Synthetic bacteriochlorins bearing polar motifs (carboxylate, phosphonate, ammonium and a short PEG). Water-solubilization, bioconjugation, and photophysical properties

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Synthetic bacteriochlorins bearing polar motifs

(carboxylate, phosphonate, ammonium and a

short PEG). Water-solubilization, bioconjugation,

and photophysical properties†

Cite this:DOI: 10.1039/c5nj00759c

Jianbing Jiang,^aEunkyung Yang,^bKanumuri Ramesh Reddy,^a

Dariusz M. Niedzwiedzki,^cChristine Kirmaier,^bDavid F. Bocian,*^dDewey Holten*^b

and Jonathan S. Lindsey*^a

Bacteriochlorins are potentially excellent chromophores for near-infrared (NIR) photochemical and

spectroscopic studies yet the intrinsically hydrophobic macrocycle core has stymied work in aqueous

media. Herein, a set of bacteriochlorins bearing distinct polar motifs is reported. The motifs include

phosphonate (pH-dependent anionic, BC1), carboxylate (pH-dependent anionic, BC2), ammonium

(permanently cationic) without (BC3) or with (BC4) a linker ester moiety, and tetraethyleneoxy (a short

PEG, polar non-ionic, BC5). The groups are located at the 3,5-positions of each of two aryl groups at

the bacteriochlorin 3,13-sites. Synthesis of the bacteriochlorins entails the Suzuki coupling of a common

3,13-dibromobacteriochlorin building block with a set of aryl boronates. Five factors were selected for

comparisons among the polar motifs upon attachment to the bacteriochlorins: (1) synthesis yield and ease

of purification, (2) amenability toward subsequent derivatization, (3) water-solubility, (4) full-width-at-half-

maximum (fwhm) of the long-wavelength (Q_y) absorption and fluorescence bands, singlet excited-state

lifetime (t_S) and fluorescence quantum yield (F_f), and (5) stability in the dark or under illumination. Water-

solubility was assessed by examination of the absorption spectra across a 1000-fold concentration range

(B0.2–0.6 mM to B200–600 mM). With the exception of BC4, all displayed good aqueous solubility,

photostability, and photophysical properties in aqueous solution (fwhm = 23–31 nm, F_f= 0.10–0.16,

t_S= 1.9–2.7 ns). The modestly lower F_fand t_Svalues for the bacteriochlorins in aqueous versus organic

(N,N-dimethylformamide) media are traced to an increased rate constant for excited-state internal conver-

sion. Upon consideration of all factors, the ammonium (short linker) and short PEG groups were most

attractive for solubilization of the bacteriochlorins in aqueous media. The studies prompted the synthesis

of two water-soluble (ammonium-substituted) bacteriochlorins bearing N-hydroxysuccinimide esters.

Received (in Montpellier, France)

26th March 2015,

Accepted 12th May 2015

DOI: 10.1039/c5nj00759c

www.rsc.org/njc

from energy sciences to photomedicine. A generic design of

hydrophilic tetrapyrroles is to introduce polar groups at syn-

Introduction

Methods for the synthesis of hydrophilic tetrapyrroles provide thetically accessible positions about the perimeter of the

an entree into diverse areas of scientific investigation ranging macrocycle.^1–5Representative polar groups include those that are

´

ionic, such as ammonium,^3,6–11sulfonate,^2,12–14carboxylate^15,16

and phosphonate,^17,18as well as those that are nonionic, such as

^aDepartment of Chemistry, North Carolina State University, Raleigh,

glycoside^19,20or polyethylene glycol.^21–24While there are numerous

NC 27695-8204, USA. E-mail: jlindsey@ncsu.edu

^bDepartment of Chemistry, Washington University, St. Louis, MO 63130-4889, USA.

reports concerning the synthesis of hydrophilic tetrapyrroles and

their use in aqueous media,^1–5as in photodynamic therapy or

E-mail: holten@wustl.edu

^cPhotosynthetic Antenna Research Center, Washington University, St. Louis,

biomedical imaging, most such reports describe one or a few

target compounds of similar structure. Independent studies of

water solubility are rarely performed, and the absence of compara-

tive studies of distinct motifs across a common molecular archi-

tecture precludes meaningful conclusions about relative merits.

Our interest in preparing hydrophilic tetrapyrroles stems

first of all from our objectives in energy sciences, where such

Missouri, 63130-4889, USA

^dDepartment of Chemistry, University of California, Riverside,

California 92521-0403, USA. E-mail: david.bocian@ucr.edu

† Electronic supplementary information (ESI) available: Attempted synthesis

of a phosphatidylcholine bacteriochlorin (BC6); mass spectrometry analysis for

BC1–BC5, BC7, and BC8; and additional data concerning the photophysical

properties of bacteriochlorins. See DOI: 10.1039/c5nj00759c

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Chart 1 Representative hydrophilic bacteriochlorins.

macrocycles can be incorporated with peptides to give self-

assembled light-harvesting architectures.^25,26The tetrapyrroles

of present interest are bacteriochlorins, which absorb strongly

in the near-infrared (NIR) region. A second objective – a spinoff

thereof – aims to use hydrophilic bacteriochlorins in biomedical

applications. A variety of bacteriochlorins with appended polar

motifs has been previously prepared; representative members

are shown in Chart 1. Compounds I,²⁷II²⁷and III²⁸were

derived by semisynthesis^29,30from bacteriochlorophyll a; IV¹³

by hydrogenation of the corresponding porphyrin;³¹and V–IX

by de novo synthesis.^32,33Methods of bacteriochlorin synthesis

have been reviewed.^34,35

Chart 2 A facially encumbered chlorin bearing a 2,6-disubstituted aryl

motif (line drawing at left; perspective illustration at right).³⁹

An attractive design element for solubilization of tetrapyrrole

Given the present challenge in constructing bacteriochlorins

macrocycles entails installation of groups that project above and that bear synthetically malleable 2,6-disubstituted aryl units

below the plane of the p-chromophore. The groups can be hydro- (mesityl groups have been installed in one instance⁴¹), we resorted

phobic to enhance solubility in nonpolar organic solvents, or quite to the use of 3,5-disubstituted aryl units. A 3,5-disubstituted arene

polar to enhance solubility in polar or even aqueous media. One is expected to have greater conformational motion about the

motif for facial encumbrance employs a 2,6-disubsituted aryl unit; carbon–carbon single bond that joins the bacteriochlorin,

the steric bulk of the 2,6-substituents suppresses rotation³⁶of enabling the polar motifs to sweep out lateral conformations

the aryl group toward coplanarity with the tetrapyrrole macro- versus the more constrained motions of the 2,6-disubstituted

cycle, thereby thrusting the 2,6-substituents above and below arenes. Unlike 2,6-disubstituted arenes, 3,5-disubstituted aryl

the tetrapyrrole plane. Such a ‘‘facial encumbrance’’ design units are readily installed via Suzuki coupling reactions.⁴⁰Nine

feature has been employed extensively with porphyrins,^37,38but such bacteriochlorins have been prepared; representative examples

to much lesser extent with chlorins, and hardly at all with are shown in Chart 3. Eight bacteriochlorins include 3,5-dicarboxy-

bacteriochlorins. A 2,6-disubstituted arene for aqueous solubi- phenyl groups at the 2,12-positions (e.g., X, XI) or 3,13-positions

lization was employed with synthetic chlorins (Chart 2),³⁹albeit (e.g., XII) of the macrocycle,⁴²whereas one contains 3,5-bis(PEG)aryl

with some diﬃculty,⁴⁰and the synthetic approach is not yet groups attached to the bacteriochlorin 15-position (XIII).⁴³(We

applicable with bacteriochlorins.

employ the term ‘‘PEG’’ here to describe a tetraethyleneoxy unit

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Chart 3 Representative hydrophilic bacteriochlorins with 3,5-disubstituted

aryl motifs.

given the widespread usage of this term.) Five of the bacterio-

chlorins contain a bioconjugatable tether, for which XII is

representative.⁴²

Chart 4 Bacteriochlorin scaﬀold and distinct polar groups.

To gain a better understanding of the virtues and limitations Suzuki coupling with a known⁴⁴3,13-dibromobacteriochlorin

of the various types of polar motifs for water-solubilization, we (BC-Br^3,13).

prepared a set of bacteriochlorins (BC1–BC5) encompassing

Herein, the synthesis of bacteriochlorins BC1 and BC3–BC5

phosphonate (BC1), carboxylate (BC2), ammonium (for BC3 are reported; the synthesis of BC2 was described earlier.⁴²The

and BC4), or PEG (BC5) moieties (Chart 4). The reaction to five bacteriochlorins are employed in a comprehensive compar-

incorporate phosphatidylcholine moieties (BC6) was incomplete. ison regarding synthetic amenability, photophysical properties

Bacteriochlorins BC3 and BC4 both bear four ammonium groups, [absorption/fluorescence bandwidths, fluorescence yield (F_f), singlet

but differ in that the former is more compact and has benzyl- excited-state lifetime (t_S)], stability and ease of derivatization.

ammonium units, whereas the latter contains alkylammonium The derivatization process of interest entails selective bromina-

groups attached via ester moieties. Each bacteriochlorin in tion at the 15-position (enabled by the distal 5-methoxy group)⁴³

the set contains a common scaffold and was derived by followed by Suzuki coupling to install a bioconjugatable tether.

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The synthetic strategy, evaluation methods, and results obtained partners were synthesized as is shown in Scheme 1. 1-Bromo-

should be applicable across the tetrapyrrole family of macrocycles. 3,5-dimethylbenzene was dibrominated with N-bromosuccin-

imide (NBS) in the presence of azobis(isobutyronitrile) (AIBN)

to give the known 1-bromo-3,5-bis(bromomethyl)benzene (1a),⁴⁵

which was prepared here at 14-fold larger scale, isolated without

chromatography, and fully characterized. Treatment of 1a with

triethyl phosphite in toluene aﬀorded the corresponding phos-

Results

I. Synthesis

A. Suzuki coupling partners. The synthesis of all the bacterio- phonate 1b in 41% yield. Pd-mediated coupling⁴⁶of 1b with

chlorins entails Suzuki coupling of the dibromobacteriochlorin⁴⁴bis(pinacolato)diboron gave diethyl phosphonate 1 in 56% yield.

BC-Br^3,13with an aryl boronate ester. Four Suzuki coupling Pd-mediated coupling of the known tert-butoxycarbonyl-protected

Scheme 1 Synthesis of Suzuki coupling partners.

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3,5-bis(aminomethyl)bromobenzene 2a (derived from 1a)⁴⁷

We also attempted to prepare a bacteriochlorin that bears

with bis(pinacolato)diboron gave the Boc-protected Suzuki phosphatidylcholine substituents as shown in Scheme 2. Suzuki

coupling partner 2 in 79% yield. The Suzuki coupling reactions coupling reaction of BC-Br^3,13with compound 4 aﬀorded the TBS-

were carried out with the palladium dichloride reagent contain- protected tetrahydroxy-bacteriochlorin (BC6a) in 81% yield. Treat-

ing a 1,1⁰-bis(diphenylphosphino)ferrocene (dppf) ligand in the ment of BC6a with tetrabutylammonium fluoride (TBAF) unveiled

presence of dimethylsulfoxide (DMSO).

all four hydroxy groups (BC6b) but attempts to incorporate the

Treatment of commercially available 5-bromoisophthalic 1,3,2-dioxaphospholane 2-oxide unit gave incomplete reaction on

acid (3a) with thionyl chloride aﬀorded 5-bromoisophthaloyl the path to the desired compound BC6 (see ESI,† Fig. S1).

dichloride, which was directly treated with N-(tert-butoxycarbonyl)- Alternative reaction with phosphorylcholine dichloride⁵¹did

ethanolamine in pyridine at 0 1C to give the Boc-protected not lead to the target bacteriochlorin BC6 (see ESI†).

3,5-diester-5-bromobenzene 3b in 83% yield for two steps.

Pd-mediated coupling of 3b with bis(pinacolato)diboron gave characterized by absorption and fluorescence spectroscopy,

Suzuki coupling partner 3 in 85% yield.

¹H NMR spectroscopy, ¹³C NMR spectroscopy (where quantity

C. Characterization. The bacteriochlorins typically were

5-Bromoisophthalic acid (3a) and 2-(tert-butyldimethyl- and solubility allowed), ³¹P NMR spectroscopy (where applicable),

siloxy)ethylamine⁴⁸were combined in the presence of 1-(3- matrix-assisted laser desorption ionization mass spectrometry

dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI) (MALDI-MS), and electrospray ionization mass spectrometry

to aﬀord the amide product 4a in 45% yield. Pd-mediated (ESI-MS). Exceptions include BC1, for which MALDI-MS spectra

coupling of 4a with bis(pinacolato)diboron gave Suzuki coupling could not be obtained, but each gave a clean ¹H NMR spectrum

partner 4 in 26% yield. This relative low coupling yield is and characteristic bacteriochlorin absorption and emission

presumably due to removal of the tert-butyldimethylsilyl (TBS) spectra. The observed charge state for the various target bacterio-

group under basic conditions at high temperature (80 1C).

chlorins upon ESI-MS analysis ranged from 1–4 (see Table S1

B. Hydrophilic bacteriochlorins. The synthesis of the set of in ESI†).

hydrophilic bacteriochlorins relied on the following strategy:

II. Incorporation of a bioconjugatable tether

A. 15-Bromination study. A potential application of hydro-

(1) introduction of the protected polar motif by Suzuki coupling

reaction of BC-Br^3,13with the above-mentioned coupling part-

ners 1–4; and (2) removal of the protecting group to give the philic bacteriochlorins entails the introduction of a conjugat-

hydrophilic bacteriochlorins directly (for BC1 and BC2), or able tether for attachment to proteins or surfaces. One strategy

followed by quaternization (for BC3 and BC4) or PEGylation in this regard relies on bromination at the 15-position of the

(for BC5).

macrocycle,⁴³which is known to occur with a high degree of

Suzuki coupling⁴⁹of BC-Br^3,13with 1 gave the protected regioselectivity for many but not all 5-methoxybacteriochlorins.

phosphono bacteriochlorin BC1a in 81% yield. The ethyl pro- The known examples of 15-bromination of 5-methoxybacterio-

tecting groups were removed by treatment with 80 equiv. of chlorins bearing diverse substituents at the b-positions (2, 12,

bromotrimethylsilane (TMSBr)^18,39in CHCl₃followed by hydro- 3, 13 positions) have been summarized.⁴²Here, bromination

lysis in methanol and water (Scheme 2). In this manner, BC1 was carried out for a set of 3,13-diarylbacteriochlorins to gauge

was obtained from the respective protected counterparts in their suitability for subsequent installation of a bioconjugat-

90% yield. The chemoselectivity and essentially quantitative able tether. The results are shown in Table 1.

nature of the protecting group cleavage reactions enabled the

resulting bacteriochlorin BC1 to be characterized and used at the 3,13-positions (BC-Ph^3,13) smoothly gave the 15-bromo-

directly without purification. bacteriochlorin as did BC2a, both of which have been examined

The parent 5-methoxybacteriochlorin bearing phenyl groups

Suzuki reaction with coupling partner 2 gave diarylbacterio- previously.⁴²BC1a under similar or somewhat more forcing

chlorin BC3a, which upon exposure to trifluoroacetic acid (TFA) bromination conditions (50 1C, 12 h) aﬀorded only a trace of

in CH₂Cl₂gave bacteriochlorin BC3b in 78% yield (Scheme 2). the desired product BC1a-Br (detected by MALDI-MS), whereas

The tetraaminobacteriochlorin provided a common precursor the majority was unreacted material. BC3a exhibited better

to both BC3 and BC5. Quaternization with iodomethane in the activity and gave an almost 1 : 1 mixture of brominated product

presence of tributylamine (Bu₃N) gave ammoniobacteriochlorin BC3a-Br and starting material, as measured by MALDI-MS

BC3 in 81% yield. Bu₃N serves to (i) neutralize the protonated assuming equal ionization eﬃciencies of these two species;

tetraamine derived from TFA, and (ii) sponge up the alkylation however, the reaction mixture could not be separated. Both

byproduct HI.⁵⁰Bu₃N was chosen versus triethylamine because BC4a and BC6a showed similar activities to that of BC2a, and

the protonated form of the former is more easily removed upon the desired 15-bromobacteriochlorins BC4a-Br and BC6a-Br

washing with tetrahydrofuran (THF). Bacteriochlorin BC3b also were isolated in good yields.

was subjected to PEGylation to give bacteriochlorin BC5 (to be

B. Bioconjugatable bacteriochlorins. Bacteriochlorins BC3

reported elsewhere). Suzuki reaction with 3 gave the 3,13-bis- and BC4 were selected for further elaboration (via BC3a-Br and

(aryl-3,5-diester)bacteriochlorin BC4a in 79% yield. Following BC4a-Br) to the bioconjugatable bacteriochlorins BC7 and BC8,

the same approach as with BC3a, deprotection of BC4a and respectively. Each target bacteriochlorin bears an NHS ester as

subsequent quaternization aﬀorded BC4 in 86% and 75% yield the bioconjugatable tether with potential application for amino-

for the two sequential steps.

protein labeling. The syntheses are displayed in Scheme 3.

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Scheme 2 Diversification of dibromobacteriochlorin BC-Br^3,13

.

The mixture obtained upon bromination of bacteriochlorin removed upon treatment with 40% TFA in CH₂Cl₂to yield BC7b

BC3a with NBS (estimated 1 : 1 ratio of BC3a-Br and BC3a, see in 99% yield. The quaternization of bacteriochlorin BC7b with

Table 1) could not be separated by column chromatography excess methyl iodide in N,N-dimethylformamide (DMF) con-

and was used directly in the Suzuki coupling reaction. Thus, taining tributylamine⁵⁰at room temperature for 24 h aﬀorded

reaction with known partner 5⁵²gave the corresponding 3,13- the ammoniobacteriochlorin BC7c in 73% yield. The cationic

diaryl-15-(carboxyalkylaryl)bacteriochlorin (BC7a) in 31% yield bacteriochlorin was precipitated by addition of diethyl ether,

for two steps. The four Boc and one tert-butyl groups were following by washing the crude precipitate with THF and

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Table 1 Bromination of 3,13-diarylbacteriochlorins^a

the phosphatidylcholine units (to form BC6) led this promising

route to be discontinued.

III. Photophysical properties

A. Spectral characteristics. Absorption and fluorescence

emission spectra were collected for each bacteriochlorin in DMF,

standard aqueous phosphate buﬀered saline (PBS; pH 7.4), and

0.5 M aqueous potassium phosphate buﬀer (PB; pH 7.0). Such

studies typically utilized bacteriochlorin concentrations of B1 mM

(e.g., A = 0.12 with e B 120 000 M^ꢀ1cm^ꢀ1for the Q_yband in a 1 cm

path); studies over a range of concentrations are described below.

All the bacteriochlorins exhibit similar absorption spectra in these

media. The spectra show characteristic bacteriochlorin features⁵³

including a strong Soret feature (with partially resolved B_yand B_x

components) in the near-ultraviolet, a modest Q_xband in the

green-yellow region, and intense Q_yband in the NIR region.

Representative spectra for BC3 in PBS and DMF are shown in

Fig. 1. Data are not given for BC1 in DMF due to minimal solubility

in that solvent. Spectral data for the bacteriochlorins in DMF and

PBS are summarized in Table 2 and in all media in Table S2 (ESI†).

As noted in the ESI† (Fig. S3), samples of the bacteriochlorins in

PBS were allowed to equilibrate after preparation prior to quanti-

tative fluorescence studies.

For each bacteriochlorin the Q_xband is at the same position

to within 1 nm in DMF, PB, and PBS, and the Q_yband to within

2 nm; the Q_yfull-width-at-half-maximum (fwhm) is the same

to within B15%. In general, the Q_yabsorption band of the

bacteriochlorins in the organic and aqueous media is quite

sharp and in the range 22–26 nm. One exception is BC4, for

which the Q_yfwhm increases from a typical 23 nm in DMF to

30–40 nm in aqueous media.

Compound R (R¹⁵= H)

Product (R¹⁵= Br) Yield (%)

BC-Ph^3,13

BC-Ph^3,13-Br

BC1a-Br

54^b

BC1a

BC2a

Trace

BC2a-Br

BC3a-Br

70^b

50^c

BC3a

BC4a

BC4a-Br

BC6a-Br

76

70

The Q_yfluorescence band of each bacteriochlorin is also quite

sharp and is typically in the range 23–26 nm (including BC4).

The fluorescence maximum is shifted from the Q_yabsorption

maximum by 6–8 nm (Table 2), corresponding to a very small

Stokes shift (Fig. 1). A weak fluorescence vibronic satellite, the

Q_y(0,1) feature, appears as a shoulder B55 nm (B800 cm^ꢀ1) to

lower energy than the Q_y(0,0) fluorescence maximum.

The eﬀect of a bioconjugatable tether on the spectral proper-

ties of the bacteriochlorins was examined for two representative

cases. BC7c (the immediate precursor to bioconjugatable, hydro-

BC6a

a

Reactions were done at 0.5 mM in THF at room temperature with

b

c

1–1.1 equiv. of NBS for 15–60 min. Ref. 42. Not isolated. Estimated

to consist of BC3a and BC3a-Br in 1 : 1 ratio by MALDI-MS assuming

equal desorption and ionization efficiencies.

diethyl ether. Coupling of the carboxylic acid of BC7c and philic bacteriochlorin BC7, which adds the tether to BC3) has

N-hydroxysuccinimide (HOSu) in the presence of N,N⁰-dicyclo- Q_yabsorption and fluorescence band positions (and fwhm) of

hexylcarbodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP) 725 nm (25 nm) and 732 nm (27 nm) in DMF, and 729 nm

aﬀorded BC7 in 80% yield (B90% purity). The same strategy (27 nm) and 735 nm (27 nm) in PBS. BC8c (the immediate

(Suzuki coupling reaction at the 15-position, deprotection to precursor to bioconjugatable, hydrophilic bacteriochlorin BC8,

remove the Boc group, introduction of the ammonium group, which adds the tether to BC4) has Q_yabsorption and fluores-

and NHS ester formation) was applied to BC4a-Br to aﬀord the cence band positions (and fwhm) of 730 nm (25 nm) and 736 nm

final target bacteriochlorin NHS ester BC8.

(27 nm) in DMF, and 727 nm (37 nm) and 736 nm (27 nm) in

Given the excellent results observed with the ammonium- PBS. Comparison with the properties of BC3 and BC4 in Table 2

substituted bacteriochlorins, comparable if not better results indicates that the tether at the 15-position aﬀects the spectral

were anticipated with the phosphatidylcholine-substituted positions by r6 nm and the bandwidths by o10%.

bacteriochlorin. Hence, in parallel with the studies of BC6a -

B. Singlet excited-state lifetime and decay-pathway yields.

BC6b - BC6, bacteriochlorin BC6a-Br also was coupled with The lowest singlet excited state (S₁,Q_y) decays by S₁- S₀

Suzuki partner 5 to give the product BC9a (39% yield; see ESI,† fluorescence, S₁- T₁intersystem crossing and S₁- S₀internal

Scheme S2); however, the (surprising) diﬃculty in fully installing conversion with yields F_f, F_iscand F_icand rate constants k_f,

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Scheme 3 Synthesis of hydrophilic bacteriochlorins bearing NHS esters.

k_iscand k_ic. All of these quantities were determined for the new 20–50% in PBS relative to that in DMF (the same disparity is

bacteriochlorins in DMF (except BC1) and PBS (Table 2). observed for BC1a; BC1 does not dissolve in DMF hence the

The hydrophilic bacteriochlorins and the corresponding hydro- comparison could not be made). The F_fvalues for three represen-

phobic precursors have similar F_fvalues in DMF, indicating that the tative cases (PBS vs. DMF) are BC2 (0.14 vs. 0.20), BC3 (0.12 vs. 0.19)

introduction of the polar motifs to bacteriochlorins does not impart and BC5 (0.16 vs. 0.20). A similar diminution was observed with

a significant adverse eﬀect on the excited-state properties (Table 2). hydrophilic chlorins³⁹and bacteriochlorins⁴²in aqueous solutions.

The F_fof bacteriochlorins BC2, BC3, and BC5 are reduced by A greater decrease in F_fis found for BC4 (0.04 vs. 0.18).

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the triplet yield, was measured using ultrafast transient absorp-

tion spectroscopy. Representative data for BC5 and BC4 in DMF

and PBS are shown in Fig. 2. The F_iscis slightly reduced for the

bacteriochlorins in aqueous solution versus DMF (Table 2). The

F

_iscvalues for (PBS vs. DMF) are as follows for BC2 (0.43 vs. 0.44),

BC3 (0.32 vs. 0.36) and BC5 (0.40 vs. 0.42). A larger reduction in

_iscis seen for BC4 (0.22 vs. 0.39). The yield of S₁- S₀internal

F

conversion (F_ic) is obtained by diﬀerence: F_ic= 1 ꢀ F_fꢀ F_isc

.

The F_icvalues (PBS vs. DMF) are as follows for BC2 (0.43 vs. 0.36),

BC3 (0.56 vs. 0.35) and BC5 (0.44 vs. 0.38). There is also

substantial increase in F_icfor BC4 (0.74 vs. 0.53).

C. Singlet excited-state decay-pathway rate constants. The

singlet excited-state lifetime (t_S) and decay yields (F_f, F_isc, F_ic)

aﬀord the corresponding rate constants (k_f, k_isc, k_ic) via the

formula k_x= F_x/t_S, where x = f, ic, isc. The latter values are listed

in the last three columns of Table 2 as the time constants (k^ꢀ1

,

in nanoseconds). The main conclusions drawn from the values

for the bacteriochlorins in PBS versus DMF are as follows:

(1) The k_fvalues do not change appreciably. The rate con-

stant for S₁- S₀spontaneous emission is related to the rate

constant for S₀- S₁stimulated absorption via the Einstein

coeﬃcients.⁵⁵As noted above (Fig. 1), each bacteriochlorin has

a similar absorption spectrum with generally similar ratios of

absorption bands in PBS and DMF, implying similar oscillator

strength of the Q_y(S₀- S₁) absorption. As such one would

expect little change in k_ffor a bacteriochlorin in the two media,

as is observed.

Fig. 1 Normalized absorption spectra (solid) and emission spectra (dashed)

of BC3 in PBS (A) or DMF (B) at room temperature.

(2) The k_iscvalues do not change appreciably for a given

bacteriochlorin in PBS versus DMF. This result is consistent

The singlet lifetime t_Sof BC1–BC5 in DMF is in the range with the expectation that the media employed should not result

1.9–3.2 ns. These values are generally similar to those for a number in an appreciable increase in spin–orbit coupling (e.g., due to an

of hydrophobic free base bacteriochlorins that have Q_ymaximum external heavy-atom effect). BC4 shows evidence for enhanced

in the same wavelength range (720–740 nm).⁵⁴The t_Svalue for (PBS vs. DMF) intermolecular interactions that could affect k_isc

each bacteriochlorin is reduced in PBS versus DMF in parallel with (and k_ic). In addition to bacteriochlorin–bacteriochlorin inter-

the F_fvalues. The t_Svalues for three representative cases (PBS vs. actions, hydrogen bonding between water and the ester-keto

DMF) are BC2 (2.1 vs. 3.1 ns), BC3 (2.2 vs. 3.1 ns) and BC5 (2.7 vs. moieties of the polar groups of BC4 could alter k_isc. However,

3.2 ns). The t_Sfor BC4 is reduced even further (1.0 vs. 2.9 ns).

a decrease rather than an increase in the rate constant would be

To understand the origin of the reduced F_fand t_Svalues, the expected if electron-withdrawing effects were the principal

yield of S₁- T₁intersystem crossing (F_isc), commonly called contributor. The result warrants further examination.

Table 2 Absorption and fluorescence properties of bacteriochlorins^a

Cmpd Solvent l_abs(nm) fwhm abs (nm) l_em(nm) fwhm em (nm) F_f

ꢀ1

F_iscF_ic

t_S(ns) (k_f)^ꢀ1(ns) (k_isc

)

(ns) (k_ic)^ꢀ1(ns)

BC1a

BC1

BC2^b

BC2

BC3a

BC3

BC4a

BC4

BC5

DMF

PBS

DMF

PBS

DMF

PBS

DMF

PBS

729

731

728

729

731

732

730

731

729

727

21

26

24

22

26

21

23

26

23

40

22

736

738

735

736

734

737

739

738

739

735

733

23

30

25

24

26

23

25

26

23

25

26

24

23

0.20

0.10

0.18

0.20

0.14

0.20

0.19

0.12

0.19

0.18

0.37 0.53 1.9

19

5

4

0.44 0.36 3.1

0.43 0.43 2.1

15

7

5

9

5

0.46 0.35 3.1

0.32 0.56 2.2

16

18

7

9

4

0.29 0.53 2.9

16

24

16

17

10

4

8

6

1

9

6

0.040 0.22 0.74 1.0

0.20

0.16

DMF

PBS

0.42 0.38 3.2

0.40 0.44 2.7

7

a

All data were acquired at room temperature. The typical errors (percent of value) of the photophysical properties are as follows: t_S(ꢁ7%),

F_f(ꢁ5%), F_isc(ꢁ15%), F_ic(ꢁ20%), k_f(ꢁ10%), k_isc(ꢁ20%), k_ic(ꢁ25%). The error bars for t_S, F_f, and F_iscwere determined from select repeat

b

measurements over a broad range of values and those for the F_ic, k_f, k_iscand k_icwere obtained from propagation of errors. Data from ref. 42.

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Fig. 2 Representative time-resolved absorption data for BC5 (A, B) and BC4 (C, D) obtained using 0.5 mJ, 100 fs excitation flashes at 730 nm.

(3) The k_icvalues are greater [the (k_ic)^ꢀ1smaller] by two-fold facilitated by use of 5% DMSO. The DMSO co-solvent was

on average for the bacteriochlorins in PBS versus DMF. An not needed in all cases, but was included in each sample for

increase in k_icwould result from inter-bacteriochlorin interac- consistency.

tions in the aqueous medium (in the ground or excited states),

The spectra for each bacteriochlorin are shown in Fig. 3

although such interactions are exhibited mainly by BC4. Although (panels B–F). While BC2 and BC4 displayed obvious band

a bathochromic shift in the Q_yband would increase k_ic,^56,57broadening indicative of some degree of aggregation, BC1,

BC1–BC5 have similar Q_ypositions and thus S₁energies. BC3 and BC5 exhibited almost unchanged spectroscopic pro-

Regardless, the combined results suggest that enhanced S₁- perties regardless of the 1000-fold change of concentration. At

S₀internal conversion primarily underlies the modest decrease all concentrations for all five bacteriochlorins, the samples

in F_fand t_Sfor the bacteriochlorins in aqueous media versus remained clear upon visual inspection indicating the absence

organic solvents. Further studies are required to elucidate the of precipitates.

mechanism(s) at play.

D. Eﬀect of concentration on spectral properties. All spectral

measurements performed above were carried out using the typical

low (micromolar) concentrations of bacteriochlorins required

Discussion

for such studies. Absorption versus concentration studies also Tetrapyrrole macrocycles are inherently hydrophobic, yet many

were conducted to assess the aqueous solution properties of the applications require their incorporation in aqueous solutions.

bacteriochlorins over a greatly expanded concentration range of Molecular design strategies that equip tetrapyrroles for solu-

1000-fold (B200–600 mM to B0.2–0.6 mM). This type of study bilization in water remain under active development.^1–5The

has been explained in detail,⁴²and the same approach was conceptual underpinning of the design explored herein is to

adopted herein. The experimental approach entails reciprocal shield the intrinsically hydrophobic faces of the tetrapyrrole

variation of concentration and cuvette pathlength (0.1–10 cm), p system from aqueous solution, by dint of steric bulk and/or

as outlined in Fig. 3 (panel A). PBS was used for BC1–BC4, electrostatic repulsion of appended polar motifs, and thereby

and neat water was used for BC5. The initial dissolution was suppress aggregation. The resulting architecture contains a

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Fig. 3 Flowchart for absorption versus concentration study (panel A). Absorption versus concentration of BC1–BC5 each over a range of 1000-fold

(panels B–F). All spectra were normalized at the Q_yband. The concentration was calculated based on absorption in the 1 cm cuvette, assuming

e(Q_y) = 120 000 M^ꢀ1cm^{ꢀ1 58}

.

hydrophilic exterior and a hydrophobic core, and as such, exhibits

Synthetic amenability is measured here by the total yield begin-

a polarity profile somewhat akin to that of a micelle. Of equal ning with the Suzuki coupling reaction of the 3,13-dibromobacterio-

importance are synthesis capabilities that together with molecular chlorin (BC-Br^3,13) and all subsequent steps. Methods for purification

design enable the desired solubility and photophysical features of compounds BC1–BC5 vary with the nature of the polar motifs.

to be attained. In the following section, we evaluate the five The rationale for emphasis on the 15-bromination is because low

solubilization motifs employed with BC1–BC5 with respect to yields in this step present a difficult separation problem; hence,

these attributes.

15-bromination can be a key bottleneck in preparation of bio-

The suitability of the solubilization motifs incorporated in conjugatable analogues.⁴²Bacteriochlorin stability is measured by

BC1–BC5 for bioconjugation studies depends on a host of the extent of bacteriochlorin (%) remaining after standing in PBS for

factors including synthetic accessibility and photophysical 48 h at 4 1C in the dark, determined by the Q_yabsorption intensity.

parameters. Five features were considered in this regard. The Bacteriochlorin stability in the light is assessed similarly in PBS

features include (1) synthesis yield from the common precursor during several hours of steady-state and time-resolved absorption

BC-Br^3,13and ease of purification, (2) ease of 15-bromination, and fluorescence studies. The results for the five bacteriochlorins

(3) water solubility, (4) fwhm of the long-wavelength (Q_y) absorp- assessed against these criteria are listed in Table 3.

tion and fluorescence bands, t_Sand F_f, and (5) stability upon

standing in the dark or in the light.

Little distinction was observed for synthesis yields, with the

range of 51–73% likely subject to improvement for any of the

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Table 3 Properties of bacteriochlorins BC1–BC5

Test Description

BC1

BC2

BC3

BC4

BC5

1a

1b

Synthesis yield^a

73%

55%

54%

51%

53%

Purification methods^bC18 chromatography Ppt, washing with

Ppt, washing with Same as BC3 Ppt, washing with

hexanes/methanol (49 : 1) Et₂O/THF (1 : 1)

Yes

G

26, 26

0.14

2.3

hexanes/CH₂Cl₂(19 : 1)

Yes, mixture

E

22, 23

0.16

3.2

95

495

2

3

4a

4b

4c

5a

5b

Derivatization^c

Water-solubility^d

fwhm (abs, flu)^e(nm)

F_f^e

No

E

26, 30

0.10

2.4

85

495

Yes, mixture

E

26,26

0.12

2.6

82

495

Yes

F

40, 26

0.04

0.9

96

495

e

t_S(ns)

Stability in dark^e(%)

Stability in light^e

96

495

a

b

c

Product of yields from all steps beginning with BC-Br^3,13until the end of the synthesis. For the final step of the synthesis. Assessed by

d

15-bromination of the relevant precursors (BC1a, BC2a, BC3a, BC4a). As measured in aqueous media by the absorption upon reciprocal change

in concentration and pathlength (E, G, F, P = excellent, good, fair, poor). For the bacteriochlorin at B1 mM in PBS.

e

bacteriochlorins via further optimization. The purification facile 15-bromination. BC4 has similarly facile 15-bromination

procedures for the final compounds vary considerably, and and stability, the broadest Q_yabsorption yet normal emission

while such consideration might seem unimportant, dyes with width, and the lowest F_fand t_Svalues. These characteristics do

charged groups can present significant difficulties with regards not bode well for use of BC4 for quantitative photochemical

to purification. The final step in the synthesis of BC1 entailed studies. BC3 and BC5 gave the narrowest absorption and

TMSBr-mediated cleavage of the ethyl phosphonate protecting fluorescence band shapes and greatest F_fand t_Svalues. BC3

groups. BC1 was purified by deprotonation of the phosphonic ranks slightly below BC5 in composite characteristics. BC3 and

acid with sodium hydroxide solution (so as to make the compound BC5 share a common precursor (BC3a), which upon 15-bromination

more polar) followed by reversed phase column chromato- yields a mixture of starting material and the desired product.

graphy (C18, 5 cm length and 1 cm diameter) using H₂O/MeOH BC3 has spectral bandwidths similar to BC1 and BC2, an inter-

(from 99 : 1 to 99 : 5) as the co-eluent. The final step in the syn- mediate F_fand slightly larger t_S, and slightly lower stability.

thesis of BC2 entailed TFA-mediated cleavage of the tert-butyl BC5 has the narrowest absorption and emission widths, the

protecting groups of the carboxylate units. BC2 was purified greatest F_fand t_S, and high stability. In general, the properties

by partitioning into the organic phase (from aqueous acid) of BC5 in PBS are the closest to those found in DMF.

followed by washing the solid with hexanes/methanol (49 : 1).⁴²

The final step in the syntheses of ammonium bacteriochlorins

BC3 and BC4 does not entail deprotection but rather quater-

nization of the free amines with methyl iodide. BC3 and BC4

Conclusions

were purified by addition of diethyl ether/THF (1 : 1) followed by The ammonium-, carboxylate-, and PEG-substituted bacterio-

sonication, centrifugation and decanting of the supernatant to chlorins all appear suitable for use with bioconjugatable tethers.

remove excessive methyl iodide and protonated tributylamine. The particular choice depends, of course, on application. For

The final step in the synthesis of BC5 also does not entail applications where fluorescence (F_f, sharpness of emission) is an

deprotection, but rather amidation of the free amines with the important parameter, the nonionic yet polar PEG–bacteriochlorin

PEG–NHS ester.

appears superior, with the incomplete 15-bromination as the

Chromatography is often regarded as the least attractive only drawback if this route is employed to install a bioconjugat-

method of purification (with regards to expense and lack of able tether.

scalability) but is widely employed in basic research. Chromato-

Much remains to be done in the area of molecular design

graphy was employed in the final step for BC1 but not for BC2– and synthetic methodology for rational and streamlined access

BC5. Consideration of the intrinsic polarity of the compounds to water-soluble tetrapyrrole macrocycles. Ultimately one goal is

suggests chromatography could have been done if needed for BC2 not only to solubilize bacteriochlorin monomers but to be able to

and BC5, but would be difficult for BC3 and BC4 given the create multipigment architectures containing bacteriochlorins that

presence of the permanently charged ammonium groups.

are water-soluble for use in diverse scientific applications.^{25,26,49,59–63}

Considering all of the factors, BC1 shows a relatively broad The design, synthesis, and physicochemical characterization

Q_yband, a lower F_fvalue and stability than others, a modest t_S, of the substituted bacteriochlorins described herein provide a

and most importantly, the precursor of BC1 is the only bacter- small step toward this goal. Yet, the 3,5-disubstituted aryl groups

iochlorin examined herein that could not be brominated. Until employed herein constitute a compromise between design objec-

methods of 15-bromination or alternative strategies for incor- tives and synthetic capabilities. One potential drawback is the

poration of a bioconjugatable tether are identified, this latter low torsional barrier for rotation about the aryl-bacteriochlorin

result at present disqualified the phosphonate motif for a variety C–C single bond, which enables motions of the polar groups

of applications. BC2 has high stability, one of the largest F_ftoward conformations more coplanar with the bacteriochlorin

values and a modest t_S, and its precursor (an aryldiester) aﬀords p framework, thereby opening the bacteriochlorin faces to

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interact with each other, or if present, with surfaces or hydro- (m with peaks at 62.42, 62.46, 62.49), 122.5, 130.0–130.3 (m

phobic patches on proteins. More attractive designs – beyond with peaks at 130.04, 130.10, 130.17), 131.2–131.5 (m with

present synthesis capabilities – would suppress or avoid such peaks at 131.38, 131.44, 131.48), 134.0–134.4 (m); ³¹P NMR

torsional motions and thereby provide more extensive facial d 25.86; ESI-MS obsd 457.0540, calcd 457.0539 [(M + H)⁺,

encumbrance.

M = C₁₆H₂₇BrO₆P₂].

2-[3,5-Bis(diethylphosphonomethyl)phenyl]-3,3,4,4-tetramethyl-

1,3,2-dioxaborolane (1). Following a general procedure,⁴⁶a mixture

of Pd(dppf)Cl₂(0.132 g, 0.180 mmol, 3 mol%), KOAc (1.76 g,

18.0 mmol) and bis(pinacolato)diboron (1.67 g, 6.60 mmol) in a

Schlenk flask was deareated under high vacuum for 20 min. Then,

a solution of DMSO (18 mL) containing 1b (2.74 g, 6.00 mmol)

(degassed for 10 min) was added under argon, and the reaction

mixture was degassed by three freeze–pump–thaw cycles. The

mixture was stirred at 80 1C for 2 h. The starting material and

product co-chromatographed upon TLC analysis [silica, ethyl

acetate/hexanes (1 : 1)]. Hence, the reaction progress was moni-

tored by ¹H NMR spectroscopy (the aromatic protons of the

product are deshielded compared with those of the starting

material) whereupon a new peak was observed at d 1.33 ppm.

The reaction mixture was allowed to cool to room temperature.

The mixture was diluted with ethyl acetate, washed with water,

dried (Na₂SO₄), and concentrated. The resulting residue was

purified by column chromatography [silica, hexanes/ethyl acetate

(1 : 1)] to obtain a light brown liquid (1.7 g, 56%): ¹H NMR (300 MHz,

CDCl₃) d 1.22–1.29 (m, 12H), 1.33 (s, 12H), 3.14 (d, J = 21.6 Hz, 4H)

3.95–4.10 (m, 8H), 7.34–7.38 (m, 1H), 7.58–7.64 (m, 2H);

¹³C NMR (75 MHz, CDCl₃) d 16.5 (d, J = 6.8 Hz), 25.0, 33.6 (d,

J = 137.2 Hz), 62.3 (d, J = 6.8 Hz), 84.0, 131.5, 133.8–134.2

(m with peaks at 133.97, 134.03, 134.09), 134.8–135.1 (m with

peaks at 134.92, 134.97, 135.02); ³¹P NMR, d 26.91; ESI-MS obsd

505.2288, calcd 505.2286 [(M + H)⁺, M = C₂₂H₃₉BO₈P₂].

Experimental section

A. Materials and methods

¹H NMR and ¹³C NMR spectroscopies were performed at room

temperature. ³¹P NMR (162 MHz) chemical shifts are reported

for the sample in CDCl₃versus the resonance of phosphoric

acid (H₃PO₄) as an external reference (insert tube). In ¹³C NMR

spectroscopy of selected compounds, not all quaternary carbons

were observed. MALDI-MS was performed with the matrix 1,4-

bis(5-phenyl-2-oxaxol-2-yl)benzene (POPOP) for bacteriochlorins,⁶⁴

except that a-cyano-4-hydroxycinnamic acid (CHCA) was used for

BC5. Silica gel (40 mm average particle size) was used for column

chromatography. All solvents were reagent grade and were used as

received unless noted otherwise. THF was freshly distilled from

sodium/benzophenone ketyl. CHCl₃was stabilized with amylenes

(r1%). Sonication was carried out in a benchtop sonication bath.

Known compounds 1a,⁴⁵2a,⁴⁷5,⁵²BC-Br^{3,13 44}BC2a,⁴²and

,

BC2⁴²were prepared following literature procedures. All other

compounds were used as received from commercial sources.

For ammoniobacteriochlorins BC3 and BC4, yield calculations

assumed the presence of the tetraiodide salt.

B. Syntheses leading to bacteriochlorins BC1–BC5, BC6

1-Bromo-3,5-bis(bromomethyl)benzene (1a). Following

a

general procedure,⁴⁵a mixture of 1-bromo-3,5-dimethylbenzene

2-[3,5-Bis(tert-butoxycarbonylaminomethyl)phenyl]-3,3,4,4-

(14.8 g, 80.0 mmol), NBS (28.5 g, 160 mmol), and AIBN (0.657 g, tetramethyl-1,3,2-dioxaborolane (2). Following a general procedure,⁴⁶

4.00 mmol, 5 mol%) in acetonitrile (400 mL) was refluxed under samples of 2a (1.25 g, 3.00 mmol), bis(pinacolato)diboron (761 mg,

argon for 5 h. The mixture was concentrated. CCl₄(75 mL) was 3.00 mmol), Pd(dppf)Cl₂(65.9 mg, 90.0 mmol), KOAc (883 mg,

added with heating to dissolve the crude product. The reaction 9.00 mmol), and DMSO (20.0 mL, deaerated by bubbling with

mixture was allowed to cool and then filtered to remove any argon for 30 min) were added to a Schlenk flask. The reaction

undissolved succinimide. The filtrate was concentrated to aﬀord the mixture was deaerated by three freeze–pump–thaw cycles. The

crude product, which upon crystallization from ethanol aﬀorded reaction mixture was stirred at 80 1C under argon for 16 h. The

1

white crystals (10.5 g, 38%): m.p. 91–93 1C; H NMR (300 MHz, reaction mixture was cooled to room temperature, diluted with

CDCl₃) d 4.40 (s, 4H), 7.32–7.35 (m, 1H), 7.47 (d, J = 1.8 Hz, 2H); ethyl acetate and washed with brine. The organic layer was

¹³C NMR (75 MHz, CDCl₃) d 31.7, 122.9, 128.5, 132.2, 140.5; anal. separated, dried (Na₂SO₄) and concentrated. Column chromato-

calcd C, 28.03; H, 2.06. Found C, 28.03; H, 1.98.

graphy [silica, hexanes/CH₂Cl₂(3 : 2)] provided a viscous liquid

1-Bromo-3,5-bis(diethylphosphonomethyl)benzene (1b). Follow- (1.10 g, 79%): ¹H NMR (400 MHz, CDCl₃) d 1.34 (s, 12H), 1.46 (s,

ing a general procedure,⁴⁵a solution of 1a (19.0 g, 55.0 mmol) and 18H), 4.32 (d, J = 5.2 Hz, 4H), 4.82 (br, 2H), 7.31 (s, 1H), 7.61 (s,

triethyl phosphite (18.6 g, 100 mmol) in toluene (30.0 mL) was 2H); ¹³C NMR (100 MHz, CDCl₃) d 25.1, 28.6, 44.8, 84.2, 129.8,

refluxed under argon for 5 h. The reaction mixture was allowed 133.0, 138.8, 156.1; ESI-MS obsd 485.2792, calcd 485.2793

to cool to room temperature whereupon toluene was removed [(M + Na)⁺, M = C₂₄H₃₉BN₂O₆].

by rotary evaporation. The resulting oily liquid was dissolved in

Bis[2-(tert-butoxycarbonylamino)ethyl] 5-bromoisophthalate

CHCl₃, washed with water, dried (Na₂SO₄), concentrated and (3b). A sample of 5-bromoisophthalic acid (3a, 490 mg, 2.00 mmol)

purified by chromatography on a short column [silica, hexanes/ in SOCl₂(5.00 mL) was stirred under argon at 65 1C for 16 h.

ethyl acetate (1 : 1)] to obtain a colorless liquid (10.5 g, 41%): The solvent was removed under vacuum. The residue was slowly

¹H NMR (300 MHz, CDCl₃) d 1.27 (t, J = 7.2 Hz, 12H), 3.09 (t, treated with a solution of N-(tert-butoxycarbonyl)ethanolamine

J = 22.2 Hz, 4H), 3.94–4.10 (m, 8H), 7.14–7.18 (m, 1H), 7.33–7.37 (800 mg, 5.00 mmol) in pyridine (4.50 mL) in an ice bath. The

(m, 2H); ¹³C NMR (75 MHz, CDCl₃) d 16.5–16.6 (m with peaks reaction mixture was stirred under argon at room temperature

at 16.51, 16.53, 16.56), 33.4 (d, J = 138 Hz), 62.4–62.5 for 16 h. The reaction mixture was diluted with ethyl acetate,

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and then washed with 1 N HCl solution and brine. The aqueous 6.65 (t, J = 4.8 Hz, 2H), 8.26 (d, J = 1.8 Hz, 2H), 8.37 (t, J = 1.8 Hz,

solution was extracted three times with ethyl acetate. The 1H); ¹³C NMR (75 MHz, CDCl₃) d ꢀ5.1, 18.5, 25.1, 26.2, 42.4,

combined organic extract was dried (Na₂SO₄), concentrated 62.0, 84.6, 129.0, 134.7, 135.6, 166.7; ESI-MS obsd 607.3762,

and chromatographed [silica, CH₂Cl₂/ethyl acetate (9 : 1)] to calcd 607.3765 [(M + H)⁺, M = C₃₀H₅₅BN₂O₆Si₂].

1

aﬀord a white sticky solid (882 mg, 83%): H NMR (400 MHz,

3,13-Bis[3,5-bis(diethylphosphonomethyl)phenyl]-5-methoxy-

CDCl₃) d 1.44 (s, 18H), 3.53–3.59 (m, 4H), 4.42 (t, J = 5.2 Hz, 4H), 8,8,18,18-tetramethylbacteriochlorin (BC1a). Following a general

5.20 (br, 2H), 8.32 (d, J = 1.6 Hz, 2H), 8.58 (s, 1H); ¹³C NMR procedure,⁴⁹samples of BC-Br^3,13(45 mg, 80 mmol), Pd(PPh₃)₄

(100 MHz, CDCl₃) d 28.6, 39.8, 65.4, 79.8, 122.8, 129.6, 132.3, (28 mg, 24 mmol) and anhydrous K₂CO₃(0.13 g, 0.96 mmol) were

136.9, 156.1, 164.6; ESI-MS obsd 553.1148, calcd 553.1156 placed in a Schlenk flask and dried under high vacuum for 1 h.

[(M + Na)⁺, M = C₂₂H₃₁O₈N₂Br].

Toluene/DMF [8.0 mL (2: 1), degassed by bubbling with argon

Bis[2-(tert-butoxycarbonylamino)ethyl] 5-(4,4,5,5-tetramethyl- for 10 min] was added along with 1 (0.12 g, 0.24 mmol), and the

1,3,2-dioxaborolan-2-yl)isophthalate (3). Following a general resulting reaction mixture was degassed by three freeze–pump–

procedure,⁴⁶samples of 3b (0.593 g, 1.12 mmol), bis(pinacolato)- thaw cycles. The reaction mixture was heated at 90 1C for 18 h.

diboron (283 mg, 1.12 mmol), Pd(pddf)Cl₂(40.8 mg, 55.8 mmol), After allowing to cool to room temperature, the toluene was

KOAc (329 mg, 3.35 mmol), and DMSO (5.6 mL, deaerated by removed by rotary evaporation. The resulting residue was

bubbling with argon for 30 min) were added to a Schlenk flask. diluted with CH₂Cl₂, and the resulting solution was washed

The reaction mixture was deaerated by three freeze–pump–thaw with aqueous NaHCO₃solution. The organic layer was separated,

cycles. The reaction mixture was stirred at 80 1C for 4 h. The dried (Na₂SO₄) and concentrated. The residue was purified by

reaction mixture was cooled to room temperature, diluted with column chromatography [silica, CH₂Cl₂/MeOH (24 : 1)] to afford

1

ethyl acetate and washed with brine. The organic layer was a green solid (75 mg, 81%): H NMR (400 MHz, CDCl₃) d ꢀ1.93

separated, dried (Na₂SO₄) and concentrated. Column chromato- (brs, 1H), ꢀ1.67 (brs, 1H), 1.30–1.40 (m, 24H), 1.95 (s, 6H), 1.97

graphy [silica, CH₂Cl₂/ethyl acetate (17 : 3)] provided a white (s, 6H), 3.37 (d, J = 15.2 Hz, 4H), 3.42 (d, J = 15.6 Hz, 4H), 3.66 (s,

1

sticky solid (548 mg, 85%): H NMR (400 MHz, CDCl₃) d 1.34 3H), 4.04–4.24 (m, 16H), 4.35 (s, 2H), 4.39 (s, 2H), 7.43 (s, 1H),

(s, 12H), 1.43 (s, 18H), 3.58 (m, 4H), 4.45 (t, J = 5.2 Hz, 4H), 5.42 7.48 (s, 1H), 7.94 (d, J = 1.6 Hz, 2H), 8.02 (s, 2H), 8.58 (d, J =

(s, 2H), 8.61 (d, J = 2.0 Hz, 2H), 8.74 (t, J = 2.0 Hz, 1H); ¹³C NMR 2.0 Hz, 1H), 8.61 (s, 1H), 8.64 (s, 1H), 8.76 (d, J = 2.0 Hz, 1H),

(100 MHz, CDCl₃) d 25.0, 28.5, 39.8, 64.9, 79.5, 84.6, 130.0, 8.80 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) d 16.7 (d, J = 3.0 Hz),

133.6, 140.2, 156.1, 165.9; ESI-MS obsd 601.2908, calcd 601.2903 31.3, 31.4, 34.0 (dd, J = 138.2 Hz & 14.5 Hz), 45.7, 45.8, 47.8,

[(M + Na)⁺, M = C₂₈H₄₃BN₂O₁₀].

52.1, 62.4 (dd, J = 16.0 Hz & 6.8 Hz), 63.3, 96.9, 122.3, 122.6,

Bis[2-(tert-butyldimethylsiloxy)ethyl] 5-bromoisophthalate (4a). 127.4, 129.9, 130.5, 131.2, 131.5, 132.9 (d, J = 11. 3 Hz), 133.3,

A sample of 5-bromoisophthalic acid (3a, 1.96 g, 8.00 mmol), 134.0, 135.2, 135.5, 135.8, 136.3, 137.0, 138.8, 154.0, 160.6, 169.1,

2-(tert-butyldimethylsiloxy)ethylamine (4.21 g, 24.0 mmol) and 169.72; ³¹P NMR d 26.9, 27.1; MALDI-MS obsd 1153.3; ESI-MS

EDCI (4.60 g, 24.0 mmol) in a mixed solvent of DMF and CH₂Cl₂obsd 577.2407, calcd 577.2409 [(M + 2H)²⁺, M = C₅₇H₈₀N₄O₁₃P₄],

(10 mL, 1 : 1) was stirred at room temperature for 16 h. The l_abs(CH₂Cl₂) 363, 510, 730 nm.

reaction mixture was diluted with CH₂Cl₂, and then washed with

2,12-Bis[3,5-bis(phosphonomethyl)phenyl]-5-methoxy-8,8,18,18-

brine for 6 times. The organic solution was dried (Na₂SO₄), con- tetramethylbacteriochlorin (BC1). Following a reported procedure,³⁹

centrated and chromatographed [silica, CH₂Cl₂/ethyl acetate (7 : 3)] a solution of BC1a (3.1 mg, 2.7 mmol) in anhydrous CHCl₃(0.14 mL)

1

to aﬀord a white sticky solid (2.00 g, 45%): H NMR (300 MHz, was treated with TMSBr (29 mL, 0.22 mmol, 80 equiv.) under

CDCl₃) d 0.05 (s, 12H), 0.88 (s, 18H), 3.51–3.56 (m, 4H), 3.76 argon, and the reaction mixture was stirred in the dark for 16 h.

(t, J = 5.7 Hz, 4H), 6.80 (t, J = 4.8 Hz, 2H), 7.98 (d, J = 1.8 Hz, 2H), Methanol (0.30 mL) was then added, and the mixture was stirred

8.06 (t, J = 1.8 Hz, 1H); ¹³C NMR (75 MHz, CDCl₃) d ꢀ5.1, 18.5, for 3 h. The solvent was removed by rotary evaporation, and the

26.1, 42.6, 61.8, 123.1, 124.2, 133.1, 137.0, 165.5; ESI-MS obsd residue was dried under high vacuum for 1 h. A solution of

559.2016, calcd 559.2018 [(M + H)⁺, M = C₂₄H₄₃BrN₂O₄Si₂].

0.25 M NaOH (1.0 mL) was added to the dried reaction mixture

Bis[2-(tert-butyldimethylsiloxy)ethyl] 5-(4,4,5,5-tetramethyl- to give a green solution. The solution was concentrated to

1,3,2-dioxaborolan-2-yl)isophthalate (4). Following a general dryness. The residue was dissolved in a minimum amount of

procedure,⁴⁶samples of 4a (2.00 g, 3.57 mmol), bis(pinacolato)- water and chromatographed [C18 silica, H₂O/MeOH (from 99 : 1

1

diboron (907 mg, 3.57 mmol), Pd(pddf)Cl₂(131 mg, 179 mmol), to 99 : 5)] to aﬀord a dark green solid (2.5 mg, 90%): H NMR

KOAc (1.05 g, 10.7 mmol), and DMSO (17.8 mL, deaerated by [300 MHz, CDCl₃/CD₃OD (1 : 1); two pyrrolic-NH and eight

bubbling with argon for 30 min) were added to a Schlenk flask. phosphonic acid protons were not observed] d 1.81 (s, 6H),

The reaction mixture was deaerated by three freeze–pump–thaw 1.87 (s, 6H), 2.96–3.06 (m, 8H), 3.63 (s, 3H), 4.32 (s, 2H), 4.51

cycles. The reaction mixture was stirred at 80 1C for 16 h. The (s, 2H), 7.41 (s, 1H), 7.47 (s, 1H), 7.73 (s, 2H), 7.93 (s, 2H), 8.69

reaction mixture was cooled to room temperature, diluted with (s, 1H), 8.73 (s, 1H), 8.75 (s, 1H), 8.85 (s, 1H), 8.95 (s, 1H); l_abs(PB)

ethyl acetate and washed with brine. The organic layer was 353, 508, 730 nm.

separated, dried (Na₂SO₄) and concentrated. Column chromato-

3,13-Bis[3,5-bis(tert-butoxycarbonylaminomethyl)phenyl]-5-

graphy [silica, CH₂Cl₂/ethyl acetate (7 : 3)] provided a white sticky methoxy-8,8,18,18-tetramethylbacteriochlorin (BC3a). Following

solid (569 mg, 26%): ¹H NMR (300 MHz, CDCl₃) d 0.08 (s, 12H), a general procedure,⁴⁹samples of BC-Br^3,13(44.6 mg, 80.0 mmol),

0.91 (s, 18H), 1.34 (s, 12H), 3.55–3.61 (m, 4H), 3.79 (t, J = 5.7 Hz, 4H), 2 (81.4 mg, 176 mmol), Pd(PPh₃)₄(55.6 mg, 48.1 mmol), Cs₂CO₃

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(156 mg, 480 mmol) and toluene/DMF [4.00 mL (2 : 1), deaerated 3.58 (s, 3H), 4.30 (s, 2H), 4.37 (s, 2H), 4.81 (s, 4H), 4.87 (s, 4H),

by bubbling with argon for 45 min] were added to a Schlenk 7.84 (s, 1H), 7.90 (s, 1H), 8.40 (s, 2H), 8.56 (s, 2H), 8.80 (s, 1H),

flask. The mixture was deaerated by three freeze–pump–thaw 8.87–8.90 (m, 2H), 8.95 (s, 1H), 9.16 (s, 1H); obsd 210.1528,

cycles. The reaction mixture was stirred at 90 1C for 18 h. The calcd 210.1530 [(M ꢀ 4I)⁴⁺, M = C₅₃H₇₆I₄N₈O]; l_abs(PB) 363,

reaction mixture was cooled to room temperature, concentrated to 519, 731 nm.

dryness, diluted with CH₂Cl₂and washed with saturated aqueous

3,13-Bis[3,5-bis(2-(tert-butoxycarbonylamino)ethoxycarbonyl)-

NaHCO₃. The organic layer was separated, dried (Na₂SO₄), con- phenyl]-5-methoxy-8,8,18,18-tetramethylbacteriochlorin (BC4a).

centrated and chromatographed [silica, CH₂Cl₂/ethyl acetate Following a general procedure,⁴⁹samples of BC-Br^3,13(44.6 mg,

(4 : 1)]. The resulting solid was treated with hexanes/ethyl acetate 80.0 mmol), 3 (44.5 mg, 134 mmol), Pd(PPh₃)₄(12.4 mg, 10.7 mmol)

(4 : 1), sonicated, and centrifuged. The supernatant was discarded and Cs₂CO₃(34.9 mg, 107 mmol) were placed in a Schlenk flask

to afford a green solid (72.7 mg, 85%): ¹H NMR (300 MHz, CDCl₃) and dried under high vacuum for 30 min. Toluene/DMF [2.70 mL

d ꢀ1.92 (s, 1H), ꢀ1.66 (s, 1H), 1.50 (s, 36H), 1.95 (s, 6H), 1.97 (s, (2 : 1), deaerated by bubbling with argon for 45 min] was added to

6H), 3.62 (s, 3H), 4.37 (s, 2H), 4.39 (s, 2H), 4.54–4.61 (m, 8H), the Schlenk flask under argon and deaerated by three freeze–

5.05 (br, 4H), 7.38 (s, 1H), 7.44 (s, 1H), 7.94 (s, 2H), 7.98 (s, 2H), pump–thaw cycles. The reaction mixture was stirred at 90 1C for

8.59–8.64 (m, 3H), 8.73 (d, J = 5.7 Hz, 2H); ¹³C NMR (100 MHz, 13 h. The reaction mixture was allowed to cool to room tempera-

CDCl₃) d 28.70, 28.76, 31.33, 31.41, 44.67, 44.79, 45.00, 45.09, ture, then concentrated to dryness, diluted with CH₂Cl₂and

45.76, 45.90, 47.9, 52.2, 63.4, 79.75, 79.82, 79.95, 84.2, 97.0, washed with saturated aqueous NaHCO₃. The organic layer was

113.6, 117.7, 122.45, 122.53, 125.2, 125.5, 125.7, 127.5, 128.4, separated, dried (Na₂SO₄), concentrated and chromatographed

129.2, 129.5, 130.2, 130.8, 133.6, 134.1, 134.86, 134.92, 134.98, [silica, CH₂Cl₂/ethyl acetate (4 :1)]. Treatment of the resulting

135.26, 135.32, 135.39, 135.6, 136.2, 136.4, 137.3, 138.8, 139.1, solid with hexanes/CH₂Cl₂(4 : 1) afforded a suspension, which

140.2, 140.4, 140.9, 150.0, 141.7, 154.2, 156.30, 156.40, 156.47, was sonicated followed by centrifugation. The supernatant was

157.6, 160.8, 169.2, 169.9; MALDI-MS obsd 1070.7; ESI-MS obsd discarded to afford a green solid (82.3 mg, 79%): ¹H NMR

1068.6043, calcd 1068.6043 (M⁺, M = C₆₁H₈₀N₈O₉); l_abs(CH₂Cl₂) (300 MHz, CDCl₃) d ꢀ1.80 (s, 1H), ꢀ1.56 (s, 1H), 1.42 (s, 36H),

363, 510, 729 nm.

1.96 (s, 6H), 1.98 (s, 6H), 3.62–3.66 (m, 11H), 4.38 (s, 2H), 4.40

3,13-Bis[3,5-bis(aminomethyl)phenyl]-5-methoxy-8,8,18,18- (s, 2H), 4.50–4.60 (m, 8H), 5.02–5.07 (m, 4H), 8.66 (s, 1H), 8.69

tetramethylbacteriochlorin (BC3b). A solution of BC3a (20.4 mg, (s, 3H), 8.87 (s, 2H), 8.92 (s, 1H), 9.02–9.04 (m, 4H); ¹³C NMR

19.1 mmol) in CH₂Cl₂(1.15 mL) was stirred under argon for 2 min, (100 MHz, CDCl₃) d 28.6, 31.30, 31.38, 40.0, 45.8, 46.0, 47.8,

followed by addition of TFA (768 mL). After 20 min, the reaction 52.2, 63.4, 65.21, 65.32, 79.9, 96.6, 97.41, 97.49, 122.6, 123.1,

mixture was diluted with CHCl₃and dried under an argon flow. 127.3, 128.3, 129.5, 129.9, 130.1, 131.44, 131.46, 134.18, 134.23,

Tributylamine (77.8 mg, 0.420 mmol) was added to the solid 135.1, 135.6, 136.32, 136.39, 136.8, 137.6, 139.3, 154.6, 156.1,

residue, and the mixture was sonicated for 3 min. A mixture of 161.3, 166.1, 166.4, 169.8, 170.5; MALDI-MS obsd 1302.7; ESI-MS

THF and hexanes (1 : 1) was then added. The resulting sus- obsd 1301.6335, calcd 1301.6340 [(M + H)⁺, M = C₆₉H₈₈N₈O₁₇];

pension was sonicated for 5 min followed by centrifugation. l_abs(CH₂Cl₂) 364, 511, 732 nm.

The supernatant was discarded to aﬀord a green solid (9.9 mg,

3,13-Bis[3,5-bis(2-aminoethoxycarbonyl)phenyl]-5-methoxy-

78%): ¹H NMR (300 MHz, CD₃OD, the four primary amine 8,8,18,18-tetramethylbacteriochlorin (BC4b). A solution of BC4a

protons and two pyrrolic-NH protons were not observed) d 1.99 (42.0 mg, 32.3 mmol) in CH₂Cl₂(1.94 mL) was stirred under

(s, 6H), 2.01 (s, 6H), 3.70 (s, 3H), 4.40 (s, 2H), 4.41 (s, 4H), 4.45 argon for 2 min, followed by addition of TFA (1.29 mL). After

(s, 2H), 4.47 (s, 4H), 7.72 (s, 1H), 7.80 (s, 1H), 8.28 (d, J = 1.8 Hz, 1 h, the reaction mixture was diluted with CHCl₃and dried

2H), 8.34 (d, J = 1.8 Hz, 2H), 8.74 (s, 1H), 8.77 (s, 1H), 8.83 (s, under an argon flow. Tributylamine (300 mL) was added to the

1H), 8.87 (s, 1H), 8.99 (s, 1H); MALDI-MS obsd 668.7; ESI-MS solid residue, and the resulting suspension was sonicated for

obsd 669.4026, calcd 669.4024 [(M + H)⁺, M = C₄₁H₄₈N₈O]; l_abs3 min. A mixture of THF and diethyl ether (1 : 1) was then

(methanol) 360, 507, 726 nm.

added, and the resulting suspension was sonicated for 5 min

3,13-Bis[3,5-bis(N,N,N-trimethylammoniomethyl)phenyl]-5- followed by centrifugation. The supernatant was discarded to

1

methoxy-8,8,18,18-tetramethylbacteriochlorin diiodide (BC3). aﬀord a green solid (25 mg, 86%): H NMR (300 MHz, CD₃OD/

A mixture of BC3b (4.0 mg, 6.0 mmol) and tributylamine (44 mg, CDCl₃; the eight primary amine protons and two pyrrolic-NH

0.24 mmol) in DMF (0.20 mL) was stirred under argon for 2 min, protons were not observed) d 1.98 (s, 6H), 2.01 (s, 6H), 3.44–4.51

followed by addition of iodomethane (45 mL, 0.72 mmol). After (m, 8H), 3.64 (s, 3H), 4.40 (s, 2H), 4.42 (s, 2H), 4.70–4.77

16 h, diethyl ether/THF (1 : 1) was added to the reaction mixture (m, 8H), 8.74 (s, 1H), 8.80 (d, J = 2.7 Hz, 2H), 8.87 (s, 1H),

to precipitate the crude product. The suspension was sonicated 8.99 (t, J = 1.5 Hz, 1H), 9.03 (t, J = 1.5 Hz, 1H), 9.06 (s, 1H), 9.10

for 3 min followed by centrifugation. The supernatant was (d, J = 1.5 Hz, 2H), 9.15 (d, J = 1.5 Hz, 2H); MALDI-MS obsd 900.4;

discarded to aﬀord a green solid. This procedure (diethyl ether obsd 301.1462, calcd 301.1463 [(M + 3H)³⁺, M = C₄₉H₅₆N₈O₉]; l_abs

and THF addition/sonication/centrifugation) was carried out (methanol) 361, 508, 727 nm.

three additional times to aﬀord a green solid (6.5 mg, 81%):

3,13-Bis[3,5-bis(2-(N,N,N-trimethylammonio)ethoxycarbonyl)-

¹H NMR (300 MHz, DMSO-d₆, the ammoniomethyl peaks were phenyl]-5-methoxy-8,8,18,18-tetramethylbacteriochlorin diiodide

overlapped with that from the moisture in the solvent) d ꢀ1.93 (BC4). A mixture of BC4b (6.2 mg, 6.9 mmol) and tributylamine

(s, 1H), ꢀ1.69 (s, 1H), 1.91 (s, 6H), 1.93 (s, 6H), 3.25 (s, 36H), (51 mg, 0.28 mmol) in DMF (0.20 mL) was stirred under argon

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for 2 min, followed by addition of iodomethane (51 mL, C. Bacteriochlorin bromination and bioconjugation

0.83 mmol). After 16 h, diethyl ether/THF (1 : 1) was added to

the reaction mixture to precipitate the crude product. The sus-

pension was sonicated for 3 min followed by centrifugation. The

supernatant was discarded to aﬀord a green solid. This procedure

Attempted synthesis of 15-bromo-3,13-bis[3,5-bis(diethyl-

phosphonomethyl)phenyl]-5-methoxy-8,8,18,18-tetramethylbacterio-

chlorin (BC1a-Br). Following a general procedure,⁴⁹a solution

of BC1a (10.0 mg, 8.70 mmol) in THF (4.35 mL) was treated with

NBS (1.54 mg, 8.70 mmol) in THF (2.17 mL) at room temperature

for 1.5 h. MALDI-MS and absorption spectroscopy showed no

(diethyl ether and THF addition/sonication/centrifugation) was

carried out three more times to aﬀord a green solid (8.2 mg, 75%):

¹H NMR (300 MHz, DMSO-d₆) d ꢀ1.87 (s, 1H), ꢀ1.62 (s, 1H), 1.91

detectable product. The reaction was then heated to 50 1C for 12 h,

(s, 6H), 1.95 (s, 6H), 3.25 (s, 36H), 3.54 (s, 3H), 3.91 (br, 8H), 4.29

whereupon a trace amount of product was detected by MALDI-MS,

(s, 2H), 4.34 (s, 2H), 4.86 (br, 8H), 8.72–8.82 (m, 3H), 8.90 (s, 1H),

but could not be isolated from the reaction mixture.

8.94–9.04 (m, 6H), 9.20 (s, 1H); obsd 268.1584, calcd 268.1585

15-Bromo-3,13-bis[3,5-bis(2-(tert-butoxycarbonylamino)ethoxy-

carbonyl)phenyl]-5-methoxy-8,8,18,18-tetramethylbacteriochlorin

[(M ꢀ 4I)⁴⁺, M = C₆₁H₈₄I₄N₈O₉]; l_abs(PB) 363, 519, 731 nm.

3,13-Bis[3,5-bis(2-(tert-butyldimethylsiloxy)ethylaminocarbonyl)-

(BC4a-Br). Following a general procedure,⁴⁹a solution of BC4a

phenyl]-5-methoxy-8,8,18,18-tetramethylbacteriochlorin (BC6a).

(42.6 mg, 32.7 mmol) in THF (65.4 mL) was treated with NBS

Following a general procedure,⁴⁹samples of BC-Br^3,13(168 mg,

(5.80 mg, 32.7 mmol) in THF (327 mL) at room temperature for

300 mmol), 4 (401 mg, 660 mmol), Pd(PPh₃)₄(104 mg, 90.0 mmol)

15 min. The reaction mixture was diluted with CH₂Cl₂and washed

and K₂CO₃(249 mg, 1.80 mmol) were placed in a Schlenk flask

with saturated aqueous NaHCO₃. The organic layer was dried

and dried under high vacuum for 30 min. Toluene/DMF [15.0 mL

(Na₂SO₄), concentrated and chromatographed [silica, CH₂Cl₂/

(2 : 1), deaerated by bubbling with argon for 45 min] was added to

ethyl acetate (7 : 3)] to aﬀord a red solid (34.3 mg, 76%): ¹H NMR

the Schlenk flask under argon and deaerated by three freeze–

(300 MHz, CDCl₃) d ꢀ1.63 (s, 1H), ꢀ1.33 (s, 1H), 1.40 (s, 18H),

pump–thaw cycles. The reaction mixture was stirred at 90 1C for

1.42 (s, 18H), 1.96 (s, 6H), 1.97 (s, 6H), 3.57–3.64 (m, 11H), 4.37

16 h. The reaction mixture was cooled to room temperature,

(s, 2H), 4.41 (s, 2H), 4.48–4.54 (m, 8H), 4.98 (br, 2H), 5.07 (br,

concentrated to dryness, diluted with CH₂Cl₂and washed with

saturated aqueous NaHCO₃. The organic layer was separated,

2H), 8.63 (s, 1H), 8.67 (s, 1H), 8.69 (d, J = 2.4 Hz, 1H), 8.74–8.76

(m, 3H), 8.89 (t, J = 1.5 Hz, 2H), 8.89 (d, J = 1.5 Hz, 2H); ¹³C NMR

(100 MHz, CDCl₃) d 28.63, 28.65, 31.5, 31.7, 40.1, 45.6, 45.9,

dried (Na₂SO₄), concentrated and chromatographed [silica,

CH₂Cl₂/ethyl acetate (9 : 1 to 13 : 7)]. Treatment of the resulting

48.2, 54.8, 63.7, 65.3, 79.9, 97.2, 97.5, 99.0, 125.1, 126.2, 129.4,

solid with hexanes/CH₂Cl₂(9 : 1) afforded a suspension, which

129.87, 129.97, 130.12, 130.3, 130.9, 132.6, 133.6, 133.8, 135.4,

was sonicated followed by centrifugation. The supernatant was

136.2, 136.7, 137.0, 138.6, 140.4, 156.2, 157.8, 160.3, 166.3,

discarded to afford a green solid (330 mg, 81%): ¹H NMR (300 MHz,

168.7, 172.0; ESI-MS obsd 1379.5458, calcd 1379.5445 [(M + H)⁺,

M = C₆₉H₈₇BrN₈O₁₇]; MALDI-MS obsd 1382.6; l_abs(CH₂Cl₂) 368,

CDCl₃) d ꢀ1.85 (s, 1H), ꢀ1.60 (s, 1H), 0.09 (s, 12H), 0.10 (s, 12H),

0.88 (s, 18H), 0.90 (s, 18H), 1.96 (s, 6H), 1.99 (s, 6H), 3.59 (s, 3H),

523, 729 nm.

3.67–3.74 (m, 8H), 3.86–3.91 (m, 8H), 4.37 (s, 2H), 4.39 (s, 2H), 6.82

15-Bromo-3,13-bis[3,5-bis(2-(tert-butyldimethylsiloxy)ethyl-

(t, J = 4.8 Hz, 4H), 8.40 (t, J = 1.5 Hz, 1H), 8.43 (t, J = 1.5 Hz, 1H), 8.65

aminocarbonyl)phenyl]-5-methoxy-8,8,18,18-tetramethylbacterio-

(s, 1H), 8.67 (s, 1H), 8.69–8.72 (m, 6H), 8.82 (d, J = 2.4 Hz, 1H);

chlorin (BC6a-Br). Following a general procedure,⁴⁹a solution of

¹³C NMR (100 MHz, CDCl₃) d ꢀ5.02, ꢀ5.01, 18.55, 18.57, 26.16,

BC6a (47.5 mg, 35.0 mmol) in THF (70.0 mL) was treated with

NBS (6.85 mg, 38.5 mmol) in THF (38.5 mL) at room temperature

for 15 min. The reaction mixture was diluted with CH₂Cl₂and

26.18, 31.37, 42.68, 42.71, 45.8, 45.9, 47.9, 52.2, 62.05, 62.12, 63.3,

96.7, 97.3, 122.84, 122.88, 124.3, 124.4, 127.3, 132.0, 132.3, 132.6,

134.3, 134.6, 134.7, 135.1, 135.6, 136.1, 136.3, 137.8, 139.3, 154.5,

washed with saturated aqueous NaHCO₃. The organic layer was

161.2, 166.9, 167.2, 169.7, 170.2; MALDI-MS obsd 1357.2; ESI-MS

obsd 1357.7680, calcd 1357.7702 [(M + H)⁺, M = C₇₃H₁₁₂N₈O₉Si₄];

dried (Na₂SO₄), concentrated and chromatographed [silica,

CH₂Cl₂/ethyl acetate (7 : 3)] to aﬀord a red solid (35.0 mg,

70%): ¹H NMR (400 MHz, CDCl₃) d ꢀ1.68 (s, 1H), ꢀ1.37 (s,

1H), 0.07 (s, 12H), 0.09 (s, 12H), 0.86 (s, 18H), 0.87 (s, 18), 1.96

(s, 6H), 1.97 (s, 6H), 3.61 (s, 3H), 3.64–3.72 (m, 8H), 3.83–3.89

(m, 8H), 4.36 (s, 2H), 4.40 (s, 2H), 6.79 (t, J = 5.6 Hz, 2H), 6.86

(t, J = 5.6 Hz, 2H), 8.41 (s, 2H), 8.44 (s, 1H), 8.61 (s, 1H),

8.66 (s, 1H), 8.67 (d, J = 2.4 Hz, 1H), 8.69 (s, 2H), 8.73 (d, J =

2.4 Hz, 2H); MALDI-MS obsd 1439.8; ESI-MS obsd 1435.6798,

calcd 1435.6807 [(M + H)⁺, M = C₇₃H₁₁₁BrN₈O₉Si₄]; l_abs(CH₂Cl₂)

368, 523, 729 nm.

3,13-Bis[3,5-bis(tert-butoxycarbonylaminomethyl)phenyl]-15-

[4-(2-(tert-butoxycarbonyl)ethyl)phenyl]-5-methoxy-8,8,18,18-tetra-

methylbacteriochlorin (BC7a). Following a general procedure,⁴⁹

a solution of BC3a (88.0 mg, 82.3 mmol) in THF (165 mL) was

treated with NBS (17.6 mg, 98.8 mmol) in THF (988 mL) at room

temperature for 1.5 h. The reaction mixture was diluted with

l

_abs(CH₂Cl₂) 364, 511, 732 nm.

3,13-Bis[3,5-bis(2-hydroxyethoxycarbonyl)phenyl]-5-methoxy-

8,8,18,18-tetramethylbacteriochlorin (BC6b). A solution of BC6a

(120 mg, 88.0 mmol) in THF (88.0 mL) was stirred under argon

for 2 min, followed by addition of 1 M TBAF solution in THF

(528 mL, 528 mmol). After 40 min, the solvent was removed

under vacuum, and the reaction residue was chromatographed

[silica, CH₂Cl₂/methanol (9 : 1 to 7 : 3)] to aﬀord a green solid

(56.0 mg, 71%): ¹H NMR (300 MHz, CD₃OD/CDCl₃; the four OH,

four amido NH, and two pyrrolic NH protons were not

observed) d 1.97 (s, 6H), 2.00 (s, 6H), 3.64 (s, 3H), 3.65–3.71

(m, 8H) 3.83–3.88 (m, 8H), 4.37 (s, 2H), 4.43 (s, 2H), 8.49 (s, 1H),

8.52 (s, 1H), 8.68 (s, 1H), 8.72 (s, 2H), 8.75 (s, 2H), 8.79 (s, 1H),

8.84 (s, 2H), 8.93 (s, 1H); MALDI-MS obsd 900.3; ESI-MS obsd

451.2159, calcd 451.2158 [(M + 2H)²⁺, M = C₄₉H₅₆N₈O₉]; l_abs

(methanol) 361, 508, 727 nm.

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CH₂Cl₂and washed with saturated aqueous NaHCO₃. The

15-[4-(2-Carboxyethyl)phenyl]-3,13-bis[3,5-bis(N,N,N-trimethyl-

organic layer was dried (Na₂SO₄), concentrated and chromato- ammoniomethyl)phenyl]-5-methoxy-8,8,18,18-tetramethylbacterio-

graphed [silica, hexanes/CH₂Cl₂(1 : 4)] to aﬀord a red solid. TLC chlorin tetraiodide (BC7c). A mixture of BC7b (12.3 mg, 15.0 mmol)

analysis was uninformative concerning the progress of the and tributylamine (112 mg, 602 mmol) in DMF (300 mL) was stirred

reaction, as no new species appeared distinct from that of under argon for 2 min, followed by addition of iodomethane

the starting material; however, MALDI-MS analysis showed (256 mL, 1.81 mmol). After 24 h, diethyl ether and THF (1 : 1) was

the presence of the starting material and the brominated pro- added to the reaction mixture to precipitate the crude product.

duct thereof (BC3a-Br): MALDI-MS obsd 1147.1, calcd 1146.5 The suspension was sonicated for 3 min followed by centrifu-

(C₆₁H₇₉BrN₈O₉); obsd 1069.2, calcd 1068.6 (C₆₁H₈₀N₈O₉). Given gation. The supernatant was discarded to aﬀord a green solid.

the inability to purify the brominated bacteriochlorin, the This procedure (diethyl ether and THF addition/sonication/

entire crude product was transferred to a Schlenk flask. Samples centrifugation) was carried out three more times to aﬀord a

of 5 (67.8 mg, 0.204 mmol), Pd(PPh₃)₄(19.2 mg, 16.6 mmol), and green solid (16.4 mg, 73%): ¹H NMR (300 MHz, CD₃OD, the

Cs₂CO₃(82.2 mg, 0.252 mmol) were placed in the Schlenk flask, carboxylic acid proton and two pyrrolic-NH protons were not

and dried under high vacuum for 30 min. Toluene/DMF [4.2 mL observed, and resonances from the four ethylene protons were

(2 : 1), deaerated by bubbling with argon for 30 min] was added overlapped with the solvent peak) d 1.84 (s, 6H), 2.01 (s, 6H), 3.21 (s,

to the Schlenk flask under argon, and the mixture was deaer- 18H), 3.41 (s, 18H), 3.72 (s, 3H), 3.88 (s, 2H), 4.37 (s, 2H), 4.93 (s,

ated by three freeze–pump–thaw cycles. The reaction mixture 8H), 7.18 (d, J = 6.0 Hz, 2H), 7.50 (d, J = 6.0 Hz, 2H), 7.60 (s, 1H), 7.75

was stirred at 90 1C for 19 h. The reaction mixture was cooled (d, J = 6.0 Hz, 2H), 8.12 (s, 1H), 8.55 (s, 2H), 8.78–8.91 (m, 4H); ESI-

to room temperature, concentrated to dryness, diluted with MS obsd 247.1662, calcd 247.1661 [(M ꢀ 4I)⁴⁺, M = C₆₂H₈₄I₄N₈O₃];

ethyl acetate and washed with saturated aqueous NaHCO₃. The

organic layer was separated, dried (Na₂SO₄), concentrated and

l

_abs(0.5 M phosphate buffer, pH 7.0) 362, 519, 731 nm.

3,13-Bis[3,5-bis(N,N,N-trimethylammoniomethyl)phenyl]-5-

chromatographed [silica, hexanes/ethyl acetate (8 : 2 to 7 : 3)] to methoxy-15-[4-(2-(N-succinimidooxycarbonyl)ethyl)phenyl]-8,8,18,18-

provide a green solid (33.0 mg, 31%): ¹H NMR (400 MHz, tetramethylbacteriochlorin tetraiodide (BC7). A mixture of BC7c

CDCl₃) d ꢀ1.61 (s, 1H), ꢀ1.24 (s, 1H), 1.48 (s, 18H), 1.50 (s, (9.8 mg, 6.5 mmol), DCC (6.7 mg, 33 mmol), and DMAP (0.20 mg,

18H), 1.52 (s, 9H), 1.83 (s, 6H), 1.97 (s, 6H), 2.59 (t, J = 7.6 Hz, 1.64 mmol) in DMF (654 mL) was treated with HOSu (3.8 mg,

2H), 2.93 (t, J = 7.6 Hz, 2H), 3.66 (s, 3H), 3.89 (s, 2H), 4.27 (d, 33 mmol) and stirred under argon at room temperature. After

J = 5.6 Hz, 4H), 4.37 (s, 2H), 4.56 (d, J = 5.6 Hz, 4H), 4.90 (br, 2H), 12 h, diethyl ether was added to the reaction mixture to preci-

5.07 (br, 2H), 6.98 (s, 1H), 7.04 (d, J = 7.6 Hz, 2H), 7.11 (s, 2H), pitate the crude product. The suspension was sonicated for

7.39–7.42 (m, 3H), 7.97 (s, 2H), 8.57 (d, J = 2.4 Hz, 1H), 8.62–8.64 3 min followed by centrifugation. The supernatant was discarded

(m, 3H); ¹³C NMR (100 MHz, CDCl₃) d 28.4, 28.7, 30.0, 30.9, to aﬀord a green solid. This procedure (diethyl ether addition/

31.3, 36.9, 44.7, 45.1, 45.2, 45.9, 47.8, 52.3, 63.5, 79.7, 80.8, sonication/centrifugation) was carried out three more times to

97.0, 97.5, 113.8, 123.1, 124.0, 125.2, 126.5, 127.2, 128.2, aﬀord a green solid (8.3 mg, B90% purity, 80% yield): ¹H NMR

129.2, 129.5, 133.6, 133.8, 134.0, 134.2, 136.2, 136.9, 137.7, (300 MHz, CD₃OD, the two pyrrolic-NH protons were not

138.8, 138.9, 139.2, 139.3, 154.9, 156.1, 156.2, 160.9, 168.9, observed, and the four ethylene protons were overlapped with

172.7; MALDI-MS obsd 1274.9; ESI-MS obsd 648.3581, calcd the solvent peak) d 1.86 (s, 6H), 2.01 (s, 6H), 3.16 (s, 4H), 3.21

648.3582 [(M + H + Na)²⁺, M = C₇₄H₉₆N₈O₁₁]; l_abs(CH₂Cl₂) 367, (s, 18H), 3.41 (s, 18H), 3.72 (s, 3H), 3.89 (s, 2H), 4.38 (s, 2H), 4.98

517, 729 nm.

(s, 8H), 7.20 (d, J = 6.0 Hz, 2H), 7.50 (d, J = 6.0 Hz, 2H), 7.60

15-[4-(2-Carboxyethyl)phenyl]-3,13-bis[3,5-bis(aminomethyl)- (s, 1H), 7.75 (d, J = 6.0 Hz, 2H), 8.12 (s, 1H), 8.55 (s, 2H), 8.78–8.91

phenyl]-5-methoxy-8,8,18,18-tetramethylbacteriochlorin (BC7b). (m, 4H); l_abs(PB) 363, 519, 731 nm.

A solution of BC7a (15.5 mg, 12.2 mmol) in CH₂Cl₂(730 mL)

3,13-Bis[3,5-bis(2-(tert-butoxycarbonylamino)ethoxycarbonyl)-

was stirred under argon for 2 min, followed by addition of TFA phenyl]-15-[4-(2-(tert-butoxycarbonyl)ethyl)phenyl]-5-methoxy-

(470 mL). After 1 h, the reaction mixture was diluted with CHCl₃8,8,18,18-tetramethylbacteriochlorin (BC8a). Following a general

and dried under an argon flow. Tributylamine (200 mL, 840 mmol) procedure,⁴⁹samples of BC4a-Br (37.0 mg, 26.8 mmol), 5 (56.3 mg,

was added to the solid residue and sonicated for 3 min. A mixture 0.257 mmol), Pd(PPh₃)₄(23.7 mg, 20.6 mmol), and Cs₂CO₃

of THF and hexanes (1 : 1) was then added, and the resulting (101 mg, 0.308 mmol) were placed in a Schlenk flask, and dried

suspension was sonicated for 5 min followed by centrifugation. under high vacuum for 30 min. Toluene/DMF [5.1 mL (2 : 1),

The supernatant was discarded to aﬀord a green solid (9.90 mg, deaerated by bubbling with argon for 30 min] was added to the

1

99%): H NMR (300 MHz, CD₃OD, the four amine protons, two Schlenk flask under argon, and the mixture was deaerated

pyrrolic-NH protons and one carboxylic acid proton were not by three freeze–pump–thaw cycles. The reaction mixture was

observed) d 1.82 (s, 6H), 1.98 (s, 6H), 2.57 (br, 2H), 2.89 (br, 2H), stirred at 90 1C for 18 h. The reaction mixture was cooled to room

3.69 (s, 3H), 3.88 (s, 2H), 4.06 (s, 4H), 4.28 (s, 4H), 4.37 (s, 2H), temperature, concentrated to dryness, diluted with ethyl acetate

7.12 (d, J = 8.1 Hz, 2H), 7.28 (s, 1H), 7.37 (s, 2H), 7.44 (d, J = 8.1 Hz, and washed with saturated aqueous NaHCO₃. The organic layer

2H), 7.64 (s, 1H), 8.20 (s, 2H), 8.67 (s, 1H), 8.74 (s, 2H), 8.77 was separated, dried (Na₂SO₄), concentrated and chromato-

(s, 1H); MALDI-MS obsd 816.0; ESI-MS obsd 409.2308, calcd graphed [silica, CH₂Cl₂/ethyl acetate (24 : 1)] to provide a red

409.2311 [(M + 2H)²⁺, M = C₅₀H₅₆N₈O₃]; l_abs(methanol) 363, solid (32.7 mg, 81%): ¹H NMR (300 MHz, CDCl₃) d ꢀ1.51 (s, 1H),

515, 726 nm.

ꢀ1.15 (s, 1H), 1.41 (s, 18H), 1.43 (s, 18H), 1.52 (s, 9H), 1.85 (s, 6H),

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1.99 (s, 6H), 2.53 (t, J = 8.7 Hz, 2H), 2.84 (t, J = 8.7 Hz, 2H), with HOSu (3.8 mg, 33 mmol) and stirred under argon at room

3.59–3.66 (m, 11H), 3.92 (s, 2H), 4.40 (s, 2H), 4.70 (t, J = 8.4 Hz, 4H), temperature. After 8 h, diethyl ether was added to the reaction

4.53 (t, J = 8.4 Hz, 4H), 5.10–5.13 (m, 4H), 6.95 (d, J = 7.8 Hz, 2H), mixture to precipitate the crude product. The suspension was

7.43 (d, J = 7.8 Hz, 2H), 8.15 (s, 2H), 8.51 (s, 1H), 8.68 (s, 3H), 8.73 sonicated for 3 min followed by centrifugation. The super-

(d, J = 2.4 Hz, 1H), 8.88 (s, 1H), 8.89 (d, J = 1.5 Hz, 2H); ¹³C NMR natant was discarded to aﬀord a green solid. This procedure

(100 MHz, CDCl₃) d 28.4, 28.6, 30.9, 31.32, 31.37, 36.9, 40.0, 45.2, (diethyl ether addition/sonication/centrifugation) was carried

46.1, 47.7, 52.3, 65.0, 65.2, 79.8, 80.9, 97.5, 97.9, 113.7, 123.2, 126.9, out three more times to aﬀord a green solid (6.4 mg, 76%):

127.5, 128.0, 128.3, 129.3, 129.5, 130.2, 131.4, 131.6, 133.5, 134.0, ¹H NMR (300 MHz, DMSO-d₆, the CO₂H was not observed, two

134.2, 135.0, 136.02, 136.13, 136.4, 136.8, 138.8, 139.0, 139.4, 139.6, ethylene protons were overlapped with the solvent peak) d ꢀ1.60 (s,

155.2, 156.1, 161.3, 165.9, 166.3, 169.46, 169.51, 172.7; MALDI-MS 1H), ꢀ1.24 (s, 1H), 1.80 (s, 6H), 1.96 (s, 6H), 2.54–2.60 (m, 6H), 2.71

obsd 1507.3; ESI-MS obsd 1505.7499, calcd 1505.7491 [(M + H)⁺, (t, J = 7.5 Hz, 2H), 3.16 (s, 18H), 3.27 (s, 18H), 3.57 (s, 3H), 3.88–3.91

M = C₈₂H₁₀₄N₈O₁₉]; l_abs(CH₂Cl₂) 368, 518, 730 nm.

(m, 10H), 4.30 (s, 2H), 4.81–4.84 (m, 8H), 6.97 (d, J = 7.5 Hz, 2H),

3,13-Bis[3,5-bis(2-aminoethoxycarbonyl)phenyl]-15-[4-(2-carboxy- 7.40 (d, J = 7.5 Hz, 2H), 8.09 (s, 2H), 8.35 (s, 1H), 8.73 (s, 1H),

ethyl)phenyl]-5-methoxy-8,8,18,18-tetramethylbacteriochlorin 8.94–9.02 (m, 6H); ESI-MS obsd 329.4254, calcd 329.4257

(BC8b). A solution of BC8a (23.7 mg, 15.7 mmol) in CH₂Cl₂(315 mL) [(M ꢀ 4I)⁴⁺, M = C₇₄H₉₅I₄O₁₃N₉]; l_abs(PB) 362, 522, 729 nm.

was stirred under argon for 2 min, followed by addition of TFA

15-[4-(2-(tert-Butoxycarbonyl)ethyl)phenyl]-3,13-bis[3,5-bis(2-

(210 mL). After 1.5 h, the reaction mixture was diluted with (tert-butyldimethylsiloxy)ethylaminocarbonyl)phenyl]-5-methoxy-

CHCl₃and dried under an argon flow. Tributylamine (400 mL) 8,8,18,18-tetramethylbacteriochlorin (BC9a). Following a general

was added to the solid residue. The resulting suspension was procedure,⁴⁹samples of BC6a-Br (35.0 mg, 24.4 mmol), 5 (40.5 mg,

sonicated for 3 min. A mixture of THF and diethyl ether (1 : 1) 121 mmol), Pd(PPh₃)₄(11.3 mg, 9.70 mmol), K₂CO₃(20.2 mg,

was then added. The resulting suspension was sonicated for 146 mmol) and toluene/DMF [2.40 mL (2 : 1), deaerated by

5 min followed by centrifugation. The supernatant was discarded bubbling with argon for 45 min] were added to a Schlenk flask

to aﬀord a green solid (15.0 mg, 91%): ¹H NMR (300 MHz, and deaerated by three freeze–pump–thaw cycles. The reaction

CD₃OD/CDCl₃, the CO₂H, NH₂and pyrrolic-NH protons were not mixture was stirred at 90 1C for 16 h. The reaction mixture was

observed) d 1.86 (s, 6H), 2.02 (s, 6H), 2.61 (t, J = 7.5 Hz, 2H), 2.87 allowed to cool to room temperature, then concentrated to

(t, J = 7.5 Hz, 2H), 3.46–3.50 (m, 8H), 3.67 (s, 3H), 3.94 (s, 2H), dryness, diluted with CH₂Cl₂and washed with saturated aqueous

4.40 (s, 2H), 4.68–4.76 (m, 8H), 7.04 (d, J = 7.8 Hz, 2H), 7.45 (d, NaHCO₃. The organic layer was separated, dried (Na₂SO₄) and

J = 7.8 Hz, 2H), 8.30 (d, J = 1.5 Hz, 2H), 8.64 (t, J = 1.2 Hz, 1H), concentrated. Column chromatography [twice, silica, CH₂Cl₂/

8.79–8.83 (m, 4H), 9.00 (d, J = 1.2 Hz, 1H), 9.12 (d, J = 1.5 Hz, 2H); ethyl acetate (7 : 3 to 11 : 9)] afforded a green solid (15.0 mg,

1

MALDI-MS obsd 1050.2; ESI-MS obsd 1049.4776, calcd 1049.4767 39%): H NMR (CDCl₃, 400 MHz) d ꢀ1.55 (br, 1H), ꢀ1.19 (br,

[(M + H)⁺, M = C₅₈H₆₄N₈O₁₁]; l_abs(methanol) 364, 515, 726 nm. 1H), 0.086 (s, 12H), 0.093 (s, 12H), 0.871 (s, 18H), 0.879 (s, 18H),

3,13-Bis[3,5-bis(2-(N,N,N-trimethylammonio)ethoxycarbonyl)- 1.50 (s, 9H), 1.85 (s, 6H), 1.99 (s, 6H), 2.55 (t, J = 5.4 Hz, 2H),

phenyl]-15-[4-(2-carboxyethyl)phenyl]-5-methoxy-8,8,18,18-tetra- 2.86 (t, J = 5.4 Hz, 2H), 3.63 (s, 3H), 3.69–3.73 (m, 8H), 3.85–3.91

methylbacteriochlorin tetraiodide (BC8c). A mixture of BC8b (m, 11H), 4.37 (s, 2H), 6.68 (t, J = 3.6 Hz, 2H), 6.88 (t, J = 3.6 Hz,

(19.0 mg, 18.1 mmol) and tributylamine (33.6 mg, 181 mmol) in 2H), 7.01 (d, J = 5.7 Hz, 2H), 7.43 (d, J = 5.7 Hz, 2H), 7.82 (s, 2H),

DMF (1.81 mL) was stirred under argon for 2 min, followed by 8.05 (s, 1H), 8.44 (s, 1H), 8.65–8.66 (m, 3H), 8.72 (s, 3H);

addition of iodomethane (257 mL, 1.81 mmol). After 44 h, a ¹³C NMR (CDCl₃, 100 MHz) d ꢀ5.030, ꢀ5.015, 18.54, 18.56,

solution of diethyl ether and THF (1 : 1) was added to the reaction 26.2, 28.4, 30.9, 31.32, 31.36, 36.8, 42.61, 42.68, 45.2, 46.0, 47.8,

mixture to precipitate the crude product. The suspension was 52.3, 62.1, 62.2, 63.4, 80.7, 97.4, 97.8, 113.7, 123.2, 123.3, 124.4,

sonicated for 3 min followed by centrifugation. The supernatant 127.1, 127.6, 128.0, 131.9, 132.6, 133.4, 133.8, 134.0, 134.1, 134.2,

was discarded to aﬀord a green solid. This procedure (diethyl ether 134.8, 135.5, 136.1, 138.96, 139.00, 139.4, 139.8, 155.1, 161.2, 166.6,

and THF addition/sonication/centrifugation) was carried out three 167.1, 169.3, 169.4, 172.6; MALDI-MS obsd 1560.0945; ESI-MS obsd

more times to aﬀord a green solid (27.0 mg, 85%): ¹H NMR 803.4280, calcd 803.4282 [(M + 2Na)²⁺, M = C₈₆H₁₂₈N₈O₁₁Si₄];

(300 MHz, DMSO-d₆; the CO₂H was not observed, and two ethylene l_abs(CH₂Cl₂) 367, 518, 731 nm.

protons were overlapped with the solvent peak) d ꢀ1.59 (s, 1H),

D. Spectroscopy

ꢀ1.20 (s, 1H), 1.82 (s, 6H), 1.99 (s, 6H), 2.74 (t, J = 7.5 Hz, 2H), 3.18

(s, 18H), 3.28 (s, 18H), 3.61 (s, 3H), 3.89–3.96 (m, 10H), 4.30 (s, 2H),

Solvents. The solvent systems employed herein are as follows:

4.82–4.89 (m, 8H), 7.01 (d, J = 7.8 Hz, 2H), 7.43 (d, J = 7.8 Hz, DMF of Z99.8% purity; PBS (phosphate buﬀered saline) is

2H), 8.11–8.19 (m, 2H), 8.27 (s, 1H), 8.76 (s, 1H), 8.94–9.03 aqueous 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄and 1.8 mM

(m, 6H); ESI-MS obsd 305.1711, calcd 305.1716 [(M ꢀ 4I)⁴⁺

,

KH₂PO₄at pH 7.4; PB is aqueous potassium phosphate buﬀer

(0.5 M, pH 7.0); and deionized water.

M = C₇₀H₉₂N₈O₁₁I₄]; l_abs(PB) 363, 519, 731 nm.

3,13-Bis[3,5-bis(2-(N,N,N-trimethylammonio)ethoxycarbonyl)-

Absorption versus concentration study. For each bacterio-

phenyl]-5-methoxy-15-[4-(2-(N-succinimidooxycarbonyl)ethyl)- chlorin, four diﬀerent solutions (A–D) in PB were prepared. The

phenyl]-8,8,18,18-tetramethylbacteriochlorin tetraiodide (BC8). concentration of solution A (B500 mM) afforded absorbance

A mixture of BC8c (8.0 mg, 4.6 mmol), DCC (6.7 mg, 33 mmol), (A) B 0.5 for the Q_ytransition measured with a 0.01 cm path-

and DMAP (0.20 mg, 1.6 mmol) in DMF (654 mL) was treated length cuvette. Successive serial dilution (10 times each) with PB

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2 I. Batinic-Haberle, J. S. Rebouças, L. Benov and I. Spasojevic,

in Handbook of Porphyrin Science, ed. K. Kadish, K. M. Smith

and R. Guilard, World Scientific, Singapore, 2011, vol. 11,

pp. 291–393.

gave bacteriochlorin concentrations as follows: [solution A] =

10 ꢂ [solution B] = 100 ꢂ [solution C] = 1000 ꢂ [solution D].

A typical experiment proceeded as follows:⁴²a stock solution

was prepared by adding a small amount of DMSO (12.5–37.5 mL,

to facilitate dissolution) to a bacteriochlorin (B0.1–0.3 mg;

B0.1–0.3 mM) in a small vial followed by addition of PB (237.5–

712.5 mL). The resulting sample was sonicated for one minute

and then filtered (poly-vinylidene difluoride high-volume low

pressure filter, pore size 0.45 mm) to obtain solution A (B500 mM).

The absorbance of solution A was measured in a 0.01 cm

pathlength cuvette. Solution B was obtained by mixing solution A

(100–200 mL) with PB (900–1800 mL) in a small vial. The

absorbance of solution B was measured in a 0.1 cm pathlength

cuvette. For further dilution, solution B (300 mL) was mixed with

PB (2.70 mL) in a 1 cm pathlength cuvette to obtain solution C

and the absorbance was measured. Solution C (2.50 mL) was

transferred into a 25 mL volumetric flask and made up to the

mark with PB. The absorbance of the resulting solution D was

measured in a 10 cm pathlength cuvette. The procedure

was followed for each bacteriochlorin BC1–BC5. Note that the

range of volumes employed depends on the initial quantity of

bacteriochlorin, which was added to the initial vial without

weighing.

Photophysical studies. Photophysical measurements were

performed as described previously^54,65and employed dilute

(mM) Ar-purged toluene solutions at room temperature. Samples

for F_fmeasurements had an absorbance o0.12 at l_excand in

the Q_yband. The F_fvalues were measured with respect to one

or two standards including free base tetraphenylporphyrin

(F_f= 0.070 in nondegassed toluene). The t_Svalues were the

average of results determined using (1) a time-resolved fluores-

cence stroboscope with a Gaussian instrument response func-

tion with B1 ns width and (2) transient absorption studies with

0.5 mJ, B100 fs excitation pulses. The latter apparatus was used

to determine F_iscvalues via the extent of ground-state bleach-

ing due to lowest singlet excited state (at early times) compared

with that due to the lowest triplet excited state (at the asymptote

of the singlet decay).

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Acknowledgements

This research was carried out as part of the Photosynthetic

Antenna Research Center (PARC), an Energy Frontier Research

Center funded by the U.S. Department of Energy, Oﬃce of Science,

Oﬃce of Basic Energy Sciences, under Award No. DE-SC0001035.

Mass spectra were obtained at the Mass Spectrometry Laboratory

for Biotechnology at North Carolina State University. Partial fund-

ing for the facility was obtained from the North Carolina Bio-

technology Center and the National Science Foundation.

21 M. F. Grahn, A. Giger, A. McGuinness, M. L. de Jode, J. C. M.

Stewart, H.-B. Ris, H. J. Altermatt and N. S. Williams, Lasers

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Article Doi

Synthetic bacteriochlorins bearing polar motifs (carboxylate, phosphonate, ammonium and a short PEG). Water-solubilization, bioconjugation, and photophysical properties

DOI: 10.1039/c5nj00759c

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Full text of DOI:10.1039/c5nj00759c

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