EDC/NHS activation mechanism of polymethacrylic acid: Anhydride versus NHS-ester

Article abstract of DOI:10.1039/c5ra13844b

Polymer brushes of polymethacrylic acid (PMAA) and PMAA-associated polymer blends of PMAA/PNIPAM (poly-N-isopropylacrylamide) were prepared on porous silicon for further investigation of the EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide) activation mechanisms by infrared spectroscopy. When the fragmentation degree of PMAA blocks in PMAA-associated polymer blends is increased, the production of anhydride wanes from dominant to recessive, whereas a complementary product of the NHS-ester waxes from recessive to dominant. The Thorpe-Ingold effect was applied to explain the formation of anhydride: the gem-dialkyl groups of PMAA next to the carboxylic acids compress the acid side chains close to each other; thus, once the intermediate of O-acylisourea forms, it will be attacked by the intramolecular neighboring acid much faster than any other nucleophiles such as NHS and water, and therefore the six-membered ring of the anhydride will be formed. All acid side chains in PMAA standing next to each other will form an anhydride, primarily due to the Thorpe-Ingold effect, unless they are sterically hindered, whereas only isolated acid side chains form the NHS-ester. The EDC/NHS activation results for four small molecules of dicarboxylic acids in aqueous media, namely, glutaric acid and 2,2-dimethyl glutaric acid, which generate disuccinimidyl ester with high yield, and succinic acid and 2,2-dimethyl succinic acid, which remain intact, can also be explained by the Thorpe-Ingold effect. A clear understanding of the EDC/NHS activation mechanisms of PMAA will take us a step closer for resolving the mechanistic ambiguity of the carbodiimide/additive coupling reactions for amide bond formation.

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EDC/NHS activation mechanism of polymethacrylic acid:
anhydride versus NHS-ester
Qin Yan,a Hong-Ning Zheng,a Chuan Jiang,a Kun Li,a and Shou-Jun Xiao*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Polymer brushes of polymethacrylic acid (PMAA) and PMAA-associated polymer blends of PMAA/PNIPAM
(poly-N-isopropylacrylamide) were prepared on porous silicon for further investigation of the EDC/NHS (1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide) activation mechanisms by infrared
spectroscopy. With increasing the fragmentation degree of PMAA blocks in PMAA-associated polymer
blends, the product of anhydride wanes from dominant to recessive, whereas a complementary product of
NHS-ester waxes from recessive to dominant. The Thorpe-Ingold effect was applied to explain the formation
of anhydride, that the gem-dialkyl groups of PMAA next to carboxylic acids compress the acid side chains
close to each other, once the intermediate of O-acylisourea forms, it will be attacked by the intramolecular
neighboring acid much faster than any other nucleophiles such as NHS and water, therefore the six-
membered ring of anhydride forms. All acid side chains in PMAA standing next to each other will form
anhydride primarily due to the Thorpe-Ingold effect, unless they are sterically hindered, whereas only the
lonely acid side chains isolated by others, form NHS-ester. The EDC/NHS activation results of four small
molecules of dicarboxylic acids in aqueous media, glutaric acid and 2,2-dimethyl glutaric acid generating
disuccinimidyl ester with high yield, succinic acid and 2,2-dimethyl succinic acid remain intact, can also be
explained by the Thorpe-Ingold effect. The clear understanding of the EDC/NHS activation mechanisms of
PMAA will be a step closer to resolve the mechanistic ambiguity of the carbodiimide/additive coupling
reactions for amide bond formation.
a.State Key Laboratory of Coordination Chemistry, School of Chemistry and
Chemical Engineering, Nanjing University, Nanjing 210093, Jiangsu, China. E-mail:
sjxiao@nju.edu.cn
†Electronic Supplementary Information (ESI) available: Supplementary tables and
figures. See DOI: 10.1039/x0xx00000x
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Introduction
Journal Name
(NIPAM) with blending ratios of 10, 20, 30, 40, 50, 60, 70, 80, 90,
Amide bond formation (or amidation) from carboxylic acid by and 100% MAA. We adapted our previous method:4b infrared
carbodiimide activation with the assistance of additives such as measurements of stepwise surface reactions, which possess the
NHS, then addition of amine sequentially in one-pot or in stepwise merit of ignoring the laborious separation and purification
reactions, is so broadly used in peptide synthesis, bio-conjugation, procedure, especially for multiple reaction steps in aqueous media.
immunochemistry, biosensing, etc., that it would be hard to 3-Amino-1-azidopropane (AAP),8 an infrared probe of the azido tag
overstate its importance.1 However the reaction mechanisms still with a strong stretching band at 2105 cm-1, was used as the
remain challenging,2 especially with debates on the intermediates, amidation reagent. We observed the complementary waxing of
and a direct evidence is the large deviation of product yields for NHS-ester and waning of anhydride with increasing the
very similar carboxylic acids under the same reaction conditions. fragmentation degree of PMAA blocks in the above-mentioned two
The initial activation intermediate is undoubtedly believed to be the polymer systems after EDC/NHS activation. Anhydride evolves from
unstable O-acylisourea,3 which goes through several transition dominant to recessive, while NHS-ester from recessive to dominant
routes and mechanisms to stable intermediates or byproducts, the complementarily with all-neighboring acid side chains to isolated
focal point for debating. Taking one of the most common activation ones by inserting other side chains. Further amidation kinetics for
recipes as an example, EDC and an additive of NHS or sulfo-NHS both PMAA anhydride and NHS-ester with AAP by means of the
with carboxylic acid in aqueous media, the initial intermediate of O- infrared absorption evolution against the reaction time was
acylisourea converts to stable intermediates or byproducts through investigated. Finally the Thorpe-Ingold effect was applied to
the following 4 pathways: 1st, nucleophilic addition of NHS to interpret the EDC/NHS activation of 4 small dicarboxylic acids of
generate NHS-ester, which forms amide with amine in a weak glutaric acid, 2,2-dimethyl glutaric acid, 2,2-dimethyl succinic acid,
alkaline solution at room temperature efficiently; 2nd, nucleophilic and succinic acid. All the experimental results helped us to
addition of carboxylic acid to form symmetrical anhydride speculate that the faster nucleophilic attack on O-acylisourea for
(suggested but a stable anhydride intermediate in aqueous media PMAA, 2,2-dimethyl succinic acid, and succinic acid is from the
was only captured and confirmed recently in the PMAA system by intramolecular neighboring acid due to the Thorpe-Ingold effect,
us4), which can react with amine directly to amide, or with thus generating anhydride; whereas the faster nucleophilic attack
nucleophilic addition of NHS to NHS-ester, or by hydrolysis to on O-acylisourea for PAA, glutaric acid, and 2,2-dimethyl glutaric
carboxylic acid; 3rd, rearrangement to a stable side product of N- acid is from the intermolecular NHS, therefore producing NHS-ester.
acyl urea; 4th, hydrolysis to urea and carboxylic acid by consuming
carbodiimide only. All the above pathways make an individual acid Experimental Section
to the product of amide complicated, for the reason in dealing with Materials
the thermodynamics and kinetics of different pathways.5
Single-side polished silicon wafers (<100>, p-type, boron-doped,
In our previous report,4b two different intermediates were observed 5.0–8.0 Ω.cm, 500 μm thick) were purchased from Hefei Kejing
in two similar acid polymers respectively after EDC/NHS activation, Materials Technology CO. LTD. N-Ethyl-N’-(3-
NHS-ester from polyacrylic acid which is normal, while anhydride (dimethylamino)propyl)carbodiimide hydrochloride (EDC) (98%), N-
from PMAA which is a surprise. We interpreted the capture of hydroxysuccinimide (NHS) (98%), sodium azide (NaN3, 98%) were
polymethacrylic anhydride with two reasons: the faster kinetic from Aladdin. Sodium methacrylate (NaMA, 99%), ω-undecylenyl
intra-molecular nucleophilic attack of carboxylic acid from
a
alcohol
(98%),
2-bromoisobutyryl
hydrobromide
bromide
(98%),
(98%),
and
3-
4-
neighboring side chain to O-acylisourea than any other pathways bromopropylamine
and the stable thermodynamic structure of polymethacrylic morpholineethanesulfonic acid hydrate (MES) were from Alfa
anhydride to avoid further nucleophilic attack from NHS and water. Aesar. N-Isopropylacrylamide (NIPAM, 99%), copper (I) bromide
Similar to NHS-ester, polymethacrylic anhydride,
a
good (CuBr, 98%), and pentamethyldiethylenetriamine (PMDETA, 98%)
bioconjugate reagent, possesses a longer shelf life time and bears were from Aldrich. Glutaric acid, 2,2-dimethyl glutaric acid, 2,2-
the metastability towards hydrolysis over hours in weak alkaline dimethyl succinic acid, and succinic acid were from TCI. PMAA (Mn
solutions. Obviously, both kinetic and thermodynamic properties of 1100) and PAA (Mn 1300) were from Polymer Source Inc. Water (18
PMAA during the EDC/NHS activation relate to the side chains of MΩ.cm) was from a Milli-Q Ultrapure water purification system.
methyl groups, which is specifically called the Thorpe-Ingold effect Fourier-transform infrared spectrometry
(or gem-dialkyl effect), one of the neighboring group effects.6 The A Bruker V80 spectrometer equipped with a deuterated triglycine
stable intermediate anhydride structure captured renders PMAA so sulfate (DTGS) detector was used to measure the spectra for the
special that further investigation on the EDC/NHS activation of stepwise reactions. In all cases, samples were mounted in a vacuum
PMAA and polyacrylic acid (PAA) will be not only beneficial for chamber and thus the interference of CO2 and water vapor from air
practical applications but also for understanding the carbodiimide- was greatly attenuated. Spectra were recorded with 100 scans at a
triggered amidation mechanisms better.
7.5 kHz velocity and a resolution of 4 cm-1, using a cleaned planar Si
In this work, we have investigated the EDC/NHS activation of two (100) chip as reference. All spectra were disposed with the OPUS
PMAA-based polymer brushes grafted on porous silicon:4b,7 1) software and spectral windows were zoomed to highlight the
reiterative EDC/NHS activation and amidation of PMAA brushes, 2) significant changes.
EDC/NHS activation of the polymer blend brushes of Preparation of porous silicon
PMAA/PNIPAM (poly-N-isopropylacrylamide) grown from mixed The wafer was cut into 15×15×0.5 mm3 pieces. Silicon chips were
monomers of methacrylic acid (MAA) and N-isopropylacrylamide sonicated in ethanol, acetone and deionized water, each for 15 min.
2 | J. Name., 2012, 00, 1-3
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Then, they were immersed in piranha solution (H2SO4/H2O2 (3/1 in and 0.25 mol/L NHS in 0.1 mol/L MES buffer (pH 6.5) at room
v/v)) at the boiling temperature for 2 h to remove any organic temperature for 1 h. For example, 15 mL freshly prepared MES
residues. The cleaned chips were rinsed extensively with water and buffer containing 2.4 g EDC (0.0125 mol) was added immediately to
ethanol and subsequently dried in a stream of nitrogen (unless a stirred 35 mL MES buffer containing glutaric acid (0.66 g, 0.005
claimed, all the following chip cleaning and drying procedure mol) and NHS (1.44 g, 0.0125 mol) at 0 0C. Then the mixture was
adopted the steps). We used the metal-assisted stain etching to stirred at room temperature for 1 h. The white suspension was
produce porous silicon.7c Briefly, the chip was immersed in 1% HF filtered, washed with water, then dissolved in dichloromethane,
aqueous solution for 15 min to remove the surface silicon oxide dried over anhydrous Na2SO4, and concentrated under vacuum. A
film. After cleaning, the hydrided chip was deposited with a thin white solid of glutaric acid di-succinimidyl ester (GA-NHS) was
metallic film of Pt (10 nm thick) using a sputter-coater (SCD 500) at obtained after recrystallization from isopropyl alcohol (1.2 g, 73%
15 mA for 180 s. The Pt-coated chip was etched in a mixture of 40% yield). Following the same protocol, a white solid of 2,2-dimethyl
HF, 30% H2O2 and anhydrous ethanol with 2:2:1 (v/v/v) ratios for 4 glutaric acid di-succinimidyl ester (DMGA-NHS) was obtained after
min in dark and sequentially immersed in 1% HF aqueous solution recrystallization with a yield of 85%. Whereas no products were
for 1 min. Rinsed with copious water and ethanol and dried with N2, obtained from either 2,2-dimethyl succinic acid or succinic acid
hydrogen-terminated porous silicon chips were gained.
Surface hydrosilylation and introduction of surface initiator4b,7c
solutions, following the same EDC/NHS activation protocol.
Preparation of 3-amino-1-azide propane (AAP)
The freshly etched silicon chips were transferred into a glass bottle AAP was prepared according to literature.8 3-Bromopropylamine
containing 10 mL neat ω-undecylenyl alcohol. The bottle was hydrobromide (15 mmol, 3.2 g) was suspended in water (10 ml),
purged with N2 for 15 min so as to vent the air. The reaction was followed by the addition of NaN3 (50 mmol, 3.2 g) in 15 ml of water.
0
performed in a CEM Discovery microwave reactor, controlled with a The mixture was heated to 80 C for 12 h, followed by the removal
dynamic mode to reach 120 0C in 10 min and held there for 20 min. of 2/3 water in vacuum. The resulting mixture was cooled in an ice
After reaction, the chip was washed sonically with anhydrous bath and diethyl ether (50 mL) and KOH pellets (4 g) were added
alcohol for 3 min and water for 3 min respectively. The undecylenyl while keeping the temperature <10 0C. The organic layer was
alcohol-modified chip with hydroxy-termini was put into a glass separated and the aqueous phase was extracted with diethyl ether
bottle containing 10 mL CH2Cl2 and 2 mL Et3N. The reaction vessel (2×30 mL), dried over K2CO3, and concentrated. The pure product
was cooled in an ice bath for 15 min, then 2 mL 2-bromoisobutyryl was obtained by silica column with CH3OH/CH2Cl2 (v:v = 1:3) to give
bromide was dropped in slowly, and finally the reaction solution a clear yellow oil (1.1 g, 73%).
stayed at room temperature for 12 h. After reaction, the chip with Amidation
the surface initiator of 2-bromoisobutyryl groups was rinsed with Generally the PMAA anhydride or NHS-ester chips were incubated
copious CH2Cl2 and ethanol, dried with a mild nitrogen stream.
Preparation of PMAA and PMAA/PNIPAM brushes4b,7c,9
in 5 mL 0.1 M NaHCO3/Na2CO3 buffer (v/v = 1/1 with DMSO, pH =
8.5) containing 0.1 mM AAP at room temperature for 1 h, whereas
Surface-initiated polymerization of NaMA was performed in a N2- for kinetic studies, the chips were taken out at 2, 20, 40, 80, and
filled vial with 1.69 g NaMA (15 mmol) and 75 μL PMDETA dissolved 120 min, rinsed with water and dried with nitrogen, and measured
in a 5 mL solution of H2O/ CH3OH (1/1 in v/v). The solution was with the infrared spectrometer.
adjusted to pH 9.0. The bottle was purged with N2 for 15 min so as
Results and Discussion
to vent the air, followed by addition of 20 mg CuBr, and held at 40
0C for 5 h. Then, the chips were removed from the vial, washed with
THF, H2O and ethanol sequentially, and dried under a stream of N2.
PMAA brushes were obtained by immersion of the chip into 0.1 M
phosphate-citric acid buffer (pH = 3.0) for 3 h to change carboxylate
anions (COO−) into carboxylic acid (COOH) species (The acidification
procedure in the reiterative reactions followed the same protocol).
The random polymer blend brushes of PMAA/PNIPAM were
prepared with the same procedure. The total amount of monomers
was 15 mmol. The 10 samples of random PMAA/PNIPAM blend
brushes with different molar ratios of NIPAM:NaMA = 9:1, 8:2, 7:3,
6:4, 5:5, 4:6, 3:7, 2:8, 1:9 were prepared respectively.
We have previously reported that the predominant product of
EDC/NHS activation on PMAA is anhydride.4 However, anhydride
has its own weakness for amidation, only half carboxylic acids can
be converted to amide, while the rest of halves return to carboxylic
acids themselves, theoretically. To overcome this weakness, we
reiterated the EDC/NHS activation for the rest of carboxylic acids
and then amidation again three times. We define the products of
the 1st, 2nd and 3rd EDC/NHS activations as 0.5G, 1.5G, and 2.5G
respectively, while the amidation products after 0.5G, 1.5G, and
2.5G as 1.0G, 2.0G and 3.0G respectively. Their respective infrared
traces are shown in Fig. 1 and the infrared peak assignments in the
carbonyl stretching region from 1500 to 2200 cm-1 are listed in
Table S1 (see Table S1 in ESI).
Surface EDC/NHS activation of PMAA and PMAA/PNIPAM brushes
The EDC/NHS activation of the chip was performed with the
optimum reaction condition reported previously by us,4b 0.1 mol/L
EDC and 0.2 mol/L NHS in 0.1 mol/L MES buffer with pH 6.5 at room
temperature for 1 h. After reaction, the chip was rinsed with water
and dried under a stream of nitrogen.
The molar concentrations of surface species can be quantitatively
estimated from their corresponding absorption bands by means of
the Beer–Lambert law: A = εbc (where A is the absorbance, ε the
molar extinction coefficient, b the thickness of polymer brushes,
and c the concentration). To execute quantitative analyses using
the infrared spectra in Fig. 1, we classified the reiterative reactions
into two groups: 0.5G, 1.5G, 2.5G and 1.0G, 2.0G, 3.0G. In the first
group, all three products in the infrared region of 1650~1850 cm-1
EDC/NHS activation of dicarboxylic acids
The EDC/NHS activation of dicarboxylic acids of glutaric acid, 2,2-
dimethyl glutaric acid, 2,2-dimethyl succinic acid, and succinic acid
were performed with 0.1 mol/L dicarboxylic acid, 0.25 mol/L EDC,
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can be fitted as shown in the right column of Fig. 1: NHS-ester αazide, we can easily derive the component compositions (see Table
(associated pink triplex infrared bands at 1738, 1780, and 1811 cm- S3 in ESI) shown in Scheme 1 under their corresponding structures
1), anhydride (associated blue doublet bands at 1760 and 1802 cm-1), respectively.
and acid residue (green curved empty single band at 1714 cm-1). The molecular structures and their stoichiometric molar ratios
Byproducts of N-acylurea will be ignored in the calculations because shown in Scheme 1 are derived from the quantitative analyses of
in the second group, non-trace in 1.0G and neglectable trace in infrared spectra. Details are illustrated: i) During the first EDC/NHS
2.0G and 3.0G of N-acylurea can be found in the carbonyl stretching activation (0.5G), anhydride is the overwhelming product (83%),
region (1700~2000 cm-1).4b The Beer–Lambert law for NHS-ester, due to the faster kinetic ring-closing mechanism and the
anhydride, and acid in the first group can be written as:
ANHS-ester = εNHS-esterbcNHS-ester
thermodynamically stable anhydride; then the left lonely acid side
chains are partly esterified to NHS-ester (10%) and partly left as
unreacted acid residues (7%), due to steric hindrance, such as the
one-end fixed backbone on silicon having less freedom of
Aanhydride = εanhydridebcanhydride
Aacid = εacidbcacid
where b has the same polymer thickness for all 3 species.
translation and rotation. ii) Successive AAP amidation (1.0G)
We adapted the linear relationship among the 3 individual molar generates amides (32%) and carboxylates (68%), which can be
extinction coefficients from our previous report: εNHS- assigned to three subunit structures. The green subunit can be
ester/εanhydride/εacid = 2 : 1.5 : 1.4b The largest peak heights of each inferred statistically if amidation occurs in an even space when half
species in Fig. 1 were used as the absorbance (
1760 cm-1 for anhydride, 1738 cm-1 for NHS-ester, and 1714 cm-1 for completely to amide and other chemical conversions rationally
carboxylic acid, individually. follow the dash arrows from 0.5G to 1.0G, an algebraic approach
A) for calculations: of anhydrides react with AAP. Supposing that NHS-ester converts
Assuming all reactants of the side chain acid groups are converted for balancing the chemical equation gives perfectly matching
to NHS-ester, anhydride, acid residue, and not to any other compositions for the subunit structures depicted in 1.0G, among
compounds, then the molar percentages (represented with β) with which the green subunit is the key structure to interpret the
an acid group as the unit converting to NHS-ester (βNHS-ester), reaction mechanisms self-consistently. iii) For the 2nd EDC/NHS
anhydride (βanhydride), and acid (βacid) can be calculated individually in activation (1.5G), the independently measured molar percentages
equations 1~3.
of anhydride (35%), NHS-ester (19%), and acid (14%), follow the
chemical reaction logistics from 1.0G perfectly. vi) Successive
amidation (2.0G) produces amides (48%) and carboxylates (52%). v)
The 3rd EDC/NHS activation (2.5G) of the left 48% acids provides
anhydride (9%), NHS-ester (24%), and acid (19%). vi) Successive AAP
amidation generates amides (62%) and carboxylate residues (38%).
Repeating the EDC/NHS activation further will generate
overwhelming NHS-ester intermediates and the AAP amidation
further will achieve over 80% amides. All subunits depicted as
monomers are suggested rationally and inferred from the green
subunits logistically.
ANHSꢀester / εNHSꢀester
βNHSꢀester
=
=
100%
100%
100%
ANHSꢀester / εNHSꢀester + 2Aanhydride / εanhydride + Aacid / εacid
(1)
(2)
(3)
2Aanhydride / εanhydride
βanhydride
ANHSꢀester / εNHSꢀester + 2A
/εanhydride + A / εacid
anhydride acid
A
/ εacid
acid
βacid
=
ANHSꢀester / εNHSꢀester + 2Aanhydride / εanhydride + Aacid / εacid
We obtained βanhydride, βNHS-ester, βacid as 83, 10, 7; 52, 28, 20; 17, 46,
37 for 0.5G 1.5G, and 2.5G respectively (see Table S2 in ESI).
For the second group of 1.0G 2.0G, and 3.0G, there are only two
,
,
products of amide and carboxylate, which can be proven from their
respective infrared traces in Fig. 1. Amide was confirmed with
amide I and II at 1650 and 1529 cm-1 respectively. Since the weak
amide II overlaps with carboxylate (1570 cm-1) and amide I is always
complicated by environments such as water vapor and its
orientation in the supporting substrate,10 we used the independent
and strong vibration band of azide at 2105 cm-1, plus carboxylate
(1570 cm-1), to calculate the amide yield (αazide) as follows:
A
cooꢀ
= εcooꢀ bccooꢀ
Aazide = εazidebcazide
A
azide
cazide
εazide
αazide
=
100% =
100%
ACOOꢀ
cazide + cCOOꢀ
A
azide
+
Fig. 1 Infrared spectra of PMAA, 0.5G, 1.0G, 1.5G, 2.0G,
εazide εCOOꢀ
2.5G and 3.0G are displayed in the left frame from top to
bottom. The region of 1650 ~ 1850 cm-1 is magnified and
fitted to have a clear view of deconvoluted anhydride
(blue doublet bands), NHS-ester (pink triplex bands) and
acid (green curved empty single band) in 0.5G, 1.5G and
2.5G respectively in the right frame.
(5)
where Aazide and ACOO- can be obtained from the peak heights of
2105 and 1570 cm-1 in Fig. 1 respectively. We adapted the molar
extinction coefficients of εazide = 1000 LM-1cm-1 11 and εCOO = 380
-
LM-1cm-1 from previous reports, therefore the amide yields were
12
obtained for 1.0G, 2.0G, and 3.0G as 32, 48, and 62% respectively.
From the above self-consistent quantitative analyses, we can
deduce the key idea about the structure-reactivity relationship: All
Combining both calculated results of βanhydride, βNHS-ester, βacid and
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acid side chains standing next to each other will form anhydride isopropylacrylamide monomers or oligomers into monomers,
primarily due to Thorpe-Ingold effect, unless they are sterically dimers, trimers, and multimers sequentially and statistically from
hindered. While the lonely acid side chains isolated by others, and low to high PMAA concentrations in the backbone. After EDC/NHS
acid side chains acting as lonely ones such as in trimer after activation, we observed the similar evolution phenomena for both
anhydride formation at either side or in tetramer after anhydride products of NHS-ester and anhydride in Fig. 2: with decreasing the
formation at the middle, form NHS-ester.
molar concentration of PMAA from 100% to 10%, anhydride evolves
To prove the above structure-reactivity statement further, we from dominant to recessive, while NHS-ester from recessive to
applied the EDC/NHS activations on another PMAA-based acid dominant.
system, random polymer blends of PMAA/PNIPAM with a series of
molar blending ratios of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%
PMAA at an increasing concentration step of 10% PMAA. In the
polymer blends, methacrylic acids are interrupted by N-
Fig. 2 Infrared spectra of the EDC/NHS activated polymer
blends of PMAA/PNIPAM with molar blending ratios of 10,
20, 30, 40, 50, 60, 70, 80, 90, 100% PMAA respectively. The
two intermediates of NHS-ester (associated triplex bands at
1738, 1780, 1807 cm-1) and anhydride (associated doublets
at 1760 and 1804 cm-1) and the acid residue (1717 cm-1) are
indicated. Amide I at 1652 cm-1 and amide II at 1535 cm-1
are from PNIPAM. A few wavenumber shifts of the same
species from Fig. 1 are due to different neighboring group
effects.
Quantitative analysis for Fig.
2 with equations 1~3 after
deconvolution (ignored) of the 10 traces in the infrared region of
1700 ~ 1900 cm-1 provide the product composition against PMAA
molar percentage in Fig. 3, without counting PNIPAM. Obviously,
10~20% acid residues remain un-reacted, which is rational from the
knowledge of chemical reactions; anhydride increases with PMAA
concentrations roughly in a linear relationship, from 6% in 10%
PMAA/PNIPAM blend up to 83% in pure PMAA; while NHS-ester,
Scheme 1 Schematic drawing of molecular structures with
three reiterative EDC/NHS activations and amidations of
PMAA (molar percentages are listed under the subunit
structures respectively). The structures in parentheses are
subunits while in brackets the units for the polymer blend.
Curved lines represent connections, but not only in this
order; subunits in parentheses with alphabet subscripts of
m, n, p, k (≥ 1) are variable in integers, meaning they can
be lonely or next to each other, while subunits in
parentheses without subscripts indicate they are lonely
and cannot be next to each other themselves. 0.5G
2.5G represent the products after the 1st , 2nd and 3rd
EDC/NHS activations respectively; 1.0G 2.0G, and 3.0G
represent the products after successive amidations
following 0.5G 1.5G 2.5G sequentially.
, 1.5G,
Fig. 3 Molar percentage of anhydride, NHS-ester, and
acid residue against PMAA molar percentage for polymer
blends of PMAA/PNIPAM after EDC/NHS activation,
without counting PNIPAM.
,
,
,
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contrast to anhydride, decreases, linearly, from 76% in 10% containing PMAA/PNIPAM blend with a content of ~60% in all acid
PMAA/PNIPAM blend to 9% in pure PMAA. In the medium PMAA derivatives, as examples to run the kinetic experiments with AAP.
concentration range (30~70%), both anhydride and NHS-ester are Although a higher NHS-ester content over 70% in all acid derivatives
generated in considerable quantities. The observed phenomena can can be derivatised from the 20% or less PMAA-containing
be explained as the same as that for the reiterative EDC/NHS PMAA/PNIPAM blend, the absolute amount of NHS-ester is too low
activations on PMAA brushes. Lonely acid side chains are dominant to precisely assay the amidation kinetics, therefore we preferred to
at lower PMAA concentrations, oligomers of MAA such as dimers, use NHS-ester from a 30% PMAA-containing PMAA/PNIPAM blend.
trimers, and tetramers are the major structures of PMAA in the Figure 4 illustrated the amidation kinetics of PMAA anhydride,
medium concentration range, while multimers (≥4) become the where Figure 4a demonstrated the spectral evolution over time and
overwhelming structures at higher PMAA concentrations. Such Figure 4b drew the absorbance curve of the height at 1760 cm-1 and
structural distributions result in the complementary waxing of NHS- 2100 cm-1 versus time, indicating the anhydride consuming rate and
ester and waning of anhydride with fragmentation of PMAA in the the amide formation rate respectively. Similarly, Figure 5 presented
polymer blend.
the amidation kinetics of NHS-ester in the PMAA/PNIPAM blend
From the above qualitative and quantitative analysis of the infrared containing 70% PNIPAM with strong and broad amide bands at
traces, we suggest the following mechanism for EDC/NHS activation 1652 cm-1 and 1535 cm-1, where Figure 5a demonstrated the
of PMAA: the acid side chain next to itself in multimer or even in spectral evolution over time and Figure 5b drew the absorbance
dimer of a polymer chain will form anhydride primarily, and the curve of the height at 1738 cm-1 and 2100 cm-1 versus time,
lonely acid side chain isolated by others will convert to NHS-ester.
indicating the NHS-ester consuming rate and the amide formation
rate respectively. From both Figures 4 and 5, we concluded that
their amidation kinetics were similar. More details are sumarised: (1)
Amidation kinetics
We have demonstrated the EDC/NHS activation product details of At the first 2 min, the amidation reactions were very fast, 50% and
PMAA with different contents in its random polymer blends. While 64% amidation products were obtained for anhydride and NHS-
the main goal of EDC/NHS activation of carboxylic acids is to couple ester respectively, whereas 30% anhydride and 50% NHS-ester
free amine-containing biomolecules, what is the kinetic behavior of were consumed. (2) As time went on to 40 min, both of the
PMAA anhydride or NHS-ester during the amidation process? To amidation reactions were close to completion, therefore the
compare their kinetics, we used an anhydride, directly derivatised reaction time of 1 h was adequate for termination of amidation
from PMAA brushes with a dominant content of ~80% in all acid reactions. (3) Trace amount of residues of anhydrides or NHS-esters
derivatives, and an NHS-ester derivatised from a 30% PMAA- at even 80 min should be due to the buried species within the
Fig. 5 Amindation kinetics of PMAA NHS-ester with AAP: a)
Fig. 4 Amindation kinetics of PMAA anhydride with AAP: a)
infrared trace against reaction time, b) absorbance height
at 1738 cm-1 and 2100 cm-1 against reaction time.
infrared trace against reaction time, b) absorbance height
at 1760 cm-1 and 2100 cm-1 against reaction time.
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polymer brushes, which would be mainly hydrolysed late because was hydrolysed to urea and carboxylic acid by consuming
they were more likely surrounded by water molecules. (4) carbodiimide only.
Hydrolysis as a competitive reaction is much slower than amidation By the way, ideal small dicarboxylic acid molecules, for investigation
at pH 8.5, for both PMAA anhydride and NHS-ester, since the water of the gem-dialkyl effect in the EDC/NHS activation, will be the gem-
molecule is a weaker nucleophile than the primary amine during dialkyl glutaric acid, i.e. 2,2,4,4-tetramethyl glutaric acid and 2,4-
the nucleophilic substitution reaction. Generally, most of dimethyl-2,4-diethyl glutaric acid, resembling the MAA dimer
anhydrides are much less stable than NHS-ester and therefore will structure in PMAA better. However these small molecules have not
be hydrolysed immediately at aqueous media, whereas PMAA yet been synthesized, maybe due to the crowded side groups.
anhydride possesses a stable structure duo to the gem-dialkyl group
effect, therefore it proceeds through a slow hydrolysis over hours in Discussion
weak alkaline solutions, comparable to NHS-ester.
From the above experimental results, we infer that, in the
analogous structures of glutaric acids including PMAA and PAA, the
initial EDC activation of the carboxylic acid to O-acylisourea is a slow
EDC/NHS activation of glutaric acids and succinic acids
Since the anhydride formation in the EDC/NHS activation of PMAA step, then the conversion of O-acylisourea to NHS-ester (the 1st
is due to the Thorpe-Ingold effect: the side methyl groups compress pathway) or to anhydride (the 2nd pathway) is a fast step. Formation
the acid side chains close to each other, therefore the formation of either NHS-ester or anhydride depends on the competition of
six-membered anhydride ring by the nucleophilic attack of the nucleophilic attack from a neighboring acid or from NHS. The gem-
intramolecular neighboring acid is much faster than any other dialkyl groups in PMAA demonstrated the typical Thorpe-Ingold
pathways for derivatisation of O-acylisourea; while for PAA, the effect in the EDC/NHS activation, that a little bit shorter distance
neighboring acid distance should be a little bit longer than that in between neighboring acid groups brought by the gem-dialkyl
PMAA, its dominant product is NHS-ester but not anhydride, due to groups in PMAA resulted in the faster nucleophilic attack on O-
the faster nucleophilic attack on O-acylisourea from NHS rather acylisourea from its neighboring acid and therefore generated
than from a neighboring acid.4 Does the Thorpe-Ingold effect also anhydride. Whereas for PAA, the nucleophilic attack on O-
play a role for small molecules in the EDC/NHS activation reaction, acylisourea from NHS was faster than its neighboring acid, thus the
with analoguous structures to the MAA dimer in PMAA? We ran the dominant EDC/NHS activation product was NHS-ester.4b It is also
EDC/NHS reactions for 4 available dicarboxylic acids: glutaric acid, reasonable to deduce that the 3rd conversion pathway of O-
2,2-dimethyl glutaric acid, succinic acid, and 2,2-dimethyl succinic acylisourea to a stable side product of N-acyl urea by intramolecular
acid. The di-NHS-ester products (GA-NHS13 and DMGA-NHS, their IR, rearrangement and the 4th pathway of hydrolysis of O-acylisourea
1H-NMR and MS are in Figs. S1-5 of ESI) were easily obtained for by water attack would be slower than the 1st and 2nd pathways. To
glutaric acid and 2,2-dimethyl glutaric acid, as white precipitates in precisely measure and distinguish the in-situ kinetics of formation
Fig. S1; whereas the di-NHS-ester products were not observed for of NHS-ester, or anhydride in molecular level is beyond the scope of
succinic acid and 2,2-dimethyl succinic acid, the reaction solutions our experimental skills. However we adapted a very rough analysis
were clear and the acids remained unreacted. Why do the similar approach to demonstrate their kinetic difference at the macroscale
dicarboxylic acids exhibit such different products? From our by following the precipitation rate of PMAA anhydride and the one
understanding on the EDC/NHS activation of PMAA and PAA, we of PAA NHS-ester with measurement of the optical density (OD) at
give our interpretation here to coordinate the observed 600 nm in Figs. S6 and S7. We observed that PMAA anhydride
phenomena together: The distance between two end carboxylic precipitated immediately after the addition of EDC to the reaction
acids in glutaric acid and 2,2-dimethyl glutaric acid is a little bit solution of PMAA and NHS, and the optical density of the solution
longer than in succinic acid and 2,2-dimethyl succinic acid, once an achieved saturation of 0.8 OD at 240 sec (or 4 min). It was very
O-acylisourea is formed, the nucleophilic attack competition likely that the OD saturation meant the completion of the reaction.
between NHS and the neighboring acid on O-acylisourea While for the precipitation of PAA NHS-ester, it started to
determines the final product. For glutaric acid and 2,2-dimethyl precipitate at 120 sec (or 2 min) after the addition of EDC, and the
glutaric acid, the nucleophilic attack from NHS is faster than from its precipitation became slower after 360 sec (or 6 min) and still
neighboring acid, therefore di-NHS-ester products are formed easily continued very slowly until our measurement time window of 1000
with high yields. While for succinic acid and 2,2-dimethyl succinic sec (or ~17 min). All the above experimental results, including the
acid, the nucleophilic attack from its neighboring acid is faster than precipitation rate measurements, render us to suggest that the
from NHS due to the Thorpe-Ingold effect, therefore the five- formation of either anhydride or NHS-ester depends on the
membered ring anhydride, succinic or 2,2-dimethyl succinic nucleophilic attack kinetics on O-acylisourea from the
anhydride, forms primarily. However both succinic anhydride and intramolecular neighboring acid or from the intermolecular NHS, in
2,2-dimethyl succinic anhydride are so vulnerable in aqueous media, the molecular level. Therefore, except for the succinic acid and
they will be hydrolysed to acids immediately. Both O-acylisourea gem-dialkyl glutaric acid structures which can be affected by the
and succinic anhydrides can react with NHS thermodynamically, Thorpe-Ingold effect obviously, all other alkyl acids with a distance
however the kinetic probability of the NHS attack cannot compete between two adjacent acids longer than glutaric acid will form the
with its competitors. Therefore the formation and hydrolysis of dominant intermediate product of NHS-esters.
succinic anhydrides during the EDC/NHS activation forms a cycle. By
the end, the only consumed reactants are EDC and water, which Conclusion
follows the 4th pathway as described in Introduction: O-acylisourea
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1995, 6, 123. (c) B. M. Prentice, J. D. Gilbert, J. R. Stutzman,
W. P. Forrest, S. A. McLuckey, J. Am. Soc. Mass Spectrom.
2013, 24, 30.
In conclusion, for the irregular EDC/NHS activation products of
PMAA, we observed the waxing of NHS-ester and the waning of
anhydride with increasing the fragmentation degree of PMAA
blocks in PMAA-associated polymer blends. It is the delicate kinetics
and thermodynamics in the molecular level that bring the
complicated production results in the macroscale world, such as
variable products and yields for different kinds of acids. Our finding
of the different EDC/NHS activation products between PMAA and
PAA and the following understanding of the Thorpe-Ingold effect on
the EDC/NHS activation of PMAA rendered us to explain the
complementary waxing of NHS-ester versus the waning of
anhydride phenomena clearly. Although the Thorpe-Ingold effect is
the key to interpret the observed phenomena, steric hindrance
should also be included, especially for the small part of NHS-ester in
the pure PMAA, and for nontrivial NHS-esters in PMAA oligomers
such as dimer, trimer, tetramer, etc. From our above speculations
about the EDC/NHS activation kinetics, we would suggest some
modifications for the well-known EDC/NHS reaction recipes used
widely. For example, the molar ratios of EDC/NHS/acid = 1.2:1.2:1,
in general reaction conditions, are commonly accepted, whereas we
suggest to add more additives of NHS, i.e. 2 to 5 or even more folds
to acid, in the reaction to obtain NHS-ester with higher yield, since
more NHS molecules will increase the probability of their
nucleophilic attack on O-acylisourea. Since the EDC/NHS activation
of PMAA is so popularly used for biomolecular immobilization and
conjugation in biomaterials, pharmaceuticals, immunology, and
food science, such a clear understanding of the reaction mechanism
suggests how to load more biomolecules while save money. Only
EDC and not any other additives such as NHS or HOBt is needed at
the first reaction to activate PMAA and synthesize amide linkages,
and then reiterating the EDC/NHS activation and amidation on the
product from the first round will increase the amidation efficiency
dramatically. We believe more precise experimental measurements
and theoretical studies of the EDC/NHS acitivation mechanisms,
both in kinetics and thermodynamics, will help to resolve the
ambiguity of the carbodiimide/additive coupling reaction for amide
bond formation better.
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Acknowledgement
This work was supported by the financial support of the National
Basic Research Program of China, No. 2013CB922101, the Natural
Science Foundation of Jiangsu Province, No. BK20130054, and
funding from the State Key Laboratory of Bioelectronics of
Southeast University.
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