

ration. Flash chromatography of the residue (25% ethyl acetate in

hexane) gave 17 (15.6 mg, 87%). ¹H NMR (CDCl₃, 200 MHz), d

9.78 (s, 1H), 3.67 (s, 3H), 2.56–2.81 (8H); HRMS (FAB) (m/z) calcd

for C₈H₁₁O₄(M⁺ꢂH) 171.0658, found 171.0656; calcd for

þ

C₈H₁₃O₅(M⁺+OH) 189.0763, found 189.0783.

þ

5.28. 5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-

pentanoic acid (6-amino-hexyl)-amide (18)

5.24. 5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-yl)-

pentanoic acid (4-amino-butyl)-amide (14)

1,6-Diaminohexane (270 mg, 2.324 mmol) in 10 mL pyridine–

H₂O (9:1, v/v) was added slowly to d-biotin p-nitrophenyl ester

(Sigma) (100 mg, 0.274 mmol) in 20 mL pyridine–H₂O (9:1,v/v).

The clear yellow solution was stirred for 24 h at room temperature.

The solvent was removed by rotary evaporation. TLC (30% NH₃-sat-

urated methanol in CHCl₃, v/v), R_f= 0.24. The crude compound was

puriﬁed by silica gel chromatography (30% methanol saturated

with NH₃in chloroform) to give 84 mg (90%) of pure 18. ¹H NMR

(CD₃OD, 400 MHz), d 4.46–4.51 (m, 1H), 4.28–4.31 (m, 1H), 3.20

(m, 1H), 3.16 (t, J = 7.2 Hz, 2H), 2.9 (dd, J = 12.4, 4.8 Hz, 1H),

2.68–2.71 (d, J = 12.4 Hz, 1H), 2.65 (t, J = 7.2 Hz, 2H), 2.19 (t,

J = 7.6 Hz, 2H), 1.34–1.7 (14H). ¹³C NMR (CD₃OD, 100 MHz), d

175.97 (CO), 166.10 (CO), 63.36 (CH), 61.60 (CH), 57.01 (CH),

42.3 (CH₂), 42.0 (CH₂), 40.2 (CH₂), 36.8 (CH₂), 33.2 (CH₂), 30.50

(CH₂), 29.8 (CH₂), 29.5 (CH₂), 28.8 (CH₂), 28.6 (CH₂), 28.0 (CH₂).

HRMS (FAB) (m/z) calcd for C₁₆H₃₁N₄O₂S⁺(MH⁺) 343.2168, found

343.2165.

Triﬂuoroacetic acid (100 lL) was added in 6 mg 13 in 400 lL

CH₂Cl₂. After stirring for 3 h, TLC (20% methanol in chloroform,

R_f= 0.1) showed the reaction was completed. The solvent was re-

moved by rotary evaporation. The yellowish residue was neutral-

ized by 1 N NaOH to pH 8.5. After removal of the solvent, the

residue was treated with methanol and the methanol extraction

was concentrated and dried to give 4 mg (90%) 14. ¹H NMR

(CD₃OD, 400 MHz), d 4.46–4.51 (m, 1H), 4.28–4.31 (m, 1H), 3.2

(m, 1H), 3.16 (t, J = 7.2 Hz, 2H), 2.9 (dd, J = 12.4, 4.8 Hz, 1H),

2.68–2.71 (d, J = 12.4 Hz, 1H), 2.65 (t, J = 7.2 Hz, 2H), 2.19 (t,

J = 7.6 Hz, 2H), 1.34–1.7 (10H).

5.25. 3-(1-{4-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-

yl)-pentanoylamino]-butyl}-1H-pyrrol-2-yl)-propionic acid 9H-

ﬂuoren-9-yl-methyl ester (15)

Amine 14 (4 mg, 0.012 mmol) in 150

DOHA-Fm (4 mg, 0.0119 mmol) in 150

l

L methanol was added in

5.29. 3-(1-{6-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-

yl)-pentanoylamino]-hexyl}-1H-pyrrol-2-yl)-propionic acid

methyl ester (19)

lL methanol. The solution

was stirred for 9 h at room temperature under argon. The solvent

was removed by rotary evaporation. TLC (5% methanol in CHCl₃):

R_f= 0.2. The crude compound was puriﬁed by silica gel chromatog-

raphy (5% methanol in chloroform) to give 6 mg (84%) of pure 15.

¹H NMR (CDCl₃, 400 MHz), d 7.70 (d, J = 7.6 Hz, 2H), 7.50 (dd,

J = 7.6, 1.2 Hz, 2H), 7.34 (dd, J = 7.6, 7.2 Hz, 2H), 7.27–7.23 (ddd,

J = 7.6, 7.2, 1.2 Hz, 2H), 6.51 (dd, J = 1.6, 2.8 Hz, 1H), 5.98 (dd,

J = 2.8, 3.6 Hz, 1H), 5.79 (m, 1H), 5.74 (m, 1H), 5.58 (s, 1H), 4.82

(s, 1H), 4.40 (m, 1H), 4.36 (d, J = 7.2 Hz, 2H), 4.20 (m, 1H), 4.15 (t,

J = 7.2 Hz, 1H), 3.76 (t, J = 7.2 Hz, 2H), 3.15 (m, 2H), 3.05 (m, 1H),

2.84–2.79 (dd, J = 4.8, 12.8 Hz, 1H), 2.78–2.76 (m, 2H), 2.71–2.66

(m, 2H), 2.64–2.61 (d, J = 12.8 Hz, 1H), 2.06–2.10 (dt, J = 3.6,

7.2 Hz, 2H), 1.72–1.36 (10H). HRMS (FAB) (m/z) calcd for

C₃₅H₄₃N₄O₄S⁺(MH⁺) 615.3005, found 615.2995.

Amine 18 (45 mg, 0.13 mmol) in 1 mL methanol was added to

DOHA-Me (23 mg, 0.13 mmol) in 1.5 mL methanol. The solution

was stirred for 9 h at room temperature under argon. The solvent

was removed by rotary evaporation. The crude compound was

puriﬁed by silica gel chromatography (5% methanol in chloroform,

TLC: R_f= 0.18) to give 47 mg (75%) of pure 19. ¹H NMR (CD₃OD,

400 MHz), d 6.52 (dd, J = 2.0, 2.8 Hz, 1H), 5.9 (dd, J = 3.2, 2.8 Hz,

1H), 5.75 (m, 1H), 4.46–4.49 (dd, J = 8.0, 4.0 Hz, 1H), 4.29 (dd,

J = 8.0, 4.8 Hz, 1H), 4.28–4.3 (m, 1H), 3.82 (t, J = 7.6 Hz, 2H), 3.65

(s, 3H), 3.1–3.2 (3H), 2.9 (dd, J = 12.4, 4.8 Hz, 1H), 2.85 (t,

J = 7.2 Hz, 2H), 2.68–2.71 (d, J = 12.4 Hz, 1H), 2.65 (t, J = 7.2 Hz,

2H), 2.19 (t, J = 7.6 Hz, 2H), 1.34–1.7 (14H).

5.26. 3-(1-{4-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-

yl)-pentanoylamino]-butyl}-1H-pyrrol-2-yl)-propionic acid (16)

5.30. 3-(1-{4-[5-(2-Oxo-hexahydro-thieno[3,4-d]imidazol-4-

yl)-pentanoylamino]-hexyl}-1H-pyrrol-2-yl)-propionic acid

(20)

5 lL DBU was added to 15 (6 mg, 0.01 mmol) in 500 lL THF and

stirred for 2 h. TLC (10% methanol in chloroform, R_f= 0.3) showed

the completion of the reaction by disappearance of the UV active

starting spot. The corresponding acid was puriﬁed by ﬂash chro-

matography (7% methanol in chloroform) to provide 3.7 mg (85%)

acid 16. ¹H NMR (CD₃OD, 400 MHz), d 6.57 (dd, J = 2.8, 1.6 Hz,

1H), 5.92 (dd, J = 3.2, 2.8 Hz, 1H), 5.79 (m, 1H), 4.60 (3H), 4.48

(m, 1H), 4.27 (m, 1H), 3.86 (t, J = 7.2 Hz, 2H), 3.19–3.15 (3H),

2.94–2.89 (dd, J = 4.8, 12.8 Hz, 1H), 2.86–2.82 (m, 2H), 2.71–2.67

(d, J = 12.8 Hz, 1H), 2.60–2.56 (m, 2H), 2.20–2.17 (t, J = 7.2 Hz,

2H), 1.72–1.36 (10H). HRMS (FAB) (m/z) calcd for C₂₁H₃₃N₄O₄S⁺

(MH⁺) 437.2222, found 437.2193; calcd for C₂₁H₃₁N₄O₃S⁺

(M⁺ꢂOH) 419.2117, found 419.2102.

Sodium hydroxide (16 mg, 0.4 mmol) was added to 19 (47 mg,

0.1 mmol) in 1 mL absolute ethanol and stirred for 4 h at room

temperature. TLC (10% methanol in chloroform, R_f= 0.3) showed

the completion of the reaction by disappearance of the starting

spot. After removal of the solvent, the residue was neutralized with

3 N HCl to pH 3 and extracted with ethyl acetate. The ethyl acetate

extract was washed with brine, dried with MgSO₄, and concen-

trated to afford 38 mg (84%) 20. ¹H NMR (CD₃OD, 400 MHz), d

6.57 (dd, J = 2.0, 2.4 Hz, 1H), 5.93 (dd, J = 2.4, 3.6 Hz, 1H), 5.80 (m,

1H), 4.46 (dd, J = 7.6, 5.2 Hz, 1H), 4.29 (dd, J = 4.8, 7.6 Hz, 1H),

3.84 (t, J = 7.6 Hz, 2H), 3.1–3.2 (3H), 2.9 (dd, J = 12.4, 4.8 Hz, 1H),

2.85 (t, J = 7.2 Hz, 2H), 2.68–2.71 (d, J = 12.4 Hz, 1H), 2.61 (t,

J = 7.2 Hz, 2H), 2.19 (t, J = 7.2 Hz, 2H), 1.34–1.7 (14H). ¹³C NMR

(CD₃OD, 100 MHz), d 176.82 (CO), 175.97 (CO), 166.10 (CO),

132.00 (C), 121.22 (CH), 107.52 (CH), 106.08 (CH), 63.36 (CH),

61.60 (CH), 57.01 (CH), 47.19 (CH₂), 41.03 (CH₂), 40.22 (CH₂),

36.82 (CH₂), 34.48 (CH₂), 32.54 (CH₂), 30.30 (CH₂), 29.78 (CH₂),

29.51 (CH₂), 27.62 (CH₂), 27.44 (CH₂), 26.95 (CH₂), 22.55 (CH₂).

5.27. 4,7-Dioxo-heptanoic acid methyl ester (DOHA-Me, 17)

Ester 1 (22.5 mg, 0.104 mmol) in 5 mL of AcOH/H₂O (3:1, v/v)

was stirred at 50 °C for 5 h. TLC (CHCl₃): R_f= 0.45 showed the com-

pletion of the reaction. The solvent was removed by rotary evapo-

L. Lu et al. / Bioorg. Med. Chem. 17 (2009) 7548–7561

7559

HRMS (FAB) (m/z) calcd for C₂₃H₃₇N₄O₄S⁺(MH⁺) 465.2530, found

465.2524.

system was stirred 20 h under Ar. After evaporation of solvent, the

crude product was puriﬁed by silica gel chromatography (10%

methanol in chloroform, TLC: R_f= 0.2) to give 40 mg (69%) of pure

23. ¹H NMR (CDCl₃, 400 MHz), d 7.70 (d, J = 7.6 Hz, 2H), 7.52 (d,

J = 7.6 Hz, 2H), 7.35 (dd, J = 7.6, 7.6 Hz, 2H), 7.25 (dd, J = 7.6,

7.6 Hz, 2H), 6.58 (m, 1H), 5.94 (m, 1H), 5.74 (m, 1H), 5.28 (m,

2H), 4.31 (d, J = 6.8 Hz, 2H), 4.14 (t, J = 6.8 Hz, 1H), 4.03–3.60

(9H), 2.82–2.78 (t, J = 7.2 Hz, 2H), 2.69–2.65 (t, J = 7.2 Hz, 2H),

2.21–2.17 (t, J = 7.2 Hz, 4H), 1.54–1.45 (m, 2H), 1.23–1.19 (24H),

0.86 (t, J = 6.8 Hz, 3H). ¹³C NMR (CDCl₃, 100 MHz), d 173.69 (CO),

173.42 (CO), 143.69 (C), 141.22 (C), 131.07 (CH), 127.77 (CH),

127.09 (CH), 125.02 (CH), 120.97 (CH), 119.98 (CH), 107.20 (CH),

105.34 (CH), 77.20 (CH) 69.07 (CH₂), 66.66 (CH₂), 65.37 (CH₂),

64.30 (CH₂), 46.65 (CH₂), 46.35 (CH), 33.97 (CH₂), 33.11 (CH₂),

31.92 (CH₂), 29.73 (CH₂), 29.67 (CH₂), 29.58 (CH₂), 29.37 (CH₂),

29.23 (CH₂), 24.83 (CH₂), 22.69 (CH₂), 21.23 (CH₂), 14.11 (CH₃).

HRMS (FAB) (m/z) calcd for C₄₁H₅₈NNaO₉P⁺(MNa⁺) 776.3904,

found 776.3939; calcd for C₄₁H₅₇NNa₂O₉P⁺(MNa₂⁺) 798.3724,

found 798.3717.

5.31. Octadec-9-enoic acid 2-[(2-{2-[2-(9H-ﬂuoren-9-

ylmethoxycarbonyl)-ethyl]-pyrrol-1-yl}-ethoxy)-hydroxy-

phosphoryloxy]-1-hexadecanoyloxymethyl-ethyl ester (21)

Triethylamine (TEA) (25

(50 mg, 0.065 mmol) in 500

0.065 mmol) in 500 L CHCl₃was added to the mixture. The sys-

lL, 0.247 mmol) was added to POPE

lL CHCl₃, then DOHAFm (22 mg,

l

tem was stirred 20 h under Argon. After evaporation of solvent,

the crude product was puriﬁed by silica gel chromatography

(10% methanol in chloroform, TLC: R_f= 0.2) to give 50 mg (76%)

of pure 21. ¹H NMR (CD₃OD/CDCl₃= 1:1, 400 MHz), d 7.74 (d,

J = 7.6 Hz, 2H), 7.53 (d, J = 7.6 Hz, 2H), 7.36 (dd, J = 7.6, 7.6 Hz,

2H), 7.27 (dd, J = 7.6, 7.6 Hz, 2H), 6.63 (dd, J = 2.8, 1.6 Hz, 1H),

5.94 (dd, J = 3.2, 2.8 Hz, 1H), 5.77 (m, 1H), 5.28 (m, 2H), 4.36 (d,

J = 6.8 Hz, 2H), 4.28 (m, 1H), 4.17 (t, J = 6.8 Hz, 1H), 4.05–3.98

(6H), 3.68 (t, J = 6.4 Hz, 2H), 2.86–2.82 (t, J = 7.2 Hz, 2H), 2.73–

2.69 (t, J = 7.2 Hz, 2H), 2.25–2.21 (4H), 2.0–1.94 (4H), 1.57 (4H),

1.21 (44H), 0.84 (t, J = 6.4 Hz, 6H). ¹³C NMR (CD₃OD/CDCl₃= 1:1,

50 MHz, APT), d 175.21 (+) (CO), 174.84 (+) (CO), 174.81 (+) (CO),

145.08 (+) (C), 142.68 (+) (C), 132.30 (+) (C), 131.24 (ꢂ) (CH),

130.95 (ꢂ) (CH), 129.13 (ꢂ) (CH), 128.53 (ꢂ) (CH), 126.31 (ꢂ)

(CH), 121.30 (ꢂ) (CH), 121.22 (ꢂ) (CH), 108.49 (ꢂ) (CH), 106.77

(ꢂ) (CH), 71.86 (ꢂ) (CH), 67.96 (+) (CH₂), 66.62 (+) (CH₂), 64.60

(+) (CH₂), 64.05 (+) (CH₂), 48.10 (+) (CH₂), 47.82 (ꢂ) (CH), 35.47

(+) (CH₂), 35.34 (+) (CH₂), 34.66 (+) (CH₂), 33.27 (+) (CH₂), 31.03

(+) (CH₂), 30.85 (+) (CH₂), 30.70 (+) (CH₂), 30.66 (+) (CH₂), 30.61

(+) (CH₂), 30.49 (+) (CH₂), 28.49 (+) (CH₂), 26.24 (+) (CH₂), 26.20

(+) (CH₂), 23.98 (+) (CH₂), 22.64 (+) (CH₂), 15.14 (ꢂ) (CH₃). HRMS

(FAB) (m/z) calcd for C₆₀H₉₁NNa₂O₁₀P⁺[(MꢂH)Na₂⁺] 1062.6177,

found 1062.6.

5.34. Hexadecanoic acid 3-([2-[2-(carboxyethyl)-pyrrol-1-yl]-

ethoxy]-hydroxy-phosphoryloxy)-2-hydroxy-propyl ester

(lysoPE-CEP, 24)

DBU (20

lL, 0.13 mmol) was added to 23 (24 mg, 0.03 mmol) in

500 L CHCl₃. After 3 h, the reaction was completed and the sys-

l

tem was diluted by 1.5 mL CHCl₃. The solution was washed with

2 mL phosphate buffer of pH 5.5. The organic part was washed

with brine, dried over magnesium sulfate and vacuum-ﬁltered,

concentrated, puriﬁed by ﬂash chromatography (CHCl₃/MeOH/

H₂O = 65:25:4, v/v, TLC: R_f= 0.18) to yield 16 mg (95%) of pure

lysoPE-CEP. ¹H NMR (CD₃OD/CDCl₃= 1:1, 400 MHz), d 6.60 (m,

1H), 5.96 (m, 1H), 5.83 (m, 1H), 4.10–4.0 (5H), 3.74–3.6 (m, 2H),

3.6–3.5 (m, 2H), 2.8–2.9 (m, 2H), 2.68–2.65 (m, 2H), 2.32 (t,

J = 6.8 Hz, 2H), 1.57 (m, 2H), 1.23 (24H), 0.85 (t, J = 7.2 Hz, 3H).

¹³C NMR (CD₃OD/CDCl₃/D₂O = 50:50:1, 100 MHz), d 175.20 (CO),

133.00 (C), 121.23 (CH), 107.36 (CH), 105.56 (CH), 70.80 (CH),

66.05 (CH₂), 65.63 (CH₂), 47.17 (CH₂), 34.61 (CH₂), 33.29 (CH₂),

32.48 (CH₂), 30.22 (CH₂), 30.05 (CH₂), 29.90 (CH₂), 29.88 (CH₂),

29.72 (CH₂), 27.04 (CH₂), 25.42 (CH₂), 23.21 (CH₂), 14.38 (CH₃).

HRMS (FAB) (m/z) calcd for C₂₇H₄₈NNaO₉P⁺(MNa⁺) 598.3121,

found 598.3053.

5.32. Octadec-9-enoic acid 2-({2-[2-(2-carboxy-ethyl]-pyrrol-1-

yl}-ethoxy)-hydroxy-phosphoryloxy]-1-hexadecanoyloxym-

ethyl-ethyl ester (PE-CEP, 22)

DBU (20

lL, 0.13 mmol) was added to 21 (24 mg, 0.025 mmol)

in 500 L CHCl₃. After 3 h, the reaction was completed and the sys-

l

tem was diluted by 1.5 mL CHCl₃. The solution was washed with

2 mL phosphate buffer of pH 5.5. The organic phase was washed

with brine, dried over magnesium sulfate, then vacuum-ﬁltered,

concentrated, puriﬁed by ﬂash chromatography (15% methanol in

chloroform, TLC: R_f= 0.18) to yield 19 mg (90%) of pure 22. ¹H

NMR (CD₃OD/CDCl₃= 1:1, 400 MHz), d 6.56 (m, 1H), 5.96 (m, 1H),

5.83 (m, 1H), 5.30 (m, 2H), 5.15 (m, 1H), 4.33 (m, 1H), 4.05–3.98

(6H), 3.81 (m, 2H), 2.83 (m, 2H), 2.58 (m, 2H), 2.27 (t, J = 7.2 Hz,

4H), 2.0–1.94 (4H), 1.57 (4H), 1.21 (44H), 0.84 (t, J = 6.4 Hz, 6H).

¹³C NMR (CD₃OD/CDCl₃= 1:1, 100 MHz), d 180.36 (COOH), 174.49

(CO), 174.08 (CO), 133.54 (C), 130.46 (CH), 130.14 (CH), 120.98

(CH), 107.26 (CH), 105.05 (CH), 71.00 (CH), 65.84 (CH₂), 64.03

(CH₂), 63.11 (CH₂), 47.29 (CH₂), 34.70 (CH₂), 34.56 (CH₂), 32.40

(CH₂), 32.42 (CH₂), 30.23 (CH₂), 30.17 (CH₂), 30.00 (CH₂), 29.80

(CH₂), 29.63 (CH₂), 27.66 (CH₂), 25.40 (CH₂), 23.13 (CH₂), 14.31

(CH₃). HRMS (FAB) (m/z) calcd for C₄₆H₈₂NNaO₁₀P⁺(MNa⁺)

862.5574, found 862.5550; calcd for C₄₆H₈₁NNa₂O₁₀P⁺[(MꢂH)-

Na₂⁺] 884.5394, found 884.5291.

5.35. 2-(9H-Fluoren-9-ylmethoxycarbonylamino)-6-{2-[2-(9H-

ﬂuoren-9-ylmethoxycarbonyl)-ethyl]-pyrrol-1-yl}-hexanoic

acid (25)

6-Amino-2-(9H-ﬂuoren-9-ylmethoxycarbonyl amino)-hexanoic

acid (40 mg, 0.1 mmol) was suspended in 10 mL methanol with

DOHAFm (30 mg, 0.089 mmol). Then 20 lL of acetic acid was

added. The suspension dissolved gradually and a light yellow oil

was generated at the bottom of the ﬂask as the reaction proceeded.

The system was stirred 24 h under Ar. After the removal of solvent,

the crude compound was puriﬁed by silica gel chromatography (4%

methanol in chloroform) to give 45 mg (75%) of light yellow oil 25.

TLC (4% methanol in chloroform): R_f= 0.15; ¹H NMR (CDCl₃,

400 MHz), d 7.75 (d, J = 7.2 Hz, 4H), 7.55 (d, J = 7.6 Hz, 4H), 7.39

(dd, J = 7.2, 7.6 Hz, 4H), 7.30 (dd, J = 7.2, 7.6 Hz, 4H), 6.57 (m, 1H),

6.06 (dd, J = 3.2, 3.2 Hz, 1H), 5.87 (m, 1H), 5.4 (m, 1H), 4.41 (d,

J = 6.8 Hz, 2H), 4.39 (m, 1H), 4.20 (t, J = 6.8 Hz, 2H), 3.78 (t,

J = 6.8 Hz, 2H), 2.85–2.73 (4H), 1.91–1.39 (6H). ¹³C NMR (CDCl₃,

100 MHz), d 176.58 (COOH), 173.08 (COO), 156.04 (CO), 143.75

(C), 143.66 (C), 141.25 (C), 130.58 (C), 127.75 (CH), 127.69 (CH),

127.07 (CH), 127.03 (CH), 125.01 (CH), 124.95 (CH), 119.99 (CH),

106.99 (CH), 105.23 (CH), 67.04 (CH₂), 66.46 (CH₂), 53.49 (CH),

5.33. Hexadecanoic acid 3-[(2-{2-[2-(9H-ﬂuoren-9-ylmeth-

oxycarbonyl)-ethyl]-pyrrol-1-yl}-ethoxy)-hydroxy-phos-

phoryloxy]-2-hydroxy-propyl ester (23)

Triethylamine (TEA) (12

(42 mg, 0.093 mmol) in 500

0.077 mmol) in 500 L CHCl₃was added to the mixture. The cloudy

l

L, 0.116 mmol) was added to lyso-PE

lL CHCl₃, then DOHAFm (26 mg,

l

7560

L. Lu et al. / Bioorg. Med. Chem. 17 (2009) 7548–7561

47.07 (CH), 46.71 (CH), 46.04 (CH₂), 33.18 (CH₂), 31.84 (CH₂), 30.73

tion of 1.0 mg/mL of p-nitrophenyl phosphate in 0.2 M Tris buffer

(Sigma–Aldrich, Milwaukee, WI, Cat. Sigma N1891) was added.

The plate was then incubated at room temperature for 20 min un-

til the maximum absorbance reached 0.6–0.8. The development

(CH₂), 22.40 (CH₂), 21.37 (CH₂). HRMS (FAB) (m/z) calcd for

C₄₂H₄₁N₂O₆(MH⁺) 669.2959, found 669.2949.

þ

was terminated by adding 3 N NaOH (50 lL) to each well before

5.36. 2-(9H-Fluoren-9-ylmethoxycarbonylamino)-6-{2-[2-(9H-

ﬂuoren-9-ylmethoxycarbonyl)-ethyl]-pyrrol-1-yl}-hexanoic

acid pentaﬂuorophenyl ester (26)

measuring the ﬁnal absorbance values. The absorbance in each

well was measured with a dual-wavelength Bio-Rad 450 micro-

plate reader with detection at 405 nm relative to 655 nm. Absor-

bance values for duplicate assays were averaged and scaled to

make the maximum curve ﬁt value close to 100 percent. The

averaged and scaled percent absorbance values were plotted

against the log of concentration. Theoretical curves for each plot

were ﬁt to the absorbance data with a four parameter logistic

function, f(x) = (a ꢂ d)/[1+(x/c)^b] + d using SigmaPlot 9.0 (Jandel

Scientiﬁc Software, San Rafael, CA). Parameter a = the asymptotic

maximum absorbance, b = slope at the inﬂection point, c = the

inhibitor concentration at the 50% absorbance value (IC₅₀), and

d = the asymptotic minimum absorbance.

Freshly distilled CH₂Cl₂(10 mL) was added to the mixture of

pentaﬂuorophenol (50 mg, 0.27 mmol), dicyclohexylcarbodiimide

(DCC, 41.5 mg, 0.201 mmol), dimethlamino pyridine (DMAP,

8 mg, 0.067 mmol) and acid 25 (45 mg, 0.067 mmol). The resulting

mixture was stirred for 72 h at room temperature. The solvent was

removed. Flash chromatography of the residue (15% ethyl acetate

in hexane, TLC: R_f= 0.2) gave low melting white solid 26 (50 mg,

90%). ¹H NMR (CDCl₃, 200 MHz) d 7.75 (dd, J = 6.8, 6.8 Hz, 4H),

7.56 (dd, J = 7.2, 7.2 Hz, 4H), 7.38 (dd, J = 7.2, 7.6 Hz, 4H), 7.29

(4H), 6.58 (dd, J = 3.2, 1.6 Hz, 1H), 6.06 (dd, J = 3.2, 3.2 Hz, 1H),

5.87 (m, 1H), 5.38 (d, J = 8.2 Hz, 1H), 4.39–4.45 (5H), 4.17–4.22

(m, 2H), 3.84 (t, J = 7.2 Hz, 2H), 2.86 (t, J = 6.8 Hz, 2H), 2.77 (t,

Acknowledgments

J = 6.8 Hz, 2H), 2.0–1.46 (6H). ¹³C NMR (CDCl₃, 100 MHz),

d

173.14 (COO), 168.96 (COO), 156.09 (COO), 143.93 (C), 143.82

(C), 141.57 (C), 141.51 (C), 130.94 (C), 128.03 (CH), 127.32 (CH),

125.22 (CH), 120.57 (CH), 120.25 (CH), 107.41 (CH), 105.62 (CH),

67.46 (CH₂), 66.73 (CH₂), 53.83 (CH), 47.32 (CH), 46.99 (CH),

46.31 (CH₂), 33.40 (CH₂), 32.18 (CH₂), 30.99 (CH₂), 22.72 (CH₂),

We are grateful for support of this work by National Institutes

of Health Grants GM21249 and HL53315, and by a State of Ohio

Third Frontier BRTT Award TECH05-064. We also thank Suresh P.

Annangudi for preparing compound 13.

21.66 (CH₂). HRMS (FAB) (m/z) calcd for C₄₈H₄₀F₅N₂O₆(MH⁺)

þ

Supplementary data

835.2801, found 835.2813.

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.bmc.2009.09.009.

5.37. Competitive ELISA for inhibition of anti-CEP antibody

binding to CEP-HSA by CEPH-BSA

References and notes

CEP-HSA was used as a coating agent and a standard, CEPH-

BSA was used as an inhibitor. A blank, a positive control contain-

ing no inhibitor, and up to 8 serial dilutions of the inhibitor and 8

serial dilutions of the CEP-HSA standard were run. Each well of

1. Gu, X.; Sun, M.; Gugiu, B.; Hazen, S.; Crabb, J. W.; Salomon, R. G. J. Org. Chem.

2003, 68, 3749.

2. Bok, D. PNAS 2002, 99, 14619.

3. Ferris, F. L., 3rd Am. J. Epidemiol. 1983, 118, 132.

the ELISA plate was coated with CEP-HSA solution (100 lL), pre-

4. Stone, E. M.; Shefﬁeld, V. C.; Hageman, G. S. Hum. Mol. Genet. 2001, 10, 2285.

5. Crabb, J. W.; Miyagi, M.; Gu, X. R.; Shadrach, K.; West, K. A.; Sakaguchi, H.;

Kamei, M.; Hasan, A.; Yan, L.; Rayborn, M. E.; Salomon, R. G.; Hollyﬁeld, J. G.

PNAS 2002, 99, 14682.

6. Gu, X.; Meer, S. G.; Miyagi, M.; Rayborn, M. E.; Hollyﬁeld, J. G.; Crabb, J. W.;

Salomon, R. G. J. Biol. Chem. 2003, 278, 42027.

pared by diluting a solution containing 187.14 nmol/mL HSA-

bound CEP in PBS to 187.14 pmol/mL with pH 7.4 PBS (10 mM).

The plate was incubated at 37 °C for 1 h, then washed with

10 mM PBS (3 ꢁ 300

37 °C with 300 L of 1% chicken ovalbuman (COA) in 10 mM

PBS. The plate was then rinsed with 0.1% COA in 10 mM PBS

(300 ). Eight serial dilutions of CEPH-BSA inhibitor or CEP-

HSA standard (120 L each with a dilution factor of 0.2) were pre-

incubated at 37 °C for 1 h with anti-CEP-KLH antibody solution

(120 ) that was prepared by adding 5 L of protein G col-

lK), and then blocked by incubating 1 h at

l

7. Ebrahem, Q.; Renganathan, K.; Sears, J.; Vasanji, A.; Gu, X. R.; Lu, L.; Salomon, R.

G.; Crabb, J. W.; Anand-Apte, B. PNAS 2006, 103, 13480.

8. Baudouin, C.; Peyman, G. A.; Fredj-Reygrobellet, D.; Gordon, W. C.; Lapalus, P.;

Gastaud, P.; Bazan, N. G. Jpn. J. Ophthalmol. 1992, 36, 443.

9. Lopez, P. F.; Grossniklaus, H. E.; Lambert, H. M.; Aaberg, T. M.; Capone, A., Jr.;

Sternberg, P., Jr.; L’Hernault, N. Am. J. Ophthalmol. 1991, 112, 647.

10. Renganathan, K.; Gu, J.; Gugiu, B.; Gu, X.; Darrow, R.; Lewis, H.; Salomon, R. G.;

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Article Doi

Synthesis and structural characterization of carboxyethylpyrrole-modified proteins: Mediators of age-related macular degeneration

DOI: 10.1016/j.bmc.2009.09.009

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Article abstract of DOI:10.1016/j.bmc.2009.09.009

Full text of DOI:10.1016/j.bmc.2009.09.009

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